This is to certify that the

thesis entitled

RESPONSE OF PLANT TISSUES AND RICE SEEDLINGS
TO TRIACONTANOL

presented by

Roger Paul Hangarter

has been accepted towards fulfillment
of the requirements for

M.S. Horticulture

degree in

/.
CF\' /,//’ "/7.
5 CAL“) x/é); K’ . / “(CC/’3

Major professor

 

 

(

 

Date //7//0/??

0-7639

 

 

RESPONSE OF PLANT TISSUES AND RICE SEEDLINGS

TO TRIACONTANOL

By

Roger Paul Hangarter

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Horticulture

1977

ABSTRACT
RESPONSE OF PLANT TISSUES AND RICE SEEDLINGS

TO TRIACONTANOL
By

Roger Paul Hangarter

Triacontanol, a straight—chain, saturated, 30 carbon alcohol
[ CH3(CH2)28CH20H ] has been shown to increase plant growth both in the
light and dark. Triacontanol had no effect on membrane permeability as

measured by the leakage of betacyanin from red beet (Beta vulgaris L.)

 

root tissue.

Studies with haploid tobacco (Nicotiana tabacum L.) cell cultures

 

established that triacontanol increased the fresh weight, dry weight,
and protein on a per tissue basis. The increase in growth was due to a
gain in structural material of the cells. Increased 02 uptake accompan-
ied the growth increase. Another long chain alcohol, octocosanol

[ CH3(CH2)26CH20H ], did not increase growth. Coefficients of variation
of 26 to 38% made tests with tobacco tissue unsuitable for further
studies.

A system employing isolated leaf segments of rice (Oryza sativa L.)

 

proved to be an excellent bioassay for triacontanol responses. Isolated
leaf segments treated with triacontanol increased in dry weight compared
with both controls and segments harvested at zero time. The response
was measured in less than 6 hr in the light and in the dark when sucrose
was added. Leaf segment experiments had coefficients of variation
around 2 to 3%.

Other growth regulators were examined for a dark response similar

Roger Paul Hangarter

to the response seen with triacontanol. Kinetin and gibberellin treated
plants were not different from controls after a 6 hr dark period while
triacontanol and indole-3—acetic acid treated plants increased in dry
weight. Rice leaf segments showed similar responses to these compounds

after 24 hr.

ACKNOWLEDGMENTS

Special thanks to Dr. Stan Ries, my major professor, for guidance
and encouragment. Thanks also to Dr. Dave Dilley and Dr. Peter Carlson
for their assistance. I also wish to thank Violet Wert and Brenda Floyd
for their aid and inspiration.

I would like to thank the other faculty and fellow graduate students

for their friendship and help.

ii

LIST OF TABLES . . . .

LIST OF FIGURES . . .

INTRODUCTION . . . .

LITERATURE REVIEW .

MATERIALS AND METHODS

RESULTS AND DISCUSSION .

SUMMARY .

LITERATURE CITED

TABLE OF

iii

CONTENTS

Page

12

LIST OF TABLES

Table Page
1. Leakage of betacyanin from red beet root tissue . . . . . . 12

2. Influence of triacontanol on growth and respiration
of tobacco callus . . . . . . . . . . . . . . . . . . . . . 14

3. Growth response of tobacco callus . . . . . . . . . .

4. Response of rice seedlings and leaf segments to
triacontanol, 1AA, 6A3, and kinetin . . . . . . . . . . . . 25

iv

LIST OF FIGURES

Figure Page

1. Response of tobacco callus to triacontanol in the

light and dark . . . . . . . . . . . . . . . . . . . . . . 15
2. Rapid response of tobacco callus to triacontanol . . . . . 16

3. Fresh and dry weights of triacontanol-treated

tobacco callus . . . . . . . . . . . . . . . . . . . . . . l7

4. Growth of tobacco callus treated with triacontanol

and octocosanol . . . . . . . . . . . . . . . . . . . . . l9

5. Influence of stage of growth of tobacco callus on

triacontanol response . . . . . . . . . . . . . . . . . . 20

6. Dose-response curve for leaf segments treated with

triacontanol . . . . . . . . . . . . . . . . . . . . . . 22

7. Change in dry weight of leaf segments treated with
triacontanol (10 ug/l) in the light and dark . . . . . . . 23

8. Change in dry weight of leaf segments treated with
triacontanol (10 Ug/l) with and without sucrose . . . . . 24

INTRODUCTION

Triacontanol, a component of the leaf wax of many plant species
(10,20,23), has been shown to stimulate dry weight accumulation in
several crops (31,32). The most interesting aspect of this dry weight
accumulation is that it occurs in the light as well as in the dark
where untreated plants lose dry weight (31). Several other compounds
have been shown to stimulate the growth of whole plants (16, 30, 46, 47),
but a search of the literature turned up no reports showing a
stimulation of dry weight accumulation during a dark period from
exogenous chemical treatment. These studies were undertaken to find
an unsophisticated experimental system for studying triacontanol
responses and to test other growth regulators for growth stimulating

activity in the dark.

LITERATURE REVIEW

Discovery of Triacontanol.

 

Triacontanol was first identified by Chibnall et al. in 1933 (7)

as the principle component of the wax of alfalfa (Medicago sativa L.)

