Carbohydrate analysis of Simmondsia chinensis(Link) Schneider and its relation to rooting

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CARBOHYDRATE ANALYSIS OF

SIMMONDSIA CHINENSIS (LINKl SCHNEIDER AND ITS RELATION TO ROOTING

. bySteven Jeffrey Reddy

A Thesis Submitted to the Faculty of the

DEPARTMENT OF PLANT SCIENCES

In Partial Fulfillment of the Requirements for the Degree ofMASTER OF SCIENCE

WITH A MAJOR IN HORTICULTURE

In the Graduate CollegeThe University of Arizona

1 9 8 0

STATEMENT BY AUTHOR

This essay has been submitted in partial fulfillment of re­quirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this essay are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed usa of the material is in the interests of scholarship. In all other instances, including reproduction of thework of art, permission must be obtained from the author.

SIGNED:

APPROVAL BY PROJECT SUPERVISOR

This essay has been approved on the date shown below:.

Q,.0 ^9:1)DAVID P^LzKILC Date

Adjunct Assistant Professor of Plant Science

ACKNOWLEDGMENTS

The author wishes to express his gratitude to Dr. LeMoyne Hogan

for his advice and interest throughout the graduate program. Special

appreciation goes to Dr. David Palzkill for his enthusiasm and critical

review of this manuscript. Appreciation is also extended to Dr. Chi Won

Lee and to Dr. Paul Bessey for their interest and suggestions.

The author would like to thank the University of Western Australia researchers: Dr. Rana Munns, Brian Attwell, Kelvin Maybury,

and Dr. Marcus Blaeklow for their assistance and for making the

Australian visit exciting and valuable. Appreciation is also expressed

to Dr. James Chute for the organization and realization of the visit.

Finally, I would like to thank my friends: William Feldman,

Carol Acuna, Bob Emrich, and Joanne Stenton for their support.

TABLE OF CONTENTS

: Page .LIST OF TABLES viLIST OF ILLUSTRATIONS , . . . „ „ . . . . . . . . . . . . . . vii

ABSTRACT . ........ . . . . . . .......... viii

INTRODUCTION . . . . . . . . . . . v . . . . . . . . . . . . . 1LITERATURE REVIEW . . . . . . . . . . . . . . . . .......... 3

Rooting Factors . . . . . . . . . . . . . . ........ . . 3Auxins . . . . . . . . . . . . . . . . . 3Rooting Cofactors ............ 4Photoperiod. . . . . . . . . . ... ® . « « « . • . » . 4Age of Shrub ............ 5Carbohydrates . . . . . . . . . . . . . . . . . . . . 5

Seasonal Carbohydrate Cycle . . . . . . . . . . . . . . . 8Trees and Shrubs . . . . . . . . . . . . . . . . . . . 8Jojoba Shrubs . . . . . . . . . . ............ 9

MATERIALS AND METHODS . . . . . . . . . . . . . .......... 1.0

Field Material ........ 10Stock Shrubs . . . . . . . . . . . . . .............. 10Cutting and Sample Collection .......... 10

Propagation Conditions . . . . . . . . . . . . . . . . . . 12Cutting Preparation . . . . . . . . . . . . . . . . . 12Greenhouse Facilities . . . . . . . . . . . . . . . . 12

Carbohydrate Analysis . ........ .. . . . 14. . Preparation of Samples ........ . . . . . . . . . . . 14

Extraction of Ethanol-Soluble Sugars . . . . . . . . . 14Extract Clearing Process . . . . . . . . . . . . . . . 14Anthrone Determination of Glucose Equivalent . . . . . 16Calculation of Sugar Percentage . . . . . . . . . . . 17Starch Extraction . . . . . . . . . . . . . . . . . . 1 7Calculation of Starch Percentage . . . . . . . . . . . 18

RESULTS AND DISCUSSION . V . . . . . . . . . . .. . . . .; . . . 19

Carbohydrate Concentration . . . . . . . . . . . 19Seasonal Carbohydrate Change . . . . . . . . . . . . . 19Individual Variation in Concentration . . . . . . . . 23

- ' iv

V

TABLE OF CONTENTS— Continued

PageLocation of Carbohydrates . . .. .................. 27Carbohydrate Change in Cuttings . . . . . . . . . . . 27

Rooting Results . . . . . . . e . . . . . . . . . . . . . 30Root Numbers and Leaf Abscission . . . . . . . . . . 30Carbohydrates and Rooting . . . . . . . . . . . . . . 33

5. SUMMARY AND CONCLUSION . . . . . . . . . . . . . 35

APPENDIX A: JOJOBA LEAF FRESH WEIGHT AND DRY WEIGHT . . . . 37

APPENDIX B: JOJOBA LEAF DRY WEIGHT AFTER EXTRACTIONw i t h ethanol . . . . . . . . . . . . . . . . . . 38

APPENDIX C: GLUCOSE STANDARD CURVE . . . . . . . . . . . . . 39

APPENDIX D: RAW CARBOHYDRATE DATA, . . . . . . . . . . . . . 40LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . . . 42

LIST OF TABLES

Table Page

1„ Carbohydrate concentration of leaves and stems of 2 jojobagroups at 4 times of the year .............. 20

2. ANOVA of stem sugar concentration of A shrubs over4 harvest dates .................. . . . . . . . . 24

3. Stem sugar concentration of individual A shrubs averagedover 4 harvest dates ............ 24

4. ANOVA of stem starch concentration of A shrubs over4 harvest d a t e s ................ 25

5. Stem starch concentration of individual A shrubs averagedover 4 harvest dates.......... 25

6. ANOVA of leaf starch concentration of B shrubs over4 harvest dates . . . . . . . . . . . . . . 26

7. Leaf starch concentration of individual B shrubs averagedover 4 harvest dates ....................... . . . . . . . . . 26

