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Thushara Sivasankaran Nair
School of Agricultural Science
University of Tasmania
Hobart
October, 2006
(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
(Preliminaries) < >.,2,; ,... .. /.>? .<,,.: . : ;.. ,.,;.. .,:*, ,* .>. ..""<,. r ,,*,3? ,,.,- .,'..,.fO. V?<.,?<d,&" ,* ,*.d??,Z.*>.~, .%:-L *,,7.P.. ?' ,:?'?;iWL+*, -,fl,~7~$7,,n;~m#4-/m@,'Px<ek?*?/fl?'~'E,&,'a.*~'J7flA
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
(Preliminaries) , : x v; ,<:: r,,,::;',/.:+.:% k, x7 ,< ,. ..*?.;A*,,<,,?, ,<~:~~':~;:~~;e,~.:~.'~;.v.:,.4 x?.,:CL!?x :+v:T,#4v,~.:74,!~s7d.*%!e*%..9A<c % ~ , w , n , ' ~ t ~ ~ * - ~ ~ ~ , & , : & < M h w ~ , * v ~ ~ ~ - , ~ ~ ~ ~ ~ , ~ , ~ ~ ~ : ~ , ' w ~ . ~ ~ ~
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
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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.
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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.
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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
Chapter-2 (Literature Review) .., , .=.. . .., . ;,. .,. -?,: ....;--,.>,:s7:<7r.-i~. w;? 't- w T,.,:* ,.... z .-,.,:.z j-.~?,;., ,s.,iz ~ ~ ~ , - ~ - + ~ ; 2 5 : ~ , ~ ~ ~ . ~ ~ w ~ ~ ~ 7 m ~ 7 ~ ~ ~ ~ . ~ , n 4 r ~ ~ e . : m ~ , ~ : v ~ ~ ~ ~ / ~ / ~ ~ ~ ~ ~ ~ ~ ~ ~ .
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
Chapter-2 (Literature Review) .,, . 6'. b,, v.4* ..,.d-,~..,'7,,< W ? . :,fi*,.Vr.4'.'e.',?;?,:.** : ~ ~ 7 ~ , ~ ' . ~ ' . C . ' ~ ~ % ? ~ ~ . T ~ . ~ ~ ~ ~ ~ ~ ~ < ~ - ~ ~ ~ ~ ~ ~ ~ < W . ~ ~ 7 ~ ~ ! , % Y ; ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ W L ~ ~ & : ~ , ~ > > ; 7 A t ~ E ~ # ; ~ , & ~ % ~ ~ ; 3 ; ' W / ~ , i * . ~ & ~ ~ ~ " ~ N L Y ~ ~ ~
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.
Chapter-2 (Literature Review) .,,::>.' ' .':Kd>+>,,,*,;7m,,.TK,,J.;-z,~.':,~L,* w ~ ~ c ; ~ ~ , ' G . , ~ ~ ; ~ ? ~ > v , v ~ & ~ ; . ~ , ~ ~ *;,-*&..;, ;z>',x,A, d v # ; ~ ~ , : w . , ~ , ~ < N , ~ ~ ~ @ . , ~ - ~ f l / ~ , ~ ~ f l ~ P ; ~ ~ ~ ~ K ~ ~ , w ; ~ / ~ / ~ / @ ~ ~ Y / ~ / & , ~ ~ N L ~ ~
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.
Chapter-3 (General Materials and Methods) .< .. .. ,: -.,a: -: ' ,'? ,.x:,+: , : ,.,,; 'r;V .fi.-::* 4 ; .,#~.-:<;,,>::,,,,.'T. ~ .~ . , *~ , ,~ ,~<~ ,~ , : ;<~ .~> . *~~~~~~~ . 7,,3.,Av,#;,;, . , 4 ~ , ~ , ~ , ' ~ , . . - " . p , ' ; ~ ~ , . ~ . ~ ~ ~ , , 7 , ~ ~ < , ~ ~ ~ ~ ~ ~ c , * ~ ~ , ~ , p / ~ ~
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).