 

leaves. It is a straight-chain, 30 carbon, saturated alcohol
[ CH3(CH2)28CH20H ]. Despite this early identification the growth
promoting properties of this compound were not discovered until
recently.

The rapid increases in the price of petroleum and natural gas
in the early 1970's resulted in increased chemical fertilizer prices
and inspired a study, in the summer of 1975, to investigate the use
of plant materials as alternative sources of fertilizers (29). These
studies led to the finding that coarsely chopped alfalfa hay applied
as a band application increased growth and yield of cucumbers (Cucumis

sativus L.), lettuce (Latuca sativa L.), tomatoes (Lycqperscion

 

 

esculentum L.), and wheat (Triticum aestivum L.). Several other crops

 

 

accumulated dry weight more rapidly following applications of small
amounts of alfalfa under greenhouse and growth chamber conditions.

It was hypothesized that the increase in growth and yield of
crops treated with low rates of chopped alfalfa was due to nitrogen
derived from the alfalfa upon decomposition (29). However, this
hypothesis was proved incorrect by further studies (32). A crystalline

substance, identified as l-triacontanol, was isolated from alfalfa

3

and was shown to increase the growth of rice (Oryza sativa L.)

 

seedlings grown in nutrient culture and the growth of corn (Zea mays L.),

barley (Hordeum vulgare L.), and tomatoes in soil (32). The response

 

of rice and tomatoes to synthetic and natural triacontanol was similar.

Triacontanol in Nature.

 

The cuticle of plants consists of a meshwork of polymerized
hydroxy fatty acids, called cutin, embedded in a complex mixture of
lipids, often in crystalline form, called plant waxes. The most
common wax components are hydrocarbons, esters, free fatty alcohols,
and acids. Ketones, secondary alcohols, diols, aldehydes, terpenes,
and flavones are also found (18). Triacontanol occurs in different
quantities in the wax of many plant species (10,20,23).

It is generally believed that wax components of the leaf are
excreted to the leaf surface as rapidly as they are synthesized, and
reabsorbtion of such water-insoluble compounds is unlikely (l9). Wax
components are thought to be end-products of metabolism insulated
from the regular metabolic functions of the plant (11). However,
Kolatukudy (18) has shown that some wax may be catabolized to a
limited extent. Occasionally, compounds considered to be cuticular
components are found in organelles such as chloroplasts (14). Moreover,
Chibnall (8.9) stated that a group of non-cuticular leaf waxes exist
which are not cuticle excretions, but constitute an integral part of
the general fat phase of leaf cells. The long chain primary components
. of the 'inside wax' include C30 compounds.

The generally recognized function of leaf wax is to maintain
the water balance of the plant. Other functions ascribed to the wax

include the prevention of loss of plant components by leaching, protection

4
from mechanical damage, excessive ultraviolet radiation, fungi, and

insects (11,23). The growth promoting effects of triacontanol (31,32)
and the possible occurence of such compounds in plant cells (8,9,14)
suggests that some components of leaf wax may also serve an active role
in growth regulating activities.

The Triacontanol Response.

 

Both foliar and root applications of low concentrations (2.3 X lO-BM)
of triacontanol increased dry weight accumulation of several species of
plants (32). Rice seedlings treated with triacontanol increase in dry
weight and leaf area within 3 hr of treatment (31). The response is
observed in the light as well as in the dark where control plants lose
and triacontanol-treated plants gain in dry weight. Triacontanol—
treated plants synthesize more protein during the dark period.

The dark response is affected by C02 and 02 concentrations (3).
Treated plants show increased dry weight in the dark only in the
presence of CO2 in the range of 200 to 400 ppm. Respiration is
increased in treated plants. The largest growth response attributable
to triacontanol was in a 52 O2 atmosphere.

Growth Stimulation of Whole Plants by_0ther Growth Regulators.

 

 

 

Grass treated with gibberellic acid is stimulated in early spring
before vigorous growth normally occurs (28,45). Two or three times
more forage than usually obtained from untreated grass is possible during
the early part of the season when growth of grass is usually slow.

'Kentuckey' blue grass (Poa pratensis) sprayed with gibberellic acid in

 

the fall when growth is unfavorable, brought about a significant
increase in both fresh and dry weights of plants (21). The increase
was largest when fertilizers were used with the gibberellic acid. In

Australia, winter application of gibberellic acid to pastures planted

5
with a mixture of Phalaris, annual grasses, and subterranean clover
showed a six-fold increase in growth (2).

Auxins at low concentrations have been shown to increase the size
and yield of several plants if applied at the proper stage of plant
growth. Huffaker (16) showed that the isopropyl ester of 2,4-D as a
spray, increased the yields of barley and wheat when the plants were
treated at the S to 7 leaf stage. The protein content of wheat was

increased by treatment. Application of 2,4-D to the foliage of

 

 

potato (Solanum tuberosum L.), pea (Pisum sativum L.), bean (Phaseolus

 

vulgaris L.), sugar beet (Beta vulgaris L.), maize, and other crops
resulted in an increase in foliage of 8 to 21% (47). The yield and
protein content of several crops were increased by application of
sublethal concentrations of simazine (30). Increased protein content
and/or yield of forage crops from setriazine applications have been
reported (1,17).