8. Carbohydrate concentration of component parts of an entirejojoba shrub harvested on July, 1979 .......... 28

9. Carbohydrate concentration of leaves of cuttings before andafter 6 weeks under propagating conditions . .............. . 29

10. Rooting and leaf abscission of cuttings collected in July,1979 from 2 groups of jojoba shrubs . . . ........ .. . . . 31

11. Rooting and leaf abscission of cuttings collected in Sep-tember^ 1979 from 2 groups of jojoba shrubs . . . . . . . . . 31

12. Rooting and leaf abscission of cuttings collected inNovember, 1979 from 2 groups of jojoba shrubs . . . . . . . . 32

13. Rooting and leaf abscission of cuttings collected in March,1980 from 2 groups of jojoba Shrubs . . . . . . .- . . . . . . 32

14. Regression analysis of root numbers and corresponding carbo­hydrate values of B shrubs for all sampling periods . . . . . 34

' ' ■ : ■

LIST OF ILLUSTRATIONS

Figure

lo Stock jojoba shrubs at Gascoyne ResearchStation9 Western Australia ..................

2* Jojoba stem cutting with roots . » „ , „ . * ,

3c Ethanol-soluble extract of jojoba leaf tissue

4o Seasonal change in sugar and starch concen­tration in stems and leaves of group A . . „ .

5o Seasonal change in sugar and starch concen­tration in stems and leaves of group B , » « «

ABSTRACT

Two groups of jojoba shrubs in Western Australia were analyzed

for sugar and starch concentration throughout a year. Cuttings from

both groups were put under propagating conditions for 6 weeks. Numbers

of roots produced and leaf abscission were recorded.

. Total carbohydrate concentration was highest in spring and

lowest in summer. The lower carbohydrate concentration in summer was : due mainly to declining starch levels <; Stem tissue was lower in sugar

but higher in starch than leaf tissue.Most shrubs were similar in carbohydrate concentration at any

particular sampling period. Variation was greatest in stem carbo-

hydrates of older plants.

Numbers of roots were higher On cuttings from younger shrubs

than from older shrubs. Spring was the period of greatest rooting Of

younger shrubs.More leaf abscission occurred from older shrubs than from

younger shrubs. Leaf abscission was consistent throughout the year.Concentration of leaf sugar was higher near stem tips than at

any other location. Roots had higher starch concentration than stems.

Carbohydrate concentration of cuttings which did not root

increased during the propagation period.

Regression analysis of carbohydrate concentration and rooting

of individual shrubs showed no significant relationship between the two.

. viii

CHAPTER 1

INTRODUCTION

Jojoba, Simmondsia chxnensls (Link) Schneider, is a member

of the Buxaceae family native to the arid regions of southwestern

United States and northern Mexico. It is an evergreen dioecious shrub

that produces one to three seeds in a capsule (Gentry, 1958)„

Jojoba has received worldwide attention due to the special properties of the liquid wax that is contained in its seeds. The

prospects of growing jojoba in arid regions, where other crops are

limited, has stimulated commercial and governmental interests.

For the potential of jojoba to be realized, Uniform, high-

producing shrubs must be developed. Because jojoba is a wild plant with considerable genetic variation, vegetative propagation methods '

have been used to increase selected plants with superior character­

istics. Many requirements for vegetative propagation have been

investigated and methods developed (Hogan et al. 1979).

Although numerous cuttings have been rooted and established

in test plots, some problems remain. A major problem is that

individual shrubs often vary greatly in rooting percentage even though

cuttings are obtained from the same area and are treated similarly.In some cases, rooting percentages have ranged from 0 to 100 between

individuals (Hogan, et al. 1979).

A possible reason for variability in rooting between plants

is that they vary in carbohydrate concentration. The objectives of

this investigation were to:1. Determine changes in concentration of starch and total sugar

in jojoba leaf and stem tissue during a season. .2. Determine whether individual jojoba shrubs differ in carbo-

hydrate concentration.

3. Determine whether carbohydrate concentration and rooting

ability of cuttings are related.

CHAPTER 2

liter a t u r e review

Cuttings from many woody plants produce roots when placed in

porous media with bottom heating and overhead misting. Rooting is often

stimulated by application of hormones to cutting bases. Although

optimum rooting conditions can enhance rooting, the condition of the shrub when cuttings are removed is also highly influential. , Rooting can be influenced by auxins and rooting cofactors, by age, by photoperiod

treatment, and by nutritional status of the shrub.

Rooting Factors

AuxinsAuxins are plant growth substances produced primarily in stem

tips. Cuttings from many broadleaf plants root well when taken just

following periods of active growth and flowering. The content and

activity of endogenous auxins at this period is thought to be the most important factor (Hartmann and Kester, 1975). The influence of auxins

on rooting of Populus nigra was studied by Nanda and Anand (1970).

They concluded that bud dormancy, and physiological factors associated with it, was responsible for rooting. They suggest that rooting was

poor in winter due to starch not being mobilized due to low bud and

auxin activity. They found that effectiveness of externally applied

auxin depended on level and activity of endogenous auxin.

Rooting Cofactors

In addition to auxins there are other naturally produced

substances called rooting cofactors that initiate rooting of cuttings. The identity and mode of action of many rooting cofactors is unknown.

