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Thushara Sivasankaran Nair

School of Agricultural Science

University of Tasmania

Hobart

October, 2006

UNIVERSITY OF TASMANIA

(Preliminaries) , ..,.,, ,-,G,,*,..,?.*G ? ".?,?,.<,.,<Wj 7. < ,., . ZV,,T<.> ,.-.,,*,. e<"<,'-,a,,,.'. ..,/*>m2.%~,*-4.-.<>;<6..;$><,'~ ? ~ 2 ~ ~ . , , , ~ f l ~ @ , : * ' . ' ? ? . < ~ ~ . ~ ~ ~ J 8 . ~ 5 ~ < ~ ~ ~ , , : ~ * ~ ~ f l , ~ ~ ~ : . ~ n , ~ a , ~ ~ ~ : ~ 7 - - / f l ~ ~ , ' < ~ , ~ ~ ~ ~ & ~ ~ , d

Declaration

I certify that this thesis contains no material which has been accepted for the

award of any other degree or diploma in any other University, and to the best of

my knowledge and belief this material contains no material previously published

or written by another person, except where due reference is made in the text of

the thesis.

Authority of Access

This thesis may be made available for loan and limited copying in accordance

with the Copyright Act 1968.

Thushara Sivasankaran Nair

School Agricultural Science

University of Tasmania

(Prelim in a ries) , /:<~,R,~~.z:>;. z ,7.,..,c. ~ . ~ ~ . ~ ; m 7 ~ , ~ ~ ~ , ~ 4 z . ~ , . : ~ > * ~ ~ , , r ~ ~ ~ ~ : . ' : , ~ . : c - . ~ ~ ~ : .,..* v-<:?.;<?~,%,*:5:?/,?P,,..,z.,,; * , " ! ~ ~ . . * , 7 ~ ~ ~ , # ~ , w , f l < : . ~ ~ < ~ ~ . ' r > ~ / k ~ ~ , . v / 9 . ~ " 0 < ~ ~ .

Table of Contents

Declaration

List of Figures

List of Plates and Tables

Acknowledgement

Abbreviations and Symbols

Species Name

Abstract

Chapter-1 General Introduction

Chapter-2 Literature Review

1. Introduction

1.1. Eucalyptus taxonomy and distribution

1.2. Eucalyptus globulus

1.3. Economic importance of E. globulus

1.4. Extent and location of E. globulus plantations

1.5. Breeding activity in E. globulus

1.6. Nursery seed production

1.7. Orchard seed description

2. Seed structure

2.1. Eucalyptus h i t and seed structure

3. Seed Quality

3.1. Seed germination and type of germination in Eucalypts

... Vll l

xii

... Xl l l

xvii

3.2. Seed vigour 15

3.2.1. Factors affecting germination and vigour 15

i. Temperature 16

ii. Light 17

iii; Moisture 18

iv. Abscisic acid (Growth inhibitor) 19

v. Age of seeds 19

3.3. Seed size 20

4. Seed Dormancy 2 1

4.1. Dormancy and its Ecological Role 2 1

4.2. Types of Dormancy 2 1

4.2.1. Primary dormancy 2 1

I. Types of Primary dormancy

i. Exogenous dormancy 2 1

ii. Endogenous dormancy 23

11. Dormancy in Eucalyptus spp. 25

4.2.2. Secondary dormancy 26

I. Environmental factors regulating secondary dormancy

i. Temperature

ii. Light and dark

iii. Water

iv. Gases

5. Methods of Improving Germination and Overcoming Dormancy

5.1. Stratification

5.2. Priming

5.3. Wetting and Drying Cycles

5.4. Smoke

5.5. Growth Hormones and other Compounds

6. Conclusion

Chapter-3 General Materials and Methods

3.1. Seedlot

(Preliminaries) i ~ . : i.. 'i.%.'.r:r *'.&LC'L;.' ' li:': :-'.*. .%r~:r*'~*. ,*' i ,~ ' i ' ' e,:"r 'p"-. A ,,v-:~~..T~I,.P.Y '~~:~ir~-i;r;r~xXi~.~~~:ir~~~~~~,r,,r,,~~~6)r6)r~~,~,~~~~n,~~~,~p~~~~~ % . I P / E , ~ F , ~ ~

3.2. Germination Conditions

3.3. Experimental Design and Statistical Analysis

Chapter-4 Water Relations in E. globulus Seed

4.1. Introduction

4.2. Materials and Methods

4.3. Results

4.4. Discussion

Chapter-5 Induction of Secondary dormancy (Dehydration and Light

Effects) 5 7

5.1. Introduction 57

5.2. Materials and Methods 5 9

5.3. Results 6 1

5.4. Discussion 69

Chapter-6 Light effects

6.1. Introduction

6.2. Materials and Methods

6.3. Results

6.4. Discussion

Chapter-7 Light Quality

7.1. Introduction

7.2. Materials and Methods

7.3. Results

7.4. Discussion

Chapter-8 General Discussion

8.1. Practical solutions

8.2. Physiological aspects

8.3. Conclusion

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Chapter-9 References

Chapter 10 Appendices

(Prelim in aries) .... ....... ....... .. < :, .: :. *.,*z .,.,.n ;7,< ..,;<,..*c,.. L....," .! <-. :*:.&,..,x .: .,;?,,*-,e>>,.r.>.<:,:v#?.;?..v,< ',2*,,fl. ~*~2~;,.7<%~~,P7~~e,"r/-~~~~w1/~~z~>'Km.~~:~?/e~~~~~

List of Figures

Fig-4.1: Water uptake with time after the start of imbibition on wet filter

paper. Data points are results of 4 replicates of 50 seeds * standard

error of the mean. ...................................................................................... 50

Fig-4.2: Water uptake with time after the start of imbibition by seedlot

"OS" in full light (A) and complete darkness (m). Data points are

results of 4 replicates of 50 seeds k standard error of the mean. .......... 51

Fig 4.3: Seed water potential with time after the start of imbibition on wet

filter paper. Data points are results of 4 replicates of 50 seeds * standard error of mean. ............................................................................ 52

Fig 4.4: Water uptake as relative water content (a) and water potential (b)

of untreated ( A ) and 30 h imbibed followed by 7 days drying (m) shown

in this figure. Data points are results of 4 replicates of 50 seeds * ...................................................................... standard error of the mean. 53

Fig 5.1 (a & b): Mean germination percentage on day 5 (o), 8 (0) and 10

(A) for seedlots 'h' (a) and 'Li' (b), germinated after soaking in

laboratory conditions at 18-20 "C for 0-48 h (1618 h of natural light/

dark cycle). Data points are results of 4 replicates of 50 seeds * standard error of the mean. ...................................................................... 63

Fig. 5.2. Mean germination percentage on days 5 ( 0 ) and 11 (0) for seed lot

'h' (a) and 'Li' (b), germinated after different periods of dark soaking

at 25 f 1 "C followed by 7 days drying back at room temperature (18-20

"C) in natural light conditions. Data points are results of 4 replicates of

50 seeds f standard error of the mean. LSDs for comparison between

soaking times on day 5 were 10.3 and 12 respectively and for day 11,

11.1 and 13.7 respectively .......................................................................... 64

Fig. 5.3: Germination in alternating lightldark (a) and darkness (b) on days

6 ( 0 , +), 10 (0, H), 13 (A, A) and 19 (0, 0 ) for seedlot 'KI' imbibed

for different time periods followed by drying back for 7 days at 18-20

"C in natural light conditions. Data points are results of 4 replicates of

50 seeds +_ standard error of the mean. .................................................... 65

(Prelim in aries) z.,,‘,* .: ..:. "< .#",:::,' L.,. ....$‘..y<%‘.<.,.?...>. :. ;r,4w,> :'......&.....# #>. , . ? Z ~ - , P ~ ~ . $ . : X T ~ ~ Z & ' >?..<..<s . ~ % ~ ~ m ~ ~ , ~ w ~ ~ w ~ w 4 ~ m ~ ~ / ~ ~ ~ 4 ~ < # 7 ~ ~ ~ ‘ %

Fig. 5.4. Mean germination (%) in alternating lightidark (a) and darkness

(b) on days 6 (0, +), 10 (0, m), 13 (A, A) and 19 (0, a) for seedlot 'h'

imbibed for different time periods followed by drying back for 7 days

at 18-20 "C in natural light conditions. Data points are results of 4

.............................. replicates of 50 seeds f standard error of the mean. 66

Fig 5.5: Glasshouse germination on days 13, 15 and 20 for seed of seedlot

'Ki' imbibed in darkness for 0 or 30 h at 25 f 1 "C and dried back and

stored for 7 days at room temperature (18-20 "C). Germination was in

alternating natural light and dark (0) or continuous dark conditions

(m) Error bars are standard errors of the mean (n=5) .......................... 67

Fig 6.3 (a & b): Germination in response to initial imbibition in darkness for

seedlot 'A' (a) and 'H' (b). Seed was imbibed for different periods of

complete darkness and transferred to light. Germination counts were

on day 5(.) and day 11 (A). Two controls were full light and constant

dark (not shown in the figure). Data points are results of 4 replicates of

50 seeds h standard error of the mean. .................................................... 80

Fig 6.4 (a & b): Germination in response to initial imbibition in light for

seed lot 'KI-1' (a) and 'H' (b). Seed was imbibed for different period of

light and transferred to constant darkness. Germination counts were

done on day 9 (0) and day 14 (A). Data points are results of 4 replicates

of 50 seeds h standard error of the means. .............................................. 81

Fig 10.1: Spectral composition of Incubator or germination cabinet (Linder

and May Model LMRIL-1-SD) lights. ................................................... 140

Fig 10.2: Light emission from the 10,000MCD Red light peaking at 625 nm.

................................................................................................................... 141

Fig 10.3: Light emission from 7000MCD White 5 mm diameter dome ...... 142

Fig 10.4: Light emission from 5000MCD Blue 5 mm diameter dome peaking

at 470 nm ................................................................................................... 143

Fig 10.5: Light emission from Far red 5 mm diameter dome peaking at 740

nm. ............................................................................................................ 144

List of Plates and Tables

Plate 1.1: Eucalyptus globulus seeds germinate uniformly and are highly

viable. Photograph shows Lannen 8 type trays, generally used in

commercial nurseries ................................................................................... 2

Plate 1.2: A Lanneno tray from a commercial nursery showing uneven

germination 5-6 weeks after sowing. .......................................................... 3

Plate 1.3: Seedling trays in a shade house. Trays in the foreground show

uneven germination evident about 4 weeks after sowing ......................... 5

Table 3.1: Details of Commercial Seedlots supplied ....................................... 37

Table 3.2: Seedlot performance test (n=4) ....................................................... 38

Plate 5.1: Photograph showing germination in glasshouse conditions.

Strongest germination was in control (untreated) seed germinated in

darkness (second from left). Treatments are D (dark germination), LID

(germination in natural light), 0 is non preimbibed seed and 30 is seed

imbibed for 30 h and dried back for 7 days before sowing .................... 68

Table 6.1 (a & b): Influence of light condition during germination on %

germination after 7 days in seedlots 'A' and 'H'. Pre-treated seed was

imbibed for 30 h in complete darkness followed by dehydration for 7

days (n=4). The control seed was not pre-treated. LSD (P=0.05) for

comparison within the table for seedlot A is 11.0 with a significant

interaction between light and pre-treatments. For seedlot 'H', there

was a significant light effect (see text) but no interaction between light

.and pre-treatment, LSD.(P=0.05) is 13. By day 10 there was no

significant treatment effects in both seedlots. ......................................... 77

Table 6.5: The effect of duration of initial light exposure on mean

germination percentage of seedlot 'h' on days 6,8 and 9 (n=4). ........... 82

Table 7.1: The effect of light quality on germination of seeds from seedlot

'KI-2' determined after 50 h (7.1 a) and 5 days (7.1 b) (n=4) ................ 97

Table 7.2: The effect of light quality following imbibition in constant

darkness for 24 h on day 5 germination percentage of seed from seedlot

'KI-2' (a) and 'H' (b) (n=4) ....................................................................... 98

4% r.,RC: .,,..; iv~. ,wi i . .Yi . ,~ ,e% ., v i , , z , ~ ~ ~ ~ ~ : ~ ~ ~ ~ - ~ ~ ~ i ~ , , ~ ~ , , , L ~ ~ ~ ~ ~ ~ , ; . ~ ~ ~ . ~ ~ r ~ a ~ . c ~ ~ ; r ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ , ~ ~ . ~ ~ ~ ~ ~ ~ . ~ . ~ f i ~ ~ . ~ ~ ~ ~ ~ ~ 1 " ~ i i o ~ , . ~ n n ~ ~ ~ ~ i d ~ b " 1 0 ~ i r / ~ ~ ~ ~ 6 ~ / * ~ / * / d ~ W ~ J I ~ 1 ~ . ~ r c d / ~ ~

Vlll

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Table 7.3: The effect of light quality following imbibition in constant

germination cabinet light for 24 h on day 5 germination of seeds from

seedlot 'KI-2'. Germination result for seeds exposed to constant

darkness and constant cabinet light from the start of the imbibition is

................................................................ included for comparison (n=4). 99

Table 7.4 (a&b): The effects of light quality during the first 24 h of

imbibition on subsequent germination of seed from seedlot 'KI-2'

under constant cabinet light or constant darkness. Germination

percentages were determined 5 days after the start of imbibition (n=4).

................................................................................................................... 100

Table 7.5 The effect of addition of 5 x 1 0 ~ M MA3 at the start of imbibition

and light quality on germination of seed from seedlot 'KI-2' (n=4). ..I01

Table 7.6 (a &b). The effect of light quality during the first 24 h of

imbibition and time of application of 5 x 1 0 - h MA3 on day 5

germination of seed from seedlot 'KI-2' (n=4) ...................................... 102

Table 7.7: The effect of light quality during the first 24 h of imbibition on

day 5 germination of seed from seedlot CKI-2' treated with 5x10-%

GA3 at the start of imbibition. Seeds were subsequently germinated in

constant darkness (n=4). ......................................................................... 103

Table: 7.8: The effects of light quality on day 5 germination of seeds from

seedlot 'KI-2' after imbibition in darkness for 24 h followed by addition

of 5x 10" M GA3 (n=4). .......................................................................... 104

Table 7.9: The effects of one or two 30-minute pulses of red light during the

first 24 h of imbibition in constant darkness on subsequent germination

of seed from seedlots 'KI-2' and 'h' under constant cabinet light. First

treatment is constant cabinet light. Germination was scored 5 days

from start of imbibition (n=4) ................................................................. 105

Table 7.10: The effects of one or two 30 minute pulses of red light during the

first 24 h of imbibition in constant darkness on subsequent germination

of seed from seedlot h under 12/12 lightldark period. Germination was

assessed on day 5 (n=4) ......................................................................... 106

... ? A ? ..:,,,.;.7 4 >$..X,.,C,.p!.'. .- ,/.,h.. ~~,;,7,,%,:Fe?,,*..::;';~,?,,k.,?:,.!,:~ <,,,* ~ , ~ ~ ~ ~ ~ : , > - ~ ' 7 . ~ ~ ~ ~ ~ , ~ ~ ' < ~ ~ ~ , , ~ ~ . ~ ~ , w . ' < ~ ! ~ . , p . ~ - , ~ ' v ~ , (Preliminaries)

1.. ~~J,,V/N/fl~flzW%V#/~>:~WlJ'AN/JI.U,ICI~ii,12:CC:';T~

Table 7.11: The effect of GA3 on germination of seed of seedlot 'h' exposed

to different light qualities (red, far red, darkness or cabinet light) for

the first 30 h of germination. 5x10" M GA3 was added at 30 h ("-"=

without GA3 and "+"= with GA3). With the exception of the continuous

cabinet light treatment, seeds were subsequently germinated under

constant darkness. Germination counts were done on day 7 (n=4). ... 107

(Preliminaries) ,. ' ,: *,' ',. -:,*, ' ..,,.:; ,..d r,.::..> v. .":- 9 7,* $Z, -'+7F'.-r/,-,,* <. , ,, .*.a ,~,4..>V,A?<W.Z~,*,~..> ~ : * ; ~ . ~ . ~ , : ~ , # ~ ~ , : , ~ ~ ~ ; < ~ ~ , " / ~ . ~ > ~ . ~ ' < < ! ~ : q , , ~ , p , , , ~ , ~ ! ~ , , ; ~ ~ p ~ 7 , ~ L

Acknowledgement

This thesis is a result of three years of hard work where I was supported by many

people and now I have the opportunity to express my gratitude to everyone.

I am deeply indebted to my supervisor Dr. Steve Wilson under whom I started

working on this project as well as I did my Postgraduate Honours. His help,

stimulating suggestions and encouragement helped me in all the time of research

and writing this thesis.

I would like to thank my co supervisor Dr. Cameron Spurr who kept an eye on

the progress of my work and was always available when I needed his advice.

I would like to thank Mr. Peter Gore and Dr. David Boomsma (Seed Energy) for

coming up with this project, financing it and supplying seeds for research. I

would like to thank Dr. Dean Williams and Mr. John Juhasz for contributing

valuable ideas in the beginning of this project.

I would like to thanks the University of Tasmania for supporting my studies and

granting an International Postgraduate Research Scholarship.

I would like to acknowledge Dr. Sarah Salardini, Mr. Bill Peterson, Ms. Angela

Richardson, Mr. Phil Andrews, Mr. Ian Cummings, Dr. Yuda Hariadi, Dr.

Chnstiane Smethurst, Ms. Leng Cover, Ms. Ifa Ridwan, Ms. Jiayin Pang, Mr.

Bhim Khatri, Mr. Sriram Padmanabhan, St. Michael's Collegiate Boarding

House staff members and many others who directly or indirectly supported me at

some point of my research.

My special gratitude towards my parents, my sister and brother in law and other

members of my family for their loving support.

(Prelim in aries) .< fi 1,' '' C.i , 2: I(",+'; -.,*'~..c,~,;A,~:,'i:i:i:'i:i:i:i:i. ii'i8r,.:,il+.il(i ,?:.?L.t'>..;?2r+C?&'r'r.Y PP 1:."1+1~~5;"0:~:d~~Y11~"~~I.~.~~:I"/d1iiiYA.W/~~BBkV'I~VaV~G~~~~~XI~W/BX~~~~.C%Yi41/fl/r

Abbreviation and Symbols

ABA

ACC

ANOVA

ATP

O c

Ca(N03)2

cm 3

co2 cm

DNA

ETH

FR

GA3

I A A

ISTA

KC1

KIN

KN03

KOH

K3P04

LSD

mm

mM

MPa

mRNA

Abscisic acid

1 -aminocyclopropane- 1 -carboxylic acid

Analysis of variance

Adenine tri phosphate

degrees Celsius

Calcium nitrate

Cubic Centimetre

Carbon dioxide

Centimetre

Deoxyribonucleic acid

Ethanol

Far red light

Gibberellic acid

Gram

Hours

Hectare

Indole Acetic Acid

International Seed Testing Association

Potassium Chloride

Kinetin

Potassium nitrate

Potassium hydroxide

Potassium phosphate

Least Significant Difference

Metre

Milli Litre

Milli Metre

Milli Molar

Megapascal

Messenger Ribonucleic acid

(Prelim in aries) > .,.>.:. ,;.,:.. ,,,,,, .: . . ,w ;,. .,<:,:,;2.,. .. -', .,..! ., K,' .,,,,.' ..., ;,r.'<, ,<-,.2.2- <.,,v<*%,, al-<;<~*.$,,,..-.$+, :?j <v,fl,x;-:<,Tx7..> ~ r ~ ~ ~ ~ ~ & ~ ~ ~ ? ~ ~ ; . ; ~ ? ~ , ~ ~ ~ : ~ ~ , m ~ ~ L 7 , u , ~ ~ ~ ~ w , a ~ a ~ 4 z : l ~

MSP

NaN03

nm

ODs

OSP

P

PEG

P fr

PIL

R

RNA

RWC

S

SEM

SPSS

STBA

STDEV

%

@

PM

I.lm

Mass Supplementary Pollination

Sodium nitrate

Nano Metre

oleoyl phosphatidal choline desaturase

One Stop Pollination

Probability

Polyethylene glycol

Far red absorbing (active) phytochrome

Private Limited

Red light

Ribonucleic acid

Relative water content

South

Standard Error of the Mean

Statistical Package for the Social Science

Southern Tree Breeding Association Inc.

Standard deviation

Percentage

Registered

Micro Molar

Micro Metre

Species Name

Common Names

Allepo pine

Alpine ash

Alpine snowgum

Ana tree

Annual ragweed

Apple

Areca palm

Barley

Beard-heath

Black box

Blackbutts

Black ironbark

Black nightshade

Blue-white clover

Brindall berry1 Malabar Tamarind

Broadleaf dock

Broad leaf ironbark

Broad-leaved peppermint

Brown barrel1 Cut tail

Bull mallee

Bull thistle

Burning bush

Cabbage gum

Calabrian pine

Capeweed

Celery

Charlock mustard

Cloeziana gum

Scientific Names

Pinus halepensis

Eucalyptus delegatensis

Eucalyptus nimphophila

Faidherbia albida

Ambrosia artemisiifolia

Malus domestica

Dypsis lutescens

Hordeum spp.

Leucopogon spp.

Eucalyptus largzflorens

Eucalyptus pilularis

Eucalyptus sideroxylon

Solanum physalifolium

Trigonella coerulea

Garcinia gummi-gutta

Rumex obtusfolius

EucalyptusJibrosa

Eucalyptus dives

Eucalyptus fastigata

Eucalyptus behriana

Cirsium vulgare (Savi) Ten.)

Dictamnus spp.

Eucalyptus pauczjlora

Pinus brutia

Arctotheca calendula

Apium graveolens

Sinapsis awensis

Eucalyptus cloeziana

, :.r\ +" , . *;..$><,*;:* ' *~. '* :4 , z#*~ , , s>~ , ~.'~,"*.~~.~8.w~~".<~~~:~~~~'~~.:~~~r. V' *" ,-: 4. >-''%: ::: : : , ~ , ~ > , " - , ~ ~ ~ : * . , ~ ~ ~ . w ~ a ~ ? ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ' w ~ : r . 9 ? ' ~ ; , ' 9 ~ ~ ~ P ' R M;;x.-&:$'~.4.*,&v@. r, Xll l

Cocklebur

Coolibah tree

Common elderberry

Common oat

Common peppermint

CornIShirley poppy

Cucumber

Empress tree

European beech

Evergreen clematis

Forest redgum

Giant-flowered phacelia

Gippsland blue gum

Good king Henry

Grevilled silky oak

Grex box

Guinea flower

Gum cistus

Hedge mustard

Iceberglprickly lettuce

Indonesian gum

Ivory lace

Jarrahl Swan river mahogany

Jointed rush

Knotweed

Kybean mallee ash

Lacy scorpion weed

Maize

Maritime pine

Meadowfoam

Mongolian Psammichloa

Xanthium strumarium

Eucalyptus microtheca

Sambucus canadensis

Avena sativa

Eucalyptus radiata

Papaver rhoeas

Cucumis sativus

Paulownia tomentosa

Fagus sylvatica

Clematis vitalba

Eucalyptus tereticornis

Phacelia grandiJlora

Eucalyptus globulus

subsp. pseudoglobulus

Chenopodium bonus-henricus

Grevillea robusta

Eucalyptus moluccana

Hibbertia amplexicaulis

Cistus ladanifer

Sisymbrium officinale

Lactuca sativa

Eucalyptus deglupta

Thryptomene calycina

Eucalyptus marginata

Juncus articularis

Polygonum species

Eucalyptus kybeanensis

Phacelia tanacetifolia

Zea mays

Pinus pinaster

Limnanthes alba

Psammochloa villosa

(Preliminaries) ;'.S-.dY>Y, , '*,%..+:. 7 @',;;.. P,& ,!w,::G,::# v,,:fc<L-,a & , " ~ . ~ , ~ ~ r ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ ~ , ~ : ~ ~ ~ ~ ~ ~ . . ~ ~ ~ ~ : ; ~ ~ P . ~ o ~ / ~ " ~ ~ . ~ ~ ~ ~ , . w ~ ~ ~ ~ ~ ~ w / 4 ~ ~ ~ ~ ~ ~ ' ~ : ~ ~ , ~ ~ ~ L ~ , ~ ~ ~ ~ ~ / ~ ~ m / ~ ~ ~ ~ ~ L ~ ~ P 4

Mottlecah Eucalyptus macrocarpa

Mountain ash Eucalyptus regnans

Mount buffalo salleel gum Eucalyptus mitchelliana

Mt. Wellington peppermint Eucalyptus coccifera

Narrowleaf plantan Plantago lanceolata

Northern bush honeysuckle Diervilla lonicera

Norway maple Acer platanoides

Oilseed rape

Parodi Burkart

Peach

Pennygrass

Pitcher plant

Poppy Anemone

Proliferating bulrush

Purple coneflower/ Echinacea

Redroot pigweed

Red bloodwood

Red-capped gum

Red elderberry

Red mallee

Red river gum

Red stringybark

River peppermint

Round leaf snow gum

Ryegrass

Salmon gum

Shining gum

Silver maple

Silver top ash

Small balsam

Sorghum

South American rush

Brassica napus

Caesalpinia paraguariensis

Prunus persica

Thlaspi awense

Sarracenia spp.

Anemone coronaria

Isolepis prolifera

Echinacea purpurea

Amaranthus retroflexus

Eucalyptus gummifera

Eucalyptus erythrocorys

Sambucus pubens

Eucalyptus oleosa

Eucalyptus camaldulensis

Eucalyptus macrorhyncha

Eucalyptus andreana

Eucalyptus perriniana

Lolium perrene

Eucalyptus salmonophloia

Eucalyptus nitens

Acer saccharinum

Eucalyptus sieberi

Impatiens parvijlora

Sorghum bicolor

Juncus microcephalus

(Prelim in aries) : , . : , , . , , : v : ; .Oi<Sr l c . . ' ~ * , ; . ~ ~ , ~ P ~ ~ , ~ p l , p l C C : # j , n Y ~ 7 W ~ % r Z f i W r i c r i c ~ ~~:I",~~I/I/W/~~/PI:~~~~W/#/B'B'B'*!?

Soybean

Spotted blue gum

Spotted gum

Spotted ladysthumb

Sticky hopbush

Striped maple

Sugar maple

Swamp gum

Swamp yate

Swift parrots

Sycamore leafed maple

Tartarian maple

Tasmania blue gum

Tassel flower

Thalla-cress

Tomato

Tropical red box

Triggerplants

Victoria blue gum

Wheat

Whispering bells

White gum

Wild lettuce

Wild oat

Winter fat

Yellow box

Yellow buttercup

Yellow horn poppy

York gum

Glycine max

Eucalyptus globulus subsp. maidenii

Eucalyptus maculata

Polygonum persicaria

Dodonaea viscosa

Acer pensylvanicum

Acer saccharum

Eucalyptus ovata

Eucalyptus occidentalis

Lathamus discolor

Acer pseudoplatanus

Acer tartaricum

Eucalyptus globulus subsp. globulus

Amaranthus caudatus

Arabidopsis spp.

Lycopersicon esculentum

Eucalyptus brachyandra

Stylidium afJine

Stylidium brunonianum

Eucalyptus globulus subsp. bicostata

Triticum aestivum

Emmenanthe penduliflora

Eucalyptus wandoo

Lactuca serriola

Avena fatua

Eurotia lanata

Eucalyptus melliodora

Hibbertia hypericoides

Glaucium flavum

Eucalyptus loxophleba

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ABSTRACT

Eucalyptus globulus is a major plantation forestry species in southern Australia.

Commercial E. globulus seedlots have high viability (95%) and uniform

germination, which is completed within 5-6 days. However commercial nurseries

have reported delays in germination with seedling emergence spread over five to

ten weeks. According to growers and seed suppliers the problem is not restricted

to particular seedlots and occurs in most nurseries. Observations have suggested

that a brief period of desiccation or high temperature between sowing and

germination may lead to the delays, but no clear cause has been established.

Further, there is nothing in the literature to indicate either cause or possible

management strategies. According to the ISTA guidelines the germination period

of E. globulus is 5-14 days under laboratory conditions and in the field,

germination should occur before 26 days. The aim of the present project was to

establish the factors responsible for this apparent induction of previously

unreported secondary dormancy in E. globulus seed and to provide a basis for

commercial management of the problem.

Initial laboratory trials concentrated on grower reports that short term desiccation

in the first few days after sowing might be responsible. A wettingdrying cycle

was found to cause an extended germination period. Initial imbibition in

darkness for periods of 18 - 36 h, followed by air drying in open storage for 7

days at 2S°C reduced subsequent germination in 12 light1 12 dark photoperiod

and slowed germination rate. Subsequent experiments demonstrated that this

effect was largely regulated by light.

As a result of the initial results, further investigations into light sensitivity in E

globulus seeds were undertaken. Seeds initially imbibed in darkness for 18 - 36 h

and then exposed to light showed decreased germination in darkness compared

with seeds exposed throughout to continuous light, continuous dark or

continuous light during imbibition followed by germination in darkness. These

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results clearly indicate that initial exposure to darkness for 18 - 36 h followed by

light caused a shift in germination behaviour.

Subsequent experiments on light quality showed that exposure to far red light

throughout imbibition and germination substantially reduced germination

percentage, whilst under some circumstances exposure to red light promoted

germination. Such a model appears to explain the commercial problem, with the

anomalous germination symptom induced by light conditions prevailing in the

hour following sowing.

Results are discussed in terms of a possible physiological basis for the induction

of an apparent light mediated secondary dormancy in this species. The practical

implications for commercial nurseries are also considered, with particular

emphasis on light management, rather than the initial ideas on water relations.

.. , .. . , . .. ,. . rJ .:.7u., .e...K-,,7., ;,by r,.s: .v:.Y,:. .px,xG,,, :rd, . <-., z~, .~7.-A-.92~:.s,~7x.~; .~ . r ~ ~ ~ ~ ~ ~ ~ . v x + ~ L ~ 7 . ~ . r ~ ~ p : Chapter-1 ~ 7 m ~ o . ~ g . ~ 7 ~ ~ 7 ~ ~ ~ ~ ~ , x m , n , ~ , ~ ~ ~ ~ m 4 ~ ~ ~ m m ~ ~ ~ ~ ~ ~ ~ ~ (General Introduction)

Chapter-I

General Introduction

Eucalyptus globulus Labill. is a major plantation forestry species in southern

Australia. Majority of plantations are grown for pulp wood production. In

addition, E. globulus is increasingly planted for producing sawn timbers, veneers

and reconstituted wood products (Raymond and Apiolaza, 2004). Most

plantations are established using seedlings grown from genetically improved

seed. Commercially, there are three more or less independent steps in

establishment, with private tree breeding organizations providing seed to

commercial nurseries, who then sell 6 month old seedlings to forestry

companies.