'Hormonal' Effects gf_Some Aliphatic Compounds.

 

 

Long chain fatty alcohols, isolated from Maryland Mammoth Tobacco

(Nicotiana tabacum L.) were shown to have a growth promoting activity in

 

the oat first-internode assay (44). The brassins, of unknown structure,
stimulate elongation of both second and third internodes of intact plants
in the bean second internode test (27). Cucumber hypocotyl elongation

can be stimulated by dihydroconiferyl alcohol (33). Growth promoting
effects of other alcohols have been reported to occur on excised wheat
roots (13). Certain synthetic aliphatic hydroxy-carboxylic acids show
promoting activities on the root growth of lettuce seedlings (24). Patents
have been obtained for the use of short chain alcohols as plant growth
stimulators (25,26).

Many long—chain carbon compounds are capable of promoting growth of

6

pea epicotyl sections (35,36,37,38,39). In this system there is an
interaction of these compounds with auxin and gibberellin action. Fatty
acid esters, alkyl lipids, natural oils, isoprenoid vitamins, alkyl
acetylenes, and insect juvenile hormones are among the compounds active
in this system. In an effort to get at the mode of action of these
compounds, Stowe and Dotts (39) found that molecular length is the most
important characteristic in common among the many compounds tested.
Their studies showed that the Optimum length is about 28 to 30 A
(approximate chain length of 20 to 22 carbon atoms) and postulated that
compounds of this length or longer are active by forcing apart membrane
lipid molecules, changing the charge distribution or chelating properties
of regulatory membranes.

Some naturally occuring aliphatic compounds possess growth inhibit-
ing activities. A C44 long—chain saturated keto-alcohol is an active
inhibitor of abnormal cell proliferation (40). Some fatty alcohols,
especially those with chain lengths of 9, 10, and 11 carbon atoms, are
active inhibitors of tobacco axillary and terminal bud growth (34).
Another compound, l—acetoxy-Z,4-dihydroxy-n-heptadeca-l6-ene, was
isolated from avocado (Persia spp.). This compound inhibits soybean
callus growth and elongation of wheat coleoptiles.

Clearly, triacontanol is one of many natural and synthetic compounds
which have been shown to affect plant growth. Numerous plant and insect
extracts have given growth promoting or inhibiting effects in a variety
of bioassays; the list of growth regulators which have been isolated
includes long-chain aliphatic compounds similar in many respects to
triacontanol. These regulators have been tested primarily in systems
using plant sections. Growth was measured by the parameters of elong-

ation, root growth or cell division. These studies may bear some

relevance to the work being done with triacontanol, particularly

in respect to properties associated with carbon chain length. The
investigation of triacontanol, however, stands out as an example of
the utilization of whole plants as a bioassay for growth stimulation.
Thus, laboratory studies may also be relevant in determing the

applicability of triacontanol for improving crop productivity.

MATERIALS AND METHODS

Treatment Application. The low solubility of triacontanol and octo—

 

cosanol in water required the development of the following procedure.
Gibberellic acid (GAB), indole-3-acetic acid (IAA), and kinetin were
handled in a similar manner.

Stock solutions of the compounds were prepared by dissolving a
measured quantity of the appropriate compound in a suitable solvent.
Triacontanol and octocosanol were dissolved in benzene for the studies
on membrane permeability and tissue culture and in chloroform for the
rice seedling and leaf segment studies. Ethanol was used for GA3 and
IAA. KOH (1N) was used for kinetin. Dilutions were prepared from the
stock solutions. All solutions were kept refrigerated and tightly
capped.

Aliquots were applied to a piece of filter paper and the solvent
was evaporated. Treatments were applied using this 'carrier' as
described for each system. In each experiment all pieces of filter
paper, including the controls, received equal volumes of the solvents
used.

Membrane Permeability. Leakage of betacyanin from red beet (Beta vulgaris

 

 

L.) root tissue was used as an assay for the effect of triacontanol on
membrane permeability. The technique was modified from Veldstra and

Boojj (43).

Beets were obtained from a local supermarket. Cylinders, 4.0 mm
in diameter, were removed from fresh beet roots with a cork borer and cut
into sections (5.0 mm) with razor blades. The sections were rinsed with
distilled water until all pigment from disrupted cells had been removed.
Sections containing large amounts of vascular tissue or those which were
exceedingly colored were removed prior to initiation of the experiment.
Ten root sections were placed in a 50 ml Erlenmeyer flask containing 20
ml of solution of the compound being tested.

Treatments were prepared by placing a piece of filter paper (1 cm2)
with the appropriate amount of the compound being tested, into each
flask containing 20 ml of distilled water with 20 ppm of penicillin-C
and allowed to sit overnight. The flasks were stoppered with a porous
plug after addition of the segments and placed in a water bath at 30 C.

Aliquots (3.0 ml) were removed from each flask at 2, 4 and 24 hr
and absorbance of the solution measured at 540 nm with a Beckman DB-G
Spectrophotometer using distilled water with 20 ppm penicillin-G as a
blank. Immediately after removing the 3.0 ml aliquot for assay, 3.0
ml fresh solution was added to the flask.