One group of cofactors has been found to have a synergistic relation­

ship with indoleacetic acid which results in increased rooting of mung

bean cuttings (Hartmann and Kester, 1975). An investigation of the role of rooting cofactors in evergreen cuttings was done by Lanphear

and Meahl (1966). They extracted methanol-soluble materials from

evergreen cuttings and tested the rooting effectiveness by the mung bean bioassay. The stage of growth was found to be the most important

rooting factor. They suggest that active vegetative growth suppresses

rooting due to a limiting or demobilizing of certain substances

(rooting cofactors) needed by the cutting. Heuser and Hess (1972)

found that juvenile English Ivy contained three lipid-like substances

that greatly increased rooting of mung bean cuttings.

PhotoperiodThe photoperiod that a plant is grown under can influence the

subsequent rooting of cuttings. Long photoperiods Can cause increased

production and storage of carbohydrates that can favor greater rooting

(Hartmann and Kester, 1975). Cuttings from dogwood Shrubs grown under short photoperiods did not respond as well as cuttings taken from dog­

wood grown under long photoperiods (Waxman, 1957). An investigation

of photoperiod and temperature on juniper shrubs and cuttings was done

by Lanphear and Meahl (.1967).. They found that rooting was reduced if

the shrub had been exposed to a long photoperiod in addition to a chilling period. They concluded that rooting in certain evergreen

shrubs is favored by conditions of dormancy and inhibited by conditions

of active growth.

Age of Shrub

In many instances, cuttings taken from younger plants root

more easily than cuttings taken from older plants. This factor seems

to be especially evident in coniferous plants or other species that are difficult to root (Hartmann and Kester, 1975). Rooting of magnolia

cuttings was found to increase in difficulty with increasing age of the

plant (Perry and Vines, 1972). They suggest that a factor associated

with juvenility, produced in buds, was responsible for increased*rooting of younger magnolia cuttings.

Carbohydrates .Carbohydrates are important in rooting cuttings because they

provide the energy necessary for respiration and for production of

new tissue. In some cases, high carbohydrate concentration of cuttings

has been shown to increase rooting. Kraus and Kraybill (1918) found

that tomato cuttings high in carbohydrates rooted while cuttings low

in carbohydrates did not. All and Westwood (1966) found that exter­

nally applied auxin caused a mobilization of carbohydrates to the area

where roots formed. They concluded that specific amounts of carbo­

hydrates are needed as an energy source for cell differentiation and

. ' 6 root production. Experiments were done on effects of girdling on

rooting of two hibiscus varieties (Howard and Sykes» 1966).. Onevariety termed "easy-to-root" accumulated much more carbohydrate above

the girdled area than did the "difficult—to-root" variety. They sug­

gested that the carbohydrate accumulation.could account for greater

rooting of the "easy-to-rbot" Variety.

The correct balance of endogenous carbohydrates and auxins hasbeen suggested as influential in rooting. Nanda et al. (1971),

working with Populus nigra, determined that correct balance of carbo­

hydrates and regulatory substances in the cutting influenced the effectiveness of externally applied auxins. Rooting was inhibited

when 1.0 mg/1 auxin and 0.01% glucose were added to the cutting media.

However, when the same auxin concentration was added with a higher

glucose concentration (1.0%) rooting was stimulated. When 1.0% glucose

was added with no auxin rooting was again inhibited. Hansen and

Eriksen (1974) found that amount of light received by the stock plant could influence rooting of cuttings. Cuttings taken from high light-

treated plants had fewer roots than cuttings taken from lower light-

treated plants. They concluded that root initiation was inhibited due

to supraoptimal carbohydrate concentration that was not in balance

with endogenous auxin. Again working with light, Hansen et al. (1978)

found that auxin production and translocation, and carbohydrate concen­

tration are influenced by the amount of light. The rooting potential,

of the cutting can be influenced by the carbohydrate—auxin balance

that results.

• ' . ' - ' : ■ ' 7Other experiments show no relationship between carbohydrates

and increased rooting. Childers and Snyder (1957) found no relation­

ship between carbohydrate concentration and rooting of American Holly, .

They concluded that cafbohydrates were not the limiting factor in the

rooting of this plant.In some cases, high carbohydrate concentration can inhibit

rooting. Endogenous sugar levels in mustard cotyledons and its effect

on rooting was investigated by Lovell et al. (1974). They found

inhibitory effects of high sugar concentration on rooting.

The influence of auxin application on mobilization and accumu­

lation of carbohydrates was examined by Altman and Wareing (1975).

Their data supports the theory that basal auxin application causes a .

downward transport of carbohydrates. They found that in bean cuttings

the most important rooting factor was the movement of carbohydrates out

of the leaves. Greenwood and Berlyn.(1973), working with pine embryo

cuttings, found evidence of increased downward movement of sucrose by application of auxin. Breen and Muraoka (1973) investigated auxin

application and movement of carbohydrates in plum cuttings. They found

that treating cutting bases with auxin caused increased rooting as well

as downward movement of carbohydrates. The auxin-treated cuttings that

formed callus accumulated more carbohydrate than non-treated cuttings.

Most of the transported carbohydrates were sucrose, glucose, fructose,

and sorbitol.

. 8Seasonal Carbohydrate Cycle

Trees and ShrubsGenerally, shrubs produce and accumulate carbohydrates in leaves

and stems when vegetative growth is slow (fall) and assimilate carbo­

hydrates when vegetative growth is great (spring). The carbohydrate concentration (throughout the season is influenced primarily by the

stage of growth (Coyne and Cook, 1970).

In pecan, starch usually has two periods of high concentration and two periods of low concentration during a season. A low point is

reached in spring following vegetative growth and a low point is reached

in winter due to starch being converted to sugars (Worley, 1979).

Similarly, starch content of peach twigs was found to reach a high point

in fall and drop with approaching winter conditions (Dowler and King,

1966). The starch drop in winter was inversely related to sugar in­

crease. The total carbohydrate of peach twigs did not decline during

winter as is the case with many trees. The upward movement of materials

from the roots was offered as a possible explanation.

