Modification of Growth and Sex Expression in Cannabis

sativa by Aminoethoxyvinylglycine and Ethephon

H. Y. MOHAN RAM and RINA SETT

Department of Botany, University of Delhi, Delhi-l10007, India

Received August 13, 1981 . Accepted November 19, 1981

Summary
Apical application of aminoethoxyvinylglycine (AVG) to female plants (5, 10, 25, 50 and
75 ~g per plant) of Cannabis sativa induced the formation of fertile male flowers on the newly
formed primary lateral branches (PLBs). 1 ~g per plant was found to be ineffective and 100 ~g
treatment proved inhibitory. In response to 5 to 50 ~g treatments the PLBs bore reduced male,
intersexual and male flowers, whereas with 75 ~g they formed only male flowers.
When AVG (25, 50 ~g) was applied to the shoot tips of male plants sprayed with ethephon
(1920 mg . I-I), the feminization effect caused by the latter was markedly curtailed.
Key words: Cannabis sativa, aminoethoxyvinylglycine, ethephon, growth regulators, reproduc-
tion, sex reversal.

Introduction
Ethephon, an ethylene releasing compound (Abeles, 1973) enhances the number of
female flowers in the cucurbits (Mc Murray and Miller, 1963; Rudich et ai., 1969,
1970; Augustine et ai., 1973) and induces female flowers in the male plants of Can-
nabis sativa (Mohan Ram and J aiswal, 1970). In these plants gibberellic acid promotes
maleness (Peterson and Anhder, 1960; Wittwer and Bukovac, 1962). It has been
suggested that flower sex expression in Cannabis is controlled by a balance between
the endogenous levels of ethylene and gibberellins (Mohan Ram and Jaiswal, 1974).
There is evidence that the silver ion is a potent inhibitor of ethylene action (Beyer,
1976) and that the cobaltous ion blocks the synthesis of ethylene (Beutelmann and
Kende, 1977). Silver nitrate and cobalt chloride have been shown to induce male
flowers on female plants of Cannabis (Mohan Ram and Sett, 1979; Sarath and Mohan
Ram, 1979), and fertile staminate flowers on plants of a strictly pistillate line of
Ricinus communis (Mohan Ram and Sett, 1980).
Recently aminoethoxyvinylglycine (AVG: L-a-amino-y-(2amino-ethoxy)-trans-l3-
butenoic acid), a structural analogue of rhizobitoxine, has been shown to inhibit
ethylene production (Bangerth, 1973; Baker et ai., 1978; Amrhein and Wenker, 1979;
Yu and Yang, 1979; Williams, 1980). The current investigation was undertaken to
ascertain the effect of A VG on the female Cannabis plants and also to investigate
whether or not the effect of ethephon on male plants can be reversed by AVG.

z. Pjlanzenphysiol. Bd. 105. S. 165-172. 1982.

166 H. Y. MOHAN RAM and RINA SETT

Material and Methods

Seedlings of Cannabis sativa growing naturally in the Delhi University Botanical Garden (in
December 1979, 1980), were transplanted at the 3- or 4-leaf stage to 25 cm earthenware pots
filled with garden soil. As the vegetative plants of either sex are indistinguishable, it was

Z. Pjlanzenphysiol. Bd. 105. S.165-172. 1982.

 Modification of sex in Cannabis sativa 167

necessary to wait until the first flowers appeared in order to separate male and female plants.
Plants of nearly uniform height (24-30 cm), bearing 5 or 6 flowering nodes, were selected for
each experiment. The main shoot tips of female plants were given an aqueous solution of
aminoethoxyvinylglycine (Hoffmann-La Roche, Nutley, New Jersey, U .S.A.) from a 0.1 ml
pipette. Tween 80 (0.01 %) was used as a surfactant. A 10 ~l drop of the test solution was
applied daily on a cotton wick placed at the shoot tip for 5 days to give the total amount. The
plants had received 1, 5, 10, 25, 50, 75 or 100 ~g of AVG by the end of the fifth day. The
controls received the surfactant solution only (Fig. 1 a, b). The male plants were first given two
foliar sprays of a freshly prepared aqueous solution of 1920 mg . 1-1 of ethephon (2-chloro-
ethyl phosphonic acid, Agromore Ltd., Bangalore) at an interval of seven days. These plants
subsequently received an apical application of either 25 ~g or 50 ~g of A VG one day after the
second spray of ethephon. Ten plants were maintained for each treatment. One set of 10 plants
served as ethephon-treated controls, besides the 10 untreated control male plants (Fig. 2 a, b)
which received only surfactant solution. The viability of pollen grains in the induced male flow-
ers was tested either by germinating in a medium consisting of 7 % sucrose and 1 % agar or by
immersion in fluorescein diacetate-sucrose solution (2 mg fluorescein diacetate dissolved in one
drop of 10 % sucrose solution) and observing their fluorescence under ultraviolet light.