A randomized complete block design split for time was used. Four
blocks were used in all experiments. Each block consisted of tissue from
one beet root to remove the variation between roots.

Tobacco Callus. Cell cultures were grown in plastic disposable petri

 

dishes (100x15) on the basic medium of mineral salts described by

Linsmaier and Skoog (22). Additions to the basal medium were, thiamine
(1.0 mg/l), inositol (100 mg/l), IAA (3.0 mg/l), kinetin (0.3 mg/l),
sucrose (3%), and agar (1%). Cell cultures remained in an undifferentiated

state on this medium. Callus was produced from pith of haploid tobacco

10

(Nicotiana tabaccum cv. Wisconsin 38) and subcultures were maintained

 

in the dark.

Aliquots (lOOul) of triacontanol or octocosanol were applied to
Whatman #1 filter paper disks (diameter 4.5 cm); controls received 100 pl
of glass-distilled benzene. The solvent was evaporated for approximately
15 min, then the papers were placed on the agar medium. Callus tissue
was broken into pieces of approximately the same size and one piece was
placed on the filter paper in each dish.

Fresh and dry weights were measured after 10 to 15 days for most
experiments. For short-term experiments the initial fresh weight was
measured and subtracted from the weights measured after 14, 24, and
36 hr. Total nitrogen was determined by the automated micro~Kje1dahl
procedure of Ferrari (12), and converted to protein using the conversion
factor of 6.25. Respiration was measured with a Gilson Differential
Respirometer according to procedures of Umbreit et a1 (42). For
experiments conducted in the light, the intensity was approximately
2.0 uW/cm2 supplied from fluorescent lamps.

Rice Seedlings. Rice seed (IR-8) were surface treated with 0.1% (w/v)

 

HgClz, planted in 77 ml plastic cups containing vermiculite and watered
with l/4-strength Hoagland's nutrient solution containing 3 mM of

nitrate nitrogen. The plants were grown under an 8 hr night at 25 C

and a 16 hr day at 30 C with 21 uW/cm2 and 8 uW/cm2 in the blue and

red spectral regions respectively (ILlSO Photometer, International

Light, Newburyport, MA). After 8 to 10 days, seedlings were transplanted
to 220 ml cups wrapped in aluminum foil which contained 180 ml of
nutrient solution. Four seedlings were suspended in the solution with

a sponge rubber disc. The nutrient solution was renewed every 2 to 3

11
days. When plants were 12 to 14 days old the l/4-strength Hoagland's
was replaced with l/2-strength Hoagland's with 6 mM nitrate nitrogen.

When about 17 days old, the plants were sorted for size and similar—
sized plants assigned to the same replicate. A randomized complete
block design with 6 blocks was used to remove the variance based on
plant size. Test cups in each replicate were assigned treatment
numbers by use of a random number table. One set of plants from each
replicate was harvested at the start of the experiment to obtain the
initial dry weight.

Test cups contained 150 ml l/2-strength Hoagland's nutrient
solution with 6 mM nitrate nitrogen and a 1 cm2 of Whatman #1 filter
paper containing the chemicals. The tests were conducted in the dark
at 30 C for 6 hr.

Rice Leaf Segments. One cm segments were removed from 17 to 20 day-

 

old rice seedlings. The segments were cut from the third and fourth
leaf with equally spaced coupled razor blades. The third leaves were
used for one experiment and the fourth leaves for another. Variation
found between different areas of a leaf was removed by using a random—
ized complete block design, blocked for the area of the leaf from which
the segments were taken.

Each experimental unit consisted of a 60 x 15 mm Petri dish
containing 10 ml of the test solution (1/2—strength Hoagland's with or
without 2% sucrose) which was allowed to sit overnight with a 0.5 cm2
of Whatman #1 filter paper containing the chemicals. Each dish
received 15 to 20 segments. One set of segments for each block was
dried at the start of the experiment for the initial dry weight.

Experiments were conducted in a growth chamber at 30 C. For

12
experiments conducted in the light, the intensity was the same as for

the plants grown in nutrient culture.

RESULTS AND DISCUSSION

Membrane Permeability. Triacontanol had no measurable effect on

 

membrane permeability of red beet root tissue at the concentrations
tested (Table l). The same experiment was repeated three other times
with similar results. The coefficient of variation for these experiments
was about 18%. Thus any changes in permeability that had occured during
the treatment were subject to a sampling error of at least this
magnitude. Clearly this is not a good system for measuring small
changes in membrane permeability. The failure to detect changes in
permeability with this system, therefore, does not establish that
triacontanol has no effect on membranes.

Table 1: Leakage of betacyanin from red beet root tissue.

 

Absorbance at 540 nm

 

 

Triacontanol Time (hr)

(us/1) 1+ 2 4 24
0.0 0.106 0.122 0.122

0.1 0.099 0.114 0.110

1.0 0.099 0.096 0.111
10.0 0.102 0.116 0.114
100.0 0.113 0.128 0.134
1000.0 0.102 0.166 0.104

 

The leakage of betacyanin from red beet root cells is due to a

disturbance of the semi-permeability of the plasmalemma and the

l3

tonoplast membrane since the betacyanin is contained within the tonoplast
(43). Triacontanol may only effect the plasmalemma or other cytoplasmic
membranes and not the tonoplast membrane. Another possibility is that
triacontanol has a more specific effect on membranes rather than a
general disturbance.