The seasonal total carbohydrate of big sagebrush was studied by

Coyne and Cook (1970). The highest concentration of carbohydrate was

reached in spring although fall also was a high period. Carbohydrate

continued to remain high in spring when other types of shrubs began to

show a carbohydrate decline. The continued high concentration could be

due to the evergreen nature of big Sagebrush. Production of new leaves

in spring is not necessary as it is in deciduous shrubs.

9Jojoba Shrubs

Little information is available on the carbohydrates, of jojoba,

A1 Ani et al„ (1972) examined both starch and sugar in leaf and stem

tissue but combined the data into total carbohydrate. They found the

highest carbohydrate content in spring when shrubs had the highest

metabolic activity. The total carbohydrate of shrubs sampled near Tucson was 14.6% in winter and 13.1% in summer. They found that carbo­

hydrate dropped quickly during summer and that the decline was in

relation to stem growth and fruit production.

Almeida (1979) investigated total carbohydrate of jojoba leaf

tissue through the year near Tucson. He also found carbohydrate to be

highest in spring (March) when photosynthesis was expected to be

greatest. Total carbohydrate reached a low in summer (July and August).

Almeida reported total carbohydrate of approximately 32.0% in winter

and approximately 16.0% in summer. He suggests that adverse summer

conditions (water stress, high temperatures) caused a decline in photo- .

synthesis and a corresponding decline in carbohydrate. A period of

vegetative growth occurred after each high point in carbohydrate was reached. Male and female shrubs had similar carbohydrate concentrations

through the year. , .

CHAPTER 3

MATERIALS AND-METHODS

Field Material

Stock Shrubs

At the Gascoyne Research Station in Western Australia were two

groups of jojoba shrubs (Figure 1). These shrubs were all started from

seed sent from Arizona. One group (A)„ contained 6 shrubs approximately

7 years of age. These shrubs were grown as a windbreak trial and had no

irrigation during their development. The stems were brittle and 'woody’

and internodes were short. The second group (B)„ contained 5 shrubs approximately 4 years of. age. This group had been irrigated and showed

vigprous growth, resilient stems, and long internodes.

Cutting and Sample Collection

At 4 times during the year (July, Sept., Nov., March) cuttings

and tissue samples were collected. From each shrub, 15 cuttings were

collected for propagation and 5 cuttings were collected for carbohy­

drate analysis. Each cutting was 15 cm in length and was taken from

stem tips. An attempt was made to select only 'semi-hardened' material

that showed new tip growthi The 15 cuttings were rinsed, rolled in

moist towelling, and placed in cool ice chests for transportation

to greenhouse facilities. The tissue samples were cooled on ice to

stop carbohydrate usage.

■ 10 .■

Figure 1. Stock jojoba shrubs at Gascoyne Research Station, Western Australia.

Propagation Conditions

Cutting PreparationPrior to sticking, flower buds and fruit were removed from the

cuttings and stem bases were recut below a node. The cuttings were

disinfected by a 1 min dip in 1% sodium hypochlorite solution.

Five randomly selected cuttings from each group were designated

as controls and had stem bases dipped (5 sec ) in distilled water.

Five additional cuttings were selected and were dipped (5 sec ) in a 4000 ppm indole-3-butyric acid solution. The remaining five cuttings

were dipped (5 sec ) in a 1000 ppm indole-3-butyric acid solution.

The cuttings from each shrub were stuck in plastic flats containing a

perlite-vermiculite medium (1:1 ratio).

Greenhouse FacilitiesA greenhouse at the University of Western Australia was used.

Flats of cuttings were placed on a bench and the entire bench enclosed

in a plastic tent to keep humidity high. During the cool months the

flats were warmed from beneath by a water bath (25°C). During the hot

summer months the plastic enclosure was air conditioned to preventotemperatures from rising above 25 C.

Watering was performed by an automatic misting system. A mist­

ing line was suspended above the cuttings and was activated once every

12 min for a duration of 8 sec.

Weekly observations were made to record leaf abscission. After

approximately 6 weeks in the greenhouse the cuttings were harvested and

roots of any length were recorded (Figure 2).

13

Figure 2. Jojoba stem cutting with roots.

14Carbohydrate Analysis

Preparation of SamplesTissue samples were taken from the ice and placed in a Dynavac

D20 vacuum desiccator for 1 week to completely dry the material.Leaves and stems were separated and ground to a fine powder in a high

speed mill. The powdered material was stored in capped glass vials.

Fresh and dry weight of jojoba leaf tissue is given in Appendix A.

Extraction of Ethanol-Soluble Sugars

The extraction procedure was similar to that used by Stoltz and

Hess (1966). A 0.1 g sample was placed in a 10 ml screw top glasstube. A 10 ml volume of 60°C ethanol (80%) was added. The tube was

capped, shaken, and then placed horizontally in a water bath (60°C) for

30 min. The tube was removed and contents centrifuged at 3000 rpm for

5 min. The supernatent containing sugars was poured into a 100 ml

flask. The extraction process was repeated twice for each sample to

remove all sugars. The final supernatent volume was 30 ml.

Extract Clearing ProcessThe supernatent had to be cleared of interfering compounds be­

fore the sugar content could be determined. Apparently, many substances

in addition to sugars were removed by the ethanol (Figure 3). The

clearing process was that used by . Smith (1969). To the 30 ml extract,

2 ml of a 10% neutral lead acetate solution was added. The flask was

filled to a volume of 50 ml with distilled water. The contents were

15

Figure 3. Ethanol-soluble extract of jojoba leaf tissue.