Chapter3 (General Materials and Methods) ~,. . ~ ~ ~ ~ . , , , ~ / P ~ ~ P , ~ ~ ~ ~ C , ; ~ , L C ~ , ~ C ~ ~ ~ , ~ * < ~ q ~ w z f i s , , .?. LZY +-- ~ ~ ~ . ~ ~ x ~ m ~ ~ 7 ~ ~ . ~ ~ ~ ~ ~ : ~ . 9 ~ . m ~ ~ ~ ~ r r ~ - . 7 ~ ~ 7 ~ . 7 ~ -.i?, :,e-:?. .,,/>A M C Q ~ 72, ,el. , ~ ~ - w ~ ~ , ~ - ~ ~ . ~ ~ - ~ ~ ~ ~ , ~ ~ ~ : v r ~ ~ ~ r~~fir,~<v,r,<fi .w,, , ;~ '~APLL~/T//T,X<~.V.-,TF~'+,:..~ -+. . ..94 , .:n.:-e- cv, >.-/.,7,/,73,'cr,--,~.v~-:*
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
Chapter-3 (General Materials and Methods) 7 > ~ " i ' w / ' ' . C i j r . ~ 7 % . i F i . " I Y : B ~ 1 - ~ C . . ~ c i d ' d ' d ' d ' Y ~ ~ I 5 ~ ~ ~ C ~ ~ ' . ~ * ~ ~ . ~ , " ~ ? ~ ! Z ~ K ~ ~ ? 7 < ~ ~ % ? + W A ~ ~ ~ 3 : - ~ N O . ~ ~ 7 ~ ' c ~ ! ~ < ~ V ~ 7 Z , ~ 4 7 ~ ~ ~ ~ ? Z ~ ~ ~ / ~ ~ f l , . ~ . ' ~ ~ V ~ H / f l / f l / ~ ' ~ ~ ~ ~ ~ ~ ' G ~ ~ ~ ~ ' # 4
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.
- $-' 2* . Chapter-1 0 (Appendices)
3 + 7 - w r .r a L. u e ~ t - i rr . r . ZLSI.. ,e .. x ,7 rn C, ~ r n PA. fl1:1:a%~& /r e,#,#,e,m,&,#m/~M,~ulcl,
4 I
4 I 4 I
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4 I 4 I
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300 . 500 700 900
Wavelength (nm)
Fig 10.2: Light emission from the 10,000MCD Red light peaking at
625 nm.
Chapter-1 0 (Appendices) ,,.", . *.,*., - ,; ,,.,..:i,.> ...,,b> ,; ,; ,.%. ,, , ,. -, *'< :.,. -: CA> ,.,.:, .+,.<.:.;,.; *?,:....".-fl'.v;,4.,:,* x.js:.~<.*<*>,p~v4J~,w,.,;~<.a~ ~ + ~ ~ ~ n ~ ~ ; , ~ ~ ~ ? ~ ~ ~ , m , z ' ~ * ~ ~ ' ~ ~ ~ ~ ' f l ~ ~ ~ ~ ~ ~ 4 ~ / m z ~ ~ ~ ~ f l ~ ~ A . ., .. . .. ,', ,
Wavelength (nm)
Fig 10.3: Light emission from 7000MCD White 5 mm diameter dome.
. .>. " Chapter-1 0 (Appendices)
,> , ,,r ',?, %.b,*, ,,: >,<? <,v,:. ,,v~,,:",#., s.*> z<.*,,w,.-v::y -;*,,,<*.>>..m,,p,Av:?,,,-< 7,pi?,;,Y,';',h<2.x,,', .?-4 ;'.T fl~/~~'.~~~~'d.'~~~'*u?~:~>;;"P.~-4m~-%.~'~~m,!~w/~~~'&~~'m~~~<
Wavelength (nm)
Fig 10.4: Light emission from 5000MCD Blue 5 mm diameter dome
peaking at 470 nm.
Chapter-1 0 (Appendices) < <.., .:. .<., .,,, ?,,. ,,.; ' .,.< - ,; .; .> ." ,,,*-:, ' ! ;c, !b. < ,,,<,*,<,,,# .:*.,< :.;:., ,: .,.y..?, v, .#.. a,,' ,< .Y': .,$. ,n:, <--:,. -;"; ' !*%.,r*,, ,? ,1,,:-,r ~ ~ ~ ~ , ~ ~ e . : ~ , ~ . 2 ~ < c ~ ~ . ~ ~ d . ~ ~ ~ ~ . ~ ~ , ~ ~ . 6 ~ ~ ~ ~
Wavelength (nm)
Fig 10.5: Light emission from Far red 5 mm diameter dome peaking
at 740 nm.