Various aliphatic compounds have been shown to promote growth in
plant systems (13, 24, 26, 27, 39, 44). Such substances enter both
chemically and centrifugally defined membrane fractions (41). It has
been suggested that molecular length of lipids is an important parameter
in the regulation of membranes. In a chemical test (5) the most
effective lengths were in the regions of C11 — C12 and C20 - C22. In
the red beet root and split-pea curvature assays (43) the most effective
lengths were C11 - C12, the C20 - 022 region was not tested. The C20 -
C22 region was the most active in the pea stem bioassay (39). The
molecular size corresponds quite well with the dimensions of a membrane
monolayer and the hypothesis that such substances are active by forcing
apart lipid molecules in biological membranes thus altering regulatory
properties. The observation that octocosanol did not affect rice
growth (32) or growth of tobacco callus supports this hypothesis. It is
possible that triacontanol affects membrane bilayers rather than
monolayers as suggested by Stowe and Dotts (39).

Research with molecules of longer molecular lengths in the
preceding systems may show another active peak in the C30 region. The
chemical nature of triacontanol and the evidence concerning molecular
length and membrane regulation still indicates that triacontanol may
affect membranes.

Tobacco callus. Triacontanol promoted the growth of tobacco callus

 

14

at concentrations as low as 0.01 pg per dish. Although the effect of
triacontanol on whole plants occurs in the light or dark (31), tobacco
callus responded only in the light (Figure 1). Response of tobacco
callus to triacontanol was not associated with any visible tissue
differentiation. Neither root nor shoot development occured in any
of the experiments and the apparent friability of the tissue remained
unaffected. Greening was uniform in treated and non—treated tissues
grown in the light.

Response of tobacco callus to triacontanol begins within the
first 14 hr of treatment (Figure 2). The increased growth was accompanied

by an increase in respiration as measured by oxygen uptake (Table 2).

Table 2. Influence of triacontanol on growth and respiration of
tobacco callus.

Fresh weight was measured at start of experiment and after 14 hr.
Gas data are averages over a 4 hr period beginning 2 hr after treatment.

 

 

Triacontanol Fresh Wt increase 02 uptake

(ugldish) ,(mgitissug), (Ml/g/hr)
0.0 38.2 215.3

0.1 44.8* 313.1*

 

*F value for comparison of control with treatment
significantly different at 0.05 level.
Triacontanol-treated tobacco callus contained 38% more protein

per tissue than controls (Table 3). This indicates that the rate of
protein synthesis increased. Also the percent dry weight was similar
in treated and non-treated tissue (Table 3) and the dry weights and
fresh weights showed similar response curves (Figure 3). These data
suggest that the increased growth caused by triacontanol was not

simply caused by water uptake and consequent cell enlargement but was

15

300

(mg)

200

Fresh wt

I00

 

O Ofll OJ l0
Triacontanol (pg)

Figure 1. Response of tobacco callus to triacontanol in the light
and dark.

‘

Initial weight of tissue was approximately 20 mg/dish. Data are
averages of 4 replicatei after 12 days growth. Light intensity was
approximately 2.0 uW/cm . * indicates F value for difference between
control and treatment significant at the .05 level.

16

Fresh wt increase (mg)

 

O 14 24
Time (hr)

Figure 2. Rapid response of tobacco callus to triacontanol (0.1 pg).

* indicates F value for difference between control and treatment
significant at the .05 level for comparisons at individual times.

17

400

300 #-

of control

 

%

200

 

I00
0 OLKN (lOl OJ L0

Triacontanol (pg)

Figure 3. Fresh and dry weights of triacontanol-treated tobacco
callus.

 

18

due to a gain in cellular structural material. Studies of free-hand

sections by light microscopy supported this conclusion.

Table 3. Growth response of tobacco callus.

Initial weight of tissue was approximately 100 mg. Data
are averages of four replicates after 15 days growth.

 

 

 

 

Triacontanol

Tissue Analysis (pg/dish)

(mg/tissue) 0.0 0.1

Fresh wt 1190.0 1620.0*

Dry wt 54.0 73.0*

Protein 22.4 32.0*
Percent

Dry wt 4.6 4.6

Protein 41.6 43.2

 

*F value for comparison of control with treatment
significantly different at the 0.05 level.

Octocosanol, a 28 carbon alcohol homolog of triacontanol did
not affect the growth of tobacco callus (Figure 4) which agrees with
the results found with whole plants (32) and supports the hypothesis
that a specific chain length may be required for activity.

In addition to the light requirement, the stage of growth of the
tissue at the time of treatment affected the response to triacontanol
(Figure 5). The tissue usually responded more to triacontanol when the
stock cultures were actively growing ('log phase') than when in the
slower growing 'plateau phase'. Response of 'plateau phase' tissue
was also more variable. The coefficients of variation for experiments
with 'plateau phase' tissue averaged 38%, enough to obscure even large
treatment effects. The coefficients of variation for experiments with

'log phase' tissue were also high (average 26%), but treatment effects

19

'300

 

 

 

00
E O
.. 200
3 Octocosano;
.C . e
U)
Q)
L:
I00 ‘
0 OJ I.O I0.0

Concentration (pg)

Figure 4. Growth of tobacco callus treated with triacontanol and
octocosanol.