16swirled and centrifuged at 3000 rpm for 5 min. The clear extract

was poured into a flask containing 0.1 g potassium oxalate. Appar­

ently, the potassium oxalate neutralizes any excess lead acetate. The

solution was refrigerated overnight and then filtered through Whatman

#42 paper.

The final extract volume was at a ratio where 1 ml equaled 2 mg

of the original dried sample. Leaf dry weight before and after ethanol

extraction is given in Appendix B.

Anthrone Determination of Glucose Equivalent

The anthrone method of sugar determination was that described by

Yemm and Willis (1954). An anthrone solution (0.1 g anthrone in 100 ml

of 76% I^SO^) was mixed and allowed to stand until clear. A 5 ml vol­ume was pipetted into a 15 X 2.5 cm test tube and the tube placed on

ice. The anthrone reagent was allowed to cool (5-10 min ) and then

a 1 ml volume of the unknown extract or standard solution was slowly

pipetted into the tube. The tube was allowed to cool 5 min more,

then was shaken. The tube was capped with a marble and placed in a

boiling water bath for 12 min - After boiling, the tube was immediately

placed on ice for 5 min to stop the color development. The samples

were allowed to reach room temperature.

The light absorbance of the samples was measured using a Bausch

and Lomb Spectronic 100 with, a wavelength setting of 625 nm. . The absorbance values of all samples were then compared to known standard

glucose curves to determine the glucose equivalent (Appendix C).

17

Calculation for Sugar Percentage

After finding the glucose equivalent from the standard curve the following equation was used to calculate the sugar percentage in the 0.1 g sample (Silveira et al. 1978):

Sugar - (Absorb. Sample). (Wt. of Glucose Equjv.) ^ pQO(% D.W.) (Absorb. Glucose) (Sample Wt.)

Starch Extraction

The method of extraction of starch from the ethanol-extracted

residue was similar to the method of Silveira et al. (1978). The residue from sugar extraction had 5 ml of distilled water added, was

shaken, then centrifuged at 3000 rpm for 5 min. The supernatant was

then discarded to remove any remaining ethanol that might inhibit later

enzyme activity. Two ml of 0.02 H acetate buffer was added to the

residue. The tubes were loosely capped and then autoclaved for 10-15

min. The solutions were allowed to reach room temperature.

A 0.3% solution of Mylase (Hallerstein Co., Deerfield, 111.) was

added and the tubes tightly capped. The caps were sealed with a putty

since the tubes were layed horizontally in a water bath (39°C) for 24

hours. Upon removal from incubation, 4 ml of water was added to liber­

ate the glucose molecules. The mixture was transferred to a flask and

the volume brought to 100 ml with water. At this point, 1 ml of extract

was equivalent to 1 mg of the original sample weight. The mixture was

filtered and then analyzed by the enthrone process as in the sugar

determination.

18Calculation of Starch Percentage

The absorbance readings of the unknown solutions were multi­

plied by 0.9 to correct for the hydrolysis of starch to glucose by the

enzyme (Pasternack and Danbury, 1970). The starch percentage in the

original 0.1 g sample was calculated as follows (Silveira et al. 1978):

Starch - (Absorb. Sample) (0.9) (Glucose Equiv. Wfr.) % iqO (% D.W.) (Absorb. Glucose Equiv.) (Sample Wt.)

CHAPTER 4

RESULTS AND DISCUSSION

Carbohydrate Concentration

Seasonal Carbohydrate Change

Sugar and starch concentrations of shrubs in both A and B

groups varied significantly depending on time of year (Table 1). Total

carbohydrate of shrubs reached its highest level in spring and its

lowest level in summer. The drop in total carbohydrate occurred when

vegetative growth and flowering were greatest. This pattern is similar

to the findings of A1 Ani et al. .(1972) and Almeida (1979).

Sugar concentration in leaves of both groups-of shrubs dropped

significantly after spring and then remained at a fairly constant level throughout the summer. However, stem sugar concentration of group A

shrubs continued to decline throughout the summer (Figures 4 and 5).

Leaf starch concentration increased sharply in spring, then

dropped quickly by summer to low levels. Stem starch concentration

showed a similar trend, but did not drop to such low levels. During

summer, an inverse relationship existed with leaf starch less than leaf

sugar, and stem starch greater than stem sugar (Figures 4 and 5). Stems

were comparatively high in carbohydrates and could be a site of carbo-

hydrate storage as they are in big sagebrush, another broadleaf ever­

green of the southwest (Coyne and Cook, 1970)i

: 19

20Table 1. Carbohydrate concentration of leaves and stems of

groups at 4 times of the year.2 jojoba

Shrub sw Sample Date Component Mean (%)

Leaf Sugar .A July 6.1 az

Shrubs September 4.9 bNovember 4.5 bMarch 4.6 b

V September 5.6 aShrubs November 4.8 b

March 4.6 bLeaf Starch

A July 3.2 aShrubs September 4.9 a

November 4.5 aMarch 0.7 b

B September 7.2 aShrubs November 6.7 a

March 1.5 bStem Sugar

A July 4.4 aShrubs September 4.0 b

November 3.0 cMarch 2.3 d

B September . 4.2 aShrubs November 2.6 b

March 2.1 bStem Starch

A July 8.9 aShrubs September 9.4 a1‘ - November 7.8 b

March 5.2 c

B September 5.5 aShrubs November 6.6 a

March 3.8 b

WA shrubs approximately 7 years old, B shrubs approximately 4 years did, Xluly data not available for all B shrubs,zMean separation by Duncan’s Multiple Range test, 0.05 level.