Germination capacity of commercial seed is over 90%, there is no evidence of

dormancy and, generally, germination is highly uniform giving an even seedling

size for plantation establishment (Plate 1.1). However commercial nurseries have

reported that germination of some seedlots extends over a period of several

weeks (Plate 1.2). This leads to variable seedling size and grading is needed to

improve seedling uniformity, substantially increasing the cost of production.

Similarly Martin-Corder et al. (1998) reported that seed size varies in eucalypt

seedlots leading to heterogeneous populations of plants in nurseries even when

seeds are from genetically improved sources. Seed quality has been suggested as

a possible cause but suppliers report that the symptom is not associated with a

particular seedlot, its age or storage conditions, and (notably) the same seedlot

may perform differently in different nurseries. Some nurseries have also reported

differences in uniformity with separate plantings of the same seedlot.

Chapter-1 (General Introduction) r___., * ~ L ~ * ~ X w , s , ~ a m ~ L . , # . w , w , w ~ w , m , ~ . ~ r s s s w , w , m w . m , w . w . ~ w , m ~ s m n L w , w ~ ~ ~ * w ~ ~ . ~ ~ w ~ ~

Plate 1.1: Eucalyptus globulus seeds germinate uniformly and are

highly viable. Photograph shows Lannen C3 type trays, generally

used in commercial nurseries.

Chapter-1 (General Zntroduction) L I - I ~ . m ~ m ~ L ~ ~ ~ ~ s . ~ , L m ~ ~ s , ~ s , ~ s , m . ~ ~ , e . , ~ s ~ s , s . ~ ~ ~ * * * m * ~ w I ~ s ~ m

Plate 1.2: A LannenB tray from a commercial nursery showing

uneven germination 5-6 weeks after sowing.

,.&.. , ::- .- ., , , :,, .. ., c.v,.,;~.9+..,. p~l:a.:C:v,.y,,/c3.,si.67.:T W/.3:.F/6em x*lc:l )s,'d ; . i : a . ~ a ~ t ; ~ v m ~ ~ ; ~ ! ~ / J t . ~ ~ e ~ ~ ~ ~ ~ . m ~ m z r : r : a / ~ r C - r C - n n ; ~ ~ : r e r , l b : m j , ~ ~ . c ~ r 7 r 7 a ~ ~ ~ ~ d a a w ~ ~ ~ Chapter-1 (General Introduction)

Seed supplied to nurseries is normally graded into various size classes to

improve germination uniformity and is routinely tested before distribution. All

seedlots showing poor germination in nurseries had displayed high viability,

uniformity and vigour in laboratory based germination tests carried out under

ISTA guidelines for the species. In several cases where uniformity was poor in

nursery plantings, germination tests were repeated, confirming initial results.

Because of these seed testing results commercial operators have considered

germination conditions as a possible contributing factor and nursery management

practises and environmental conditions during and immediately after

germination have been investigated by one of the seed supply companies (Gore

pers. comrn. 2003). 3

Seed germination practices vary between nurseries and details were difficult to

obtain. Most sow in December-January to have seedlings ready for winter

planting and the following steps seem to apply fairly generally across all

nurseries.

Some nurseries sow fresh dry seed others may soak for 20 minutes and

seeds that float are discarded.

If necessary, seeds are surface dried and machine sown direct into

Lannan trays.

After sowing trays are stacked plastic wrapped and incubated in a

temperature controlled room, set at 20 to 25 OC.

Trays are uncovered after 3 to 4 days and transferred to benches in a

shade house (Plate-1.3) or outside.

Good quality seeds germinate in about 5 days under optimal conditions.

Some nurseries reported but did not detail a potassium nitrate priming treatment

and details of sowing depth, potting mix, and handling after sowing were not

available.

Chapter-1 (General Introduction) ~ ~ ~ , ~ ~ , s , ~ ~ s , ~ ~ ~ r . ~ r , s . s s ~ I X * . ~ , ~ s ~ , m r ~ , s , ~ ~ ~ s ~ ~ ~ , s , ~ ~ ~ ~ , m ~ ~ ~ . ~ m m ~ ~ , ~ . ~ r , n ~ ~ ~ s ~ ~

Plate 1.3: Seedling trays in a shade house. Trays in the foreground

show uneven germination evident about 4 weeks after sowing.

Chapter-1 (General introduction) '.+5 '-: '3 . ' r r:, ;,x -:,, C: z .! ..VA. l :<; :'. L'i ' I'm;"lt- ',P.%i&'.~1il:il:Ccx.L> .,I<?Lv ,,,~~@,:FdCfi.Yn;eP7 ~ ~ ~ ~ ~ ~ ~ ~ ~ Y Y ~ ~ Q / ~ < C I ~ ~ ~ ; ~ W ~ ~ ~ ~ ~ ~ R ~ A . A . M L ~ & ~ R : ~ ~ I R / ~ / E . K ~ D A

The present project investigated the cause and physiology of this apparent

delayed germination in E. globulus. It aimed to identify limiting factors in the

seed germination environment and to develop appropriate strategies for

commercial nurseries to improve uniformity of seed germination. An informal

survey of commercial nurseries and detailed discussions with the seed supply

company prior to the commencement of experiments failed to identify any

consistent links between expression of the symptom and seed lot or any

particular nursery or nursery activity. However, two possible lines of

investigation did emerge. Firstly, some breeding lines appeared less likely to

suffer uneven germination. Secondly, as noted above, some variation in

uniformity within the same planting in a larger nursery was reported. In the latter

case, Williams (pers. com 2003) noted that the problem seemed worse near the

end of travel of an overhead irrigation gantry. Investigations suggested reduced

water application on the affected cells.

As control of the problem through selective breeding is a long term prospect,

management conditions during germination, starting with a possible link with

water relations, were the focus of this study.

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Chapter-2

Literature Review

1. Introduction

1.1. Eucalyptus taxonomy and distribution

The genus Eucalyptus (Myrtaceae) contains almost 700 species, among which

many are still unnamed or unidentified (Williams and Brooker, 1997). Eucalypts

cover a wide range of geographic and environmental gradients and have the

ability to withstand a range of temperature and rainfall regmes. Hence they are

seen growing from tropical to subtropical to temperate regions. Eucalypts were

originally introduced to different parts of the world due to their economic

importance and ornamental purposes. In Australia, Eucalypts dominate forests

and woodlands of the coastal regions and temperate cooler regions of Southern

Australia. They regenerate in sun light but not under shade (Stoneman and Dell,

1993).

The genus is divided into the monocalyptus and symphyomyrtus subgenera

(Cliffe, 1997). In Tasmania both monocalyptus (1 7 taxa) and symphyomyrtus

(1 2 taxa) are found (Pryor, 1976; Kantivilas, 1996). The difference between the

two subgenuses are that symphyomyrtus species have higher seed viability and

germinate earlier, and have a higher root to shoot ratio (Davidson and Reid,

1980) and faster early growth (Noble, 1989). The surface of seed of the

symphyomyrtus species is reticulate and the testa is formed from a single

integument. Seed of monocalyptus species has a smooth or irregularly sculptured

surface. The testa is formed from two integuments and the cotyledons are

emarginate (Kantivilas, 1996). Compared to monocalypts, symphyomyrts are

generally drought and flood tolerant, resistant to frost, especially under

waterlogged conditions, and more tolerant to saline conditions (Noble, 1989).

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Generally symphyomyrts species are better at adapting and surviving in diverse

environments (Anekonda et al., 1999).

1.2. Eucalyptus globulus

Eucalyptus globulus Labill or Tasmanian blue gum as it is known locally

belongs to syrnphyomyrtus subgenus (Young, 2002). It is a fast growing

hardwood tree and is the most widely cultivated Eucalypt throughout the world

(Groenendaal, 1983; Young, 2002). It has smooth brown coloured bark that peels

off in large pieces revealing a bluish white inner layer of bark. Juvenile foliage is

grey green, thick, smooth, waxy and rounded after germination but later on it

acquires an elliptical or narrowly lanceolate shape (Hall et al., 1970; Young,

2002). At maturity E. globulus can grow to 46 to 55 m tall with a diameter of 1.2

to 2.1 m (Bean and Russo, 2000). They produce grey, brown or black coloured

seeds (Boland et al., 1980).

E. globulus is mainly divided into four subspecies, globulus, bicostata,

pseudoglobulus and maidenii (Wang et al., 1988; Jordan et al., 1993; Dutkowski

and Potts, 1999). The subspecies are mainly identified by their reproductive

traits. Subspecies maidenii has seven fruits per umbel with small capsules.

Subspecies globulus has solitary flowers with the largest capsules. Subspecies

bicostata and psuedoglobulus are three fruited and pseudoglobulus has smaller

capsules with longer pedicels than bicostata (Jordan et al., 1993).

In Tasmania, the native range of E. globulus extends over 400,000 ha mainly in

the northeast and central east coasts and in the Midlands (Kirkpatrick and

Gilfedder, 1999). It is also distributed along the southeast coast and in small

areas of the west coast of Tasmania and the Bass Strait islands to the north of

Tasmania. It is also found in the Cape Otaway and Wilson's Promontory areas of

Victoria (Young, 2002). E. globulus are reserved at Maria Island and Freycinet

National Park for the conservation of swift parrots (Lathamus discolour)

(Kirkpatrick and Gilfedder, 1999).

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Generally E. globulus trees exist in 250-1 100 m altitudes (Donald and Jacobs,

1993). It can grow in a variety of soil types including heavy but well drained

soils and good quality loams with ample moisture content (Hall et al., 1970).

Temperature range for the germination and seedling survival is 13-33 "C with an

optimum of about 28 "C (Lopez et al., 2000). E. globulus seedlings cannot

tolerate cold, frost, drought or harsh winds. E. globulus. subsp. globulus is

planted in warm environments on the coast between sea level and 450 m above

sea level.

1.3 Economic importance of E. globulus

E. globulus is cultivated principally for timber and for paper making. Compared

with other Eucalypts it has superior growth rates (5-10 years to maturity and

harvesting) and excellent pulp properties (Jordan et al., 1993). Timber is used for

heavy and light constructions and it is mainly used where bending is necessary

(Hall et al., 1970). E. globulus subspecies globulus is most preferred due to its

ability to grow faster outside its native geographic zone (Wang et al., 1988). E.

globulus is also cultivated for land reclamation especially to prevent soil erosion,

as a wind break and in several places they are planted for their ornamental value.

It is used as fuel wood for human consumption. In addition, E. globulus flowers

are used for honey production and the leaf oils are used for producing aromatic

soaps, candles and essential oils etc.

Due to its value as a plantation species, E. globulus is now geographically

widespread with 1,700,OO ha planted worldwide (Dutkowski and Potts, 1999)

and 350,000 ha planted in Australia (Greaves et al., 2004). It is grown in

California, Hawaii, Southeast Asia, Southern Europe, Brazil, Ethiopia, Argentina

and Portugal (Young, 2002). Due to its importance as a plantation species there

is a significant nursery industry based on production of E. globulus seedlings for

plantation establishment. Reliable seedling production depends on a sound

understanding of germination behaviour of E. globulus seeds.

. ,. . ,. ,

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1.4. Extent and location of E. globulus plantations

In Australia E. globulus are mainly planted commercially in western Australia,

Victoria, Tasmania and southern Australia (Jones and Potts, 2000). In Tasmania,

the principle area is 41'30'-43'30's and including Victoria it occurs at 38'30'-

43'30's latitude with a rainfall of 500-2000 mm (Hall et al., 1970; Tibbits et al.,

1997).

1.5. Breeding activity in E. globulus

The importance of E. globulus as a plantation species has meant that it is the

subject of significant breeding activity. E globulus breeding program utilizes

well-established scientific techniques such as molecular genetics and

bioinformatics (McRae, 2004) with improvement achieved through controlled

cross pollination of selected parents. The main objectives of E. globulus tree

improvement through breeding program is to maximise the value of the species

for production of trees for krafi pulp (McRae et al., 2004). Main traits for

improvement are wood basic density and kraft pulp yield (McRae et al., 2004).

Another objective is to improve disease resistance (Thamarus, 2000) and to

decrease susceptibility of trees towards herbivorous pests (Jones and Potts,

2000). Breeding is largely done by cross pollination to avoid inbreeding

depression and promote heterozygosity, genetic variation and genetic exchanges

(Moncur et al., 1995).

1.6. Nursery seed production

Under typical plantation conditions about 0.1% of Eucalypt seeds establish

(Schmidt, 2000). Nursery stock are therefore used in plantation establishment

(Boland et al., 1980). Seeds are planted in the winter season in nurseries and

seedlings are planted out after 6 months. Seeds are collected in the previous

summer (Sasse et al., 2003) approximately one year after flowering (Boland et

al., 1980). Germination varies according to the seedlot due to provenance

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difference (Lopez et al., 2000), occasionally leading to non uniform germination

in the nursery.

Generally commercial E. globulus seeds are sown in seedling trays containing 64

to 12 1 cells, commonly nurseries prefer 64 seedlings in a tray. A number of tray

types are commonly used in Eucalypt nurseries including LannenB (81 cell),

Kwik Pot@ (64 cell), Col-Max@ (64 cell). Each cell is designed in such a way it

has enclosed sidewalls that taper slightly with depth and an air root-pruning base.

Typical cell volume range from 49-85 cm3 (Mullan and White, 200 1).

Potting mixture is prepared from materials that provide rapid drainage; ample

aeration and high water holding capacity such as a mixture of peat moss, coarse

sand and sandy loam. A slow releasing fertilizer OsmocoteB or MagampB is

mixed along with the potting mix. The mixture is sterilized and then spread in

the trays after which they are irrigated. A precision sower is used to sow seed on

the surface of the mixture. One seed is sown per cell. A layer of vermiculate is

added to cover the cells and the trays are then irrigated again.

Before sowing many nurseries pre-treat the seed, usually soaking and priming in

KN03, although specific pre-treatments vary between nurseries. Following

sowing the trays are stacked on top of each other and left for incubation (Boland

et al., 1980) after covering with a black plastic in a germination room with

optimum temperature and light conditions and high humidity. Seeds of E.

globulus start germinating on day 5, so from day 3 onwards trays are transferred

to the shade house benches where the seedlings are grown to 15 cm and

hardened before transplanting to the field (Mullan and White, 2001).

1.7. Orchard seed description

E. globulus seeds grown in nurseries come from seed orchards with the objective

of producing superior quality seeds (Pound et al., 2002a). Eucalypt seed orchards

generally consist of cloned trees or seedlings of selected trees chosen for optimal

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cross-pollination, production of abundant high quality seeds at an early stage and

maintenance of genetic purity. Eucalypt trees are difficult to manage in their

natural condition due to their height, so in orchards these trees are kept short

through the use of grafted rootstocks for easy seed harvesting and shade

avoidance. Irrigation, nutrition, insect or diseases and weeds are managed to

promote optimum seed production (Boland et al., 1980).

According to the Southern Tree Breeding Association Inc. (STBA), Australia

uses cross pollination of superior female flowers with elite male pollen to

produce high performing progeny with improved growth and superior wood

quality (McRae, 2005). Seed capsules are collected at maturity, one year after

flowering. Seeds are mature when the h i t s turn brown and hard. Seed maturity

can be tested by cutting fruits to check that the seeds have a white embryo and

chaff is present towards the top of the capsule. Several methods are used to

collect seeds, depending on the amount of seed required, the value of the seed

and the height of the trees. Capsules are hand picked from standing trees by

climbing or use of a cherry picker or from branches cut or pulled down from the

canopy. Seed is also collected from trees felled during commercial logging

(Boland et al., 1980).

After collection, the capsules are dried depending on the climatic conditions. In

hot, dry climates open drying is done whilst in cooler, humid climates artificial

heating is used. In Tasmania heated, forced air drying kilns are used. In response

to drying, the capsules open and release the seeds, which fall through the

perforated walls of the kiln for collection. Separating seeds from the chaff is

difficult but sowing pure seeds enables precise sowing and minimises freight

costs. Therefore after extraction seeds are cleaned to remove impurities such as

chaff, grubs and leafy materials. Cleaning is done by sieving or use of aspirators,

specific gravity separators. Specific gravity separators divide seeds according to

their weight and size (Boland et al., 1980; McDonald, 1998). After cleaning, E.

globulus seeds are graded through various sizes of sieves with graduated mesh to

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improve uniformity of germination in the nursery. After grading, seeds are stored

in air tight containers at low temperatures prior to the delivery to the nurseries.

2. Seed structure

2.1. Eucalyptus fruit and seed structure

E. globulus produces yellowish whitish flowers that appear feathery with a lot of

stamens arising from the calyx (Skolmen and Ledig, 2002; Young, 2002). The

ovary is surmounted by the style and stigma, and is embedded in the hypanthium

consisting of the chambers that contain ovules. These chambers contain central,

vertical, basally attached columns that bear placentae, on which ovular structures

are borne in transverse or longitudinal rows.

Carr and Carr (1962) observed that the ovular structures towards the base of

placenta initiate last and mostly become viable seeds whilst ovular structures

towards the top initiate first and form sterile structures called ovulodes (Boland

et al., 1980; Sedgley, 1989). The non-fertilised ovules and ovulodes develop into

chaff (Boland et al., 1980; Williams and Brooker, 1997). Thus the viable seeds

form deep inside the ovule and shed after the chaff. Williams and Woinarski

(1997) reported that E. globulus seeds can produce 100 seeds per capsule. The

ovary matures within the hypanthium to form a woody capsule after pollination.

Once the capsule dries the top of the capsule splits radially and ruptures locular

walls (Williams and Brooker, 1997). The seed sheds through these openings at

the top of the capsules and is dispersed through wind (Young, 2002; Raymond

and Apiolaza, 2004).

Eucalyptus species generally produce seeds without endosperm (Boden, 1957;

Sedgley, 1989). The Eucalyptus seed consist of an embryo inside a seed coat

derived from the ovule integuments. Initial growth of the seedlings depends on

the embryo reserve and then on the cotyledons through photosynthesis (Cremer

et al., 1978; Boland et al., 1980; Mulligan and Patrick, 1985; Williams and

Brooker, 1997; Close and Wilson, 2002).

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3. Seed Quality

Seed quality is a broad term encompassing seed lot characteristics such as

germination and vigour potential, genetic quality, mechanical purity such as

undesirable weed seeds or other crop seeds, and fieedom fiom pathogens and

pests (Turnbull and Doran, 1987). Quality of the seed can be affected during

seed collection, cleaning, drying, packaging and storage (Mulawaman et al.,

2003). The genetic quality of a seed is affected by provenance and the genetic

constitution and seed size influences tree growth and yield in nurseries (Turnbull

and Doran, 1987). Good quality seeds have high germination percentage, rapid

and uniform germination.

3.1 Seed germination and type of germination in Eucalypts

Germination occurs under a favourable set of environmental conditions such as

moisture availability, temperature and light that are species dependant. It

commences with water uptake by the seeds and ends with the emergence of the

radicle (Bewley and Black, 1982). After germination, seedlings are structurally

divided into hypocotyl, epicotyl and radicle. There are two types of germination

in angiosperms. In epigeal germination the hypocotyl elongates and pushes the

cotyledons above ground (Moro et al., 2001), often with seed coat attached. As

the hypocotyl elongates, the cotyledons separate showing unexpanded leaves

(Schmidt, 2000). During hypogeal germination the epicotyl emerges above the

ground first and expansion of the hypocotyl is minimal so that cotyledons remain

in the seed coat and stay below the soil surface (Schmidt, 2000; Parolin, 2001).

On top of the epicotyl, leaf tissues are present, on elongation of the epicotyl the

young leaves protrude upward and the leaves begin to unfold. In Eucalyptus

epigeal germination takes place (Boland et al., 1980; Schmidt, 2000).

Germination capacity of a seed lot can be determined by number of pure seeds

that produce normal seedling in a laboratory test, seeds that produce weak and

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abnormal seedlings are rejected. An abnormal seedling normally has a short

radicle, pale hypocotyl, and the cotyledons are unable to unfold and shed the

seed coat in eucalypts (Boland, 1977; Boland et al., 1980). Viability of

Ezicalyptus species is lost during storage due to the ageing process that is

governed by the respiration rate. However viability can be adjusted by

controlling oxygen availability, moisture content and temperature (Lockett,

1991).

3.2 Seed vigour

Seed vigour determines rapid, uniform emergence and development of the

normal seedling under ideal and adverse conditions (Turnbull and Doran, 1987;

McDonald and Copeland, 1997). Highly vigorous seeds have shorter germination

time and are able to germinate under less favourable conditions, ensuring that

they are less susceptible to variable environmental conditions during

establishment. High seed vigour increases the chance of obtaining uniform

germination and effective competition with weeds. Loss of vigour results in

slower seed germination and more erratic germination of the seed lot (Heydecker

et a!. , 1975).

Vigour testing is considered to be more useful than the standard germination test

as it gives better results regarding quality and planting value of the seedlots

(Wang et al., 1996). Two vigour tests commonly used are accelerated aging and

the conductivity test (Kolasinska et al., 2000). In accelerated aging seeds are

subjected to high temperature and high humidity stress for short duration prior to

germination testing. Lower quality seeds deteriorate more quickly than higher

quality seeds. In conductivity test lower quality seeds leak ions more profusely

compared to higher quality seeds (McDonald, 1998).

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3.2.1. Factors affecting germination and vigour

Germination and vigour are influenced by a range of environmental factors such

as moisture availability, temperature, light, gas exchange and nutrient

availability (Bradford, 1995) and physical factors such as seed age and seed size

(Skolmen and Ledig, 2002).

i. Temperature

Temperature is a critical factor in seed germination, with sub or supra-optimal

temperatures leading to impaired germination or induction of dormancy. When

germination occurs at lowest temperature it is known as minimum or base

temperature. At optimum temperature germination is the maximum and rapid

and above maximum or ceiling temperature germination does not occur

(Alvarado and Bradford, 2002). Seed metabolism depends on temperature, as

temperature increases imbibition increases accordingly. High quality seeds can

generally germinate across a broader range of sub or supra-optimal temperatures

compared to low quality seeds (Copeland and McDonald, 1995). Exposure of

some seeds such as soybean, lima bean, and cotton to low temperature induced

chilling injury that affected biochemical and metabolic activities of the seeds.

Different studies have shown chilling injury reduced ATPase activity, altered

respiration activity, protein synthesis, reduced ethylene production and ACC

oxidase activity (Posmyk et al., 2001).

Eucalyptus seeds generally germinate under a relatively wide range of

temperatures. Donald and Jacobs (1993) germinated E. globulus subsp. bicostata

at 12 'C, 17 "C, 22 'C and 27 "C and recommended 17-22 "C as ideal temperature

range. They found more dormant seeds at lower temperature but little difference

between other temperatures in terms of germination percentage. Lopez et al.

(2000) germinated E. globulus seeds at 13 "C, 18 "C, 24 "C, 28 "C and 33 O C and

found that complete germination took place at 28 "C, above and below this

temperature germination capacity declined drastically. At lower temperatures

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reduced germination was associated with lower rates of imbibition (Lopez et al.,

2000). Earlier studies based on E. wandoo Blakely and E. oleosa F. Muell.

suggest that the optimum range of temperatures for germination was 10-20 OC

and 15-20 "C respectively. E. salmonophloia germinated within the range of 10-

35 OC at any time of the year after heavy rainfall (Yates et al., 1996). Subalpine

and lowland E. pauczflora seed germinated between 15-20 "C but higher

temperatures caused physiological damage (Beardsell and Mullet, 1984). Three

species of Eucalyptus were studied at 5 OC, 10 OC, 15 OC, 20 OC, 25 OC, 30 OC

and 35 OC, where E. oleosa percentage germination was high between 10 OC to

20 OC, E. wandoo percentage germination was high at 25 OC and E. rudis

germination was higher at 30 OC (Bell and Bellairs, 1992).

ii. Light

Germination, survival of the seedlings and their further development depend on

light. In many species light is an important environmental cue for regulation of

seed dormancy (Pons, 2000). The amount of light received by a seed depends on

its position in the soil, characteristics of the seed coat and any other structures

surrounding it (Pons, 2000). Seeds on the soil surface behave differently from

the seeds buried at different depths in the soil (Atwell et al., 1999). Deep burial

of seeds of oilseed rape increased their chance of entering secondary dormancy

in the absence of light and decreased the chance of subsequent germination

(Pekrun et al., 1997b). Imbibition of meadowform seeds in light induced

secondary dormancy (Nyunt and Grabe, 1987). For Atriplex cordobensis, a

native shrub of semiarid central region of Argentina, germination was found to

be inhibited in darkness at higher temperature especially in winter harvested

seeds (Aiazzi et al., 2000). Some studies show that at in certain plants failure to

germinate at low temperature under complete darkness can be overcome by

exposure to light (Zohar et al., 1975).

Generally temperate tree species require light to germinate, with germination

promoted by red light and inhibited by far red light (Toole, 1973). Eucalypt

., ,.... . . - . ,-, . . . . ,, . Chapter-2 (Literature Review)

.. ,. . ,..4Y:,.. .,. .,..).~,r. ?,C,<EIEI ZC:.-?:X C - L C ~ ~ ~ ~ ~ I ~ ~ . ~ ~ , . : ~ J U , ~ : O O , ~ O O O O , O ~ ( ~ ~ ~ ~ ~ O O ~ : C : ~ ; ~ ~ : ~ ~ ~ ~ ~ ~ ~ ~ ' ~ F . ; ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ . T ~ ~ ~ ~ ~ Y ~ : P ~ ( T ~ R ~ ~ / ~ ~ ~ X ~ ~ ~ ~ ~ " / I . ~ ~ N : ~ X X ~ ~ ~ ' ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ I :

seeds have light requirements that appear to vary according to genotype (Pons,

2000), between seedlots (Grose and Zimmer, 1958) and according to the age of

the seeds (Zohar et al., 1975). E. globulus seeds are recommended to be

germinated in the presence of light by Langkamp (1987). The light requirement

of seeds of certain Eucalyptus species could be related to seed size. It has been

noticed that other myrtaceous species such as Melaleuca and Leptospermum

have smaller seeds and they shed their seeds after wildfires when light is not a

limiting factor (Beardsell and Richards, 1987).

iii. Moisture

A minimum state of hydration is required for seeds to germinate. Moisture is

important for maintaining cell turgor, for activation of enzymes, and in the

breakdown, translocation and storage of reserve material (Copeland and

McDonald, 1995). In seeds, initiation of cell elongation is the most sensitive

stage to water stress (Hegarty and Ross, 1980181). Soybean seeds soaked at 10-

15 "C for 2-8 days before planting did not affect germination but when they were

soaked at 25-30 "C, germination reduced significantly as the length of soaking

increased i.e. 4 days soaking significantly affected germination (Wuebker et al.,

2001).

Availability of water can affect germination and induction of dormancy through

imbibition damage, low moisture stress, and creation of anaerobic conditions.

Imbibing seeds in excess water causes anaerobic condition; during anaerobic

respiration ethanol is produced fiom the pyruvate conversion. Generally short

term anaerobic conditions are not harmful to seeds but if ethanol is produced in

excess it damages membrane function and prevents germination (Booth, 1992).

Turnball and Doran (1987) showed that E. delegatensis and E. regnans seeds

were sensitive to excess water during germination.

At high or low temperature rapid water uptake can cause imbibitional damage,

reducing the rate of germination. Rapid water flow into the dry seed can damage

membranes causing leakage of ions, proteins, carbohydrates and other substances

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(Veselova et al., 2003). In Soybean (Glycine max) rapid water uptake caused

epidermal cracks that reduced the survival of the seedlings (Booth, 1992).

Hypoxia is another case caused under excess water, where seeds consume

oxygen rapidly and diffuse oxygen slowly through the seed coat (Veselova et al.,

2003).

iv. Abscisic acid (Growth inhibitor)

Abscisic acid is the primary inhibitor of germination in many seeds. In

particular, high levels of ABA have been shown to induce deep dormancy in

some seeds (Pinfield and Gwarazimba, 1992). Increased level of ABA helps

developmental stages of embryo maturation but adversely affects seedling

emergence (Davies, 1990). Hilhorst and Karsseen (1 992) suggested that seeds

become dormant only when the embryo initiates the production of ABA.

Feurtado et al. (2004) noticed the production of ABA was mainly concentrated

in the megagametophyte. It develops in response to stress factors such as water

stress during seed development and germination (Yoshioka et al., 1995). In dry

seeds the amount of ABA is very low. Debeaujon and Koornneef (2000)

suggested that ABA induces dormancy during late phase of seed maturation and

is synthesized in the seed embryo. Oxygen deficiency increased the level of

endogenous ABA and decreased the amount of GA3 and cytokinin in maize

seeds (Prasad et al., 1983). After ripening has been shown to reduce the levels of

ABA in seeds (Grappin et al., 2000).

v. Age of the seeds

Seed vigour declines during storage (Lovato and Balboni, 2002). Seed ageing

delays radicle emergence, seedling growth, total germination in a seed lot or seed

population, and increases the chances of development of abnormal seedlings at

sub optimal temperature conditions (Veselova et al., 2003). Seed vigour can be

lost due to imbibitional damage caused by seed ageing where rapid ion leakage

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takes place due to semi permeability of membranes as cells deteriorate (Zeng et

al., 1998).

High temperature during storage can lead to fungal growth that kills seeds or

deteriorates seed vigour. Eucalypt seeds can generally be stored at room

temperature but seeds of E. delgupta and E. microtheca need to be stored at or

below freezing point to maintain viability and vigour (Turnbull and Doran,

1987). E. paucijlora seed germination decreased markedly with storage period.

After extraction germination was found to be 80% but after three months storage

it was 33% and five weeks later it was 20%. Similar trends were noticed with E.

dives (Boden, 1957).

3.3 Seed size

In many species seedling establishment, growth and yield are affected by seed

size, leading to size grading of seed for commercial use. Larger seeded species

generally have higher survival rate compared with smaller seeded species,

although time taken to reach reproductive maturity is generally slower (Moles

and Westoby, 2006). Bell et al. (1995) studied germination responses of 43

species native to western Australia and noticed that smaller seed species,

understorey and obligated seeder species generally germinate in a narrow range

of temperatures compared with larger seed, overstorey and resprouter species.

Most of the myrtaceous species have small seeds, so they require optimum

condition for germination (Turnbull and Doran, 1987). Amongst the Eucalypts,

species with larger seeds and seedlings survive better, especially in soil with low

nutrient content (Milberg et al., 1998).