Initial weight of tissue was approximately 20 mg/dish. Data
are averages of 4 replicates after 12 days growth. Light intensity
was approximately 2.0 pW/cmz. ** indicates F value for difference
between control and treatment significant at the .01 level.

20

SOCIE: I I I I :;

 

 

 

\0?» °
9 0 0

40C)*-
’u‘o
E
E
.c 300 A’ateau
‘53 W
L:

20C)

0 Ol)| OJ L0 I01)

Triacontanol (pg)

Figure 5. Influence of stage of growth of tobacco callus on
triacontanol response.

Initial weight of tissue was approximately 100 mg/dish. Data
are averages of 4 replicates after 15 days growth. Light intensity
was approximately 2.0 pW/cmz.

21

were statistically significant. The large amount of variability observed
in the callus growth and the lack of a response in the dark made the
tobacco callus system unsuitable for use in studying the mode of action
of triacontanol.
.EAEE.EEE£ Segments. Rice leaf segments incubated in the light in the
presence of triacontanol increased in dry weight over the controls at a
concentration of 10 pg/l (Figure 6). The increase in dry weight at this
concentration is in agreement with the optimum rate effective with
intact rice seedlings (32), however, the response appears to be limited
to a much narrower range of concentrations. The leaf segments do not
respond to triacontanol in the dark and the response in the light is
not as rapid as it is in the whole plant system (Figure 7).

The segments in the light are provided with a source of energy
from photosynthesis while those in the dark are not. The level of
soluble carbohydrates was found to decrease in plants treated with
triacontanol (Ries and Wert, unpublished data). Leaf segments
incubated in the dark with sucrose increased in dry weight when
treated with triacontanol in less than 6 hr while the controls lost
weight (Figure 8). Treated and untreated segments in the absence of
sucrose both lost weight. The dark response of the segments in the
presence of sucrose is similar to the dark response of intact plants (31)
and suggests that the carbohydrates stored in the intact plant are
required for triacontanol to be effective.

The similar response patterns between the intact plant and the
isolated leaf segment system, the low coefficient of variation (2-3%),
and the small amount of time required to conduct an experiment indicate

that this system may be useful in further studies.

22

1.25

Dry wt (mg /segment)
5
o

H
H
O"

1.10

 

0 0.001 0.01 0.1 1.0 10.0

Triacontanol (mg/l)

Figure 6. Dose-response curve for leaf segments treated with

triacontanol.

** indicates F value for difference between control and treatment
significant at .01 level.

23

0.20

)

.0
H
O

A from Zero Time (mg/segment

-0.05

 

Time (hr)

Figure 7. Change in dry weight of leaf segments treated with

triacontanol (10 pg/l) in the light and dark.
** indicates F value for difference between treatments
significant at the .01 level between comparisons at individual
times for each light condition. A a light control; B - light +
triacontanol; C - dark + triacontanol; D - dark control.

24

+SUCI'OSG — - —
005 —sucrose *

A **

——————-‘>

fi————--

/’

DIX)
I3

0 ----—---——--—m

A

B

A from Zero Time (mg /segment)

-QOS

 

Time (hr)

Figure 8. Change in dry weight of leaf segments treated with
triacontanol (10 pg/l) with and without sucrose.

*and ** indicate F value for difference between treatments
significant at the .05 and .01 levels respectively between comparisons
at individual times for each sucrose level. A = triacontanol; B =
control.

25

Other Growth Rggulators. Intact rice plants treated with 0.1 mg/l of

 

triacontanol gained in dry weight over initial dry weight and controls
which lost dry weight during a 6 hr dark period (Table 4). Plants

treated with GA and kinetin (0.1 mg/l) lost dry weight along with

3
control plants. IAA (0.1 mg/l) treated plants, however, gained weight.
The same experiment conducted with leaf segments in the presence of
sucrose showed that both triacontanol and IAA stimulated dry weight

accumulation in the dark while 0A3 and kinetin had no activity after

24 hr (Table 4).

Table 4. Response of rice seedlings and leaf segments to triacontanol,
IAA, GA3’ and kinetin.

Rice seedlings received 0.1 mg/l of the compounds and were
harvested after a 6 hr dark treatment. Leaf segments received 0.01 mg/l
of the compounds and were harvested after a 24 hr dark treatment.

 

 

 

 

 

 

Dry wt
Seedlings Segments

Treatment (mg/seedling) (mg/segment)
Zero time 66.1 0.98
Control 64.3 0.95
Triacontanol 67.8 1.04
IAA 67.0 1.04
6A3 64.4 0.95
Kinetin 63.9 0.94

LSD.05 1.2 0.02

LSD.01 1.6 0.03

These experiments show that triacontanol is not unique in its
ability to stimulate plants to increase their dry matter production in
the dark. IAA can also elicit this response. Reports on hormonal

activities of other fatty compounds indicate that their activity may

26

be linked to auxin induced processes (37,40,44). These data suggest

that there may be a link between triacontanol and an auxin induced

process.

 

-~.‘.""'" I) 151’ ”mars-r

x1“.