CHO

(%)

CHO

(%)21

10.0Leaf Tissue

Sugar

Starch

12.0Stem Tissue

10.0 Starch

Sugar

M J J A S O N D J F M A

MonthFigure 4. Seasonal change in sugar and starch concen­

tration in stems and leaves of group A.

CHO

(%)

CHO

(%)

22

10.0 -

8.0 -

6.0 -■

4.0

2.0 --

12.0

10.0 -

8.0 -

6.0 •-

4.0 -

2.0 -

Figure 5

Leaf TissueStarch

Sugar

Stem Tissue

Starch

Sugar

J J A S O N D J F M A

MonthSeasonal change in sugar and starch concen­tration of stems and leaves in group B

23Plants of groups A and B were similar in sugar concentration

throughout the year. However, leaf starch was higher in shrubs of

group B than in shrubs of group A, while stem starch was higher in

shrubs of group A. The inverse starch relationship between the groups

could be due to differences in growth. Plants of group B were growing more vigorously and could have been photosynthesizing faster than plants

of group A, leading to starch accumulation in the leaves.

Individual Variation in ConcentrationBecause plants of groups A and B were physically different,

statistical analysis of variance was performed on each separately.

Carbohydrate values for each shrub, for the entire year, were compared

to determine whether individual shrubs differed. Within both groups,

most shrubs were similar in carbohydrate concentration. However, a few shrubs had starch or sugar concentrations that were significantly

different from the others in their group. Analysis of variance revealed

differences in stem sugar concentration of individual shrubs of group A

(Tables 2 and 3). Variance was also found in stem starch of group A

shrubs (Tables 4 and 5). In group B, variance was. found in leaf starch

concentration (Table 6). Mean separation revealed that only shrub B 2

was significantly different (higher), in leaf starch than the other

shrubs of the group (Table 7).The greater variability of carbohydrate levels of shrubs in

group A was possibly due to sample material being older and less suit­

able for analysis. Plants of group B appeared more uniform in growth .

Table 2o ANOVA of stem sugar concentration of A shrubs over 4 harvest dates o

Sourcew S. S. D.F. M.S. F. Sig.

Main 20.19 8 ' 2.52 44.98 .001Season 16.90 3 5.63 100.41 .001yIndividual 3.28 5 .65 11.72 .001yResidual 00 15 .05

Total 21.03 23 .91

^Analyzed on Randomized Complete Block design« ^Within OoOl significance level.

Table 3. Stem sugar concentration of over 4 harvest dates.

individual A shrubs averaged

Shrub Stem Sugar (.%)

A 3 4.05 azA 4 3.80 abA 6 3.58 bA 2 3.18 cA 1 3.15 c

. A 5 3.05 G

ZMean separation by Duncan?s Multiple Range test, 0.05 level.

25Table 4. ANOVA of

dates.stem starch concentration of A shrubs over 4 harvest

SourceW S. S. D.F. M.S. F. Sig.

Main 76.12 8 9.51 20.85 .001Season 61.81 3 20.60 45.15 .001'Individual 14.30 5 2.86 6.27 .002'Residual 6.84 15 .45Total 82.96 23 ' 3.60

^Analyzed on Randomized Complete Block design. ^Within 0.01 significance level.

Table 5. Stem starch concentration of over 4 harvest dates.

individual A shrubs averaged

Shrub Stem Starch (%1

A 4 8.53 azA 3 8.50 aA 6 8.28 aA 1 8.25 abA 5 7.28 beA 2 6.43 c

Z ’Mean separation by Duncanrs Multiple Range test, 0.05 level.

26Table 6. ANOVA of leaf starch, concentration of B shrubs over 4 harvest

dates.

„ w Source S. S. D . F. M.S. ■ F. sig.

Main 127.95 6 21.32 37.27 .001Season 99.42 2 49.71 86.88 .001?Individual 28.52 4 7.13 12.46 . .002?Residual. 4.5.7 8 .57Total 132.53 . 14 9,46

^Analyzed on Randomized Complete Block design. ^Within 0.01 Significance level.

Table 7. Leaf starch concentration of individual B shrubs averaged over 4 harvest dates.

Shrub Leaf Starch (%).

B 2 7.83 ZaB 1 5.10 bB 6 4.50 bB 8 4.40 bB10 4.00 b

Mean separation by Duncan’s Multiple Range test, 0.05 level.

27and development and could have supplied more consistent data. Carbohy­drate values for individual shrubs for each sampling period is given in Appendix D.

Location of Carbohydrates

An entire shrub was harvested in Western Australia on July,

1979 and divided into various components (leaves, stems, roots) for

sugar and starch analysis,(Table 8). Generally, sugar concentration in leaves was highest at the apex and lowest at the stem base. Leaf starch

did not show a change in concentration with location on the shrub.

Stem tissue was analyzed as a combined sample and was similar to leaves

in sugar concentration but was lower in starch. The root sample was

similar to leaves in sugar and in starch.

Sugar may have been higher in younger leaves because they are more active and act as a sink for photosynthate. Roots were compara­

tively high in starch, perhaps reflecting their role as a storage site

for carbohydrates during winter.

Carbohydrate Change in Cuttings

Leaf carbohydrates were analyzed on cuttings that had been under

propagating conditions for 6 weeks and had not rooted. Sugar and starch

concentration of most cuttings had increased over the initial levels

(Table 9). In some cases, carbohydrate concentration after 6 weeks was

more than doubled the original values.Apparently, cuttings continue photosynthesis while under prop­

agating conditions and accumulate more carbohydrate than is used for

28Table 8. Carbohydrate

jojoba shrubconcentration of component parts of an (23 cm height) harvested on July, 1979,

entire

Plant Part Sugar (%) Starch (%)

Leaves, Node lw 6.1 4.6

2 5.5 5.3ft 3 5.6 7.3ff 4 5.6 5.6If 5 5.1 5.4If 6 4.7 4.7

7 5.2 1.7II 8 5.5 5.3II 9 4.8 3.6

Stem 5.1 2.2

Root 5.3 5.5 ■

wNodes numbered from apex to base.