Grose in 1963 cited in Cremer et al. (1978) observed that in E. delegatensis,

larger seeds present in a seedlot germinated faster and had a lower degree of

dormancy. Humara et al. (2002) found that, during water stress, E. globulus

produced uneven seedlings with the small sized seeds producing the larger

seedlings. In contrast, in the presence of excess water, germination of smaller

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seeds decreased. Lopez et al. (2000) found that in E. globulus germination varied

at different temperatures according to seed size. Within the myrtaceous genera it

has been noticed that smaller seeds are most susceptible to damping off caused

by Pythium, Rhizoctonia or Phytopthora (Turnbull and Doran, 1987).

4. Seed dormancy

4.1. Dormancy and its Ecological Role

Dormancy is a mechanism of prevention of germination of seeds when

environmental conditions are unfavourable for seedling establishment

(Bouwmeester and Karssen, 1992; Bonner et al., 1994). Absence of germination

in a mature intact seed under favourable conditions such as light, temperature,

water and oxygen indicates the presence of dormancy in seeds (Foley and

Fennimore, 1998; Martinez-Gbmez and Dicenta, 2001).

4.2. Types of Dormancy

4.2.1. Primary dormancy

Primary or innate dormancy has been reviewed at length by Baskin and Baskin

(1998). Primary dormancy is induced in seeds whilst on the mother plant to

prevent precocious germination or germination after shedding (Bewley and

Downie, 1996; Fenner, 2000). The length of primary dormancy can vary from

one month to several years (Gbehounou et al., 2000).

I. Types of primary dormancy

Primary dormancy is classified according to the nature of the restriction on

germination into the following groups.

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i. Exogenous dormancy

Exogenous dormancy refers to inhibition of germination by a restrictive factor

present outside the embryo, ie. in the seed coat or endosperm. The two most

common forms of exogenous dormancy are termed physical dormancy and

chemical dormancy.

The primary reason for lack of germination in seeds with physical dormancy is

the impermeability of the seed coat to water (Baskin, 2003; Baskin et al., 2004)

Seed coat impermeability is usually associated with the presence of one or more

layers of impermeable palisade cells or malphigian cells located in the epidermal

layer (Bell, 1999; Baskin et al., 2000). Under natural conditions seed coat ,

imposed physical dormancy is broken by low fluctuating temperatures (Baskin,

2003), fire, desiccation, freezing and thawing cycles or passage through the

digestive tract of an animal (Baskin and Baskin, 1998; Adkins et al., 2002). Once

the seed coat becomes permeable it does not revert to an impermeable state.

Physical dormancy was broken in Dodonaea viscosa seeds by mechanical

scarification (Baskin et al., 2004) and in some Rhamnaceae species found in

Australia, physical dormancy was broken by hot water treatment (Turner et al.,

2005), however hot water treatment reduced germination in D. viscosa seeds

(Baskin et al., 2004). Baskin and Baskin (1998) reported that physical dormancy

is present in fifteen families of angiosperms.

Chemically dormant seeds do not germinate because of the presence of inhibitors

in the pericarp or seed tissues (Baskin and Baskin, 2004). Baskin and Baskin

(1998) extend this definition to cover compounds produced in or translocated to

the seed that inhibit germination. ABA and coumarin are the most common

inhibitors isolated (Adkins et al., 2002). Plummer et al. (1995) reported that

chemical inhibitors caused seed coat dormancy in Lomandra sonderi

(Dasypogonaceae). This dormancy can be broken by removal of the pericarp

(Baskin and Baskin, 1998) or leaching by prolonged rainfall (Adkins et al.,

2002).

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Mechanical dormancy is an additional form of exogenous dormancy. Seeds with

mechanical dormancy cannot germinate because embryo development is

physically restricted due to the presence of a hard enclosing structure.

Mechanical dormancy differs from physical dormancy in that water and oxygen

uptake are not necessarily impeded (Schmidt, 2000). Bachelard (1967) reported

that mechanical dormancy occurred in E. paucijlora and E. delegatensis seeds

where the seed coat restricted embryo development.

ii. Endogenous dormancy

There are two principle forms of endogenous dormancy; morphological

dormancy and physiological dormancy. Morphologically dormant seeds have

underdeveloped embryos at the time of seed dissemination (Geneve, 2003).

Morphological dormancy is mainly seen in species originating from temperate

regions (Baskin and Baskin, 1998). Morphological dormancy can be broken by

warm or cold stratification, seed priming or treatment with chemicals such as

potassium nitrate or gibbereliic acid (Geneve, 2003).

Physiological dormancy involves changes taking place inside the embryo that

inhibit germination (Baskin and Baskin, 1998; Geneve, 2003). Chemical

inhibitors associated with physiological dormancy include phenolic compounds

and ABA (Bewley and Black, 1982). In most species, seeds with physiological

dormancy remain permeable to water. Nikolaeva (1977) cited in Baskin and

Baskin (1998) classified physiological dormancy into three types non-deep,

intermediate and deep. Mature seeds with non-deep physiological dormancy fail

to germinate at any temperature or germinate in a narrow range of temperatures

(Baskin and Baskin, 2004). Non-deep physiological dormancy typically occurs

in seeds of weeds, vegetables, grasses and flower crops (Geneve, 1998). It is

generally overcome by a period of cold (0.5-10 "C) or warm (215 "C)

stratification depending on the species (Baskin et al., 2002). In Psammochloa

villosa non deep physiological dormancy was overcome by 4 weeks of cold

stratification at 3-5 "C in darkness (Huang et al., 2004). Several reports indicate

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that non-deep physiological dormancy can also be overcome by the treatment

with chemicals such as potassium nitrate, thiurea, kinetin, ethylene or

gibberellins (Baskin and Baskin, 1998) and dry, cool storage should also break

non-deep physiological dormancy (Geneve, 2003).

Intermediate physiological dormancy is a deeper form of dormancy typical of

seeds from temperate zones (Geneve, 1998) for example Pinus sp. (Geneve,

2003) and Polygonum sp. (Timson, 1966). Intermediate physiological dormancy

can generally be broken by cold stratification, dry storage at room temperature

(Geneve, 2003) or in some species treatment with growth regulators such as GA

or Ethephon (Baskin and Baskin, 2004). Embryos of non deep and

intermediately dormant seeds usually germinate if seed coat or endosperm is

removed (Geneve, 2003; Baskin and Baskin, 2004).

Deep physiological dormancy occurs in species fiom tropical, subtropical and

temperate zones, e.g. seeds of some species of Prunus and Dictamnus (Geneve,

2003). In general embryos excised from seeds with deep physiological dormancy

fail to germinate or produce abnormal seedlings (Baskin and Baskin, 2004),

suggesting that at least some of the factors regulating this form of dormancy are

present in the embryo. Relatively long periods of cold stratification, for example

7 weeks in Acer platanoides (Pinfield et al., 1974) and 3-4 months in western

white pine (Pinus monticola Dougl. ExD. Don) (Feurtado et al., 2004) were

required to overcome deep physiological dormancy. Application of GA and

fluridon also broke deep dormancy in western white pine (Feurtado et al., 2004).

A third (combinational) form of endogenous dormancy is added,

morphophysiological dormancy, represents a state in which morphological

dormancy is associated with physiological dormancy (Baskin and Baskin, 1998;

Baskin and Baskin, 2004). It has been reported in a number of species including

Hibbertia hypericoides (Dilleniaceae) (Schatral, 1996), the weedy facultative

winter annual Papaver rhoeas (Baskin et al., 2002), and in six woody Hawaiian

endemic lobelioids (Campanulaceae) (Baskin et al., 2005). In some species with

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morphophysiological dormancy, warm stratification or treatment with GA3 has

been effective in promoting embryo growth and germination (Hidayati et al.,

2000). In Sambucus canadensis and Sambucus. pubens (Caprifoliaceae)

morphophysiological dormancy was broken by warm stratification followed by

cold stratification (Hidayati et al., 2000).

11. Dormancy in Eucalyptus species

Trees belonging to the Myrtaceae family generally have non-dormant seeds at

dispersal because they undergo a long period of after-ripening in the capsule

before they are released (Baskin and Baskin, 1998). Despite this, Grose (1957a)

reported both full (complete inhibition of germination) and partial (delayed

germination) dormancy in eucalypts. Fully dormant seed was observed in

seedlots of E. delegatensis, E. nimphophila, E. kybeanensis and E. mitchelliana.

Partially dormant seed was observed in E.nitens, E. andreana, and E. fastiga.

High summer time temperatures have been shown to promote primary dormancy

in maturing seed of E. delegatensis (Grose, 1957a). Beardsell and Mullet (1984)

noticed a dormancy in E. pauciflora seeds from higher altitudes compared with

low land, that was broken by stratification. Seed coat dormancy has been

reported in E. sieberi probably due to inhibition of water uptake by inner

integument (Gibson and Bachelard, 1986). In E. pauciflora, seed coat imposed

dormancy developed during long term storage. In this instance it was broken by

moist stratification and alternating day-night temperature of 25 "C and 3 "C,

soaking in water at room temperature for 48 h, or soaking in 2% KN03 for 24 h

(Boden, 1957). In some species of monocalypts seed coat impermeability to

oxygen has been reported (Richards and Beardsell, 1987). No reports of primary

seed dormancy in E. globulus were found in the literature.

Boden (1957) reported that E. paucijZora seed germination was spread over

several weeks under nursery conditions. Uneven germination under nursery

conditions has also been reported for E. fastigata, E. robertsoni, E.

macrorrhyncha and E. dives however, similar symptoms were not observed in E.

bicostata, E. melliodora and E. gummifera. As already noted, delay or spread in

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germination of E. globulus is a significant problem in commercial nurseries.

Although Boden (1957) suggested that these symptoms were likely to be due to

seed coat imposed dormancy that developed as a result of prolonged storage,

fi-esh seedlots of E. globulus have shown the same problem. It seems that in

some of these instances the delay in germination may therefore be due to

induction of secondary dormancy during germination.

4.2.2. Secondary dormancy

Secondary dormancy occurs in mature, nondormant or germinable seeds, after

seed shedding and in some seeds after the inhibition of primary dormancy

(Bewley and Black, 1994). It is induced due to unfavorable conditions during

germination such as water, temperature, oxygen or nitrate stress or inappropriate

light conditions (Bewley and Black, 1994; Hilhorst, 1998; Kepczynski and

Bihun, 2002). Induction of secondary dormancy varies according to the species

and also between individual seeds within a seedlot (Kebreab and Murdoch,

1999a). The length of secondary dormancy increases with the duration of

unfavourable conditions (Nyunt and Grabe, 1987). The differences in the

physiological factors of primary and secondary remain unresolved. In Avena

fatua secondary dormancy induced by anoxia differed fiom primary dormancy in

that it could be overcome by the application of nitrate, whereas breaking of

primary dormancy required both after-ripening and nitrate treatments (Hilhorst,

1998).

I. Environmental factors regulating secondary dormancy

i Temperature

Thermodormancy can occur when seeds are exposed to high temperature stress

during germination (Kepczynski and Bihun, 2002). It has been recorded in

several species such as apple (Malus), lettuce (Lactuca), and celery (Apium). In

these species exposure to temperatures above 25 "C induced secondary

dormancy, and subsequent exposure to optimum temperature failed to induce

Chapter-2 (Literature Review) :.., ,;,-,, ., . >, ,., . y,.,,,.9L7 2vs ?,4 ,s:c* 3,,?,>-~,&-.,.,,>,~~:,;,,,~v w,,.,'~,. <e:q$-e+x ,.+.%'I.;*.'* * ,.:.w.27,-. W ~ : ~ " . ~ ~ . . . ~ ~ ~ ~ . : , : ~ U V ~ , , W ~ < ~ / U / ~ ~ C W : ; ~ * . ~ * ; ' W ~ G V ~ ' ~ ~ . .

germination (Geneve, 2003). Similarly in Amaranthus caudatus exposure to 45

OC induced secondary dormancy, when it was returned to optimum temperature

of 25-35 "C seeds failed to germinate (Kepczynski and Bihun, 2002). The degree

of thermo-dormancy increases with increase in temperature. Low temperatures

can also lead to induction of secondary dormancy. Generally, low temperature

induction of secondary dormancy is slower than high temperature induction

(Bradford and Somasco, 1994). Prolonged cold treatment at 4 OC for 19-25

weeks induced secondary dormancy in Polygonum perscicaria L. seeds

(Bouwmeester and Karssen, 1992).

Kepczynski and Bihun (2002) suggested that higher temperature stress reduces

seed coat permeability to oxygen. Small and Gutterman (1992b) reported that

dormancy caused by higher temperatures resulted in low respiratory rate,

ethylene and protein synthesis and reduced ATP production in Lactuca serriola

seeds. In wheat seeds Walker-Simmons (1988) found that high temperature

inhibition of germination was associated with increased ABA synthesis in the

embryo.

ii. Light and dark

Skotodormancy is another form of secondary dormancy occurring in light

requiring primary dormant seeds due to prolonged imbibition in darkness (Desai

et al., 1997). Skotodormancy has been documented in a range of species. For

example longer periods of dark stratification of Lolium rigidum. seeds induced

light sensitive dormancy (Steadman, 2004). Empress tree seeds entered

secondary dormancy in the absence of sufficient light. This was broken by

alternating temperature, nitrate application and exposure to a range of light

pulses (Grubisic and Konjevic, 1992). Non dormant seeds of Ambrosia

artemisiifolia lost the ability to germinate at lower temperatures in complete

darkness due to the induction of secondary dormancy but later on when seeds

were exposed to normal temperatures in the presence of light seeds failed to

germinate (Baskin and Baskin, 1980).

Chapter-2 (Literature Review) ., . .,.?:,,, ., , ; ,,,p<,.pA&!. # > ~,-r~,;*,.*;;:.:.v,;,h.flfir*" ,,y wP,&>:<?;.*7.&%<,! ,,,*,?--*-,,,;>. .:we$?;, 7*Y.,!,9,,+:$3, < ~ ~ ~ , ~ v / ~ ~ ~ ~ ~ ~ # , ~ ~ , ' ~ ~ < ~ ; ~ , : ~ . : ~ V & ~ ~ / ~ ' ~ / ~ : ~ N ~ ~ ~ ~ ~ ' ~ V / ~ / m < . ' . ~

In many species (cucumber and lettuce) there is evidence for a link between

phytochrome and regulation of secondary dormancy. In cucumber seeds,

following FR irradiation with R irradiation reversed the inhibitory effect of FR

irradiation on germination. In' cocklebur seed, darkness and far red light together

caused secondary dormancy (Yoshioka et al., 1995). For lettuce, secondary

dormancy induced by high temperature was prevented by exposure to red light

(Khan and Karssen, 1980). Similarly, in many species, secondary dormancy was

induced by exposure to low temperature. In cocklebur seed, darkness and far red

light together caused secondary dormancy (Yoshioka et al., 1995).

iii. Water

Water potential is an important factor in determining secondary dormancy.

Secondary dormancy was induced in oilseed rape when seeds were imbibed in a

solution with a water potential of -1 5 bars (Pekrun et al., 1997a). Yoshioka et al.

(1995) found that in giant foxtail seeds, however COz promoted germination,

under conditions of water stress, C 0 2 caused secondary dormancy. Various

studies have shown that seeds under water stress or under oxygen deprivation

undergo secondary dormancy in many species. In rapeseeds induction of

secondary dormancy under oxygen stress was not as effective as under water

stress (Pekrun et al., 1997a). Rapeseeds were found to be light sensitive after

continued imbibition under water stress (Pekrun et al., 1997a).

iv. Gases

Several reports suggest that ethylene is actively involved in inhibiting primary

dormancy in many seeds (Kepczynski and Kepczynska, 1997). Corbineau et al.

(1988) suggested that induction of dormancy in sunflower at high temperature is

due to their inability to convert 1 -aminocyclopropane- 1 -carboxylic acid to

ethylene under high temperature stress. In Amaranthus caudatus, higher

temperature (20' - 30 "C) caused seed coat impermeability to oxygen, resulting

in induction of secondary dormancy. Increasing the concentration of oxygen

Chapter-2 (Literature Review) ,. ., ., . . .,:: r . ?*. VF ..'.;-,,-,; ;r,-+, L...'zs,&>%.4~!..-?,<~., v.e;;:2 ..::#,?,.*.:,< ..,. <.,&:<T ;., : ~ , ~ , ~ < < ~ ~ . : * - , ~ < ~ ~ ~ ~ ~ ~ , ~ ~ , / ~ , ~ , ~ ~ ~ & , . ~ ~ , , ~ ~ f l , ~ ~ ~ " / ~ , ~ , ~ ~ ~ , ~ ~ r f l ~ ~ , ~ . ~ . ~ , %

prevented induction of secondary dormancy. This may also be related to the

production of ethylene in seeds, which requires oxygen to convert ACC to

ethylene (Kepczynski and Bihun, 2002).

5. Methods for Improving Germination and Overcoming

Dormancy

Dormancy is an important issue in nursery and forestry industries. Therefore it

becomes important to understand means of overcoming dormancy where a high

level of uniform rapid establishment is necessary for economical seedling

production. A range of practices is used to obtain uniform germination. These

are outlined below, with particular reference to their application to eucalypt

seeds.

5.1. Stratification

Stratification is a technique used principally to break morphological,

physiological and morphophysiological primary dormancy (Geneve, 2003). In

some species, such as oilseed rape (Pekrun et al., 1997b), Glaucium flavum

(Thanos et al., 1989) and Pinus brutia (Skordilis and Thanos, 1995) stratification

is also effective in breaking secondary dormancy.

The process of stratification involves incubating seeds in moist conditions at low

temperature. Effective temperatures are usually between 0 and 10 "C, with 5 OC

being optimal for many species (Nikolaeva, 1969 cited in Baskin and Baskin,

1998). The effectiveness of stratification is species dependant (Vincent and

Roberts, 1977; Andersson and Milberg, 1998), with generally longer periods of

cold stratification required for seeds of plants fi-om high altitudes compared to

lowland coastal areas (Richards and Beardsell, 1987; Cavieres and Arroyo,

2000).

Chapter-2 (Literature Review) -a: . . > -. /-' '- Y-. :.,1_1-~(1.. X Z ~ ~ ~ - ~ . ~ S ~ : S ~ ' ~ - ' X ~ ~ ~ ; G ? S : . P , . C . N ~ ?n'%ZR;s;C,F,b.b.i3-i3-M~7:F-Prr-~:~ ~ ~ ~ ~ C C ~ C ~ / ~ , ~ . : K I I ~ ~ ; W / U ~ ' ~ ~ ~ / ~ I ~ W / ~ ~ / ~ ~ * ~ I ~ ' . ~ ' ~ S I ~ ~ ~ ~ ~ ~ ~ : ~ ~ ~ ~ W ~ . ' ~ ~ A ! L

Several mechanisms for overcoming dormancy through stratification have been

suggested. These include a role for stratification in mobilisation of food reserves

required for germination (Bell, 1999), promotion of GA synthesis (Moore et al.,

1994; Mikulik and Vinter, 200 1 -2002), decreasing ABA levels (Mikulik and

Vinter, 2001 -2002) increasing integument permeability and embryo maturity in

seeds (Hennion and Walton, 1997), and promoting radicle protrusion by

weakening the surrounding structures (Downie et al., 1997). In seeds with seed

coat and embryo dormancy, the stratification requirement can be reduced by

removing the seed coat (Richards and Beardsell, 1987). In Pinus brutia longer

period of stratification improved germination in far red light induced secondary

dormant seeds (Skordilis and Thanos, 1995).

Stratification has been shown to be effective in overcoming dormancy in a range

of Etlcalyptus species including E. camaldulensis and E. radiata (Zohar et al.,

1975), E. delegatensis (Battaglia, 1993, 1996) and E. paucijlora (Boden, 1957;

Beardsell and Mullet, 1984) It has also been observed to be effective in non

commercial species such as E. coccifera and E. perrineana (Lockett, 1991). The

duration of stratification required depends both on the species of eucalypt and

the initial level of seed dormancy.

In some Eucalyptus species stratification increased the range of temperatures

over which germination occurred. For example non stratified seeds of E.

delegatensis did not germinate above 21 "C however following four weeks of

stratification it germinated at 29.4 "C (Grose, 1957a), whilst longer periods of

stratification of E. delegatensis seeds up to 7 to 8 weeks increased germination

capacity at suboptimal temperatures (Battaglia, 1993, 1996). In E. camaldulensis

seeds, stratification reduced the requirement for light during germination (Grose

and Zimmer, 1958). However temperatures above 25 "C induced secondary

dormancy in stratified seeds of E. pauczj7ora from two subalpine populations

(Beardsell and Mullet, 1984). No published information on the effect of

stratification on germination of E. globulus seed or the effectiveness of

stratification for breaking secondary dormancy in eucalypt seed was found.

5.2. Priming

Priming or osmoconditioning is a pre-sowing hydration technique, used for

obtaining uniform germination (Turnbull and Doran, 1987). Seeds in contact

with priming solution uptakes water in normal way but stops as soon as it

reaches equilibrium with the osmotic potential of the solution (Heydecker et al.,

1975). It allows seeds to go through all the essential processes required for

germination but prevents cell elongation and emergence of radicle (Heydecker et

al., 1975). For priming generally substances that are metabolically inert, non

poisonous, chemically inert and water soluble are used to lower the water

potential of the solution (Heydecker et al., 1975). The main solutes used for

priming are mannitol and polyethylene glycol (PEG) (Woodstock, 1988) and

some nutrient salt solution such as KNO3, K3P04, NaN03, Ca(N03)2 (Krygsman

et al., 1995). Priming has been used to break primary dormancy in Pacific silver

fir (Abies amabilis [Dougl .ex.Loud.] Dougl. .ex. Forbes), subalpine fir (A.

lasiocarpa [Hook] Nutt.), Noble fir (A. procera Rehd.) (Ma et al., 2003),

Echinacea purpurea (L.) Moench (Pill et al., 1994), morphological dormancy in

vegetable seed (Dearman et al., 1987) and secondary dormancy in lettuce seeds

(Bonina, 2005).

Seed priming is thought to exert a positive effect on germination by allowing

mobilization of reserve materials, changes in enzyme activity, DNA and RNA

synthesis, and repair to damaged membranes at water potentials below which

germination can occur (Bray, 1995). Limited published information is available

regarding priming of eucalypt seeds, although it is used extensively in some

nurseries to obtain uniform germination, particularly with E. nitens seed (Juhaz,

2004). E. delegatensis seeds pre-imbibed in a solution of -2MPa obtained rapid

and synchronous germination compared with control seeds (Battaglia, 1993).

Haigh (1997) noticed that in the laboratory E. tereticornis, E. moluccana, E.

fibrosa and E. sideroxylon seeds improved germination after priming with PEG

solution compared with KN03 solution. Krygsman et al. (1995) noticed that

priming in 2% KN03 solution resulted in higher germination of E. globulus

Chapter-;! (Literature Review) . . '7.t.: ;-: 5,. ,?'.'"p.:'.':;L'nr ;."~P)i?~:i~;?~,'.5~'~~,Y.;Y.';Y;Y~b~2?~~~~CC'C~i3;L'.~2,.~~~77~,>,IIYYi.i.C~YPY~7~~~ ~ ~ ~ ~ + P @ / 4 ~ ~ ~ ~ O . Q ~ X X X ~ , Z ~ I r ~ ; G r / ~ ~ R / r O / 4 ~ ~ ~ / C ~ 4 4 ~ ~ ~ / i B i B f f / & . / I ? r ? r : d " l i

seeds under polyhouse and field conditions compared with priming in PEG. Seed

vigour of E. globulus was increased by treating with 2% KN03 solution for 5

days at 22 "C (Krygsman et al., 1995).

5.3 Wetting and Drying Cycles

In several seed types a cycle of wetting and drying has been reported to increase

germination. Wetting and drying increased germination in the seeds of E. sieberi

(Battaglia, 1993; Yates et al., 1996) but it did not improve germination in E.

salmonophloia and E. delegatensis seeds (Hilhorst and Karssen, 1992). Wetting

and drying is discussed at length in chapter 5.

5.4 Smoke

Recently, smoke or smoke extracts generated from burning branches or foliage

of native species have been shown to induce germination in seed of a range of

species, particularly those growing naturally in places where flora regenerates in

response to fire. Dixon et al. (1995) used smoke to test 94 species from 30

families of native Western Australian plants that did not germinate or germinated

erratically in the laboratory or field conditions. Among these smoke enhanced

germination in 45 native species in the glasshouse but Eucalyptus species were

not tested. Smoke treatments were prepared by using fresh or dry plant materials

collected from native Banksia-Eucalyptus woodland sites near Perth (Dixon et

al., 1995).

Among the species studied, smoke has been used to improve germination and

break dormancy in both positively and negatively photoblastic seeds (Brown and

Van Staden, 1997). Smoke has also been used to promote germination in species

that are not adapted to fire. For example, Drewes et a1 (2000) used smoke to

break dormancy and it replaced light requirement in lettuce seeds. In a recent

study Merritt et al. (2005) isolated butenolide 3-methyl-2H-furo[2,3-clpyran-2-

one from the smoke, and demonstrated that this compound increased seed

sensitivity towards GA and increased germination in darkness.

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There have been few reports regarding improvement of germination in E.

globulus using smoke. In one study, however aqueous ash solution did not

increase germination in E. globulus Labill. (Reyes and Casal, 1998)

5.5. Growth hormones and other compounds

Hormones play an important role in the seed germination and are often used to

break dormancy. Gibberellins, auxins, cytokinins and indole acetic acid (IAA)

are endogenously present in the seeds (Davies, 1990) and all have been

implicated in regulation of germination processes. Gibberelic acids are the most

commonly used growth hormones to break dormancy in many seeds and

biologically active forms used to break dormancy are GAI, GA3, GA4 and GA7

(Ma et al., 2003). Studies on GA suggest that GA hydrolysis of storage material

present in the endosperm mobilises reserve food material in seeds (Tieu et al.,

1999). In addition, growth hormones have been shown to induce a weakening of

the tissues surrounding the embryo and to stimulate germination of immature

embryos (White and Rivin, 2000).

Nitrogenous compounds enhance germination in many seeds. ISTA recommends

0.2% KN03 in its germination protocols for many seeds (ISTA, 1999). Nitrate at

10 mM increased germination in Emmenanthe pendulzora and Phacelia

grandiflora (Thanos and Rundel, 1995) and lower concentration of NO donor

sodium nitroprusside broke dormancy in Arabidopsis (Libourel et al., 2005).

Alboresi et a1 (2005) suggested that addition of larger amounts of nitrate to the

mother plant reduces dormancy in the seeds produced. They also noticed that

nitrate broke dormancy but stratification was a better method to overcome

dormancy. Hilhorst and Karssen (1989) suggested that in Sisymbrium officinale

L. nitrate activated germination through GA synthesis. Khan et al. (1981) cited

in Arin and Kiyak (2003) suggested that presence of nitrate during imbibition

supplements additional substrate for amino acid and protein synthesis.

..... , . s >,. 7 ?':,? ~ ~ ~ ~ - ~ ~ ~ , ~ ~ . ~ ~ . ~ ~ . . , ~ ~ . ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ . ~ , ~ ~ . y ~ ~ ~ ~ ~ . . L ~ ~ ~ ~ y ~ ~ ~ ~ . ~ . ~ . ~ . , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 4 p . ~ ~ ~ . . . ~ ~ m ~ ~ ~ ~ t ~ , ~ 7 , ~ 7 w ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ , ~ , ~ ~ ~ ~ . ~ , ~ ~ z , 2 x ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ . o 4 Chapter-2 (Literature Review)

Ethylene is another phytohormone that enhances germination. It is produced in

seeds during the early stages of imbibition (Matilla, 2000). Ethylene plays an

important role in regulation of cell growth, seed maturation and might be

involved in breaking of primary dormancy (Matilla, 2000). Weaver, 1972 cited

in Heydecker and Coolbear (1977) that ethephon releases ethylene in the plant

tissues especially in lettuce seeds. Several studies have shown improvements in

cold stratification if the seeds are imbibed at ambient temperature prior to

exposure to the chilling treatment (Posmyk et al., 2001). One possible

explanation proposed by Posmyk et al. (2001) is that the initial imbibition period

results in increased production of ethylene.

A number of small molecular weight organic compounds with lipophilic

properties such as acetaldehyde, acetone, chloroform, and ethanol have been

shown to interact with phytochrome in breaking seed dormancy. For example, in

cucumber seeds, secondary dormancy caused by exposure to far red light was

broken by treatment with ethanol or acetone (Amritphale et al., 1993). Several

reports suggest that these compounds have an anaesthetic property at the

membrane level, specifically ethanol that has been shown to act as a respiratory

substrate in stimulating germination (Amritphale et al., 1993).

Limited work on the effects of growth regulators and other dormancy breaking

compounds on germination of eucalypt seed has been published. As noted earlier

in the review KN03 pre-treatments are widely and successfully used to promote

nursery germination in a range of eucalypts including E. nitens and E. globulus.

Similarly, E. pauciflora dormancy was broken by exposure to thiourea and

potassium nitrate (Boden, 1957). Bachelard (1967) found that GA up to a

concentration of 50mgll promoted germination of E. paucijora seeds whereas

kinetin inhibited germination. Combination of GA and light improved

germination in E. regnans and E. fastigata (Bachelard, 1967). The relationship

between GA treatment and light in covered in greater depth in Chapter 7.

Chapter-2 (Literature Review) %.. "' a I.,.* ?:$"iD .2:.P;r?ii:,.W'<.&.-)a C:/%M.)!C"%'~P,~:Y;~77T)r?<a'%-W,, ,//n ?>, C:%i:> a ~ ~ ~ ~ , ~ Y y & ~ ~ , ~ , ~ / g ~ r p 4 ~ ~ ~ Y ~ y , ' b : ~ ~ ~ ~ ~ ~ ~ I p ~ ~ ~ r C Y ~ ~ I ~ / ~ / 1 ~ ~ ~ ~ ~ # / ~ ~

6. Conclusion

This literature review has examined factors affecting seed germination and

dormancy with particular reference to Eucalyptus in order to identify factors that

may contribute to the variable germination of E. globulus seed in commercial

nurseries. No published information on induction of dormancy in E. globulus

was found. Whilst water deficit has been shown to delay germination in E.

globulus seeds (Lopez et al., 2000) no evidence was presented in this study of

induction of dormancy. Although uneven germination has been attributed to

variation in seed size within a seedlot, E. globulus seeds sown in nurseries are

size graded. Therefore, seed size variation does not explain the problem of

delayed or variable germination observed in commercial nurseries.

Based on the findings of studies using other Eucalyptus species, it appears

possible that a range of environmental factors including temperature stress, water

stress or light conditions during germination are likely to play a role in delaying

germination in commercial nurseries. The involvement of some of these factors

is examined in the research outlined in the following chapters, with a view to the

development of strategies to improve uniformity of E. globulus seedling

establishment in commercial nurseries.

