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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