29Table 9. Carbohydrate concentration of leaves of cuttings before and

after 6 weeks under propagating conditions.

Sugar (%) Starch (%)

Shrub Initial Final Change (%) Initial Final Change (%)

A 1 4.2 6.8 + 62 8.5 13.1 + 54A 2 5.0 5.7 + 14 3.7 7.5 +103

A 3 4.6 7.2 + 57 2.9 5.9 +103A 4 5.6 6.3 + 13 5.1 A A

A 5 4.8 3.5 - 27 3.2 4.9 + 53

A 6 5.2 6,1 + 17 6.2 4.9 - 21

B 1 5.2 6.1 + 17 6.4 7.3 ' + 14

B 2 5.0 8.1 + 62 10.4 8.0 - 23

B 6 6.6 14.0 +112 7.1 . A A

B 8 6.0 6.1 . + 2 5.2 12.9 +148

BIO .5.5 6.0 + 9 7.2 7.7 + 7

"Material unavailable for analysis.

. 30respiration. Since these non-rooted cuttings contained high carbohy­

drate concentrations, carbohydrate level was apparently not the limiting factor for rooting.

Rooting Results

Root Numbers and Leaf Abscission

For each sampling period, the number of roots produced by each

cutting was recorded (Tables 10 through 13). Due to low rooting re­

sponse of all cuttings, root numbers for control and the two auxin-

treatments were combined to give total root numbers per shrub.

In every sampling period, cuttings of group B shrubs produced

more roots than cuttings of group A shr,ubs. The condition and age of

cutting material probably influenced rooting more than any other factor.

No cuttings from the March sample produced roots. Cuttings from this

period appeared pale and desiccated when removed from shrubs and all

died before .6 weeks under propagating conditions. Rooting of cuttings

from individual shrubs varied from one sampling period to the next. Cuttings from shrub B 8 produced 111 roots during September but produced

0 roots during November.Considerably more leaves abscised from cuttings of group A

during propagating than from cuttings of group B,(Tables 10 through 13).

In this study, high leaf abscission from cuttings of group A was prob­

ably due to the poor quality of the cutting material. Between shrubs,

the relative amount of abscission appeared to be consistent throughout

the year.

31Table 10. Rooting and

1979 from 2leaf abscission of cuttings collected in groups of jojoba shrubs.

July

Shrubs No. of Roots No. of Leaves Abscised

A 1 7 82A 2 0 71A 3 48 48A 4 0 66A 5 0 123A 6 0 117

„ *B 1B 2B 6B 8BIO

'VB shrubs not sampled.

Table 11. Rooting and leaf abscission of cuttings collected in Septem­ber 1979 from 2 groups of jojoba shrubs.

Shrubs No. of Roots No. of Leaves Abscised

A 1 0 86A 2 0 62

• A 3 0 34A 4 0 29A 5 0 118A 6 ■ 3 124

B 1 2 7B 2 4 . 24B 6 26 1 12B 8 111 0BIO 45 : 0

32Table 12.. Rooting and leaf abscission of cuttings collected in Novem­

ber, 1979 from 2 groups of jojoba shrubs.

Shrub No. of Roots No. of Leaves Abscised

A 1 0 66A 2 0 68A 3 O ' . 27A 4 0 84A 5 0 82A 6 14 110

. B 1 18 1B 2 . 13 ' 5B 6 0 6B 8 o 15BIO 25 6

Table 13. Rooting and leaf abscission of cuttings, collected in March, 1980 from 2 groups of jojoba shrubs.

Shrub No. of Roots No. of Leaves Abscised

A 1 0 154A 2 0 98A 3 0 91A 4 0 13A 5 0 30A 6 0 128

B 1 0 0B 2 0 ' 0B 6 0 0B 8 0 16BIO 0 0

33Carbohydrates and Rooting

Regression analysis was used to detect any significant relation­

ship between carbohydrate concentration and root numbers. In no case did carbohydrate concentration of an individual shrub correlate with root numbers of cuttings taken from that shrub (Table 14).. Those shrubs highest in sugars or starch did not show any increase in root numbers on

cuttings. Conversely? cuttings from shrubs relatively low in carbohy­

drates did not show any less production of roots.Generally, carbohydrates and rooting were highest in spring

(September sample) and lowest in summer (March sample). However, this

trend did not apply to individual shrubs.

34Table 14. Regression analysis of root, numbers and corresponding carbo­

hydrate values of B shrubs for all sampling periods.

VariablesCorrelationCoefficient Significance

Roots by Total Leaf CH0W 0.304 nsz '

Roots by Total Stem CH0W 0.273 ns

Roots by Leaf Sugar 0.579 ns

Roots by Leaf Starch 0.210 ns

Roots by Stem Sugar 0.272 nsRoots by Stem Starch 0.180 ns

wSum of sugar and starch concentrations.^Determined on 14 degrees of freedom at 0.01 level.

CHAPTER 5

SUMMARY AND CONCLUSION

Total carbohydrates in jojoba shrubs in Western Australia peaked

in spring and dropped in summer. Starch concentration fluctuated more than sugar concentration. It was primarily a decrease in starch that caused reduced total carbohydrate in summer. A possible reason why

sugar concentration remained constant through summer is that starch

was being converted to sugars. As suggested by Almeida (1979)9 summer

conditions could be inhibiting photosynthesis and preventing a total

carbohydrate increase.