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General Materials and Methods

Materials and methods are detailed in each experimental chapter, but several

aspects of the study were common throughout and are detailed here.

3.1. Seedlot

A range of commercial E. globulus seedlots were used and the Materials and

Methods for each experiment detail the seed lot(s) in each case. All seed was

open pollinated seed orchard material of known female parentage. Reports fiom

commercial nurseries at the beginning of the study suggested some variability

between seedlots in expression of the uneven germination symptom.

Unfortunately supplies of individual seedlots after commercial distribution were

limited and it was not possible to concentrate research on those showing

strongest field expression. Consequently, many of the key experiments were

repeated across two or more seedlots, where possible using at least one known to

show uneven germination.

To simplify layout of experiments in the germination chamber experiments

repeated on different seedlots were analysed separately for treatment responses.

Seed lot differences and seed lot by treatment interactions were not compared

statistically. Seed was supplied by Seed Energy P/L and details of source,

location, date of collection etc. are given in Table-3.1. Seed was delivered dry in

polythene bags with no pre-treatment (apart from cleaning and grading) and

stored at 4 "C prior to use. All seed supplied was substantially free fiom chaff

and other impurities and size graded to commercial specifications. Unless

otherwise stated germination tests used 50 seeds counted with a Contador seed

counter (Baumann Saatzuchtbedarf D-74638, Waldenburg).

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Table 3.1 : Details of Commercial Seedlots supplied

Seedlot Year Provenance Size (pm) 1000 seed weight (g)

'h' 2001 Heath Seed Orchard 1000-1210 1.155

'H' 2001 Heath Seed Orchard 1588-1 936 2.795

'Li' 2001 Heath King Island 1588-1936 2.659

'KI' 2003 Heath Seed Orchard 1693-1954 1.873

'Ki' 2003 Heath King Island 1954-2183 2.581

'A' 2003 Heath Top Level 1690- 1950 1.94 1

'KI- 1 ' 2004 Level 4 120 King Island Bawdens 1500-1 700 2.727

'KI-2' 2004 King Island 133 1500-1 700 2.007

Bawdens + Heath

'0s' 199 1 Hobart Native Forest Ungraded

Chapter-3 (General Materials and Methods) .>, .. .,.d. :;*.. r;,x: . ~ ~ ~ ~ ~ ~ ~ . ~ ~ . ~ ~ ~ - ~ ~ ~ ~ ~ ~ " ~ ~ . ~ ~ , ~ ~ : ~ , . ~ , ; ~ . ~ ~ ~ ~ ~ ~ ~ : ~ ; : ~ . ~ ~ ~ ~ ~ , : ~ ~ ~ ~ ~ . ~ ! . ~ ~ , ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ : ~ , . ~ ~ ' ~ ~ ~ ~ , ~ ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ / ~ ~ ~ ~ ~ ~ ~ ~ ~ / ~ / ~ ~ ~ ~

All seedlots were from E. globulus orchards located near Mount Gambier in

South Australia. The orchard contained trees originating from throughout the

geographical range of E. globulus sp. globulus and populations integrading with

other E. globulus subspecies. The majority (approximately 90%) of trees present

in the orchard were from the Western Otways and Strezelecki ranges, the

Furneaux islands and south-eastern Tasmanian races. For further details on the

racial classification refer to Dutkowski and Potts (1 999).

At the beginning of the study tests were carried out at 25 O C in 12/12 photoperiod

(ISTA, 1999) on all seedlots subsequently used in experiments. Germination

counts were done on day 7 and results were as follows.

Table 3.2: Seedlot performance test (n=4)

Seedlots Mean Germination (%)

'h' 90

' H ' 9 1

'Li' 8 7

'KI' 99

' Ki ' 99

'A' 99

'Ki-1 ' 96

'Ki-2' 93

'0s' 80

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3.2. Germination Conditions

For germination tests in most experiments, seeds were spread uniformly on top

of two layers of Type 2, Advantec, 80 mm diameter filter paper in a petri plate.

The papers were initially moistened with 5-7 mL of deionised water with

additional deionised water added during the test period to avoid drying. In later

experiments with treatments requiring covering for light exclusion difficulties

with water supply necessitated some modifications to this method. These are

noted in the relevant sections.

Laboratory experiments were carried out according to ISTA (1999) guidelines,

which recommend 25 "C and 14 days of germination for E. globulus. Preliminary

trials (not reported) showed minimal incidence of fungal growth during

germination and in the trials reported there was no fungicide pretreatment.

Except where noted, all experiments were conducted at a set temperature of 25

"C in a Linder and May Model LMRIL-1-SD germination cabinet with 12/12

photoperiod where light was supplied by the two GE fluorescent tubes

(F30W133). Measurements using "TinyTalk" data loggers (Hasting Data Loggers

P/L) at various times during the study confirmed that the temperature remained

at 25 + I "C throughout. Except for the light quality trials (Chapter 7) three

alternative light conditions were used. The cabinet lights were used to provide

either constant light or 12/12 h of light1 dark. Constant darkness was achieved by

covering petri plates with two layers of domestic aluminium foil. In trials

comparing foil covered dark treatments with a light environment provided by the

cabinet light, exposed plates were wrapped in clear polyethylene "zip lock" bags

to provide similar evaporative conditions.

In trials designed to provide a germination profile and counts were done daily,

seeds were considered as germinated when the radicle protruded more than 4

mm and cotyledons were spread widely (Boland et al. 1980). In later

experiments, where progressive counts would have risked compromising the

light environment, germination was taken as the number of seeds with a radicle

length of 4 mm or greater on day 5. This form of assessment was chosen because

Chapter-3 (General Materials and Methods) ..JA.s,:?',., ,% j . : ~ ~ < * ~ ~ ~ . ~ ~ ~ . ~ , ~ * ? ~ ~ ~ ~ ~ > P ~ ~ , ~ ~ , . ~ ~ . ~ ~ . , ~ ~ ~ . & .,,. B,rn,?..* .#,.-7@,&-:,t*.'3 v~~~~'r'*~~~~m~~~~4~m~~~~~~~,~/m,~,m/~!~.'~,~u/#/e,n~w~w~~rr~~v#/#/~~

it enabled rapid completion of experiments with sufficient resolution of

treatments with respect to delayed germination.

In trials determining germination profiles, a squash test (Yates et al., 1996) was

pertbrmed to determine the viability of ungerminated seeds at the end of each

germination test period. Seeds with white or green embryos were considered as

viable (Baskin and Baskin, 1998).

3.3. Experimental Design and Statistical Analysis

In all experiments, seed pre-treatment (eg hydrationldehydration cycle, and light

conditions during imbibition) were carried out in true replicates to avoid

pseudoreplication (Morrison and Morris, 2000). All experiments were

randomised complete block designs unless specified otherwise, with blocks

arranged according to position in the germination cabinet. Results were analysed

with ANOVA using the general linear models package of SPSS (12.0.1, 1989-

2003) and for comparison of means, Fisher's (Steel and Torrie, 1981) LSD was

calculated at the 0.05 level of probability unless otherwise specified. As already

noted, individual seedlots were subjected to separate analyses of variance. Error

bars shown on graphs are standard errors of the mean (SEM). For time course

studies, results were analysed either using separate analyses of variance to

compare treatments at selected times or as repeated measures analysis using the

same SPSS package. In some experiments means are reported with standard

deviations in the text.

Although almost all results were germination percentages, tests for normality and

heterogeneity of variance failed to give cause for concern and all analysis were

carried out on untransformed data.

, .., Chapter-4 (Water Relations in E. globulus Seed)

" ,?,>;, ?, ....,.,,, <,,< .,.. <.,,.,-,.. .,'..,: --.,- .. .o-,c ,,,% :.; .,,, T..:.. .,'..p.,.*r...Lp ,~/..>.,~,,:<<~<<-,,.-&%,:. .,.,.. ,*...,m ~~:,-,,.-.~~~.~",~?~~,'~"',~~x,.,@.~~<~'~~~~~~w,",.c.w.~~~~~R,~<..;~~

Chapter-4

Water Relations in E. globulus Seed

4.1. Introduction

The observations on field factors that might induce delayed germination in

commercial nurseries suggested a possible link with available water.

Consequently this initial study aimed to establish the basic water uptake

characteristics of E.globulus seeds. Further, the water uptake pattern and related

onset of respiration provide the major physiological markers for the germination

process, necessary for interpretation of, and comparison between germination

studies.

The first step in germination is water uptake (imbibition) from the available soil

moisture. Seeds imbibe 2-3 times their dry weight before radicle emergence.

Water uptake then continues with seedling development and growth. Water

diffuses into the seeds in response to a difference between seed and soil water

potentials (Akhalkatsi and Losch, 2001). Seeds generally imbibe water

regardless of dormancy status, but water movement is governed by factors such

as seed size, seed coat permeability, oxygen uptake, water content and

temperature (Bewley and Black, 1994; Edelstein et al., 1995; Akhalkatsi and

Losch, 2001). Some species are sensitive to rapid water uptake, which along with

low or high temperature, lack of oxygen, carbon dioxide and ethylene, may

reduce germination (Hosnedl and Honsov6, 2002). Vesalova et al. (2003)

observed that pea seeds reaching 60-70% moisture content after 20 h of

imbibition produced normal seedlings whereas peas seeds that reached 90-100%

moisture content in the same period produced either abnormal seedlings or lost

their capacity to develop radicle.

Bachelard (1985), observed that germination of E. seiberi, E. pilularis and E.

maculata was reduced at soil matric potential of -0.001 to -0.003 MPa, compared

Chapter-4 (Water Relations in E. globulus Seed) z , , . *:9:, ,.,, ,.=..<- ~.:,'<m ,/= .,,. .w,;*7e.'fi!. <. ,?.s.>z7*- P ;.- c r ' - ~ e / A r . ~ ~ ~ ~ ~ ~ ~ ~ . ~ : . : - ~ ~ ~ ~ , * ' , ~ ~ ~ , ~ ~ . f l , L - , ~ ~ , , , z : G ~ ~ ~ 'C, ~ ~ ~ ' ~ ~ ~ , ~ ~ ~ ~ ~ r ~ ~ , f l ~ ~ d ~ , ~ / & / @ ~ & ~ m ~ ' ~ ~ ~ n ~ ~ , ~ / ~ / N ~

with -0.01 and -0.02 MPa. Similarly E. camaldulensis and E. regnans

germination decreased with decreasing osmotic potential and increasing moisture

stress. E. camaldulensis germination decreased below -0.6 MPa and E. regnans

germination decreased below -0.8 MPa (Edgar, 1977). In southwestern Australia,

E. macrocarpa, E. tetragona, E. loxophleba and E. wandoo germination was

inhibited at -1.0 MPa (Schutz et al., 2002). E. globulus Labill seed germination

was sensitive to water potential of 0 to -0.75 MPa and germination was inhibited

below -0.25 MPa of water potential (Lopez et al., 2000).

When exposed to water, dry seeds follow a characteristic triphasic curve of

imbibition. The duration and contribution to total water uptake of the three

phases varies according to seed size, weight, structure, path of water entry,

permeability of seedcoat, chemical composition of seed, temperature and seed-

water contact areas (Obroucheva and Antipova, 1997). The first phase is

generally a period of rapid water uptake followed by second, equilibrium phase,

with little change in total water content, before a final period of rapid uptake as

the radicle emerges and germination is considered as complete.

In Phase I, water movement is driven predominantly by the matric potential of

the seed structure (Obroucheva and Antipova, 1997). Lettuce seeds (Lactuca

sativa L. cv Empire) completed phase I in 3 h (Bradford, 1990) Lettuce has been

used in many germination studies as it is sensitive to factors such as plant growth

regulators, light, temperature and water availability has been used in many

studies (Matilla, 2000). In Trigonella coerulea seeds radicle emergence occurred

after 48 h (Akhalkatsi and Losch, 2001). It has been suggested in several reports

that seeds with high initial moisture content can imbibe more rapidly than seeds

with low initial moisture content due to changes in the seed permeability

(Vertucci, 1989). As water uptake continues, cells rehydrate, metabolic function

resumes and respiratory activity begins (Bewley, 1997). Obroucheva and

Antipova (1997) concluded that two steps were involved in the activation of

respiration. The first step was activation of cytoplasmic enzymes of glycolysis

instead of mitochondrial enzyme and second step was completion of

mitochondrial biogenesis. They observed that in soybean seeds respiration

Chapter-4 (Water Relations in E. globulus Seed) -.,. .:. ..? - , .. -.., ...? ,, , ,@.... <.?&. ,-,!.. 7,e,:;m, c ~ 9 ~ ~ : . ~ . ~ ~ ~ . : . ~ ~ ~ 4 ~ ~ ~ ~ : . ~ ~ ~ , ~ ~ ~ ; ~ ~ ~ , ; ~ : ~ ~ ~ ~ , : ~ , 2 ~ ~ . ~ ~ ~ z ~ v ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , . ~ , v a : ~ ~ ~ e ~ ~ ~ ~ ~ : ~ ~ w , w ~ ~ ~ ~ w ~ ~ ~ e ~ ~ ~ / ~ ~ ~ ~ z ~ . .. . , .

commences at 20% moisture content and then increases markedly at 40-50%

moisture content growing tissues. They believed that mitochondria activation

was responsible for an increase in respiration at this stage.

Phase I1 of water uptake, in which the seed structure reaches saturation, is known

as the lag or plateau phase. The duration of phase I1 does not determine whether

the seed is dormant or not rather, a long phase I1 is a symptom of dormancy

(Bradford, 1990, 1995). During this phase initiation and completion of

endosperm weakening takes place in lettuce seeds (Bradford, 1990). Fully

imbibed non dormant seeds then enter phase 111. Phase I11 incorporates cell

expansion (Obroucheva and Antipova, 1997), cell division, DNA synthesis and

radicle elongation. The driving potential for resumed water uptake in phase I11

could be solute accumulation during hydrolysis of polymeric reserves in the

radicle and/or changes in elasticity with weakening of tissues surrounding the

embryo or the radicle tip itself (Welbaum and Bradford, 1990; Bewley, 1997).

As a result of water influx, radicle turgor increases and extension commences.

In some species, such as rape (Brassica napus), the testa cracks and the radicle

emerges during imbibition, but in others the fully imbibed embryo remains

inside the testa or endosperm for an extended period (Welbaum et al., 1998).

Various studies have shown that this condition occurs when embryo growth

potential is insufficient to overcome the restraint caused by covering external

tissues. To overcome such external barriers, the embryo has to increase turgor to

a critical "yield point" as a result of accumulation of solutes and associated

osmotically driven water uptake (Welbaum and Bradford, 1990).

In imbibing seeds, respiration tracks the triphasic water uptake, reflecting

metabolic changes with an initial increase, followed by a plateau phase and then

a rapid increase with germination. Bewley and Black (1994) associated increase

of respiration at the commencement of phase 111. of water uptake with increased

gaseous diffusion as seed coat is breached. Botha et al. (1992) concluded that

Chapter-4 (Water Relations in E. globulus Seed) . .1 ' - .., " , 7 2 ;. .,/. .,..,?,.. .y,.,,<.,- ,, :, :- * .,: . . .< .7.7 ,? ,.. .+ ,-v ., ..:.. , ?,,,,,7 * ~ 7 ; ~ . ? ; ~ ~ z . v , ~ . ~ : ~ , * , . < * ~ * ~ T,'7~L5.~y~,,E,0,~~,fl.;r.,g,mA7<R.Ar..mm37.

anoxia, and ethanol biosynthesis, may restrict respiration in the period from full

hydration to splitting of the seed coat, just prior to radicle emergence.

Exposure of photoblastic seeds to red light promotes respiration (Evenari et al.,

1955). Respiration rate of tobacco seeds imbibed in darkness increased during

imbibition, but decreased when imbibition was complete. Subsequent exposure

of the fully imbibed seeds to light promoted respiration Kip (1927), cited in

Toole et al., (1956) indicating that tobacco seeds require light during

germination. In some species, the effect of light on respiration appears

temperature dependant. For example, a short burst of red light increased oxygen

uptake in lettuce seeds at 25 OC but had no effect at 30 OC (Pecket and Al-

Charchafchi, 1979).

Most published work on water uptake has used seeds in free water or on

saturated paper. Water uptake by seeds in soil is a more complex process linked

to the behaviour of water within the soil structure. Soil matric potential is related

to surface and colloidal forces in the soil matrix (Woodstock, 1988), but rate of

imbibition of seeds in soil was reported by Vertucci (1989) to have little

relationship to the potential gradient between seed and soil. This suggests a role

for hydraulic resistance at the seedl soil interface, leading to the conclusion that

seed size plays an important role in rate of water uptake with the potential

difference between seed and soil determining the degree of hydration. Smaller

seeds generally have better contact with the soil than bigger seeds, due to a larger

surface to volume ratio, and, as a result the latter, take more time to reach

equilibrium water potential with the soil and thus for germination to start (Boyd

and Van Acker, 2004). That is, for biologically similar seed, differences in size

alone may change the rate of imbibition and could affect the time to complete

germination.

Several studies have suggested that germination is affected by evaporation of

water fkom the soil surface. Multiple hydrationldehydration cycles may result in

a loss of viability or induced secondary dormancy (Downs and Cavers, 2000;

Chapter-4 (Water Relations in E. globulus Seed) - .. - . ,..+ ,, .- ;". .,. :S.:-ir ,..,, <;,".'.,h..<';';.?,.1 ?Y ,: , .*i"..~,.?.i".i..7rt,.~",,TI,~ *,,:u <,:*,..,,~C".";,?II:C.i:q#-r.CV~ ,̂ :',m.a~~.",~~L~~.,;~*r;C~,AAAA(.(.,zi~,'d~~P,~'T,wii~Gi . . .

Kagaya et al., 2005). Natural populations of seeds germinating in areas with

unreliable rainfall or higher evaporation rate, tend to germinate quickly, when

moisture is readily available (Yirdaw and Leinonen, 2002).

Although the water potential gradient between seed and soil, along with the

hydraulic resistance to water movement, determines both the rate and degree of

hydration, aspects of seed water potential remain poorly understood and detailed

studies have been limited by the heterogeneous structure of the seed itself. As

noted in a range of reviews (Bewley and Black, 1994), in seeds the relative

contributions of the three elements of the Schollander (Scholander et al., 1964)

model of plant tissue water potential, change with time from predominantly

matric at the start of imbibition to osmotic and pressure potential as germination

proceeds. Because of the complex structure of the seed, there have been few

attempts to measure the components, although there have been several reports of

measurement of total potential in excised embryos (Haigh and Barlow, 1987).

For intact seeds, total potential has generally been measured psychrometrically

(Haigh and Barlow, 1987) but the method presents significant difficulties. In

particular, seeds need to be removed from water and surface dried (Welbaum and

Bradford, 1990) before measurement, leading to potential errors relating to the

drying method and to redistribution of water in the surface dried seed during

measurement. Unfortunately, simply measuring water content, relative to seed

dry weight suffers from a similar difficulty with effectiveness of surface drying

also impacting on water content, particularly at the lower end of the scale. This

inaccuracy is likely to be greatest in small, high surface-to-volume ratio, seeds

with adherent surface water likely to make a greater contribution to both

measured potential and gravimetric water content. Regardless of these

difficulties an estimate of the timing of the various phases of water uptake was

considered essential to a better understanding of the light responses reported later

in the present study.

. - . .,, ..._, Chapter-4 (Water Relations in E. globulus Seed)

, ,, , .,..., A ~ , ~ ~ : . ~ . . , . . . ~ ! ~ , , . .\ , .. . -..- a ,. ,c:~:y;.: \T :, ~ ~ , z ~ ~ ~ ~ e ~ . + - , ~ ~ ~ ~ ~ ~;>,.e~,>;;.~'>.r~~;,:,,? , W ~ ; L - ~ ~ ~ , W ~ - . ; : : , ; ~ : : ~ Y ~ ~ ~ A - , ~ ~ ; ~ ~ ~ ~ , ~ ~ ~ ~ N A V , ~ ; % ~ ~ ~ @!?$w~?@'~Vfl.&!:*%

Two extended water uptake determinations (Experiments 4.1 and 4.2) were

carried out under laboratory conditions. In the first, designed simply to establish

the duration of phases I and 11, no treatments were applied. The second was

designed to investigate light effects on imbibition. In both experiments water

uptake was determined as changes in relative water content with time. Based on

the results of these two experiments experiment 4.3 was designed to show

changes in water potential and verify the duration of phase I under the same

imbibition conditions. An additional experiment 4.4 investigated changes in

phase I following a hydrationldehydration cycle of the type suggested as a

possible contributing factor to delayed germination. The final experiment was a

simple respiration study intended to determine the delay between the start of

imbibition free water and commencement of respiration.

4.2. Materials and Methods

All experiments except the measurement of respiration used seedlot "0s" (Table

3.1). Imbibition was 'carried out in the germination cabinet set at 25 "C with each

plot consisting of 100 seeds on two layers of wet filter paper in a petri dish.

For water content determination, weights of air-dried seeds were recorded before

the start of each experiment. After each period of imbibition, wet weight was

recorded after the. wet seeds were removed from the petri plates and surface dried

by placing them on a paper towel on the laboratory bench for 5 minutes.

Moisture percentage was then calculated after Kader and Jutzi (2002) using the

formula:

Relative water content = ((Wet weight- Dry weight)/Dry weight) x 100

Seed water potential was measured on similarly surface dried seeds, using a

Decagon SCl OA Thermocouple Psychrometer and sample chamber. The unit

was calibrated using a standard solution of KC1 and all measurements were taken

after a routine equilibration time of 30 minutes regardless of the estimated water

Chapter-4 (Water Relations in E. globulus Seed) . . ? .,*.e. ?<...'.> :*? <.-. ,.,~--~>~,~.:~~,",<~,~<~,..~.<.~~~:~<~~..~.~:~r.;~c?p~? < q . , f i , ~ ~ , v . V . . c-%r,F . ‘ ,.. . ,,...,+.. -. ... ?-.- . ', v , m ~ ~ ' ~ , ! ~ , ~ ~ / m : ' ~ w ? ~ ~ / n ~ ~ r n m - ~ ~ ~ ~ ~ 7 / ~ ~ < ~ ~ 4 ~ > ~ ~

potential. Calibration and all measurements were carried out at a laboratory

temperature of 17*1 "C.

Experiment 4.1.

Seed was allowed to imbibe water for 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 12,

18, 21, 24, 27, 30, 33, 36 and 39 h in 12/12 photoperiod. Timing of the start of

imbibition relative to photoperiod was not recorded. There were four replicates

for each time and mean relative water contents were plotted (with standard

errors) against time.

Experiment 4.2.

As for experiment 4.1, water uptake was measured as changes in relative water

content for 48 h but in this case there were two light treatments, complete

darkness (see General Materials and Methods) and continuous light with

constant exposure to the germination cabinet lights. Imbibition treatments were

2,4, 6, 8, 10, 12, 14, 16, 18,20,22, 24,26,28, 30, 32, 34, 36, 38,40,42,44and

48 h. There were 8 replicates at each time and results were plotted as mean

relative water content (with standard errors) against time. Separate ANOVA

analyses were carried out to test light treatment effects at times showing an

apparent difference between light treatments.

Experiment 4.3.

Seed was allowed to imbibe for 2, 4, 6, 8, 10, and 12 h in a 12/12 photoperiod.

Seed water potential was measured after each 2 h interval and means and

standard errors plotted against time. There were four replicates at each time.

Again the start of imbibition relative to photoperiod was not recorded.

Experiment 4.4.

A control (stock seed with no pre-treatment) was compared with seed subjected

to one hydrationldehydration cycle. For the latter, seed was imbibed for 30 h in

complete darkness in separate replicates (see Chapter-3, General Materials and

, Y , T ~ . ~,.e .s,,~;,A- :*;:'>:=h**.,s .YX ~ , ~ ~ ~ ~ ~ : : ~ ~ ~ ~ ~ ~ ~ , ~ . ~ , ~ : ~ : ~ . ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ : : i ~ ~ ~ ~ ~ ~ w ~ ~ ~ . : ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ , m w ~ ~ ~ ~ ~ ~ : ~ ; ~ ~ w ~ ~ ~ ~ ~ : ~ - ~ , ~ ~ ~ , , ~ ~ ~ ~ ~ . ~ m ~ ~ ~ m ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ # ~ m m ; 47

Chapter-4 (Water Relations in E. globulus Seed) ~'.~.;~,~.',:;'.~j<::.,~, P'"i:. ~ ~ ~ ; ~ , ~ , , ~ . ~ v : ~ : ~ , ~ . : ~ ~ . ~ ~ , , ; ~ f i ~ c . ~ . ~ ? $ : # ~ ~ ~ ~ v , * ~ > w ~ ~ . . ~ ~ : ; : ~ w ..:e:fP:.*<'~,~~~a.~.~,~~.~w~~<~~:7,~7~~K4':~.~m~n,5~~~.'~~m~a~m~~"~~:fl/d7w%w~~~~~

Methods), before being air dried under normal laboratory conditions

(approximately 12/12 laboratory lighting and 18-20 "C room temperature) for 7

days, before the experiment was carried out. Both relative water content and

water potential were measured at hourly intervals from 1 to 7 h after the start of

imbibition. There were 4 replicates of the two pre-treatments. The results were

analysed as repeated measures (over time) using the repeated measures package

in SPSS.

Experiment 4.5.

Respiration rate during imbibition was measured using Warburg's respirometry

as described by Sestak et al. (1971). Seeds from seedlot "KI-2" were added to

the manometric flask. A small piece of filter paper dipped in 10% KOH solution

was placed in the centre of the flasks as a carbon dioxide absorber. 5 mL of

deionised water was added to the 200 seeds to start imbibition. The experiment

was conducted at 20 21 O C . There were two treatments with 4 replicates in a

completely randomized design. The laboratory lights were left on to give a

continuous light treatment with foil wrapped flasks giving the dark treatment. It

was not possible to completely exclude light in the dark treatment due to light

leakage through the glass connections in the manometer. Measurements were

commenced after 6 h and continued at 2 h intervals until there was a

displacement of the two columns, indicating that respiration had started. Results

were recorded as the time from start of imbibition. An analysis of variance was

carried out to compare the effects of light and dark.

4.3 Results

As shown in Fig 4.1, relative water content increased rapidly in the first 6 h after

the start of imbibition, reaching a mean relative water content of 61%. There was

a brief plateau during this initial increase with only a small increase between 2.5

and 5 h. After 6 h, water uptake reduced to a consistently slower rate, which

continued through to 40 h. Radicle emergence was noted in some seeds after 36

h of imbibition. At this stage mean relative water content had reached 80%.

Chapter-4 (Water Relations in E. globulus Seed) ,-:, .>T .,;: ':;*';,?,,~,-~;~.~ . .-<<.,..e,,v-,,>* ::.;,,>,~.~~,p7'::,.~,+- ~ , ~ ~ 5 ~ ~ ~ , - / ~ 4 ~ ~ ~ ~ , ~ , m ~ ~ : ~ ~ ~ ~ A " . ~ ~ M , ~ , ~ < ~ , : ~ ~ ? > 7 ~ x ~ . : ~ , ~ ~ ~ , n m ~ . ' ~ ~ ~ f ~ ~ , ~ ~ ~ ~ ~ ~ L ~ / ~ , & 7 ~ / ~ / # / ~ ~

There was no significant difference in relative water content between seeds

imbibed in the dark and in light throughout the 48 h of imbibition in experiment

4.2 (Fig 4.2). Water uptake was again rapid in the first 6 h with a transition to a

stable slower rate from 11 h onwards. Some radicle emergence was noted at 38 h

in constant darkness, whilst in constant light radicle emergence was first noted at

42 h. Germination counts were not made and apparent differences not tested

statistically.

In Experiment 4.3, water potential increased rapidly to around -0.5 MPa after 6 h

and remained relatively constant thereafter (Fig 4.3). As shown in Fig 4.4 a, seed

that had been allowed to imbibe and dehydrate before imbibition in this

experiment had a relative water content curve not significantly different to

control seed. For seed water potential, there was no significant interaction

between time of imbibition and treatment (Fig 4.4b). In Experiment 4.5 there

was a significant (P<0.05) effect of treatment on time to commencement of

respiration. Seeds started respiring after 11 h in complete darkness but in

constant light there was no measurable respiration until 17 h after the start of

imbibition.

Chapter-4 (Water Relations in E. globulus Seed) ~ , ~ . ~ ~ ~ C - ~ , ~ v , ~ : ~ ,,~,,~:~:~,:,ir,l.;(7-;E~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ , r ~ ~ ~ ~ ~ ~ ~ ~ R ~ ~ ~ / i C C C p . ~ ~ p . ~ : ~ 7 ' . ~ I Z,VrnR,;n 6Zjr.,mLv ~~.~.*;O."#~~~/~T.~-LRRWOGPI~"*~Z~~W.F/LI/~~~,U:~~:'~R.R~~~'.~I.~I~~~~:

1 I I I I I I -I

0 5 10 15 20 2 5 3 0 3 5 40 Water uptake (hours)

Fig-4.1: Water uptake with time after the start of imbibition on wet

filter paper. Data points are results of 4 replicates of 50 seeds i

standard error of the mean.

Chapter-4 (Water Relations in E. globulus Seed) ..7 .?*??,,* v.<r . , s - -.&'d s,.,.,.; :>%r''?A;:.&:,.: ?,PC;- '*7'-i-,- ., ~P>'<~..,./'w.~+- .,. F,<, ~:~~.*:;*,>,,>?~',Jo? .~~~,.~z~:?.T~.r,~m::.~..-~~.~~c~~~~~~;~*5?~~,#~~~4w~~7'.,A~~4.7'a~*vm4

Water uptake (hours)

Fig-4.2: Water uptake with time after the start of imbibition by seed

lot "0s" in full light (A) and complete darkness (w) . Data points

are results of 4 replicates of 50 seeds & standard error of the

mean.