SUMMARY

Triacontanol had no measureable effect on membrane permeability
of red beet root tissue. Tobacco callus increased in fresh weight,
dry weight and protein on a per tissue basis in the light in response
to triacontanol. Oxygen uptake also increased. Tobacco callus
growth was highly variable and did not respond to triacontanol in the
dark making it unsuitable for further studies. Isolated leaf segments
of rice, when treated with triacontanol, increased in dry weight in the
light and in the dark in the presence of sucrose. Variability in this
system was very low. The similarity in response patterns between leaf
segments and intact plants indicate that the leaf segment assay may be
useful for further studies.

Indole—3—acetic acid applied to intact rice seedlings and isolated
leaf segments in the dark stimulated dry matter accumulation similar to
the stimulation observed with triacontanol. Stimulation of dark CO2

fixation by IAA has been indicated by observations on malate accumulation

and IAA stimulated growth in Avena sativa coleoptile tissue (15). This

 

dark 002 fixation was suggested to be related to increased PEP carboxylase
activity due to higher cytOplasmic pH values generated by IAA stimulated
H+ secretion. Triacontanol may stimulate dark CO2 fixation (31), however,

other studies refute this hypothesis (3). The fatty nature of triacontanol

.suggests that it interacts with cellular membranes. Perhaps triacontanol

27

28

interacts with membranes in such a way as to stimulate H+ secretion
resulting in an increased PEP carboxylase activity and consequent dark
C02 fixation. The source of the triacontanol—stimulated dry weight
increase, CO2 or another source, must be determined before the mechanism

of action can be identified.

 

LITERATURE CITED

10.

ll.

12.

LITERATURE CITED

Allinson, D. W., and R. A. Peters. 1970. Influence of simazine
on crude protein and cellulose content and yield of forage
grasses. Agron. J. 62:246-250.

Arnold, G. W., D. Bennet, and C. N. Williams. 1967. The promotion
of winter growth in pastures through growth substances and
photoperiod. Aust. J. Agr. 18:245-257.

Bittenbender, H. C., D. R. Dilley, V. Wert, and S. K. Ries. 1977.
Environmental parameters affecting dark response of rice
seedlings to triacontanol. Supplement to: Plant Physiol. 59:46.

Bittner, S., S. Gazitand, and A. Blumenfeld. 1971. Isolation
and identification of a plant growth inhibitor from avocado.
Phytochem. 10:1417—1421.

Booij, H. L., and H. G. Bungenberg De Jong. 1949. Researches on
plant growth regulators XV. The influence of fatty acids on
soapcoacervates. Biochem. Biophys. Acta. 3:242-259.

Brown, A. W., B. Hill, and I. J. Dymock. 1977. The relationship
between dark carbon dioxide fixation and IAA stimulated H+
secretion. Supplement to: Plant Physiol. 59:41.

Chibnall, A. C., E. F. Williams, A. L. Latner, and S. H. Piper.
1933. The isolation of n-triacontanol from lucerne wax.
Biochem. J. 27:1885—1888.

Chibnall, A. C. 1934. The constitution of the primary alcohols,
fatty acids and paraffins present in plant and insect waxes.
Biochem. J. 28:2189—2208.

Chibnall, A. C. 1934. The metabolism of plant and insect waxes.
Biochem. J. 28:2209-2219.

Eglinton C., A. G. Gonzalez, R. J. Hamilton, and R. A. Raphael.
1962. Hydrocarbon constituents of the wax coatings of plant

leaves: A taxonomic survey. Phytochem. 1:89—102.

Eglinton, G., R. J. Hamilton. 1967. Leaf epicuticular waxes.
Science. 156:1322-1334.

Ferrari, A. 1960. Nitrogen determined by a continuous digestion
and analysis system. N. Y. Acad. Sci. 87:792-800.

29

13.
14.

15.

l6.

l7.

18.

19.
20.
21.

22.

23.

24.

25.
26.

27.

30

Gudjonsdottir, S., and H. Burstrom. 1962. Growth-promoting
effects of alcohols on excised wheat roots. Physiol. Plant.
15:498-504.

Gulz, P. G. 1968. Normale und verzweigte alknae in Chloro-

plastenpraparaten und Blattern von Antirrhinum majus. Phytochem.
7:1009—1017.

 

Haschke, H. P., and U. Luttage. 1975. Stoichiomeiric+correlation
of malate accumulation with auxin—dependent K - H exchange

and growth in Avena coleoptile segments. Plant Physiol. 56:
696—698.

Huffaker, R. C., M. D. Miller, K. G. Baghott, F. L. Smith, C. W.
Schaller. 1967. Effects of field application of 2,4-D and iron
supplements on yield and protein content of wheat and barley
and yield of beans. Crop Sci. 7:17-19.

Kay, B. L. 1971. Atrazine and simazine increase yield and quality
of range forage. Weed Sci. 19:370-371.

Kolattukudy, P. E. 1969. Plant Waxes. Lipids. 5:259-275.

Kolattukudy, P. E. 1970. Biosynthesis of cuticular lipids. Annu.
Rev. Plant Physiol. 21:163-192.

Kolattukudy, P. E., and T. J. Walton. 1972. The biochemistry of
plant cuticular lipids. Prog. Chem. 13:121—175.