Cuttings from older A shrubs produced few roofs regardless of

sampling period. The ’woody’ condition of the cuttings could have

caused poor rooting and excessive leaf abscission. Cuttings taken

during mid summer failed to produce roots. Cuttings from younger B

shrubs during spring produced more foots than cuttings taken in the

summer. There was no consistency in rooting ability of specific shrubs

from one sampling period to the next.Analysis of various portions of a jojoba shrub revealed highest

leaf sugar and starch concentrations were found closest to stem tips.

The roots also contained relatively high sugar and starch concentration.

Cuttings in propagating beds apparently continue to photosyn­

thesize and accumulate carbohydrates. High levels of sugar and starch

were found in cuttings that had not rooted after 6 weeks in the

' ■ ■ , 35 .

• 36greenhouse. These cuttings apparently required some other factor than

carbohydrates for rooting since carbohydrates were not limiting.

Variation in carbohydrate concentration was greatest in stem

tissue of the A group. The greater age and 'woody* condition of the

tissue could have caused the variation.

Statistical correlation could not be found between carbohydrate concentration and rooting. Individual shrubs, such as B 2 could be

quite high in carbohydrates and yet not root better than other shrubs

of lower carbohydrate levels.Jojoba cuttings are probably influenced by a number of inter­

related factors. Carbohydrates are probably an important factor but not

the most influential one. A project investigating interaction of carbo­

hydrates and auxins might reveal rooting correlations.The effectiveness of this study in investigating carbohydrate-

rooting correlation was hampered by lack of cutting material. Possibly,

an expanded study using more replication could reveal a correlation.

. APPENDIX A

JOJOBA LEAF FRESH WEIGHT AND DRY WEIGHT

Table A.I. Fresh and dry weights of jojoba leaves from the 1st node (apex) to the 6th node.

Leaves Fresh Weight (g) Dry Weight (g) . Dry Weight (%)

1* 0.009 0.003 36.72 0.014 0.005 36.83 . 0.115 0.038 33.04 0.105 0.035 33.3

5 0.111 0.039 34.86 0.107 0.038 35.2

. 7 0.151 0.056 36.9:8 0.106 0.040 37.7

9 0.228 0.085 37.0 :10 0.278 0.103 36.9

11 0.403 0.142 35.112 0.269 0.098 36.7

*Each node contains”2 leaves

37

APPENDIX B

JOJOBA LEAF DRY WEIGHT AFTER EXTRACTION WITH ETHANOL

Table B.l. Percentage dry weight remaining after 3 extractions with ethanol on 5 replications.

Sample Before Extraction (g) After Extraction (g) Remaining (%)

1 0.1 0.056 56.02 0.1 ' 0.056 56.03 0.1 0.055 55.04 0.1 0.055 55.0

5 . 0.1 0.054 54.0

38

Absorbance at

625 nm

APPENDIX C

GLUCOSE STANDARD CURVE

0.9

0.4

250200150100

Glucose (micro gm/ml)

39

APPENDIX D

RAW CARBOHYDRATE DATA

Table D.l. Sugar concentration (%) of A and B shrubs for all sampling periods.

Sample Period

Shrub Tissue July . Sept. Nov. March

A 1 Leaf : 5.5 4.2 4.1 5.3Stem 4.0 4.0 2.7 1.9

A 2 Leaf 6.2 5.0 5.4 5.1Stem 4.1 . 3.7 2.8 2.1

A 3 Leaf 5.2 4.6 4.3 4.7. Stem 5.0 5.0 3.5 2,7

A 4 Leaf 7.8 5.6 4.9 4.6Stem 5.2 4.0 3.4 2.6

A 5 Leaf 6.1 4.8 4.5. 4.2Stem 3.8 3.6 2.8 2.0

A 6 Leaf 6.0 . 5.2 4.2 4.1Stem 4.7 3.9 3.1 2.6

B 1 Leaf * 5.2 4.8 4.7Stem 5.0 2.1 2.0

B 2 Leaf * 5.0 4.2 5.0Stem 4.5 2.7 2.3

B 6 Leaf . A 6.6 5.0 4.2Stem 4.3 2.9 2.0

B 8 Leaf A 6.0 5.0 4.6Stem 3.4 2.7 2.2

BIO Leaf A 5.5 4.7 4.6Stem 4.2 2.8 2.0

"kB shrubs not sampled.

40

41.Table D.2. Starch

periodsconcentration (%) of A and B shrubs for all sampling

Shrub . Tissue

Sampling Period

July Sept. Nov. March

A 1 Leaf 3.8 8.5 7.2 0.9Stem 4.6 6.5 5.5 3.8

A 2 Leaf 3.2 3.7 5.3 0.7Stem 4.1 4.7 3.1 1.1

A 3 Leaf 2.1 2.9 4.9 0.8Stem 4.9 4.8 5.2 2.9

A 4 Leaf 3.7 5.1 4.8 0.7Stem 4.2 5.9 5. 7 2.5

A 5 Leaf . 3.6 . 3,2 3.2 1.0 .Stem 4.6 4.0 4.3 4.0

A 6 Leaf 3.1 6.2 1.9 0.2Stem 4.5 6.5 4.9 2.9

B 1 Leaf * 6.4 7.6 1.3Stem 5.3 7.7 4.1

B 2 Leaf * 10.4 9.1 4.0Stem 5.6 6.6 6.1

B 6 Leaf * 7.1 ' 5.5 : 0.9Stem 5.6 6.8 • 3.3

B 8 Leaf * 5.2 6.0 0.8Stem 5.5 6.3 2.3

BIO Leaf * 7.2 5.3 0.7Stem 5.6 5.6 3.2

B shrubs not sampled.

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