Chapter-4 (Water Relations in E. globulus Seed) , r,,; ,..,,. 7 ... < .*./ ,-,/-,'.' ..Yk>-<<,.:.~ ,c.< ,v,*,*t,,,.->*. ,.,< r .,,'W'.,..*," L v , ~ . ~ , ~ ~ . . ~ ' : " ~ ~ ~ ~ + ~ ~ , ~ ~ , ~ ~ . ,',, "~,,~~<~.~,~;~,~,~T~~,~~4.~fi~~/~~~~~,~*7~~6~~;~~~&,/p+F/~'~.~~7o;s,n

Water uptake (hours)

z - v, ae Fig 4.3: Seed water potential with time after the start of imbibition on

wet filter paper. Data points are results of 4 replicates of 50 22 z

seeds standard error of mean. c'5

Chapter-4 (Water Relations in E. globulus Seed) 9,; ?<..fi2<.*.+,.,6 ,G<">"P,.~G...,. ..~,P;v;P;,4., .P,/,,&., r . ~ ~ . v w , A , ~ ~ : y ~ . : ~ : ~ ~ - / d ~ 4 P : ~ ~ s , ~ ; " . - ~ C , 2-*'i.,,+,,G, m ~ . ~ , ~ ~ , < : ? ~ s , ~ ~ . ~ / ~ ~ ~ ! ~ ~ ~ w ~ ~ ~ ~ , f l / ~ ~ ~ ~ / ~ ~ ~ ~ F , w ~ ' e ~ ~ ~ ~ / m ~ ~ / ~ <

a

Water uptake (hours)

Fig 4.4: Water uptake as relative water content (a) and water

potential (b) of untreated ( A ) and 30 h imbibed followed by 7

days drying (m) shown in this figure. Data points are results of 4

replicates of 50 seeds * standard error of the mean.

:,. . ' ?: ,. .... Chapter-4 (Water Relations in E. globulus Seed)

!. . . ..,.., 4, .&,,,,',a:- :> ,;-,a:..,. .: ,~,,.fi;:p.-2,~',~,.~~~x~&;~"?;'>.:,; :,,a, --,a>.;,-,' .,,, ?r:,.Y,,-?J,?!,t~. <<*'W>.4YS ~.&::*~~~4;~w85;~rG$~,c/>.Av/~/@,.F~'47x~.~~..;~~:.

4.4. Discussion

In each of the experiments in which water uptake was measured, the initial rapid

uptake corresponding with what is generally accepted as phase I of imbibition

lasted for approximately 6 h. By this time seeds were 45-50% hydrated. Battaglia

(1993) noticed that water uptake was rapid in E. delegatensis until 40% of

hydration was achieved, and this was reported as necessary for seed germination

to occur successfully. He also noted that for seed germinating in solutions with

water potentials above -1 MPa, 40% percent relative water content (RWC) was

achieved before 24 h of imbibition.

The marked discontinuity from 2.5 to 5 h in the half hourly measurements during

initial uptake in experiment 4.1 was not evident in later experiments. All showed

a relatively smooth transition into phase I1 after 5-6 h at a relative water content

of around 55%. It is notable that the 6 h relative water content reading in

experiment 4.1 is somewhat higher than would be expected from the other

experiments in the series, suggesting that some experimental error at this point

may have emphasised an apparent discontinuity.

In each of the experiments in which imbibition was continued for a substantial

period beyond the end of phase I, relative water content increased steadily from

55% at the start of phase 11. In experiment 4.1 it reached around 80% before the

end of readings at 39 h. The increase in experiment 4.2 was also consistent, but

by the end of measurements at 48 h both light treatments had reached a little over

60%. The rise to 65% RWC during phase I1 compares with a report by Lopez et

al. (2000) who noted that, in E. globulus, radicle emergence occurred when the

relative water content neared lo+-5% in laboratory conditions.

In both of these trials radicle emergence was noted after about 36 h but this did

not correspond with a marked upswing in RWC normally associated with the

start of phase 111. Similar results were obtained by Donovan (2001) with Radiata

pine seeds, where the radicle emerged without any rise in water content. A

possible explanation for these observations is that the force required to split testa

Chapter-4 (Water Relations in E. globulus Seed) j.,? ,:,,,r,c.,>J~&~,;~.;:,*,@ ;+...*,,..5.:*.e,y .,;:,y.; ,,* >?,.~?:*L>,.e,&>~v<-,>,,mA.'..*?~%. , - ~ . . * : ~ ~ , < . ' . ~ - ~ ? , v 2 < , ~ 4 ! ~ f l ~ - ~ ~ ~ ~ ' % ~ Y & > ~ ~ & ~ ~ ~ ~ L ~ ~ 7 ~ ~ ' @ : ~ 7 . w L e , ' & ~ ~ / # / f i . ~ ~ w . w ~ ~ ~

was small. In tomato seeds, phase I11 also started without any noticeable change

in water uptake and water potential (Haigh and Barlow, 1987).

Water potential increased above -0.5 MPa after 6 h of imbibition in experiment

4.3, remaining relatively stable until the final measurements at 12 h. In

experiment 4.4 non-pretreated seeds showed a similar rise, reaching -0.5 MPa

after 4 h but continued to increase with time, albeit more slowly, reaching -0.1

MPa after 7 h. The apparent end to the rapid uptake period of phase I of

imbibition was much clearer in the water potential measurements, with phase I

clearly concluded at about 6 h. In contrast, the RWC data showed a more gradual

change, starting at 6 h and continuing to 11 h, before the slower rate of uptake in

stage I1 was clearly established.

When exposed to water, pre-imbibed and desiccated seed hydrated faster than

non-pretreated seed reaching -0.5 MPa between 2 and 3 h after the start of

imbibition and rising to -0.1 MPa within 5 h. When ryegrass seed pre-treated in

the same manner (wetting and drying) was rehydrated, water uptake was faster

compared with control seeds and germination was completed earlier (Lush et al.,

1981). Several reports have noted that rehydration of dehydrated seeds increased

both the respiration rate during germination and germination capacity (Hegarty,

1978).

Respiration in seeds imbibed in darkness commenced after 11 h, 5-6 h after the

end of phase I in the earlier experiments. Although these respiration and water

uptake measurements were made on different seedlots, it is notable that 11 h

corresponds closely with the end of the transition between rapid uptake of phase

I and the slow increase in RWC that appears to correspond with phase 11.

Referring to these data, RWC at 11 h was around 55% to 65% according to

experiment 4.1 with a water potential between -0.4 and -0.1 MPa. Lopez et al.

(2000) reported that E. globzilus germination ceased below -0.25 MPa.

Furthermore, the water content figures agree with results of Obroucheva and

Chapter-4 (Water Relations in E. globulus Seed) 5 ...?.>..:,r7~<,::5:... . 'c. Zz~~~.:'.~ ;?.C?A',?YL?, d ~ , ~ ~ ~ ~ ~ ~ , ~ ~ ; . Z Z . ' ~ c : ~ ' ~ ~ r , ~ ~ : ~ . V ~ % ~ ~ ~ V . ; ~ .? :p,,%.,-, 6~;:~+v,W~r~~~,~7V~~,%2:~WS/~~n,.T/a&fiW,O/~nF/~~&~,~J<~~G-~€~L~~~ZVX2

Antipova (1997) who reported that, in pea, respiration increased at 45-50%

hydration due to tissue growth.

There was a notably later start to respiration in seeds exposed to constant light

during imbibition. There are few reports of such a response in the literature, but

Kipp (1927) cited in Toole et al., (1956) noticed that in tobacco seed, respiration

was increased in darkness. The observation, in experiment 4.2, that radicle

protrusion appeared to start earlier in dark germinated seeds may be linked to the

observed change in respiration in darkness compared with light. An early start to

both respiration and radicle emergence in the dark suggests that exposure to light

at this stage of germination may delay development. Unfortunately most of the

water uptake experiments were carried out in a 12/12 lightldark cycle and as

timing in relation to the start of experiments was not recorded the light condition

during the first 12 h was unknown. In experiment 4.2, where imbibition was

carried out in either constant light or dark, the two hour sampling interval may

not have been sufficient to show any differences in water uptake, if present in

this seedlot.

In summary the results indicate completion of phase I at about 6 h followed by a

transition into phase I1 with an ongoing near linear increase in relative water

content from about 11 h. There was no change in this linear trend to mark the

start of phase 111, in spite of radicle protrusion at 32-48 h. There was an

apparently earlier start to radicle emergence in seeds germinated in darkness and

a correspondingly earlier commencement to respiration.

Chapter-5 (Induction of Secondary Dormancy) .,;.; ..: d . < ,<,r .,;7>7 .T:$ .,.r;::m ,",,+!? zs,; ~~~,.,-~~~~:,":~"~.;~<~~.~$~~*.~/~".~.q,~,,.~.o ~ , ~ , , ; y , , ~ . ; ~ . w + . ~ ~ - ~ ~ . ~ : . : ~ . ~ ~ ~ ~ ~ ~ ~ : ; ~ , , ~ , p / ~ ~ ~ ~ ~ ~ ~ , ~ ~ , ~ ~ ~ ~ ~ ~ ~ & , ~ , ~ ~ ; ~ , m , ~ ~ ~ ~ ~ ' ~ ~ ~ , ~ ~

Induction of Secondary Dormancy (Dehydration and

Light Effects)

5.1. Introduction

As noted in Chapter 1, initial observations suggested that uneven germination of

E.globu1u.s seedlings might be associated with a period of dehydration shortly

after sowing. In a natural environment, most seeds germinate near the soil

surface and may undergo several cycles of wetting and drying before completing

germination (Downs and Cavers, 2000). In some seeds such a hydration

dehydration cycle has induced secondary dormancy (Ren and Tao, 2003),

delayed germination in non dormant seeds (Baskin and Baskin, 1982; Downs

and Cavers, 2000) or caused loss of viability. It is often unclear how many

hydration dehydration cycles are required for seeds to lose their viability or

germinability (Ren and Tao, 2000).

Commercially, germination of seeds of some species, for example tomato and

oats (Berrie and Drennan, 1971), wheat (Hanson, 1973) and Aster kantoensis

(Kagaya, et al., 2005), is improved by priming treatments that involve a

hydration-dehydration treatment or to prepare seeds to germinate during adverse

conditions (Bharati et al., 1983) such as drought (May et al., 1962). Hegarty

(1978) reported that seeds imbibed and then dried back germinate rapidly

compared with freshly imbibed seed, but there is a negative effect on

germination if cell elongation was initiated during imbibition. In some vegetable

seeds germination is more sensitive to water stress during seed germination

compared with seedling radicle growth (Hegarty and Ross, 198018 1).

Following hydration-dehydration cycles in Aster kantoensis, germination

decreased as the number of cycles was increased, but shorter period of

dehydration were less damaging (Kagaya et al., 2005). Longer periods of

Chapter-5 (Induction of Secondary Dormancy) z-,< . .+..: -::..,r~b. -".-::-m.~,.w 'TG. 2;. .K ;,.%..-.L~~;:Z:;.+'#:JX,:~V. + +,74:'..c~ W : ~ - V ' J ; ; ~ W , . L ~ < ; ~ 0 ~ ~ . 2 r . ~ . ~ ~ v m / ~ ~ ~ ~ ~ ~ 4 w ~ m . s ? ~ ~ ~ ~ ~ 7 ~ ~ ~ ~ w , ~ ~ ~ ~ / ~ ~ / n ~ ~ ~ f f ~ ~ ~ w ~ ~ ~ x ~ ~ ~ ~ ~ ~ ~ 4

hydration increased germination in some lots of tomato seed but in others it was

decreased (Penaloza and Eira, 1993). Baskin and Baskin (1982) suggested the

length of imbibition period affects germination and not the drying period. One

cycle of wetting and drying did not affect bull thistle (Cirsium vulgare) but when

seeds were subjected to two or more cycles, germination was delayed (Downs

and Cavers, 2000).

During imbibition, enzymes necessary for germination are produced and stored

reserves are mobilised (Bewley and Black, 1994). When seeds undergo

hydration followed by dehydration, the progress of metabolic activity is partially

reversed, but the net effect may be to hasten germination on subsequent exposure

to water (Hanson, 1973; Bharati et al., 1983). Uneven germination in some

soybean lines was reduced by prior hydration at higher temperature followed by

germination at lower temperature, instead of hydration followed by drying and

storage (Bharati et al., 1983).

There are few reports of alternate wet/dry cycles on the germination of eucalypt

seeds. Wetted and dried seeds of E. sieberi germinated rapidly compared with

fkeshly imbibed seeds (Gibson and Bachelard, 1986; 1988). E. salmonophloia

seed viability was not affected by one cycle of wetting and drying (Yates et al.,

1996). Wilson et al. (2005) reported that E. ovata seed, hydrated and

subsequently dehydrated during pelleting, exhibited what appeared to be a type

of secondary dormancy broken by germination in constant darkness.

Interactions between hydration/dehydration treatment and light conditions have

been reported in other species. Prolonged imbibition of seeds in darkness under

water stress developed light sensitivity and induced secondary dormancy in

Brassica napus seeds (Pekrun et al., 1997a). The germination response of seeds

of bull thistle to wetting and drying cycles differed according to whether the

seeds were held under constant darkness or alternating light and dark (Downs

and Cavers, 2000). Ryegrass seed subjected to a wetting and drying treatment

Chapter-5 (Induction of Secondary Dormancy) .r,yez.b m.. - ,r r:. :-w ~p.~m~,~~~~~~.:r.m.e;:~.;:~~~t~t,~t~~~.:.:~~.~.~~~~.~~-~~i~ r:e;*-<+. r4*l.irve : T L ~ Z c ~ r ) ? d W Y I I I ) ~ I ) ~ ~ ( . < ~ L I * r + O o ' r S I r S I ~ / U W / X X X C I / ~ B / L . / ~ ~ ~ ~ ~ R / f l / / j r j r ~ ~ C I . ~ ~ :

showed reduced dark dormancy if the treatment was imposed in light (Lush et

al., 1981).

The experiments reported in this chapter investigate whether hydration-

dehydration treatments carried out under laboratory conditions could induce

secondary dormancy or delayed germination similar to the uneven pattern of

germination observed in commercial nurseries. In view of previous work by

Wilson et al. (2005), showing a likely interaction between a hydration-

dehydration cycle and light conditions during germination in E.ovata, several

different light treatments were included in combination with dehydration.

5.2. Materials and Methods

Pre-soaking, imbibition desiccation and germination

In the first four of the seven experiments reported in this chapter, seed was

soaked in containers with enough water to cover the seed to a depth of

approximately 10 mm. Replicates of fifty seeds were soaked in separate vials of

distilled water under ambient laboratory conditions, with a temperature of 18-20

"C and a normal diurnal lightldark cycle. The distilled water was changed every

6 h to prevent hypoxia. For germination testing, soaked seed was surface dried

with paper towel before being placed on filter paper in petri dishes and

transferred immediately to the germination cabinet, For the remaining

experiments in this and later chapters, imbibition was on wet filter paper in petri

plates.

For desiccation treatments, seed was removed from the soaking vials or from wet

filter paper, surface dried with paper towel and placed on two layers of dry filter

paper in new petri plates. These were left open for 5-6 h, before the lids were

replaced. The dishes were then placed on a laboratory bench for the times gven

(usually 7 days). The laboratory was temperature controlled within the range 18-

20 "C. Lighting was a mixture of natural daylight and supplementary fluorescent

Chapter-5 (Induction of Secondary Dormancy) < :. .... z,,. .. n: !-A*;.!3 #,-;.5>,z,,,w,,..,A..~.,.* < > ~ ~ > . ~ : z ~ : ~ ~ ~ ~ , ~ , ~ ~ ~ ~ . ; - ~ ! ~ , ~ ~ $ ~ , ' ~ ~ w < ~ ix.:,%,;/*~:~~~.~,z~~,v~v~.v,~r~~,.~,'~,m,~~&:~<m;~,~~,~'~~~~~@/~/~,m/~/~/fl~

tubes to give an illumination period of approximately sixteen h. After drying,

distilled water was added to wet the filter papers to start germination.

All experiments were randomised complete block designs, with blocks arranged

according to position in the germination cabinet. Each trial consisted of four

replicates.

Experiments

In a preliminary experiment, seedlot 'h' (Experiment 5.la) and seedlot 'Li'

(Experiment 5.1 b) were surface sterilised with 10 mL of 12% commercial bleach

before being rinsed thoroughly 4 - 5 times in distilled water and blotted dry with

a paper towel. Seedlots were then soaked in deionized water under natural light

and dark conditions (approximately 1618 h) for 0 (control), 6, 12, 24 and 48 h

before transferring to the germination cabinet. Germination was scored on days

5, 8 and 10 after the end of pre-soaking.

In experiments 5.2a and 5.2b, seeds of seed lot 'h' and seedlot 'Li' (respectively)

were soaked for 0 (control), 18,24 and 30 h in constant darkness at 25 "C before

being allowed to desiccate for 7 days as described above. After desiccation the

seeds were germinated under 12/12 photoperiod, with germination scored on

days 5 and 1 1.

In experiment 5.3 a and b seeds of seedlot 'KI' and experiment 5.4 a and b

seedlot 'h' (respectively) were pre-imbibed for 0 (control), 7, 14,24,30 and 35 h

in the dark on wet filter paper in a petri plate at 25+1 "C in a germination cabinet,

before being allowed to desiccate for 7 days as described above. Germination

was carried out in the germination cabinet in either constant darkness or 12/12

photoperiod. Germination counts were done on days 6, 10, 13 and 19. The foil

wrapped (constant darkness) plates were unwrapped in a dark room and scored

using a green safe light. The experiment was analysed as a factorial design with

six times of imbibition by two light treatments.

Chapter-5 (Induction of Secondary Dormancy) 3.:'- .:, . i. : D.~YE,,TF.+V,T n ,.:u:~~:+?:.:.za;v,s~' r;~~w~m,c~.;,~~?-;n,?p~,~~i),? ~fi~crr~n~i~~~.~~,~d1111010~~1~6~~n~~,'1c;~%yr,:1;i:c~:~~~/nn'i,:1~~ 'RIR:!rr:nAV;lr7m,:w/n,N/fl,a/I

In experiment 5.5, seeds of seedlot 'Ki' were imbibed for 30 h in the dark at

25f l°C, and allowed to desiccate for 7 days. After desiccation, seeds were

transferred to Lanneno trays filled with Richgroo seed raising mixture. Each

plot consisted of 12 cells with 5 seeds per cell. After sowing, the trays were

irrigated. One half of each tray was then covered with an opaque reflective

insulation sheet, and the other half covered with transparent polythene, providing

light and dark conditions with similar rates of evaporation. Sensors attached to

"Tiny Talk" temperature loggers (Gemini Data Logger, Chichester, West Sussex,

UK) were inserted just below the surface of the potting mix in a tray of both

treatments to record temperatures near the germinating seeds. A non-imbibed

control treatment was also included. The experimental design was a randomised

complete block with two 24 cell trays with two treatments in each block and 6

replicates. Germinated seeds were scored daily between days 13 and 20 in

natural light conditions. Seeds germinating in the dark were re-covered

immediately after each count.

5.3. Results

As shown in Fig 5.1, soaking seeds under the natural light/dark cycle for more

than 24 h reduced day 5 germination significantly (P<0.05) compared with the

control in seedlot 'h'. On day 8, germination remained significantly (P<0.05)

lower for seeds soaked for more than 24 h. Soaking did not adversely affect

germination in seedlot 'Li'.

Dark soaking for 18 h or more, followed by drying, reduced germination on day

5 significantly (P<0.05) in each of the seedlots (Fig 5.2 a and b). By day 11,

germination had improved, but in seed lot 'h', there was still a significant

negative treatment effect on germination for soaking periods of 24 h or more

followed by drying back. In seedlot 'Li', 24 h of dark soaking followed by 7

days drying significantly reduced germination compared with untreated seeds,

. . ..,<., . .',-A*. ;?, ., ,,. :... ,.., . ., ,, . Chapter-5 (Induction of Secondary Dormancy) . + ... ,-, ,?<'+,'#'-..*r<WA:.!: ~>,.:'V>>:V ?,.?.<: 2 /*:;', ' - ,. .:.~~.~.'~:2;-Y.~:i?dz~.7:&;.~?:1~.~~~#~ 1 , ~ <..d,ma. V ~ ~ : ~ ; & ; ; V L ~ A ~ ~ I ~ ~ W ~ ~ ; ~ ~ . b~>r~%m/fl~q~:

but seeds imbibed for 30 h in darkness followed by 7 days drying did not show

impaired germination on day 1 1.

In experiment 5.3 (a and b) and 5.4 (a and b) there was a significant (P<0.05)

interaction between light conditions during germination and imbibition time

when germination was recorded on day 6 in both seedlots 'KI' and 'h'.

As shown in Figs 5.3(a) and 5.4(a), germination on day 6 decreased markedly

with increasing time of imbibition for seed germinated in alternating light dark

conditions. In contrast, seed germinated in the dark showed no significant

response to the soaking and drying pre-treatment (Figs 5.3b and 5.4 b). In seedlot

'KI', 30 and 35 h of imbibition followed by 7 days drying reduced germination

on day 10 significantly (Pi0.05) in alternating light and dark, whereas in seedlot

'h' imbibing seeds for 30 and 35 h followed by drying reduced germination

significantly (P<0.05) on day 10 in both alternating light and dark conditions as

well as constant darkness. There were no significant effects of pre-treatment or

germination conditions on germination on days 13 and 19 in either seedlot.

In the glasshouse trial (Fig 5.5) germination was significantly (P<0.05) higher in

darkness compared with natural light. There was a marked difference in

germination between dark pre-soaked and non-treated seed, with dark pre-

soaking causing a significant (P<0.05) reduction in germination in both darkness

and natural light. However, there was no significant interaction between the two

treatments. Although temperature measurements were not replicated, there were

some notable differences. Over the course of the experiment, daytime maxima

under transparent plastic covering, reached 47 to 50 OC with night minima of 9 to

10 "C. Under the opaque cover, maxima were lower, at 38 to 40 OC with similar

minima of 9 to 10 OC.

Chapter-5 (Induction of Secondary Dormancy) s , - .'.. .-,.: .,~.&,~,::'i,~~ *;,t;. : ?>,*.,.:'.r.'v g.; .,,. 7.:,,.:.w,, 7 *7./;, ra.;<-'>"J :*':,$~P.L<:. t >z:rfi~<~>.* ~*~~~7~,~:w,;7~~,f7.*~~.'.~~~~-"~/&~~w,m,~~~m~w~>z~,N/~~m~

V

0 10 20 3 0 40 5 0 Time (h)

Fig 5.1 (a & b): Mean germination percentage on day 5 (O), 8 (0)

and 10 (A) for seedlots 'h' (a) and 'Li' (b), germinated after

soaking in laboratory conditions at 18-20 "C for 0-48 h (1 618 h of

natural light1 dark cycle). Data points are results of 4 replicates

of 50 seeds * standard error of the mean.

Chapter-5 (Induction of Secondary Dormancy) :.,:-,4wc ,... -,+z,.~~~,,; :k +: ,,~$:,,.d,,~c~.7, ,?'*J ~ T ~ , ~ ~ " ~ ~ , ~ - ; F ~ ' ~ ; ; ~ , + , : & ~ ~ : ~ + , ~ A : , ' , . . ~ , - ~ ~ ~ *.,',r J%,%?A., w., -;. ~ b ? , ~ , ; , . ~ ~ , ~ , , ~ x ! > ? , f l ~ 3 ; ' ~ ~ V . ~ S : ~ , ~ . : ~ ~ f l , ~ ~ % ~ % < ~ X ~ , , V f l 7 ~ S ~ ~ ? A W / S V ~ ~ 4 ' %

I I I I 1 I I

0 5 10 15 20 2 5 30 3 5 Soaking time (hours)

Fig. 5.2 (a & b). Mean germination percentage on days 5 (0) and 11

(0) for seedlots 'h' (a) and 'Li' (b), germinated after different

periods of dark soaking at 25 f 1 "C followed by 7 days drying

back at room temperature (18-20 "C) in natural light conditions.

Data points are results of 4 replicates of 50 seeds + standard

error of the mean. LSDs for comparison between soaking times

on day 5 were 10.3 and 12 respectively and for day 1 1, 1 1.1 and

13.7 respectively.

Chapter-5 (Induction of Secondary Dormancy) ,.>'.?::.,,'\ *,;.',,<<.,s7,# .&.%'TR,$,'% <*.:'#;.r*L7.:9,'z ,fl,<.,~;:%.,,..*~..'e~.'>:?;:,4Ke,A>;~!,&,z:.? 'ey2:?vd~~T,%~;~~~&~~~,~~,w~*~<~~~w,,*&~y~~~m/~~,~w.'e5x~~~:~~-,~,mw~

V

0 10 20 30 40 Imbibition period (hours)

Fig. 5.3: Germination in alternating lightldark (a) and darkness (b) on

days 6 (0, +), 10 (0, M), 13 (A, A) and 19 (0 , e) for seedlot 'KI'

imbibed for different time periods followed by drying back for 7

days at 18-20 O C in natural light conditions. Data points are

results of 4 replicates of 50 seeds + standard error of the mean.

..-.-, ,. ,, , .? .;vF%,.*,,,,r7,,w.L7iTrd.F,F,-7 .$<.*.,a .?, ~.-2v~~,,:57~,...,a;~:e.,~T.c..:3qx Chapter-5 < , . I . ~ ~ . ~ ~ , ~ ~ 3,.,ri (Induction . ~ . Q ~ w , x , , ~ ~ . . : ~ z . , . . ~ ~ ~ ~ , ~ s ~ ~ ~ i ~ . , . ~ , B , ~ ~ ~ ~ m ~ ~ ~ ~ ~ m ~ . ~ ~ ~ ~ ~ ~ ~ F , w , ~ , : ~ ~ ~ . of Secondary Dormancy)

10 20 30 40 Imbibition period (hours)

Fig. 5.4. Mean germination (%) in alternating lightldark (a) and

darkness (b) on days 6 (0, +), 10 (0, a), 13 (A, A) and 19 (0 , 0 )

for seedlot 'h' imbibed for different time periods followed by

drying back for 7 days at 18-20 "C in natural light conditions.

Data points are results of 4 replicates of 50 seeds f standard

error of the mean.

Chapter-5 (Induction of Secondary Dormancy) 7"f c ,>,.+.'n,~,'?*',,.%, ~ ~ ~ z . ' ~ ? , ~ 4 ~ ~ ~ ~ . ~ - ; > ~ " ; , ~ ~ ~ ~ . , ~ ~ ~ ~ . ' ~ ~ ~ . ~ ~ ~ ~ . . . ~ ~ ~ ~ ~ ~ * . ~ . ? ? ~ > 7 ~ ~ , ~ ~ ~ ~ . & ~ ' ~ < . ~ : ~ . ~ . v w < ~ . ; z ; ~ < P , R 8 ~ , . ~ 4 . > 7 x , ~ ~ * ~ ~ ~ / ~ ~ ; ~ ~ ~ ~ ~ ; w , ~ ~ ~ ~ ~ / ~ w ~ ~ / ~ / ~ / ~ ~ w ~

Imbibition period (hours)

Fig 5.5: Glasshouse germination on days 13, 15 and 20 for seed

from seedlot 'Ki' imbibed in darkness for 0 or 30 h at 25 + 1 "C

and dried back and stored for 7 days at room temperature (1 8-

20 "C). Germination was in alternating natural light and dark (0)

or continuous dark conditions (m). Error bars are standard

errors of the mean (n=5).

Chapter-5 (Induction of Secondary Dormancy) ~ ~ . . * , z ~ z z w , m z . , ~ ~ w . z z * , ~ , ~ ~ , ~ ~ ~ ~ , w ' z ~ . ~ # , ~ , ~ , # , ~ , ~ ~ ~ . , ~ ~ w , z s ~ . ~

Plate 5.1: Photograph showing germination in glasshouse

conditions. Strongest germination was in control (untreated) seed

germinated in darkness (second from left). Treatments are D (dark

germination), LID (germination in natural light), 0 is non preimbibed

seed and 30 is seed imbibed for 30 h and dried back for 7 days

before sowing.

. Chapter-5 (Induction of Secondary Dormancy) 1 ... I? r'<Z'W: Lib.:.'< *:. .','.'~.,r~,ii' dti.''..d'. 1 %<?,+.. C"4'.,:5 0, N ,rri<Tn*, <. r: 3'~3':<,IiJCICIC17~~AIN.C;i+Z:~.L'5555~~ X ~ ~ ~ , 1 " 1 " ~ R ~ p ~ , ~ I I ~ , 0 C e . ~ ~ / * F , * F ~ ~ , ~ M ~ l a i * : ~ . ~ / C L : f l , J ~ # ~

5.4. Discussion

The decrease in germination percentage after 24 h dark soaking observed in this

work is consistent with results from previous studies on germination with a range

of other species including Acer pensylvanicum (Bourgoin and Simpson, 2004),

Caesalpinia paraguariensis (Abraham de Noir et al., 2004) and wheat (May et

al., 1962).

Pre-treatment by imbibition in the dark followed by desiccation and storage

induced delayed germination in both seedlots, consistent with an earlier study

reporting that dehydration of E. delegatensis seeds after 48 h reduced

germination capacity (Battaglia, 1993). The near linear reduction in initial

germination with increased soak time up to 25 h suggests that the response is

related to progressive changes in the seed during the early stages of germination,

rather than any damage to the embryo or seed structure occurring after a

threshold period of soaking. This contrasts with tomato seeds subjected to

similar treatment. Germination was reduced when seeds were subjected to

dehydration after 25 h imbibition but if dehydration commenced before 25 h of

imbibition, there was no effect on germination (Berrie and Drennan, 1971).

Most published reports indicate that generally, a single hydrationldehydration

cycle increases germination rate in both laboratory and field studies, but there

have been reports of adverse effects on germination. Gibson and Bachelard

(1988) reported that E. sieberi seeds did not germinate after five cycles of

wetting and drying. Senaratna and McKersie (1983) reported that prolonged

imbibition of soybean seeds, followed by dehydration to 10% moisture, reduced

germination. Becwar et al. (1982) also showed viability loss in silver maple and

areca palm seeds, which was attributed to excess electrolyte leakage caused by

dehydration stress.

In the present study it was notable that one cycle of hydration and dehydration

appeared to delay light or light/dark germination in both the laboratory and the

glasshouse. Germination in constant darkness followed a normal pattern, close to

Chapter-5 (Induction of Secondary Dormancy) ,&. . < .',;+,., .>,:, .,' ;, *'d',,'* ,p,*:,'...., < ; , . : ~ ~ , / ~ " " ~ , $ 7 , . . ~ ~ . ; , , ? , ~ : ~ , ~ ~ , ~ ~ . ; ~ ? , , .,<,,,'":%', ,.,, <",>',">\'."., .f,"<.y<*;*,P .,' *?<#7,?e,d:?.p, ~>.~rs,#fi~*%:~~~#'~-w,~~.~..~,d7.~#~,~:;,~?~~~fl!*~:,.9,~~~

non-pretreated seed. As noted in a recent conference paper, (Thanos et al., 2005),

light inhibition of germination is relatively unusual, but studies carried out by

Bell et al. (1 995; 1999) with E. marginata and Wilson et al. (2005) with E. ovata

demonstrated that darkness may improve germination in Eucalyptus species

under some circumstances. However, (Schutz et al., 2002) reported that

germination rate of E. loxophleba and E. wandoo at high temperature (28 OC)

was lower in darkness compared with continuous light. Similarly, Downs and

Cavers (2000) observed that, after eight cycles of wetting and drying, bull thistle

germination was decreased in continuous darkness compared with alternate light

and dark.