Leben, C., and L. V. Barton. 1957. Effects of gibberellic acid
on growth of Kentucky blue grass. Science. 125:494-495.

Linsmaier, E., and F. Skoog. 1965. Organic growth factor
requirements of tobacco tissue cultures. Physiol. Plant.
18:100-127.

Martin, J. T., and B. E. Juniper. 1970. The cuticles of plants.
Edward Arnold Ltd. N. Y. 347pp.

Mikami, Y., H. Takahara, H. Imura, A. Suzuki, and S. Tamura. 1970.
Several synthetic hydroxy—acids as plant growth regulators.
Agr. Biol. Chem. 34:977-978.

Miller, G. T. 1969. Method of increasing the amount of fruit. U. S.
Patent No. 3,472,647.

Miller, G. T. 1973. Method for stimulating plant growth. U. S.
Patent No. 3,764,294.

Mitchell, J. W., N. Mandava, J. F. Worley, and J. R. Plimmer. 1970.

'Brassins — a new family of plant hormones from rape pollen. Nature.

225:1065-1066.

 

 

 

 

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

31

Morgan, D. G., and G. C. Mees. 1956. Gibberellic acid and the
growth of crop plants. Nature. 178:1356-1357.

Ries, S. K., H. Bittenbender, R. Hangarter, L. Kolker, G. Morris,
and V. Wert. 1976. Improved cr0p growth and yield from organic
supplements. 12} W. Lokeretz, ed. Energy and Agriculture. Acad.
Press, N. Y. (In Press).

Ries, S. K., C. J. Schweizer, and H. Chmeil. 1968. The increase
in protein content and yield of simazine-treated crops in
Michigan and Costa Rica. BioScience. 18:205-208.

Ries, S. K., and V. Wert. 1977. Growth responses of rice seedlings
to triacontanol in light and dark. Planta. 135:77-82.

Ries, S. K., V. Wert, C. C. Sweeley, and R. A. Leavitt. 1977.
Triacontanol: A new naturally occuring growth regulator. Science.
195:1339-1341.

Sakuri, N., K. Shibata, and S. Kamisaka. 1974. Stimulation of
cucumber hypocotyl elongation by dihydroconiferyl alcohol.

Interactions between dihydroconiferyl alcohol and auxin or
CA. Plant and Cell Physiol. 15:709-716.

 

Steffens, G. L., T. C. T30, and D. W. Spaulding. 1967. Fatty I
alcohol inhibition of tobacco axillary and terminal bud growth. '
J. Agr. Food Chem. 15:972-975.

Stowe, B. B. 1958. Growth promotion in pea epicotyl sections by
fatty acid esters. Science. 12:421-423.

Stowe, B. B. 1960. Growth promotion in pea stem sections. 1.
Stimulation of auxin and GA action by alkyl lipids. Plant
Physiol. 35:262-269.

Stowe, B. B., and J. B. Obreiter. 1962. Growth promotion in pea
stem sections. II. By natural oils and isoprenoid vitamins.
Plant Physiol. 37:158-164.

Stowe, B. B., and V. W. Hudson. 1969. Growth promotion in pea
stem sections. III. By alkyl nitriles, alkyl acetelenes and
insect juvenile hormones. Plant Physiol. 41:1051-1057.

Stowe, B. B., and M. A. Dotts. 1971. Probing a membrane matrix
regulating hormone action. Plant Physiol. 48:559-565.

Struckmeyer, B. E., and R. H. Roberts. 1955. The inhibition of
abnormal cell proliferation with anti-auxin. Amer. J. Bot.
42:401-405.

Tinoco, J., and D. J. McIntosh. 1970. Interactions between
cholesterol and lecithin in monolayers at the air-water
interface. Chem. Phys. Lipids. 4:72-84.

42.

43.

44.

45.

46.

47.

32

Umbriet, W. W., R. H. Burris, and J. F. Stauffer. 1972. Manometric
techniques. Fifth ed. Burgess Publishing Co., Minneapolis,
Minn. 439 pp.

Veldstra, H., and H. L. Booij. 1949. Researches on plant growth
regulators. XVII. Structure and activity on the mechanism of
action. Biochim. Biophys. Acta. 3:278—312.

Vlitos, A. J., and D. G. Crosby. 1959. Isolation of fatty alcohols
with plant-growth promoting activity from Maryland Mammoth
tobacco. Nature. 184:462-463.

Wittwer, S. H., and M. J. Bukovac. 1957. Gibberellin and higher
plants. V. Promotion of growth in grass at low temperatures.
Quar. Bull. Mich. Agr. Exp. Sta. 39:682—686.

Wort, D. J. 1966. Growth, yield, composition, and metabolism of
various crop plants as affected by the application of 2,4-D-
nutrient dusts and sprays. Proc. Int. Symp. Plant Stimulation,
Sofia. 341-353.

Wort, D. J., and K. M. Patel. 1970. Response of plants to
naphthenic and cycloalkenecarboxylic acids. Agron. J. 62:644-646.

 

 

 

 

 

IIIIIIIIII'

I

448

III

II

V”
U"
I

o
3
o
3
9
2
1

IIIIIII

III

3