The present results clearly indicate that delayed germination can be induced by

pre-treatment, and that continuous darkness during germination overcomes the

change in germination rate and uniformity. Using Baskin and Baskin's (1998)

definition this can be described as "dormancy". Definitions of secondary, or

induced, dormancy vary in the literature (Bewley and Black, 1994; Pekrun et al.,

1997a; Kebreab and Murdoch, 1999a) but Baskin and Baskin (1998) cited

Harper (1957) in defining induced dormancy as failure of non dormant seeds to

germinate normally in response to particular environmental factors. Delayed

germination induced by pre-treatment subsequently overcome by light conditions

during germination appear to be correctly classified as "induced dormancy",

using this definition. Thus, in spite of Baskin and Baskin's suggestion that there

is no reason to use the term, induced dormancy is used throughout this thesis to

describe delayed germination in response to pre-treatment.

With seed germinated in the glasshouse, temperatures were higher in the light

exposed treatment, but the germination pattern was consistent with laboratory

studies at lower and constant temperatures. That is, the results do not indicate a

temperature effect on germination. However, a study on lettuce reported that

high temperature initiates a light requirement in dark germinating seeds (Kristie

et al., 1981), perhaps suggesting that more evidence is required to completely

eliminate a temperature effect in the symptom reported in commercial nurseries.

.. . . - , . . ,., ,. , . , .. ..* Y..~'..- ..j,.~..,:w.d7. O,..VT.C T. .. ., &A C,<A. ....,P Chapter4 em.,+.> % <. .~7~=.fl,,w,m4-,~,~~.,--,~,~w,~.,~,~,,z (Induction of . ~ ~ ~ ~ ~ . ~ . ~ , ~ , W ~ ~ V ~ C ~ W ~ ~ ~ ~ ~ , W , ~ ~ ~ . W , ~ ~ ~ ~ ~ ~ Secondary Dormancy)

Overall, the experiments described show that pre-treatment by allowing the seed

to imbibe water in darkness, for around 20 to 30 h, followed by desiccation and

storage, leads to induced dormancy in this species. From the results obtained in

the first experiment, notably the significant reduction in germination with

soaking without subsequent desiccation, it is difficult to separate soaking (or

perhaps light conditions during soaking) from the desiccation treatment. The

response to light during germination indicates that the induced dormancy is

broken by germination in darkness. This induction of dark responsive dormancy

suggests some re-examination of the possible influences of environmental

conditions, particularly during early germination, in seed testing and dormancy

studies. The results of experiment 5.3 showing a significant interaction between

pre-treatment and continuous light or continuous dark emphasise the importance

of light as a critical factor for further study. From a practical point of view for

commercial nurseries, appropriate management of light during the early stages of

germination may have a marked influence on uniformity of germination and is

likely to represent a relatively inexpensive change in management practice.

Chapter-6 (Light Effects) ., ' ,,., .,,.. ,.,. +, ,..,<. $.,:$,,+-,,,- !?;!:, p 4 a ~ ; . ? . ~ ~ ~ ~ . ~ , ; . ? i ~ ; : 9 ~ ~ c , A . " ~ . W ~ < . ~ ' , : . ~ '<9,,.%%?,:? :.'., '.,><> :,,"?r<3-,7, 'p+:,",,r,O.'W,d; ~ J ~ R W & ~ $ ~ Z @ ~ S V < P , ~ - ~ J , ; ~ ~ ~ < ' ~ : < ~ ' & ~ ' ~ V ~ ~ > Y ~ ?f..:l , .. . .>.. ...

Chapter-6

Light Effects

6.1. Introduction

In the previous chapter, experiments aimed at demonstrating a decrease in

germination in response to a hydrationldehydrationlrehydration cycle suggested

that the observed effects might have been due to light conditions during

germination rather than the hydration cycle. In particular a period of darkness

during early imbibition appeared to induce lower or delayed germination. Light

is essential for some species during germination, and for survival and further

development of seedlings in all species. Plants have developed various

mechanisms to adapt to different light conditions (Toyomasu et al., 1998) and

the complex processes involve several photoreceptors, with phytochromes the

most studied (Chory et al., 1996).

At germination, the amount of light received by a seed depends on its position in

the soil and the characteristics of its seed coat and any other structures

surrounding it (Pons, 2000). It is generally agreed that light can pass through the

top 4-6 mm of soil (Thanos et al., 1989) but, within this depth, there is a change

in light quality. Seeds on the soil surface 'may therefore behave differently

compared with seeds buried at various depths (Atwell et al., 1999). Within the

light spectra, different wavelengths cause different germination responses across

a wide range of species (Bell, 1993; Atwell et al., 1999).

In the absence of light, seeds develop etiolated seedlings, with a long hypocotyl,

apical stem and unopened cotyledons. On exposure to light, de-etiolation starts

as hypocotyl growth decreases, the apical hook opens, cotyledons expand and

chloroplast development begins (Frankhauser and Chory, 1997). Studies on light

influences on some Eucalyptus species by Clifford (1953) suggested that light

requirements vary according to the age of the seeds. For example, E. nitens seeds

Chapter-6 (Light Effects) : 7 . ,"iv..,* +.,;%,;,:., ri ,. - w. .~.%cI. h . . ~ i * 1117(.iv:if-&w%t: ..''zv,c*~s fit~:.1~~(.?..4idx'& *;A$';% ~ % A ~ - ~ & ~ ' C S ~ ~ & ~ ~ % ~ ~ ~ ~ ~ ~ K ~ Y ~ J ~ , ~ ~ ~ ~ ~ I D I / ~ M ~ R ~ ~ ~ ~ ~ ~ ~ : . P ~ ~ V * Y * Y & * Y ~ ~ . ~ ~ ~ V I :

collected from green capsules required light to germinate but seeds collected

fkom brown capsules did not require light. Similarly, E. sieberiana seeds

collected from immature capsules and stored for 7 weeks required light for

germination, but after 13 weeks, germinated in darkness. Observations such as

these led to the suggestion that seeds of some Eucalyptus species undergo

changes when inside the capsule for longer periods that enable then to germinate

in the absence of light (Clifford, 1953).

In many studies, light conditions have been shown to influence seed dormancy.

Dormancy responses to light are classified into three categories (Pons, 2000).

Positively photoblastic seeds germinate only in white light, negatively

photoblastic seeds germinate in darkness, and light insensitive seeds can

germinate in both white light and darkness (Takaki, 2001). Boundaries between

these categories may not be clear. For example, several studies have reported

that seeds of Phacelia tanacetfolia are negatively photoblastic with germination

inhibited by continuous white light (Piravano et al., 1996). However, in the early

stages of imbibition (up to 24 h), exposure to light or darkness had no apparent

effect on the levels of DNA, RNA or proteins in seeds of this species, (Piravano

et al., (1996). After 24 h mRNA levels declined markedly in darkness but

increased in light, suggesting a possible link between light inhibition of

germination and mRNA level.

Grose and Zirnmer (1957b) classified Eucalyptus species into four categories

according to their light requirement for germination. These are: a) those that

require light to germinate (for example E. camaldulensis, E. regnans and E.

behriana); those that require continuous darkness (for example E. dives and

E.sideroxylon), those that require periods of both light and darkness (for example

E. largiflorens and E. occidentalis (Zohar et al., 1975)) and those that are

insensitive to light or dark (for example E. macrorrhyncha). In E. occidentalis

light requirement i.e. the 1ight:darkness ratio increased with increasing

temperature between 15-30'~ (Zohar et al., 1975). This temperature response

was linked to phytochrome by Fielding et al. (1992) cited in Benvenuti et al.

Chapter-6 (Light Effects) :. . . .: -.z?.rr..,r,;. ;,, . ../;:r/%? .., irJ:.':.;li*.IC;n?l;.:+:l- :r,/r:':.U f i , ~ . ' r : @ l & s ; : < q jr%t,~+I f l ~ ~ ~ ~ . : : i t ~ C : , ~ p I X ~ . ~ : ~ ~ : t I i i ' / r R C : 4 < ~ ~ ~ ~ n n ~ : < ~ ' ~ C I ' i : ~ : 5 : I I < 1 1 ~ ~ W ~ ~ ~ ~ ' r ~ ~ r C C @ a X Y ~ ~ ~ ~ ,

(2001) who suggested that high temperature destroys Pfr thus reducing

germination by removing the promotive effect of the active form of

phytochrome.

This chapter reports experiments that examine interactions between light and

pre-germination hydratioddehydration cycles aimed at clarifying the suggestion

from Chapter 5 that light rather than hydratioddehydration cycle may have been

responsible for delayed germination or induced secondary dormancy. Subsequent

experiments reported in this chapter examine the influences of duration of the

dark imbibition period on this induced dormancy.

6.2. Materials and Methods

Two similar experiments (6.la and b) using different seedlots compared pre-

treatment (imbibition for 30 h in darkness, followed by 7 days air drying under

laboratory conditions) and germination in continuous light or darkness in a

factorial design. The remaining experiments examined similar light treatments,

but without the pre-imbibition/desiccation cycle.

Experiment 6.1 (a and 6)

Seedlots 'A' (a) and 'h' (b) were imbibed in complete darkness for 30 h, before

the aluminium foil covering was removed, and then allowed to desiccate on the

laboratory bench under ambient light and evaporative conditions as described

previously. Drying was allowed to continue for seven days before water was

added and the dishes returned to the germination cabinet for germination. At this

stage two lightldark treatments were applied with light treatment exposed to

constant cabinet lights and the dark treatment rewrapped in aluminium foil.

Fresh (non pre-treated) seed was also included to give a two pre-treatment by

two sets of germination conditions factorial design. Germination counts were

done on day five. There were four replicates of 50 seeds in each plot. Results for

each seedlot were analysed separately.

Chapter-6 (Light Effects) ;:" ;*<$ > :,,:r ~:.*m;Y~Y.?>:.?.~7..~~.:%.~~~~~Tfif~@, ~-:!<:'c-?.7,?@ : ~ ? ~ ~ Y ~ ~ ' F ~ ~ ~ ' ~ ~ P ~ ' N / ~ , ' O , ' ~ Z ~ ~ ~ ~ & ~ Z ~ ~ . . ! ~ ~ & / A ~ ~ D , ~ ! ~ Z ~ ~ ~ X ~ & < Z ' G : V ~ ~ ~ ~ , ~ ~ / & ~ T ~ % Z ~ ~ ~ # / & ~ ' ~ / ~ ~ ~ ~ ~ ~ ~ ~ / ~ ; W / A ~ <

Experiment 6.2 (a and b)

Seedlot 'KI-2' (a) and 'h' (b) were imbibed in constant light or constant dark for

30 h after which they were transferred to either constant dark or constant light

for the remainder of germination. Germination counts were carried out on day 6

for seed lot 'KI-2' and on day 6, 8 and 10 for seed lot 'h'. In both trials there

were four replicates of 50 seeds in each plot. Design was a randomised complete

block in a two pre-treatment by two-germination treatment factorial. Separate

analyses of variance were carried out for each seed lot.

Experiment 6.3 (a and b)

Again, two experiments with identical treatment designs were carried out on

different seedlots. Seedlots 'A' (a) and 'H' (b) were imbibed in darkness for 3,6,

9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 h and then exposed to light until

germination counts were done on day 5. Two control treatments were kept in

either continuous light or dark throughout. A second germination count was done

on all treatments on day 11. Dark counts at the first time in the continuous dark

treatment were done under a green safe light and the petri plates rewrapped with

foil. for the remainder of the germination period. A randomised complete block

design was used with four replicates of 50 seeds in each plot. Seedlots were

subjected to separate analysis of variance.

Experiment 6.4 (a and 6)

This experiment was designed to verify the earlier observation (6.3 a&b) that

extended darkness after initial imbibition in light was likely to have little

influence on germination capacity or mean germination rate. Seedlots 'KI-1' (a)

and 'H' (b) were allowed to imbibe in light for 3, 6,9, 12, 15, 18,21,24,28, 30,

33 and 36 h and then kept in darkness till the first count was done on day 9. Two

controls were either exposed continuously to the germination cabinet light or

kept in darkness throughout. A second count was done on day 14. The

Chapter-6 (Light Effects) ,, ... . ..* -, ; ,::.:,:. ,, P .; . ,:,,,.-,,4*,#<:+<,::'s:,... .,.+ce.?- 6~'p,.,.:.:.,,<,~: ~~.*.,-,z.,v ,d"L+,. 5 w*, . ,~\ ;~.>"7,fp, .~~,"~*&.4~ :,,.* >O,'@ ,~;*,,~,.,,,\"~$w?~,p,~~~m,m4a,:w/@,~&,~.q,~~

experiment was a randomised complete block design with four replicates with 50

seeds in each plot. A separate analysis of variance was done for the two seedlots.

Experiment 6.5 (a and b)

This experiment examined the influence, on germination, of timing of the start of

imbibition relative to a diurnal (lighudark) cycle. Using a germination cabinet set

at 1211 2 lighudark, imbibition was commenced 3 ,6 ,9 and 12 h into the light half

of the cycle. The trial was a randomised complete block design with the four

starts of imbibition times as treatments. Seedlots 'KI-2' (a) and 'h' (b) were used

in the two identical trial designs and counting was done on day 6 and 8 for

seedlot 'KI-2' and for seedlot 'h' counts were done on day 6, 8, 10 and 12.

Results were analysed separately for the seedlots and count times

Chapter-6 (Light Effects) .: ,. ,JV, ., .. , r .,<,? ,, *z:,~pd$b.<.,w, ,.,. a,, :,. $,,,>,$e.,. *?as:<+ <C27~0?,;>,<<,4",,<7.*~' /#.5 ~ / ~ , ~ f l , . Z E ' & ~ / , X < ~ : ~ 6 ! J ~ , W , ' ~ ~ & ~ ~ / ~ # & W f l / & ~ N / ~ ~ ~ @ % ~ / ~ < i ~ ~ , ~ ' # ' < # ' ~ ~ ' ~ ~ ~ ~ ~ / ~

6.3. Results

Experiment 6.1 (a and b)

Table 6.1 (a & b): Influence of light condition during germination on

% germination after 7 days in seedlots 'A' and 'H'. Pre-treated seed

was imbibed for 30 h in complete darkness followed by dehydration

for 7 days (n=4). The control seed was not pre-treated. LSD (P=0.05)

for comparison within the table for seedlot A is 11.0 with a

significant interaction between light and pre-treatments. For seedlot

'H', there was a significant light effect (see text) but no interaction

between light and pre-treatment, LSD.(P=0.05) is 13. By day 10 there

was no significant treatment effects in both seedlots.

Mean Germination (%)

Seed lot 'A' (a) Seed lot 'H' (b)

Day 7 Light Dark Light Dark

Control 8 3 95 66 84

Pretreatment 43 74 54 82

Day 10

Control 96 96 93 8 8

Pretreatment 93 8 8 90 89

Germination improved significantly in light and dark conditions at Pi0.05 in

seedlot 'A' (Table 6.1 a) on day 7. Germination percentage increased in

complete darkness in both non pre-imbibed and pre-imbibed and redried seeds.

By day 10 there were no significant treatment effects. In seedlot 'H', there was

no interaction (P>0.05) between light and pre-treatment for day 7 germination

and no effect of pre-treatment. There was however a significant (Pi0.05)

difference between light treatments with seeds exposed to continuous dark

Chapter-6 (Light Effects) 9 : - h *t, ?* '.?.@7'.4S.Y .+:,km' .?,?,,,.,+'9 ' :$? . '~?f i .&,:> ~T,"",P~~d~~X%W~;.'#.VJh#~~'~'~n~~>?L<.7A,~W.~ ? . f i7~~">+* .~~, :#~~,W,@LV<%<<! '~~~N*&~~5'~~~M~~'R~N/#~~~~#f l /M~R&-L

having a germination percentage of 83% and seeds exposed to constant light

having a germination percentage of 60.3 %. By day 10 most seeds had

germinated and there were no significant treatment effects (results not shown).

Experiment 6.2 (a and b)

In seedlot 'KI-2', there was no significant interaction between treatments. There

was a small but significant difference between imbibition treatments (P10.05).

When seeds were imbibed for 30 h in constant light, day 6 germination was

98*2% compared with 93*7% for initial imbibition in darkness. For seedlot h

there was no significant interaction but there was a significantly greater

germination for seeds imbibed in constant light (P10.05). Seed imbibing in light

gave 68*5% germination compared with 39.5*8% for dark imbibed seed. On

days 8 and 10 there was no significant difference in germination between

treatments, with germination values ranging from 78 to 93%. By day 8 and 10,

germination improved in seed lot 'h', hence results are not shown.

Experiment 6.3(a and b)

As shown in the Fig 6.3 both seedlots showed a similar response to increasing

length of the period of dark imbibition. From 18 to 36 h, germination was

significantly (P10.05) reduced compared with germination in both full light and

complete darkness for both seedlots. In seedlot 'A' 98 to 99% seeds germinated

in full light and complete darkness. In seedlot 'H', there was a significant

(P<0.05) difference in germination between the constant light and dark

treatments with 100% germination in the dark, but only 75*11% germination in

constant light.

By day 11, all slow germinating seeds had germinated and there was no

significant difference between any of the treatments. In seedlot 'A' the mean day

11 germination percentages of seeds held under constant light or complete

darkness were not significantly different, with germination at 99*1% and 98*2%

.,. . ....,, -,,"" Chapter-6 (Light Effects) I . /,'.*/irL *..'.>st. =/@ X ,'il:.<t,,,::, L'.rtrtrtrtrtrt ?555ri ':.% ,%.< :r*,i-i."% 7..7.7.7.?":I:(I,~(I, (I;* d. fin *:I* C C ~ ~ ' ~ ~ ~ ' ~ ' . I I - W I I ~ % ~ ' Y F V ~ ~ J / ~ C / ~ C < P A ~ ~ S C ~ ~ ~ R ; ~ % < ~ ~ I " I ~ / ~ ~ ~ ~ ~ E ~ / ~ . I ~ . T ~

whereas in seed lot'H', day 11 germination was 95k3% in full light and 100% of

seeds germinated in complete darkness.

Experiment 6.4 (a and b)

As shown in Fig 6.4, day 9 germination in both 'KI-1' and 'H' was above 80%

but there were significant (Pc0.05) reductions in constant light compared with

treatments involving a shift to dark afier initial exposure to light. In seedlot 'KI-

1 ' on day 9, 83% of seeds germinated in full light and 93% of seeds germinated

in complete darkness. Seeds exposed initially to light and then transferred to dark

showed no significant effect of duration of initial exposure to light on

germination. By day 14, most seeds germinated, with no significant differences

observed between any treatments (results not shown).

Experiment 6.5 (a and b)

Timing of the start of imbibition relative to the diurnal light cycle had no

significant effect on day 6 germination percentages in seedlot 'h', with all

treatments giving a germination percentage below 11% (Table 6.5). By day 8

there was a significantly (PFO.01) reduced germination percentage for seed that

has commenced imbibition early in the 12 h light period. This effect persisted,

and on day 10, seed, which had commenced imbibition 3 h into the light period,

still showed significantly reduced germination. By day 12 the treatments were

not significantly different (data not shown). In seedlot 'KI-2' (a) there was no

significant difference between treatments at any stage of germination (data not

shown).

. ., .,. ,.. .-. , .:.E:7.,,v,e:T'+,"M' O v ~ . . ? .-Ll.i..ys +,.tl~Ux,,~~i,!;,l;~fl~~wi~~:.KIli.lili;~, F . ~ ~ , ~ : ~ ~ 7 T 4 Y I ~ ~ , U , U : f ru~~~,':l"W~~7~~#"fRic.,Zrii;~?~?.~~~~*r/n?i.PYd/iP.~'K~'~~,Z2 Chapter-6 (Light Effects)

0 1 I I 1 1 I I I I

0 5 10 15 20 25 30 3 5 40

Duration of dark (hours)

Fig 6.3 (a & b): Germination in response to initial imbibition in

darkness for seedlot 'A' (a) and 'H' (b). Seed was imbibed for

different periods of complete darkness and transferred to light.

Germination counts were on day 5(.) and day 11 (A). Two

controls were full light and constant dark (not shown in the

figure). Data points are results of 4 replicates of 50 seeds A

standard error of the mean.

Chapter-6 (Light Effects) , .... ~~z,,aL.,e..,r .,-,,* e~ yv.c..,e,..,,;4,~;~.,~ ~~.~e~,,,e,,t:,z~'~!,m~<; ~ ~ ~ ~ ~ ~ c . ~ . ~ ~ ~ ~ ~ ~ , ~ ~ ~ , ; v ~ ~ ~ c ~ ~ ~ ~ ; n ~ ~ ~ ~ ~ . w ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ , : . r . ~ , ~ ~ ~ ~ ~ ~ ~ r ~ ~ $ ~ ~ e * ~ ~ ~ ~ : ~ ~ ~ ~ ~ ~ ~ ~ / ~ ~ ~ @ ~ w ~ ~ ~ ; w ~ ! ~ ~ ~ ~ ~

0 10 20 30 40 Duration of light (hours)

Fig 6.4 (a & b): Germination in response to initial imbibition in light

for seedlot 'KI-1' (a) and 'H' (b). Seed was imbibed for different

period of light and transferred to constant darkness.

Germination counts were done on day 9 (0) and day 14 (A). Data

points are results of 4 replicates of 50 seeds standard error of

the means.

Chapter-6 (Light Effects) < ~ ~ y ~ , & - . p ~ ~ , , ~ ~ ~ < ~ , ~ A ~ ~ r ~ ~ ~ ~ P / ~ ~ ~ " . w ; < ! ~ ~ ' ~ . ~ < ' : : , ~ " : . ; ? < < + : , ~ . ~ : ~ ; ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ - ~ ~ Y ~ " . a ~ T . ~ . ~ t ~ ! > ! 2 ~ ~ ~ ~ ~ m ~ ~ < ? : ~ ~ ~ ; & ~ ~ ~ * ~ ~ ~ ~ m < . ~ , ~ ~ ~ ~ ' ~ ~ 7 : m m w ~ ~ , ~ ~ ~ ~ . 7 ~ . ~ w ~ ~ ~ ~ 5 ~

Table 6.5: The effect of duration of initial light exposure on mean

germination percentage of seedlot 'h' on days 6 ,8 and 9 (n=4).

Mean Germination (%)

Duration of light Day 6 Day 8 Day 10

(h)

3 11 72 84

6 9 67 8 3

9 8 45 67

12 10 54 8 1

LSD N S 15.7 7.5

6.4. Discussion

It was noted in Chapter 5, that germination was higher in constant darkness

compared with pre-imbibition and desiccation of the seed under natural light and

dark conditions. In experiment 6.1 germination was enhanced, in the absence of

light, in fresh as well as dehydrated seeds, whereas exposure to light extended

the period of germination regardless of pre-treatment. Both seedlots showed the

same pattern of germination in response to light. This result suggests that earlier

indications that pre-imbibition and desiccation may lead to reduced or delayed

germination may have been misleading (Chapter 5). The present results

demonstrate a direct effect of light and delayed germination in hydrated and

dehydrated seeds germinated in light could have been associated with light

during initial imbibition rather than a desiccation effect. There are several reports

of light conditions during imbibition influencing germination. For example,

Pirovano et al. (1 996) demonstrated that light inhibited germination in Phacelia

tanacetifolia seeds imbibed in the dark. The results may also be consistent with

Chapter-6 (Light Effects) 7 . '>>,< ..)-,r,.).,~:.~,:.,Y-,~~. ririri.,,<* fir. <:.,,.;.r,,.,:l. ,< ,". ;%c.l - r: , 7: ,".1. 'i L - I . d ; l i'., .r:.,:a.r:,n?c>.r:,.*1 ~3~,r' / ' :~~~3'/::~~,~-~:~>'I . ' I , ' I t?~*",,~~~'i i~~J~R,~~,m?.m?L-~

similar lightldark germination responses reported by Wilson et al. (2005) on E.

ovata, in which the hydrationldehydration effects of pelleting were not separated

from possible light influences during the production of large multi-seed pellets.

The importance of light exposure during imbibition was confirmed in experiment

6.2. When seeds were allowed to imbibe for 30 h in either light or dark and then

allowed to complete germination in either dark or light, initial exposure to light

resulted in higher day 6 germination. The effect was present in both seedlots but

more marked in seed lot 'h'. This result thus suggests that initial imbibition in

darkness may be responsible for delaying germination on subsequent exposure to

light.

Experiment 6.3 confirmed this observation. As the dark imbibition period

increased in duration from around 6 h to near the 30 h used in experiment 6.2,

the germination response also increased. The decline in germination with

increased duration of the dark period appears to commence from the end of

phase I of imbibition, based on the time scale for water uptake established in

Chapter 4. It is notable that, in this trial, day 5 germination after imbibition for

18 to 36 h was significantly lower than for seeds germinated in constant

darkness. Further, for the seedlots used, there was no significant difference in

germination between constant light and constant dark. Consequently, these

results suggest that initial dark imbibition from around 6 to around 30 h may

induce a degree of light sensitivity in the germinating seed. That is, whilst

germination may be reduced in continuous darkness, in some seedlots the effect

is exacerbated if germination is allowed to continue in light after an initial dark

imbibition period. Results from experiment 6.4 showing that exposure to light

followed by darkness did not influence germination suggest that the response

was not simply due to changing light conditions but requires a specific shift from

initial dark into light.

Experiment 6.5 places this darkllight effect on germination into the context of a

natural diurnal cycle. For one of the seedlots, seeds starting imbibition within

... .. . Chapter-6 (Light Effects)

-.A , 7 f . .?<Y j ~ ~ . : ~ ~ ~ ~ ~ ~ ~ ~ ~ W . ~ . ~ . , ~ ~ ~ ~ ~ ~ ~ ~ . ' ~ ~ . ' . ~ , ~ ~ ~ ~ 4 ~ : : ' 'C?~:?,M+-*~/A .A:.,L,.'?,P,F <-:a . ~ ~ . ~ ~ ~ ' > , ~ ~ ~ ~ / ~ . ~ , ~ ~ ~ & . M ~ : @ ~ ~ , ~ ~ ~ . L W ~ ~ ~ ~ ~ ~ ~ / N ~ ; . S 7 ~ & ~ ~ ~ ~ ~ ~ ~ N i ~ ! W ~ ~ ~ / & ~ ~ ~

three hours of day light in a 12/12 cycle showed a significant delay in

germination compared with seeds with imbibition started later in the day. It

appears that these seeds had completed phase I of imbibition before entering a 12

h dark period, thus behaving in a manner similar to seed exposed to 21 to 24 h

dark imbibition in experiment 6.3.

This result has major practical implications for commercial nurseries and such a

diurnal influence on seed germination does not appear to have been previously

recorded in the scientific literature. Practically, this effect seems to explain the

highly variable germination obtained for some seedlots in some nurseries. The

variability may simply be associated with the time of day the growing medium is

irrigated enough to commence imbibition. Consequently the first step in

managing this variability may be to either provide constant light for the first two

days after sowing, or avoid sowing and/or irrigation in the early morning. Local

seed testers who use 100% humidity, 25 OC temperature and constant light for

germinating E. globulus seeds in the laboratory report 95% and above

germination. Clifford (1 953) suggested that E. globulus requires darkness for

germination. Turnbull and Doran, (Appendix 3E in Langkamp, (1987)

mentioned that E. globulus ssps. globulus and bicostata require light and 25 OC

for optimal germination. In many nurseries sown seeds are irrigated, covered

with black plastic and incubated in a room with optimum temperature for 3 days

before transferring to a shade house.

Overall there were some differences between seedlots in response to continuous

light or dark, with some requiring light for normal germination, suggesting that

any generalisation about the species being positively photoblastic may be

incorrect (Clifford, 1953; Langkamp, 1987). Further, the induction of a light

sensitive secondary dormancy in response to a period of darkness early in phase

I1 of imbibition has not previously been reported for this or other species. Thus

from both a scientific and practical point of view, further investigation is

warranted.

,... ?,,..- r. ,*-.,A,,...., .,,,.& A:,; ,,.* ,,.: .,.,,,, ...,.,, :?.. ,.&. %- .. .. c....2,",. ‘,;- .." .2v ,c .,>,:, (.,'! :, ,* 2 ,>. ,,,; 2,,m,d.~yy2: Chapter- * . ,~,m!;fl,..~9,~5,,v4pe~jw,r,~~,~4w~r~fl,~i,+~,pd, 7 (Light Quality)

Light Quality Effects

7.1. Introduction

Experiments in the previous chapter demonstrated that E. globulus seed imbibed

in the darkness becomes light sensitive later in germination, suffering a dark

induced, light responsive secondary dormancy. As noted in a recent review paper

by Thanos et al. (2005) light inhibition of germination is not common. There are

few studies on either the ecological significance or control mechanisms of this

phenomenon. Light quality has been implicated in many studies on light

promotion of germination and light quality (as well as duration) may be a

significant factor in nursery management of secondary dormancy in E. globulus.

The phytochrome system, and its responses to wavelengths at the red end of the

visible spectrum, is the most widely studied control mechanism in seed

germination light responses. Red light is known to promote while far red light

inhibits germination. A detailed review, particularly of recent advances

associated with gene expression, is beyond the scope of the present study and the

following is intended as a general background on phytochrome mediated

responses to red and far red light, with the emphasis on seed germination. The

possibility that the response observed in Chapter 6 may have been blue light

(cryptochrome) mediated was also considered.

Plants absorb red and far red light from sunlight (Atwell et al., 1999). In a dense

forest, light at the soil surface is generally less than 100% of h l l sun due to

shading by the leaf canopy. There is also a . shift in light quality, as the

photosynthetically active range of the spectrum is preferentially absorbed by

chlorophyll resulting in reduced total light energy and a shift in wavelength.

Light passing through the canopy mainly is depleted in the red (R) band of the

spectrum with relatively little reduction of far red (FR) (Smith, 1995; Chung and

3;**1"L-.f..7,,-Zli'.,."i%tr,:-x'l~.z .%' Chapter- 7 (Light Quality)

. . ,.W.."XV.J -'P~Cc::'..,:Z9~.<'.',>:ij7~.17'-/; : ' : 6 ~ ' $ ' % . : R W I C . : C . ~ , 7 K ~ RjiWf4;j&r=i CrcrB>%(;fl,'O,rC>7/N/rF Ftl...E.E4~WaCfi~F~iDZP,:~Z47

Paek, 2003). In controlled environment studies, Borthwick (1957) noted that

incandescent filament light and sunlight contain high levels of both red and far

red light whereas domestic fluorescent light was low in far red and relatively

high in red.

This change in RIFR ratio has a marked effect on seed germination in some

species. For example, seeds of Cordia africana did not germinate under a leaf

canopy as RIFR ratio was too low (Yirdaw and Leinonen, 2002). Similarly,

Muntingia calabura L. seed failed to germinate in shade but germinated in open

conditions with full light (de A. Leite and Takaki, 2001). Germination was also

increased in Glaucium. flavum seeds by irradiating for a short period of red light

(Thanos et al., 1989). The positive effect of a short burst of red light decreased

according to the depth of burial in Rumex. obtusifolius seeds (Benvenuti et al.,

2001).

Lettuce seed has been widely used to study red light effects on germination and

several papers (Toyomasu et al., 1993) have reported that irradiation with red

light for a brief period induces germination. Irradiance with far red inhibits

germination and cancels the effect of red. Further, when red light was given

immediately after far red it cancelled the far red effect and induced germination.

Ungerminated Glaucium flavum treated with white light earlier, on transfer to

darkness germinated whereas ungerminated seeds treated with blue and green

light where blue and green light contained considerable amount of far red light

germinated poorly on transfer to darkness (Thanos et al., 1989).

A range of specialized red light photoreceptors fiom Phytochrome A through

Phytochrome E have been identified in Arabidopsis thaliana seeds, along with

cryptochromes and phototropins that mainly absorb light fiom UV-A and blue

light (Magliano and Casal, 2004). In response to far red light, PhyA causes

hypocotyl elongation during germination whilst PhyB inhibits hypocotyl

elongation in response to red and white light (Elich and Chory, 1994; Franklin

and Whitelam, 2003).

. . Chapter- 7 (Light Quality)

,., , . , . .,-".< . ., .-%,a,..,. *:>,>:~, ,7*,.7:, ,.,.2.n%,:";La * ?',.7'3 + : x ~ ~ ~ ~ f i > . . . : < ~ . * ~ ~ , ~ < < . , ~ ; ~ , . w A ~ ~ ~ ~ . ? , ~ ~ : ~ w m ~ r : . . % v : a ~ ~ ; ~ ~ N / # ~ ~ / * ~ m , ~ ~ , n . w / ~ / ~ ~ f i ~ / & / ~ ~ s / ~ ~ ~ ? ' ~ ~

Phytochromes exist in two interconvertible forms, Pr with a light absorption

peak at 660 nm (red) and a physiologically active form Pfi at 730 nm (far red)

(Benvenuti and Macchia, 1997; Batlla and Benech-Arnold, 2005). Normally

synthesised in the Pr form, phytochrome is converted into the Pfi from when it

absorbs red light (Bell, 1999). On irradiation with far red light Pfi reverts into Pr.

Pfi is considered a promoter of seed germination, whilst Pr has an inhibitory

effect on germination (Pons, 2000; Fankhauser, 2001; Chen et al., 2004).

Benvenuti and Macchia (1997) suggested that in the absence of germination

promoters or stimulators, a minimum quantity of Pfi present in seeds will act as a

gemination activator.

Kendrick et al. (1969) studied the appearance or synthesis of phytochrome in

Amaranthus. caudatus seeds during imbibition. They noticed an increase in

phytochrome on hydration and a second phase of increase in phytochrome after

10 h of hydration, showing synthesis of phytochrome at this stage. In

Arabidopsis. thaliana seeds phyB develops after 3 h of start of imbibition

(Shinomura et al., 1994) whereas in soybean seeds, phytochrome activation

occurs at 17- 19% hydration (Obroucheva and Antipova, 1997). Casal and

Sanchez (1998a), in their review suggested that phytochromes are formed during

seed formation in Pr or Pfr form. Formation of Pr or Pfi depends on the seed or

fruit covering, and also if fruits are shaded by green leaves as green leaves

absorb light from the Red region compared with the far red region.

PhyB is mainly responsible for control of seed germination (Chung and Paek,

2003) and in the Pfi form, phyB is responsible for promoting germination in

darkness (Casal and Sanchez, 1998a; Takaki, 2001). Generally Phy B is the

predominant form in dry seeds and phyA appears after imbibition and in all

mature seeds Pfr is present before exposure to light (Casal and Sanchez, 1998a).

Shinomura et al. (1994) showed that in Arabidopsis. thaliana seed germination is

mainly controlled by Phy B. Bell (1 999) reported that, in darkness, phytochrome

is present in the Pr form which, on exposure to red light converts to Pfi.

Chapter- 7 (Light Quality) .. " . I ^ ,. 2,'. , I.-." i <11- 6, -:.- .; ~.<m"*.,.;?2"3:d~,', ,: r.,̂ .":('L".;j.,li .."a:..' <C .,.jl,i ,/< ,.,. ,,lS L>,>.',' ,+p$j,~'.g":?.;.# :~'~":~~,.~i..~,.,~~;n",y~~~.l;w.*4,Ih*,;i*V~~~n:*y~yY~

Links between phytochrome and gibberellin in control of germination have been

reported. For example, Thomas (1992) suggested that phytochrome responses are

related to GA3. Phytochrome has been reported to act promoting GA3 synthesis,

increasing the level of response of seeds towards endogenous GA3 and

suppressing the development of inhibitors (Garcia-Martinez and Gil, 2001). Da

Silva et al. (2005) noted that exogenous GA may replace other environmental

requirements needed for germination and Plummer et al. (1997) reported that red

light promotes GA biosynthesis. In Argania spinosa seeds final germination

percentage was delayed, germination rate decreased and the interval of

germination was increased in darkness but when soaked in GA3 solution for 24 h

germination did not overcome light requirement (Alouani and Bani-Aameur,

2003). Addition of GA in lettuce seed replaced the promotive effect of red light

and red light treatment increased the production of endogenous GA level

(Toyomasu et al., 1994). Further support for a role for GA in mediation of

phytochrome responses is provided by a number of studies that have shown

inhibition of light induced germination by paclobutrazol (a synthetic GA

inhibitor) (Plummer et al., 1997). Inhibition of germination by paclobutrazol can

be prevented by addition of GA3 (Plummer et al., 1997).

In relation to the present study, light and GA interactions have been reported in

several eucalypts. Germination of E. marginata was suppressed in light

compared to darkness, but the inhibitory effect of light was prevented by

exogenous GA3 (Bell et al., 1995). Earlier studies by Bachelard (1 967) suggested

that addition of GA3 overcame light requirements in E. delegatensis R T Baker

and E. pauczjZora Sieb. No studies of Eucalyptus seed were found that described

mechanisms that would readily explain the darkllight induced dormancy noted in

the earlier chapters of this thesis.

Although there is little in the literature to indicate how the induction of

secondary dormancy in E.globulus seeds observed in this work may be regulated,

it is clear that none of the pigment systems should be eliminated at this stage.

. Chapter- 7 (Light Quality) ? ..7- 7:. -:,..". ,..:., ~~*&,*:v,:;.? .$7,.z~0,:.*7:?.~*:. : ;x,~, . j~~:, ,~.Y,, '-',,*..:<;-4,. < ~ ; , & ! ~ ~ ~ ! ~ , z ~ ~ . & - ~ ~ ~ ~ , ~ ~ ~ , ~ ~ ~ , . ~ % ~ ~ ~ x ~ ~ : ~ ~ , ~ , ~ ~ , ~ , f l s / ~ ~ ~ , ~ ~ / ~ ~ . ~ < ~ ~ , ~ , ~ / ~ , ~ ~

Consequently, this chapter contains a series of experiments using various

combinations of white, red, blue, and far red light and darkness during and after

imbibition. Exposure times and combinations of treatments were similar to

experiments in earlier chapters. It was anticipated that the results of this work

might have significant practical implications for the design of nursery lighting

for germination of this species.

In contrast to the generally accepted mechanism involving red light promotion of

germination, several studies have reported an inhibitory effect of red light and a

promotive effect of blue, far red light and complete darkness on germination.

Bell (1993) reported that Trachyandra divaricata seed germinated in darkness

and far red light but germination was inhibited in orange and red light. Pfi

reverts to Pr in complete darkness in imbibed seeds, causing inhibition of

germination (Pons, 2000). In contrast, several studies have suggested that Pfi is

present in dark germinating seeds (Benvenuti and Macchia, 1997; Casal and

Sanchez, 1998a). The report by da Silva et al. (2005) that GA inhibited

germination in coffee seeds whilst complete darkness promoted germination may

also implicate phytochrome in light inhibition of germination.

7.2. Materials and Methods

Seedlot 'KI-2' was used in most of the experiments unless noted otherwise.

Imbibition and germination treatments were carried out on petri plates as

described earlier. Light sources were single red, blue, white, or far red light

emitting diodes inserted through the centre of petri dish covers. Because of the

potential sensitivity of control systems like phytochrome to short term exposure

to particular wavelengths, treated plates were wrapped in aluminium foil to

exclude external light and in case of increase evaporation under the lights, the

two filter papers g were placed on a layer of padded absorbent paper holding

approximately 10 mL of distilled water. The red, blue and white LEDs supplied

by JayCar PIL (Hobart, Tasmania, Australia) were all 5 mm diameter domes

Chapter- 7 (Light Quality) ..... ,,,.,< *>, ,77c,z ,;,;: <; ,:, G y<.c.a6 -:>cs.~~:.,g. ,* V : ~ , ~ ~ ~ . : ~ Z ~ . ~ ~ ~ G ~ L - X ~ ~ ~ ~ ~ : ~ . ~ , ~ ~ ~ ~ , ~ w ~ - ~ ~ ~ ~ ~ ~ ~ e ~ ~ : ~ ~ ~ - ~ r ~ ~ . w , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ a ~ ~ ~ ~ ~ m ~ ~ . ~ ~ ~ ! w m ~ ~ ~ ~ x ; ~

with reference number and specifications as follows: 10,000MCD red peak at

625 nm, 7000MCD white, no wavelength specified, 5000MCD blue peak at 470

nm. Far red diodes were supplied by ROITHNER LASERTECHNIK GmbH,

Vienna, Austria, as part number (ELD-740-524) with a specified peak

wavelength of 740 nm. Wavelengths were checked using LI-1800

(SPECTROPHOTORADIOMETER) (LI-COR, USA). For further information

refer to Chapter-10. All diodes were connected in parallel to a 2.5 V DC supply.

Because only four plates of each light were prepared, experiments were limited

to four replicates of each light treatment, necessitating a series of small

separately analysed experiments. Timing of light and the addition of gibberellic

acid were based on earlier experiments with the end of phase I of imbibition

taken as around 6 h from the start, and maximum induction of secondary

dormancy occurring with 24 to 30 h exposure to dark (beginning at the start of

imbibition).

Experiments

Experiments 7.lb to 7.8 are summarised in the following table followed by table

explanation:

Chapter- 7 (Light Quality) 5.J .3 . .- ':..'.>:-,,1..<.8..5>: .'?A. ,4vx':'.- z ~.~..~~:,~~~,w.":n~,~~'~~?!~~~.w<~~~~~,s~,~'~*~.:-~.%.~~<,-~.~:~4>;~~,?~~.~~.~~~~~~~~~~~~;~~~~w,~~m.~~~~~,z~~.~w~~.~N,@~~,m,~,w~&-,~?<

Experiments Seedlots Light

treatments

0-24 h

7.1 a 'Kl-2' Constant

red, white,

blue, far

red,

cabinet

light or

constant

darkness

7.1 b 'KI-2' As for

7.1 a

7.2 a 'KI-2' Constant

darkness

7.2 b 'H' As for

7.2 a

7.3 'KI-2' Constant

light

7.4 a 'KI-2' As for

7.1 a

Light

treatments

day 2-5

Constant

red, white,

blue, far

red,

cabinet

light or

constant

darkness

As for

7.1 a

As for

7.1 a

As for

7.2 a

As for

7.2 a

Constant

darkness

GA3

application

Not applied

Not applied

Not applied

Not applied

Not applied

Not applied

Additional

treatments

None

None

Constant

cabinet

light or

constant

darkness

(t=day 0-5)

As for

7.2 a

Constant

darkness

(t=day 0-5)

Constant

cabinet

light or

constant

darkness

(t=day 0-5)

. . Chapter- 7 (Light Quality)

. . ., .,: :< .', ,;: - t ;; , 9 : , , ' ~ , ~ ~ ~ . , ~ ' , ~ , ~ ~ ~ ; < ~ ~ : . > ' , " , ~ ~ I : ' ~ ~ : ~ <?.;,:47>:?;F, +.',-L?,% <:. .'. ,.. ' ' ~ + L ~ ~ , Z :%-&,.%; .r,j-,;Y ~ ? ~ . ~ ; ; ~ : < F * ~ . ; , r , * , Y ~ . , ~ v - ~ ~ & ? , & ~ % r ~ ~ A ~ w ~ ~ < ~ , ~ ~ ~ ~ , ~ e / ~ ? ~ :

Experiments Seedlots Light Light GA3 Additional

treatments treatments application treatments

0-24 h day 2-5

7.4 b 'KI-2' As for Constant Not applied As for

7.1 a light 7.4 a

7.5 'KI-2' As for Applied

7.1 a As for None

7.1 a

7.6 a 'KI-2' Constant Constant Applied Constant

red, white, cabinet after 24 h cabinet

blue, far light light with

red, & without

cabinet GA3

light

7.6 b 'KI-2' As for As for Applied at As for

7.6 a 7.6 a the 7.6 a

beginning

7.7 'KI-2' Constant Constant As for Constant

red, white, darkness 7.6 b darkness

blue, far with &

red,

constant

without

GA3

darkness

7.8 'KI-2' Complete Constant Applied Constant

darkness red, white, after 24 h darkness

blue, far without

red, GA3

cabinet

light

Chapter- 7 (Light Quality) ? .> :, ,- 2 .r4.,<:, .. ,%?-,.. . -. <. (.+ e;.:* :: .#. <.<< ,,7,*.4;'6,'*. .,r;-x:. . :.; 7. * ", . :J c.%:'e.r, . : = . ~ . ~ . , ' . : : ~ & : ~ , ~ . . ~ . - ~ . ~ ~ c ~ P , F , ~ ' 7 ~ t z ' ~ 4 * ' ~ ~ s ~ ~ ~ - ~ ~ v ~ ~ : . v / ~ ~ ~ ~ ~ 7 ~ ~ ~ ~ A

In the first experiment (7.1 a) seeds were exposed continuously to red, white,

blue, far-red diodes, germination cabinet lights or constant darkness for 50 h

from the start of imbibition. The second experiment used the same treatments as

experiment 7.1 a except here (7.1 b) they were continued until germination counts

were' done on day 5. In both trials there were four replicates in a randomised

complete block design.

Experiment 7.2 a and b used two seedlots ('KI-2' and 'H') to test light responses

after imbibition in darkness. In both experiments, seeds were imbibed in constant

darkness for 24 h and then exposed to either continuous red, blue, white, far red

or cabinet light until germination counts were done on day 5. In both cases, two

controls, constant cabinet light and constant darkness were included. These

experiments were arranged in a randomised complete block design with four

replicates.

Experiment 7.3, tested light responses after imbibition in constant light

(germination cabinet lights) for 24 h. After imbibition the treatments were

constant darkness, and continuous exposure to red, blue, white, far red or

gemination cabinet lights. A treatment in which seeds were exposed to constant

darkness throughout imbibition and germination was also included. This

experiment was arranged in a randomised complete block design.

In Experiments 7.4a and 7.4b, seeds were imbibed in red, white, blue, far red or

cabinet lights or constant darkness for 24 h and then transferred to constant

darkness (Experiment 7.4a) or constant cabinet lighting (Experiment 7.4b) until

germination counts were completed on day 5. Two controls, constant exposure to

cabinet lights or darkness (through imbibition and germination), were also

included in both trials. These experiments were arranged in a randomised

complete block design.

The next set of experiments examined the influence of exogenous GA3 on

germination responses to light quality. A stock solution of GA3 was prepared by

Chapter- 7 (Light Quality) c , , . ,4.,?,,..:.*-*,,w* $ ,.~, ,.-,*,;*e $,.,.,., .*,*",:2/>; ,p;. .x.7,2.,;> .: ,,-z+>:<-.;,- *.~,~~,,,'.,~\.<:. 3;,,'r,,w.~,*', ?*?,f?,<,9,,,;,# c~,~;c,;';?*?:%7,~ y..~.'~>+~.,<vA~?L:?,6.?2.7k?%,;?.'*,j../&?

adding 0.0173g GA3 in 100 mL 0.1N NaOH. 10 mL of this stock solution was

then used to prepare a 5x lo-' M GA3 test solution, which was then used in place

of distilled water at the start of imbibition. For trials where imbibition in water

preceded addition of GA, the filter paper and absorbent paper were first wet with

deionised water and 3 mL of GA3 test solution was added at the appropriate time.

GA3 addition to treatments involving continuous dark or where seed was

transferred from darkness to a specific light treatment was done under a green

safelight.

Experiment 7.5, was similar in design to 7.1, but GA3 was added at the

beginning of imbibition and germination was then counted after 5 days in

constant darkness or constant red, blue, white, far red or cabinet light.

In Experiment 7.6 a, imbibition was in constant red, blue, white, far red or

cabinet light for 24 h before GA3 was added. The remainder of experiment to day

5 was then completed in constant cabinet light. Experiment 7.6 b was identical to

experiment 7.6 a except that GA3 was added at the beginning of imbibition. In

both experiments there were constant light treatments (throughout imbibition and

germination) with and without the addition of GA3. In experiment 7.7, GA3 was

added at the start of imbibition and seeds were then exposed to red, blue, white,

far red, or constant darkness. After 24 h, the lights were switched off and the

seeds left in darkness until germination counts were done on day 5. A constant

dark control (no GA3) was also included.

In experiment 7.8, seeds were imbibed in constant darkness for 24 h, before GA3

was added. Germination then continued in red, white, blue, far-red or cabinet

light until germination counts were done on day 5. A control treatment consisting

of constant darkness for the duration of the experiment without the addition of

GA3 was also included.

In experiment 7.9, seedlots 'KI-2' and 'h' were used to test the effects of short

duration exposure to red light. There were six treatments. Two untreated controls

Chapter-7 (Light Quality) 7 .$'?,-7 ,>:?.>,...<.> .?V# 0,;V 1 ~ ~ < ~ ~ ' 6 ~ ~ ~ , 4 ~ ~ J . , ~ ~ ~ > ~ # ~ , ~ f l , W ~ P . 3 ~ ~ : ' ' ~ : 4 - V ~ ~ 2 ~ 7 ~ . ~ ~ ~ ~ 6 ~ ~ ~ : G ~ ~ ~ ' 4 ~ ~ ~ 7 + W ~ ~ ~ ~ W & / ~ A V Y ~ ~ ' & : ~ ~ M W + ~ < G ~ # L m ~ ~ ; k r ~ f l / m ~ ~ 7 H ~ ~ , ~ f l A ~ V / & 5 ~ , . W , ~ ~ # ~ ~ N d

were exposed to constant light and constant darkness and in a third 'control'

seeds were imbibed in constant darkness for 24 h and germinated in constant

cabinet light. In the red light treatments, seeds were exposed to the following

sequences of light conditions from the onset of imbibition:

. constant darkness for 23.5 h, red light for 0.5 h, germination cabinet

lights until completion of the experiment;

constant darkness for 5.5 h, red light for 0.5 h, constant darkness for 18 h,

germination cabinet lights until completion of the experiment;

constant darkness for 5.5 h, red light for 0.5 h, constant darkness for 17.5

h, red light for 0.5 h, germination cabinet lights until completion of the

experiment.

Germination counts were done on day 5 in 'KI-2' seedlot and day 7 for seedlot

'h'.

Experiment 7.10 is surnmarised in a table format. Seedlot 'h' was used to test the

effects of short duration exposure to red or far red light followed by 12/12 light/

dark cycle.

Treatment Dark (h) RedIFar Dark (h) RedIFar 12 light/

red (h) red (h) 12 dark

period

1 23.5 0 0 0.5 Yes

2 5.5 0.5 18 0 Yes

3 5.5 0.5 17.5 0.5 Yes

4 5.5 0.5 0 0 Yes

5 0 0 0 0 Yes

There were nine treatments in total. In treatment 1 seeds were imbibed for 23.5

hin constant darkness followed by 0.5 h of red or far red light before germinating

in 1211 2 photoperiod with transfer taking place at the beginning of 12 light112

dark period. In treatment 2 seeds were imbibed in constant darkness for 5.5 h

Chapter- 7 (Light Quality) ;-- '.,<,, ;7:.9,..>!.~: 0.C' ,. ,,.. ,P;,F~'77...~-,,,w.~.,'.:~m,~#<8*.,P!e .rq:r,.": k:,:$ ~-7.,j~2.-~,*,.<:?.~ T?L",>< <I,', ' ,<,, .%:r . ~ ~ < , ' , ' , ~ ~ : p ~ ~ - ~ ~ ' ~ ~ ~ ~ , ; , + : , e , ~ # r , ~ < ~ , ' ~ ? 4 . ~ , < * % ~ , ~ ? ; ~ ~ & , F , ~ , ~ . ~ , ' ~ ? ~ < ~ ~ <

followed by 0.5 h red or far red light followed by 18 h of constant darkness

before germinating in 1211 2 photoperiod with transfer taking place at beginning

of 12 light/l2 dark period. In treatment 3, seeds were imbibed in constant

darkness for 5.5 h followed by 0.5 h of red or far red light. Seeds were then left

in constant darkness for 17.5 h followed by another 0.5 h of red or far red light

before germinating in 12 light/ 12 dark photoperiod with transfer taking place at

12 light/l2 dark period. In treatment 4 seeds were imbibed in constant darkness

for 5.5 h followed by 0.5 h exposure to red light before germinating in 12/12

photoperiod with transfer taking place at 12 light11 2 dark period. A 12 light/ 12

dark photoperiod control (treatment 5) was included.

Experiment 7.1 1, was designed to determine the effect of GA3 on far red light

inhibited seeds from seedlot 'h'. The experiment was a factorial design

consisting of two GA3 treatments (with and' without GA3) by four light

treatments. The light treatments were red light, far red light, complete darkness

and constant cabinet light was applied from the start of imbibition for 30 h. After

the light treatments were completed, germination was allowed to continue in

darkness until day 7 when final germination counts were done. Two additional

treatments were included in which seeds were exposed to constant cabinet lights

with or without GA3 application 30 h after commencement of imbibition.

7.3. Results

In germination tests assessed at day 7, marked effects of light quality on seedling

appearance were observed. Seedlings from red or white light treatments

resembled seedlings germinating under cabinet' light. They were stout with a

reddish hypocotyl with red or green open and well-spread cotyledons. In contrast

seedlings from seed germinated in far red light resembled those germinated in

the absence of light being etiolated and lacking chlorophyll.

Chapter- 7 (Light Quality) f,'#.,;7: !,'"?,'* .fl:,v ~ : ~ ~ ~ . : ~ ~ ~ ~ : . ' ~ , ~ ~ : ~ > , ~ . ~ < ~ ~ ~ ~ ~ ~ W . ' ' . ' ~ ' ~ ~ ~ ~ ~ ! , ; ~ W < ' ~ ~ > ~ ~ ? ~ - ~ . 2 ~ , ~ ~ ~ ~ m * - ~ ~ . ~ ' ~ ~ ~ d ? ~ . 7 : T , ' w e / m ~ ~ , i ~ ~ / ~ , < ~ ~ ~ . t ~ L ~ / ~ * , ~ w . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , ~ , ~ ~ ~ , ~ / f l , w c z ~ ~

In all experiments, germination was reduced or delayed by extended exposure to

far red light. In experiment 7.1 (a) constant far-red light significantly reduced

radicle protrusion at 50 h (Pc0.05) and in Experiment 7.1 (b) the delay was still

evident on day 5 (Table 7.1). Radicle protrusion was significantly earlier

(Pc0.05) in constant darkness than in any other treatment. The trend was still

apparent on day 5 when germination of seed exposed to constant red light was

significantly (P<0.05) greater than germination of seed from all other treatments

except constant exposure to cabinet lights.

Table 7.1: The effect of light quality on germination of seeds from

seedlot 'KI-2' determined after 50 h (7.1 a) and 5 days (7.1 b) (n=4).

Treatment Mean Germination (%)

(a) (b)

Cabinet light 37 90

Dark 54 86

Red 45 98

White 43 7 8

Blue 45 85

Far red 6 24

LSD (P=0.05) 7.5 8.5

After 24 h of dark imbibition, exposure to far red light until day 5 significantly

(P<0.05) reduced germination compared with all other light treatments in

seedlots 'KI-2' and 'H' (Table 7.2). Results were similar for both seedlots with

no other treatment responses observed. Seed imbibed in cabinet lights for 24 h

and then exposed to far red light showed a similarly reduced germination

Chapter- 7 (Light Quality) :<F.,",,z,:. .7:-<-:<.G5r:e;.-$',..;,<?&?; .c'$: .TS..O &YL?:,<, 2:,;<, ,;L*... :::7~~.~~>,,,'.~~~~-.~?~~,.~~"7~'~5~~ (7.: 'V ~ : . : > ~ ~ , ~ ~ v ! . ~ . ~ w ~ Q ; ~ A u / ~ A ~ . ~ ~ m ~ ~ ~ ~ ~ m ~ ~ ~ . ~ . ' ~ ~ ~ ~ z ~

compared with other light treatments following imbibition in cabinet lights

(Table 7.3).

Table 7.2: The effect of light quality following imbibition in constant

darkness for 24 h on day 5 germination percentage of seed from

seedlot 'KI-2' (a) and 'H' (b) (n=4).

Treatment Mean Germination (%)

Seedlot 'KI-2' (a) Seedlot 'H' (b)

Cabinet light 9 1 82

Dark

Red

White 93 8 9

Blue

Far red

LSD (P=0.05) 1 1 . 1 19

Chapter- 7 (Light Quality) Cli' . . ' . V , L S V . ; ' L .:l'l,r.'Zb?iFYP,n.:2'r'r7dCN,,iVe-~hli/SSs! L>A:<'9,;n'P'H. ?~~S~~ .W~~ . '~70 ,1 '~ 'C /Y~~ ! ~ .P /~~ ,~X~17UiL i7 ;~~~~~7~~~~ / rP i 'A /d iW/W/Co 'J~ :YYyW~~ i7 /~WI :

Table 7.3: The effect of light quality following imbibition in constant

germination cabinet light for 24 h on day 5 germination of seeds

from seedlot 'KI-2'. Germination result for seeds exposed to

constant darkness and constant cabinet light from the start of the

imbibition is included for comparison (n=4).

Treatment Mean Germination (%)

Constant cabinet light 95

Constant darkness

Dark

Red

White

Blue

Far red 33

LSD (P=0.5) 5.8

In Experiment 7.4a, initial imbibition in far red light for 24 h before transferring

to constant darkness significantly (W0.05) reduced day 5 germination compared

with all other treatments, except for blue light (Table 7.4). There was no effect of

light quality on germination of seed initially imbibed for 24 h in cabinet lights

and then exposed to, different wavelengths of light. In contrast, imbibition in

light followed by transfer to darkness for the remainder of the five days resulted

in a significantly (P<0.05) higher germination percentage than any other

treatment.

"., ..+.,, . ., ,. #.,.v. ..;+> .<,,: ,e,-,-,,(G? !.cci5,qi%~v vF:;L,c '$ ?e.?p,,v a*;.c%wfl,G:742< +,,v.;..~~w,+-,q,.~p ~ ~ ~ ~ i ~ , ~ , ~ ~ , ~ ~ . r ~ 4 v ~ ~ , , ~ , , ~ ~ ~ w , e , ~ , A ~ ~ ~ ~ , ~ ~ ~ e , ~ , w , ~ , ~ A w , ~ , ~ , ~ ~ ~ ~ N , ~ 4 Chapter- 7 (Light Quality)

Table 7.4 (a&b): The effects of light quality during the first 24 h of

imbibition on subsequent germination of seed from seedlot 'KI-2'

under constant cabinet light or constant darkness. Germination

percentages were determined 5 days after the start of imbibition

(n=4).

Treatment Mean Germination Mean Germination

(%) darkness (%) light

(a) (b)

Constant cabinet light 97 97

Constant darkness 9 1 90

Cabinet light 95 -

Dark - 99

Red 98 9 5

White 94 9 8

Blue 89 97

Far red 86 9 8

LSD (P=0.05) 3.8 2.7

Experiment 7.5 (Table 7.9, examined the effects of application of GA3 at the

start of imbibition and exposure to different wavelengths of light for the duration

of germination. In this experiment exposure to far red light also caused a marked

reduction in germination percentage with or without GA3 application or

irrespective of GA3 application. In addition, exposure to blue light in

combination with GA3 also caused a small but significant reduction in

germination percentage compared with seeds germinating in constant cabinet

light without GA3.

Chapter- 7 (Light Quality) :/. .v. - , .7!! '..z.d.: ,..;<". c7..q: ..> ~: :~ .~4:~T~." :~7c: -~~;~~, , i ' ;~~~%74?: .?L~'~~~~ d,:;.<-; ,.. ,' ,. , .. .* ..... ,>.c ."%-,,% z7,<, \ ~~~~;~~~~-!~=~~~,:;,~",~~/o.<'~ ~7,#,,~,;~*~v<,/~;%:;6>,~4~.27,~4/?

Table 7.5 The effect of addition of 5x10-= M G& at the start of

imbibition and light quality on germination of seed from seedlot 'KI-

2' (n=4).

Treatment Mean Germination (%)

Constant cabinet light 93

Constant darkness 86

Cabinet light 94

Darkness 86

Red 93

White 88

Blue 8 3

Far red 24

LSD (P=0.05) 7.1

Exposing seeds to different wavelengths of light for the first 24 h of imbibition,

before or after GA3 addition, had no effect on subsequent germination under

cabinet lights (Experiment 7.6, Table 7.6). Significant differences (P<0.05) in

seed germination percentage were observed when seeds were treated with GA3

and then exposed to different light treatments for 24 h prior to completing

germination in constant darkness (Experiment 7.7, Table 7.7). In this set of

treatments, exposure to far red light resulted in a small but significant reduction

in germination percentage compared with exposure to red or white light. There

was no significant difference in germination percentage of seed exposed to far

red light, blue light or constant darkness, with or without GA3 application.

Chapter-7 (Light Quality) , :, ,..- .,,> , ,..,,r~ ,3: .:.~*>.%*:-.;,r,t7 S:::C.; p. $-A,?,,r ,.w,.?i?:*-/<X:: . V..: ,:W.'..-.~~; ~9.V. '0~~$?~N~SZ%~7/ .~~;~~A*~, 'W>~~~0<3!#~ 'W9T~'W/P~f l ; f l2H~X~f l . 'U /0 ,W,W, ' f l~~<

Table 7.6 (a &b). The effect of light quality during the first 24 h of imbibition

and time of application of 5x10-= M G& on day 5 germination of seed from

seedlot 'KI-2' (n=4).

Treatment Mean Germination (%) Mean Germination (%)

with GA added 24h after with GA added before

start of imbibition imbibition

(a) (b)

Cabinet light (no GA) 96 96

Cabinet light 97 97

24 h of red light 97 99

24 h of white light 97 96

24 h of blue light 99 9 8

24 h of far red light 96 9 8

LSD (P10.05) NS NS

Imbibition for 24 h in complete darkness followed by GA3 treatment and then

exposure to different light treatments (Experiment 7.8, Table 7.8) resulted in

significant (Pc0.05) treatment effects. A marked reduction in day 5 germination

percentage for the far red treatment compared with all others was observed. The

germination percentage of seed exposed to constant darkness was also slightly

(P<0.05) lower than seed exposed to red and white light.

. .. Chapter- 7 (Light Quality)

. .. . - ' . 8 . ." .- .',-.:'. t ..T .w ., ,.,*c:,.r: 'r~--d,7'$,S:.? ;, ,-<..,-:.: ;\;,:.,<:,L><w ,,,.;.:c7 ,. s,(,,- ,.,. ,*<.+- k" *4r.2,, <z,,:,,~.,~r#;fl,*,.,~,~,p,",~m,~~*.,#~#,~~**~-/

Table 7.7: The effect of light quality during the first 24 h of

imbibition on day 5 germination of seed from seedlot 'KI-2' treated

with 5x10-= M GA3 at the start of imbibition. Seeds were

subsequently germinated in constant darkness (n=4).

Treatment Mean Germination (O/O)

Constant darkness (No GA) 90

Dark 9 1

Red light 97

White light 96

Blue light 9 1

Far red light 84

LSD (P=0.05) 8

Treatments involving a short exposure to red light (Experiment 7.9, Table 7.9)

did not affect germination in seedlot 'KI-2'. In contrast, a small but significant

reduction (compared with all other treatments) in germination percentage on day

7 was observed for seed fiom seed lot 'H' imbibed in the cabinet lights before

transfer to darkness for the remainder of germination. There were however no

other significant treatment effects.

Experiment 7.10 examined the effects of short duration exposure to red or far red

light followed by 12/12 light/ dark cycle. Seeds from seed lot 'h' germinating in

a 12/12 photoperiod (the control treatment) had a significantly (Pc0.05) reduced

day 7 germination (54%) compared with all other treatments. There were several

other small but significant treatment effects. The highest germination (95%)

occurred in seed imbibed in constant darkness for 0.5 h with a 18 h exposure to

red light followed by 12 light/ 12 dark period. Imbibition in constant darkness

for 5.5 h followed by 0.5 h far red light before transfer to 12/12 photperiod

" ,- .. Chapter- 7 (Light Quality)

, ., .T,r!''*;.&,?<7~.S ~ ~ : ~ , ~ . ~ ~ ~ : ~ ~ V ~ ~ : , ; ~ ~ ~ ~ . J . ~ ~ : ~ > . : ~ ~ ~ ' ~ V ~ ~ ~ ~ < . ~ A ~ ~ . Z ~ ~ : ~ ~ . ~ Z ~ : ~ ~ * . Y ~ ~ V A ~ : + ? < ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D ? ~ ~ ~ , ~ ? ~ ; U / R , ~ . T ~ ~ ~ C ~ ~ W ~ E , N ~ M , ~ N , ~ / ~ ~ < ~ ~ / ~ ~ ~ W , ~ ~ ~ S Z

reduced germination percentage. There was no significant difference between the

treatments were short pulses of far red light was given.

There was a significant (P50.05) interaction between light and GA3 treatments

on day 7 germination percentage of seed lot h in the final experiment (Table

7.1 1). Exposure to far red light for 30 h followed by darkness for the remaining

germination period markedly reduced germination irrespective of GA3 treatment.

Addition of GA3 significantly reduced gemination in seed exposed constant

dark, and to an initial 30 h of cabinet lights or red light. The most marked

inhibitory effect of GA was on seeds germinated in constant dark. Addition of

GA3 did not improve germination in far red light.

Table: 7.8: The effects of light quality on day 5 germination of seeds

from seedlot 'KI-2' after imbibition in darkness for 24 h followed by

addition of 5x10" M GA3 (n=4).

Light + GA3 treatment following Mean Germination %

24 h imbibition in darkness

Constant dark (no GA) 90

Cabinet light (+ GA) 96

Red light

White light

Blue light 96

Far red light 5 0

LSD (P=0.05) 6

.. ". ..-, . Chapter-7 (Light Quality)

.. L.. +.'.? ~..,r7,7,.-.,~.,-&0 6. -,r... - : . . ,,:*;+,-,,,,'s ~ , ' " ~ ~ ~ 7 ~ ~ ; ' ~ , ,<-~~~.$~:,v7,*.;r: ',+"T,',- >?Z*flL.?+7?,S " " , ~ ~ , ~ 7 : < , ~ ~ w ~ : ~ . r ? . " ~ ~ / ~ ~ n ~ ~ ~ , , m ~ m * ~ ~ , ; ~ ~ ~ w , m ~ . w , n / ~ , ~ , ~ v ~ : ~ ~ <

Table 7.9: The effects of one or two 30-minute pulses of red light

during the first 24 h of imbibition in constant darkness on

subsequent germination of seed from seedlots 'KI-2' and 'h' under

constant cabinet light. First treatment is constant cabinet light.

Germination was scored 5 days from start of imbibition (n=4).

Mean Germination (%)

Light Treatments Seedlot61U- Seedlot'h'

Dark Red Dark Red 2'

0 0 0 0 90 75

24 0 0 0 92 86

0 24 0 0 96 8 5

0 23.5 0.5 0 92 84

5.5 0.5 18.0 0 9 1 86

5.5 0.5 17.5 0.5 93 9 1

LSD (P=0.05) NS 8.5

.,.- ..- ... ....J. -, gf14,...,-: . - c . z , , ~ . . . ~ + x ~ ~ , ; ~ . L . ~L..~,~~.~G~x,.;,.P,-,.:~ ~ , : r , ~ ~ m ~ ~ . ~ r c ~ : : o . ~ ~ ~ . ~ w ~ ~ * : ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ V C . ~ ~ ~ , ~ : F ~ ~ , ~ ~ ~ , ~ : K , ~ ~ ~ ~ ~ Z ~ ~ ~ ~ ~ , ~ ~ ~ D L ~ W , W ~ ~ ~ Chapter- 7 (Light Quality)

Table 7.1 0: The effects of one or two 30 minute pulses of red light

during the first 24 h of imbibition in constant darkness on

subsequent germination of seed from seedlot h under 12/12

lightldark period. Germination was assessed on day 5 (n=4).

Mean Germination %

Light Treatments

Seedlot 'h' Dark Red Dark Red

Dark Far red Dark Far red

23.5 0 0 0.5 9 1 5.5 0.5 18 0 8 8 5.5 0.5 17.5 0.5 8 7 5.5 0.5 0 0 9 1

Control (1211 2 lightldark period) 54

LSD (P<0.05) 6.2

Chapter- 7 (Light Quality) ...~+?:;:..>~~;..z,+. .: ~ ~ : 4 r ~ ~ , ~ : ~ . ~ . ~ ~ t ~ ~ ~ ~ , ~ + ~ ~ , . * . ~ . ~ ~ ~ ~ ~ ~ e ~ ~ ~ . ~ w . ~ , , + ~ ~ + ~ ~ ~ ~ ~ : ~ ~ . ~ , ~ ~ ~ ~ ~ ~ . ~ ~ ~ * ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ' c r ~ ~ ~ ' w ~ ~ ~ ~ ~ ~ ~ ~ ~ m ; ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ o ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ,

Table 7.11: The effect of G& on germination of seed of seedlot 'h'

exposed to different light qualities (red, far red, darkness or cabinet

light) for the first 30 h of germination. 5x10" M G& was added at 30

h ("-"= without G& and "+"= with G&). With the exception of the

continuous cabinet light treatment, seeds were subsequently

germinated under constant darkness. Germination counts were

done on day 7 (n=4).

Gibberellic Acid Light Mean Germination (%)

- Constant cabinet lights 8 5

- Dark 92

- Cabinet lights 9 1

- Red 89

- Far red 19

+ Constant cabinet lights 78

+ Dark 62

+ Cabinet lights 8 3

+ Red 7 3

+ Far red 17

LSD (P=0.05) 7.3

7.4. Discussion

As reported in many other species, these experiments show that, for E. globulus,

far red light reduced or inhibited germination and that, under some

circumstances, red light promoted germination. These two responses confirm a

.... Chapter- 7 (Light Quality) ,'. .AS. C.3#4, B,Z.;Ly%0.':4 ' ~ ' ~ ~ ~ . W ~ ~ W ~ ~ ~ W , ~ ~ > X ~ ~ . ~ > : ~ ~ ~ ~ ~ L ~ ~ ~ ' ~ C ~ ' ~ : ~ ~ ~ ~ - ' ~ ~ ~ ~ ~ ~ ~ > ~ ~ ~ ~ ~ ~ / ~ ~ ~ ~ ~ / ~ ~ ~ ~ ~ W ~ ~ ~ ~ ~ ~ % ' M ' ~ ~ ~ ~ ~ . N ~ & . ' ~ ~ / ~ ~ ~ ~ ' ~ ~ ~ W ~ ~ ~ W ~ ~ ~ . ' W ~ ~ ~ & ~ ~ / ~ ~ ~ ~ ' ~ A

control system attributed to phytochrome (Toole, 1973; Bell, 1993). However,

the timing of responses relative to stage of germination suggests a more complex

system than reported in the literature. Further, there is no clear reason why initial

imbibition in the dark should lead to the light sensitivity reported in the previous

chapter, and the results do not suggest an obvious model for control of this

induction of secondary dormancy. In fact, the light response noted earlier

appears to contradict the apparent phytochrome control indicated here.

A notable result from Experiment 7.1 was the earlier radicle emergence in dark

treated seeds, and this corresponds with an early commencement of respiration in

the dark noted in Chapter 4. As shown in the second experiment, by day 5 there

was no difference in germination between dark and light germinated seed

indicating that this early difference in radicle emergence is lost as development

continues from 2 to 5 days after the start of imbibition. This early radicle

emergence in dark imbibed seeds may account for the differences in light

response noted earlier.

Experiments 7.2 and 7.3, in which far red exposure after 24 h markedly reduced

germination, indicate that sensitivity to far red is apparent fiom 24 h after the

start of imbibition, even in the strongly germinating 'KI-2' seedlot. In contrast,

whilst exposure to far red in the first 24 h (Experiment 7.4) reduced day 5

germination, the reduction was relatively small. This change in relative

sensitivity to far red at around 24 h after the start of imbibition was also evident

in the combined GA and light experiments. Experiment 7.5 confirmed an overall

effect of far red, but exposure in the first 24 h followed by cabinet lights in

experiment 7.6 failed to show any treatment differences and there was only a

small far red response in experiment 7.7. In contrast, exposure to far red after 24

h in experiment 7.8 resulted in a marked reduction in day 5 germination. In the

final experiment (7.11) pre-treatment was extended to 30 h in this slower

germinating seedlot ('h') and there was a marked reduction in day 5 germination.

Combined, these results indicate a far red sensitive period commencing 24 h

(perhaps a little earlier) and extending to at least 30 h after the start of

imbibition. These times correspond closely with the time scale for the dark

influence on germination shown in the previous chapter. That is, seeds imbibing

in the dark from around the end of phase I of imbibition through to thirty hours

showed decreasing day 5 germination when exposed to constant light for the

remaining germination period. Although inhibition of germination by white light

has been reporteded in E. marginata and E. erythrocorys (Bell et al., 1995),

Anemone coronaria L. (Bullowa et al., 1975) and Glaucium. Jlavum (Thanos et

al., 199 1) and inhibition by far red light was reported by Bell (1 993) in Juncus

articularis, J. microcephalus and Isolepis prolifera, and specific timing of

exposure was reported by Negbi et al. (1968) in lettuce seeds and consequent

effects.

Gibberellic acid has been used to promote germination in several Australian

native species. Plummer and Bell (1995) used GA3 to replace a red light

requirement in germination and addition of GA3 overcame white light inhibition

of germination of seed of E. marginata and E. erythrocorys (Bell et al., 1995).

The present trials were designed primarily to test whether GA3 was able to

overcome the inhibitory effect of far red light, demonstrated in the earlier

experiments. The results clearly indicate that GA3 addition before or during

germination did not reverse the effect of far red light. Furthermore, there was

some evidence that GA3 itself might induce secondary dormancy. As shown in

Table 7.1 1, Addition of GA3 after 30 h imbibition resulted in a marked reduction

in 5 day germination of seeds in constant darkness, There were similar, smaller

but significant reductions with GA addition after 30 h of cabinet and red light

before transferring to dark for the remainder of the 5 day germination period.

Spectral composition graphs (Chapter-10 Fig 10.3) show that the incubator and

white LED lights had almost similar broad peaks at around 580 nrn, which

extended up to about 670 nm. The red and far red LEDs had narrow peaks,

approximately 60nm in width centred on 610 and 750 nm respectively. Thus

both the cabinet lights and white LEDs had relatively weak emissions at the far

red end of the spectrum with a balance strongly towards red.

Chapter- 7 (Light Quality) ?.fi>,.<A&.,.,<.* '-,,"::'*,e%r.~... ,,; m<w47,>n,.F>3.<*>,-<4 ..a< 'w:-*..Z?Lr'"-,x; ...,-v,7 . :,.>:',':%***,3,v,~:q .*t.!Vd?> w ' % . ~ . * , ~ ~ ! . ~ . ~ - , : ~ . . - < ~ ~ ~ . ~ , * - / * ~ ~ , ~ r ~ ~ 7 < ~ , ~ ~ # ~ ' ~ , ~ v n a v ' % ~ > ~ ~ ~ + ,

Whilst exposure to far red light following imbibiton in light delayed

germination, the spectral range in the cabinet lights (in particular) suggest that

the darwlight induced dormancy reported in earlier chapters may not be

mediated by far red light. Although phytochrome control has been related to GA

activity (Thomas, 1992), failure of GA3 to reduce or eliminate the far red effect

on germination and, particularly, reduced germination with some GA3 treatments

also give rise to questions about the role phytochrome plays in regulating

germination of E. globulus seed.

Chapter-8 (General Discussion) ~ ~ ~ r ~ . r ~ ~ ~ / ~ - ' . ~ . ' ~ ~ . - ~ ~ ~ ~ . - ~ * ~ ~ ~ , ~ ~ : ~ < " / . ~ < . < ; , , ~ : . . ~ ~ : , , ; ~ ~ ~ < ~ .;.'*,~<=<~.~.~w*,L.',=,<.,*,%.. % - . ~ ~ ~ ~ ~ & : : ~ P ' ~ ' L ~ o , ~ ~ ~ ; : ~ ~ m ~ ~ o ~ ~ ~ / ~ ~ < ? ~ ~ ~ ~ ~ ~ . ~ ~ . ~ o , ~ * / ~ , ~ ~ ~ ~ ~ . . ~ . m ~ a ~ z z ~ ~ <

General Discussion

As noted in the Introduction (Chapter-1), uneven and unpredictable germination

of E.globulus in commercial nurseries in southern Australia resulting in some

sowings taking several weeks to germinate has caused significant economic

losses due to uneven seedling size. The study has shown that conditions during

germination may induce marked changes in germination measured 5-7 days after

the start of imbibition. Management of these conditions, particularly the light

climate during the first 24 h after initial wetting, appears likely to reduce

variability in germination. The study has also suggested that endogenous control

of germination and the response of the control system to external stimuli may be

more complex than the models based on published studies on lettuce,

Arabidopsis and other commonly reported species. Industry reports suggested

that germination was erratic due to alternating temperature and moisture stress at

the time of sowing leading to delayed germination. Hence the aim of this project

was to identify the causes leading to delayed germination in nurseries and to

understand the physiology of the seeds. Practically, attention to nursery light

management appears to offer a solution to the commercial problem, but the

complexity of the physiology suggests that other environmental factors may be

implicated.

8.1. Practical solutions

Contrary to the industry suggestion that short term water stress might induce

secondary dormancy, the results provide firm evidence that light, as light/dark

cycles, shortly after the start of imbibition may be responsible for the type of

symptom noted in commercial nurseries. Experiments 6.5 and 7.10 clearly

demonstrated that the time of day when sown seeds are able to commence water

uptake may have a significant influence on day five germination. Although the

results of this laboratory experiment may be difficult to translate to a commercial

nursery scale, there is a strong suggestion that, if seed is given access to

sufficient water to commence normal imbibition in the morning, it will generate

a light sensitive secondary dormancy. Similarly treated seed with water supply

commencing in the afternoon germinated normally. Clearly there are day-length

and water availability issues likely to impact on aspects of this overall picture.

Nevertheless, there appears to be an inescapable general conclusion, that

darkness from 6 through to about 24 to 30 h from the start of imbibition followed

by light exposure will influence uniformity of germination. Detailed information

on commercial nursery management was difficult to obtain due, in part, to a lack

of appropriate records and to commercial confidentiality issues. Few nurseries

contacted had any record of timing (relative to daylnight) of their various

activities and none were able to provide information that might support this

conclusion. The critical involvement of time of initial wetting (and perhaps

continuity of the early stages of imbibition) may however explain the early

association of uneven germination with a wetting drying cycle by commercial

nursery managers. This suggestion was apparently associated with malfunction

of an irrigation gantry (Gore pers. corn.), and rather than a wetldry cycle, the

malfunction may have simply resulted in some seeds commencing imbibition at

a different time to others and thus being exposed to different 1ightJdark cycles.

Differences between seedlots, in their response to dark induced secondary

dormancy, were consistent with industry experience. Typically, uniform

germinating seedlots, such as 'KI-2', were generally less susceptible to

laboratory induced secondary dormancy. On the other hand, seedlot 'H', noted

for demonstrating uneven germination in some sowings in commercial nurseries,

showed readily inducible secondary dormancy in most experiments.

The results raise questions about seed testing. The ISTA guidelines do not

specify light conditions for seed testing in E. globulus and local seed laboratories

generally carry out germination tests under continuous light. For adequate

reporting of seed lot quality, light conditions during testing should be specified

. - Chapter-8 (General Discussion) ,*,$ ., : : ~ ~ ~ n . ~ t ~ ~ ~ ~ ~ : r ~ . ~ ~ L W ~ ~ r .-A*,. t, '*. a:..,+Y.;,:e7e .%%+;a: -.A*- ,f>i,;-,,. .Ye+;:+ ~ ~ ~ d , ~ ' ~ > ~ 3 ; ' S ~ . ~ W ~ v ~ ~ 8 * ;:ye .'-Z/Pz 9:>f,4?,BV~/##~&7*'N%'*P,%~7'W,~'W4~Z~T?W,&q:%3YPH,Z3

and test results should perhaps carry an explanation that test results may vary

according to light conditions.

It was anticipated that experiments in Chapter 7, might provide low cost, easily

managed solutions to dark induced dormancy, or provide a basis for treatments

to reduce the risk of dormancy induction. Unfortunately neither of the two

treatments, considered likely to be commercially useful, GA treatment or

provision of red light during germination, produced results with immediate

application. As discussed below, this chapter underlined the apparently

anomalous responses associated with the red light receptor phytochrome, and

further work is needed to clarify whether manipulation of this and associated

control systems may be useful as a practical management tool.

Thus, whilst further work is also needed to establish the range of issues

potentially involved in induction of light sensitive secondary dormancy, the

study does offer some directions for commercial management of E. globulus

germination. Regardless of seed test results, these should include:

As far as possible seed should be exposed to either constant light or

constant dark during the first 36 h from initial wetting.

If this is not possible seed should be wet at a time of day that avoids a

dark period starting from around 9-12 h after initial wetting and

extending for 12 or more h.

If these conditions cannot be reliably avoided, nurseries should select

seedlots known to be relatively immune fi-om these light responses.

8.2. Physiological aspects

The suggestion by Baskin and Baskin (1998) that induced secondary dormancy

may be an inappropriate or redundant term, underlines the lack of published

reports on the type of response to light reported here. The unusual nature of the

response is also emphasized by the comment by Thanos (2005) on the

Chapter-8 (General Discussion) ?L?P'.*,.s>.':C,:>,~.~~''<,'.,*% %bZ%-,,r'.'#,S?~-#:*<e,, 2,,.%;,974%&' ~j;,,WO~~5d?,fi;2."~9:~P,.~,~$~C/?~:7 ;S:,-.>?V,L e7.WL.i ,?s>&,'s,, ,p,8+fl,&,~p,w,~L@,&'p,,~,~/fl/&,&,@,~/~,fl,~w/~,~$

comparative rarity of light related dormancy, most light responsive seeds being

induced to germinate on exposure to light rather than the reverse. Consequently

the change in constant light or constant dark germination pattern induced by

initial dark imbibition followed by constant light does not appear to fit any of the

conventional models [light promotion: Arabidopsis thaliana (Botto et al., 1993),

Datura ferox (Casal et al., 1991), light inhibition: E. marginata and Trymalium

floribundum (Bell et al., 1995)l for control of germination.

The light response appears to be biphasic, with light exposure early in

germination delaying the start of respiration and radicle emergence. From the

experiments reported there was no way of determining whether this was an

active response to light exposure or whether the earlier appearance of the radicle

in dark grown seed was an active response to darkness. Paradoxically, the

difference in time of radicle emergence did not result in any difference between

dark and light germinated seed when germination was counted on day 5. The

second of the two phases in the light response is the light induced delay in the

completion of germination when dark imbibed seed is exposed to light after

around 24-30 h of imbibition.

The initial delay in radicle emergence in dark imbibed seeds was not noted until

the latter stages of the study, when some difficulties with availability of seedlots

and consistency in results as seedlots aged, were beginning to emerge. For

example experiment 7.3, carried out using three-year seed old seedlot 'h' seed,

failed to give the expected dark induced dormancy and although germination

rates were high in all treatments, variability was also unusually high. Across all

experiments, there were also practical difficulties measuring germination in

constant dark or light wavelength treatments. Because spectral responses were

unknown, germination counts were limited to a single time and there was no

opportunity to develop full germination profiles.

Reduced germination in response to extended exposure to far red light indicates

that there is a phytochrome control system similar to that described for lettuce

Chapter-8 (General Discussion) 5 ,:', :,8 .?:.,,C,,<,*":;%,,?. C'.,-!,,+-'CLC ,e..:&*'i%e5'..>,.'>c?W , ; ? Z * . . ~ ~ G T . Y ? P~.A~~.P,;I:;~~LV->~YK..?,~;::~?&~,::~,~~(-V~,~,~' *;.,#<W,E,&,,~/~~~G;P~,-*,S~XXX,;V,~~'C~,P,~*W,:*~:*IY<

(Kristie et al., 1981; Toyomasu et al., 1994; Hsiao and Quick, 1997) and other

species. That is, exposure to far red light mimics after imbibition (Negbi et al.,

1968) a shaded environment in a natural ecosystem and germination is reduced

or delayed (Bell, 1999). Under similar models, such a marked response to far red

is also present if seeds are imbibed in the dark in suboptimal conditions

(Benvenuti and Macchia, 1997). In this study however seed exposed to constant

dark germinated normally in most cases. Based on the assumption that Pfi is the

active form of phytochrome, this suggests that there is some protective

mechanism in E. globulus preventing reversion of Pfr to Pr in darkness.

Considering the germination responses to light in more detail (i.e. early

respiration and radicle emergence in darkness and the dark induced secondary

dormancy), the experiments in Chapter 7 provide no clear evidence of a

phytochrome control system in the germination responses described in detail in

Chapter 6. Thus, although the response to far red confirms the presence of a

phytochrome system with potential to control germination inconsistencies in the

results in this chapter do not give support for speculation on control of the

induced secondary dormancy. Several issues, including the apparent reduction in

germination in response to added GA in experiment 7.1 1, suggest that a Pfr

mediated inhibition may be operating. The very low far red levels in the cabinet

lights, which initiated the photosensitive response in dark imbibed seeds also

suggests that a light mediated Pfi to Pr shift is unlikely to be involved.

A major difficulty in comparing these results with other published work on

eucalypts (and other species) is that, frequently, aspects of seed treatment are not

adequately described or controlled. For example germination was delayed when

E. delegatensis seeds were imbibed for 24 h or more followed by desiccation

(Battaglia, 1993) but desiccation conditions were not described in detail. Earlier

work done by Wilson et al. (2005) also suggested a darktlight cycle during

imbibition and delayed germination may have been responsible for a similar

induced secondary dormancy in E. ovata, but the treatments were not sufficiently

controlled to make a direct comparison.

Chapter-8 (General Discussion) +.. ~ .: , .,, ,.2..& .. ?4 :.:,.< ,A ;'7.',;7,,.5:. '.,?& ;WW,<.T<:. ,pT5',:i':<,78X<~,V: .?,N,':AL >LC;:;: <~~.P,>.V~.F+>C.XX. :*?;+ '>,7L<vL:ri&,<&;"#; ~ ~ ~ Q ~ # , ~ W ~ N / . ~ ~ W . ~ / ~ A ' # , W ~ ~ ~ ~ ~ / @ ~ F A

The ecological significance of this induced secondary dormancy is unclear.

However, the timing of the cycle effective in the induction of secondary

dormancy is close to natural daylnight cycles, suggesting that it may be a method

of delaying germination in summer when days are longest and water is less

available. As the seed is so small and the volume of water for full hydration is

minimal, it also seems possible that it could be a method of minimizing

germination in response to night time dew formation. That is, germination is

most likely to proceed if seed starts imbibition from around the middle of the day

onwards. Availability of water at this time of the day will almost certainly be as

a result of rain rather than dew.

8.3 Conclusion

The results obtained here give a plausible explanation for the variable

germination patterns obtained with E. globulus in commercial nurseries and

revised management approaches have been suggested. The physiology of the

apparently anomalous responses to light require further exploration, as does the

ecological significance. The light responses were not totally consistent between

seedlots and (perhaps) age of seed suggesting that seed testing protocols for this

and possibly other eucalypts are in need of revision. Further, selection of

particular genetic lines may need to take this germination characteristic into

account.

Chapter-9 (References) d. . 4'. + ... ' *.:;u ,ed,,.*, z. '?.<:< P.e+.:.;,: P, :: i ~ ~ , . ~ ~ ~ ~ ; r ~ ~ ~ : ~ d ~ ~ ~ Y ~ ~ ; * ~ ~ ~ ~ 9 ~ ~ ~ ~ ~ ~ ? , z ~ ~ ~ ~ 4 ' ~ ~ / ~ ~ ~ ~ . ~ ~ ~ X ~ 2 - ~ ~ ~ " ~ W ~ : ~ < ~ ' ~ ~ ~ ~ P ~ ~ ~ ~ & ~ ' ~ ~ ~ / ~ 7 A ~ ~ ~ . ~ ~ < ~ : ~ # ~ / ~ ~ i 7 ~ Z ~ ~ ~ N / ~ W ~ W / ~ ~ / ~ ~ ~ ~ ~ ~ I

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Chapter-1 0 (Appendices) *.v,.%.<7.+-ALY,~".;.%#;. A? 4.7-*,~-;.;7.# .~*,~~.~. .~, .d~. '+X." , .~~. ,>'~~:~.9,,,., A> 7 ?5-.,<>7P.. .?, <>,d? ~ > : < * ~ , ~ ~ < ~ ; , ' * ~ ' ~ ~ W , * ; ~ ? ~ P , ~ , ~ : V ~ ~ ~ ~ . ~ < , ~ . A ~ ' ~ , ~ ~ ; # ~ ~ ~ ~ ~ / ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ # - ~ ~ W,.'e/&.

Chapter-I 0

Appendices

300 400 500 600 700 800

Wavelength (nm)

Fig 10.1: Spectral composition of Incubator or germination cabinet

(Linder and May Model LMRIL-1-SD) lights.

- $-' 2* . Chapter-1 0 (Appendices)

3 + 7 - w r .r a L. u e ~ t - i rr . r . ZLSI.. ,e .. x ,7 rn C, ~ r n PA. fl1:1:a%~& /r e,#,#,e,m,&,#m/~M,~ulcl,

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Wavelength (nm)

Fig 10.2: Light emission from the 10,000MCD Red light peaking at

625 nm.

Chapter-1 0 (Appendices) ,,.", . *.,*., - ,; ,,.,..:i,.> ...,,b> ,; ,; ,.%. ,, , ,. -, *'< :.,. -: CA> ,.,.:, .+,.<.:.;,.; *?,:....".-fl'.v;,4.,:,* x.js:.~<.*<*>,p~v4J~,w,.,;~<.a~ ~ + ~ ~ ~ n ~ ~ ; , ~ ~ ~ ? ~ ~ ~ , m , z ' ~ * ~ ~ ' ~ ~ ~ ~ ' f l ~ ~ ~ ~ ~ ~ 4 ~ / m z ~ ~ ~ ~ f l ~ ~ A . ., .. . .. ,', ,

Wavelength (nm)

Fig 10.3: Light emission from 7000MCD White 5 mm diameter dome.

. .>. " Chapter-1 0 (Appendices)

,> , ,,r ',?, %.b,*, ,,: >,<? <,v,:. ,,v~,,:",#., s.*> z<.*,,w,.-v::y -;*,,,<*.>>..m,,p,Av:?,,,-< 7,pi?,;,Y,';',h<2.x,,', .?-4 ;'.T fl~/~~'.~~~~'d.'~~~'*u?~:~>;;"P.~-4m~-%.~'~~m,!~w/~~~'&~~'m~~~<

Wavelength (nm)

Fig 10.4: Light emission from 5000MCD Blue 5 mm diameter dome

peaking at 470 nm.

Chapter-1 0 (Appendices) < <.., .:. .<., .,,, ?,,. ,,.; ' .,.< - ,; .; .> ." ,,,*-:, ' ! ;c, !b. < ,,,<,*,<,,,# .:*.,< :.;:., ,: .,.y..?, v, .#.. a,,' ,< .Y': .,$. ,n:, <--:,. -;"; ' !*%.,r*,, ,? ,1,,:-,r ~ ~ ~ ~ , ~ ~ e . : ~ , ~ . 2 ~ < c ~ ~ . ~ ~ d . ~ ~ ~ ~ . ~ ~ , ~ ~ . 6 ~ ~ ~ ~

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Fig 10.5: Light emission from Far red 5 mm diameter dome peaking

at 740 nm.