Results
Studies on Female Plants
The first visible response to treatment with A VG (excepting 1 J.1g) was the decolor-
isation of the shoot tip and the young leaves. With 5 and 10 J.1g the apical meristem
was not damaged and the growth of the main shoot was resumed after a short period.
The shoot tip was killed by higher amounts (25 -100 J.1g per plant). The plants receiv-
ing 5 to 100 J.1g were significantly shorter than the controls, the reduction in height
being dosage-dependent. There was, however, no difference in the increase in the
number of nodes following treatment with 5 and 10 J.1g. No new nodes were
produced in the plants receiving 25 J.1g or higher amounts, but some increase in their
length occurred owing to internodal elongation.
Treatment with 5 to 50 J.1g of AVG caused the formation of female (9, flower with
a carpel surrounded by a large bract), intersexual (q', flower bearing both carpel and

Figures 1- 5: Cannabis sativa: 1 a, 2 a: Terminal portions of a control female and male plant,
respectively, xO.57. - 1 b shows an enlarged female flower, x2.3. - 2 b is a magnified view of
the inflorescence with male flowers and buds, x 1.6. -3: Modified flowers from AVG-treated
female plant (50 ~g), x5. - 3 a-c are intersexual flowers, each with a characteristic bract, 3 d, e
are reduced male flowers, and 3 f is a fully altered male flower (see text for details). - 4 a:
Terminal portion of a female plant treated with 75 ~g of AVG. Note the dried shoot tip
(arrow) and two long PLBs bearing induced male flowers towards their tips (photographed 48
days after treatment), xO.2. - 4 b: shows an enlargement of the terminal portion of one of the
PLBs, x 0.7. - 5 a: Terminal portion of a male plant treated with ethephon (1920 mg .1- 1) +
AVG (50 ~g). Note the dried shoot tip (arrow) and the three PLBs bearing a few intersexual and
a large number of male flowers (mf) (photographed 35 days after treatment), xO.5. - 5 b: A
magnified view of the tip of a PLB, x 1. (br = bract; mf = male flower; PLB = primary lateral
branch; st = stigma; sta = stamen; tp = tepa!.)

z. Pjlanzenpbysiol. Bd. 105. S. 165-172. 1982.

168 H. Y. MOHAN RAM and RINA SETT

stamens Fig. 3 a-c), reduced male (RO', flower having four or fewer stamens Fig. 3 d,
e), and male (0', bearing five tepals and five stamens with a copious amount of pollen
grains Fig. 3 f) flowers in the newly formed primary lateral branches (PLBs). The
nodes that developed on the main axis, subsequent to treatment with 5 and 10 IJg also
produced flowers of altered sex, along with normal female flowers. The data in
Table 1 indicate that the percentage of flowers of altered sex (\1, RO', 0') in each lat-
eral branch increases with the amount of the chemical applied. The data for flower
number were collected from the first three newly formed PLBs, since this was the
minimum number present in all the treated plants, excepting those receiving 75 IJg
(in which only two PLBs developed and these were longer than the PLBs formed in
response to the other treatments and in the controls). Interestingly, in plants treated
with 75 IJg of AVG, no intersexual flowers or reduced male flowers were formed and
the branches bore fully altered male flowers with a few female flowers in the basal
nodes (Fig. 4 a, b).
In plants treated with 100 IJg AVG, the main axis did not produce new nodes since

Table 1: Effect of AVG on shoot growth, length of primary lateral branches (PLBs) and flower
sex expression in female plants of Cannabis l ).
Treatments Increment in Increment in Position Length of Flower numberlPLB Percentage
(Ilg per plant) height (em) no. of nodes ofPLB') PLB of altered
x CI x CI x CI '< Ro- O- sex')
0 2.11 0.42 3.56 '"
- Nil
(control) 42.00 5.6 5.50 0.83 NS 2.11 0.42 3.11 - Nil
2.28 0.67 3.67 - Nil
2.20 0.42 3.78 - Nil
40.20 4.78 NS 5.70 0.59 NS 2.06 0.42 3.00 - Nil
2.25 0.58 3.30 - Nil
3.67 1.63 3.11 3.00 2.00 3.10 72.26
31.28 5.06*) 5.11 1.46NS 1.94 0.89 1.89 1.83 1.60 3.60 77.94
1.63 0.94 1.50 3.65 2.10 3.10 85.51
8.55 2.12 3.55 4.00 1.00 4.10 72.22
10 26.95 6.73*) 5.20 l.20 NS 5.05 1.78 2.90 3.10 2.10 3.60 75.21
3.88 0.99 1.80 3.20 1.10 2.10 78.05
9.78 4.27 2.27 5.70 2.10 12.30 89.85
25 15.78 3.51*) D 9.56 4.02 1.70 3.30 1.00 14.70 91.79
14.71 6.91 2.80 3.00 3.20 15.50 88.57
10.44 7.61 7.30 5.70 2.00 19.00 78.53
50 12.40 2.92*) D 11.33 6.40 6.80 4.80 6.50 23.40 83.61
14.86 2.69 3.40 5.50 8.00 21.40 91.12
1 21.50 4.08 3.80 - 30.50 88.92
75 11.00 2.19*) D 2 23.00 0.86 2.50 - 41.40 94.31

100 4.80 1.64*) D

l) Average of 10 plants, 48 days after treatment; CI confidence interval at P ~ 0.05; D dried

shoot tip.
2) Percentage of flowers of altered sex out of the total number of flowers present on each PLB.
J) Numbers indicate position from base upwards.
- Indicates absence of flowers.
*) Highly significant over control at P ~ 0.Q1.
NS Not significant relative to the control.

Z. Pjlanzenphysiol. Bd. 105. S. 165-172. 1982.

 Modification of sex in Cannabis sativa 169

the apical meristem was killed. The young leaves became yellow and abscised. How-
ever, as a result of internodal elongation a slight increase in shoot length was
recorded. No new lateral branches developed in most of the treated plants. Occasion-
ally, one or two PLBs were noticed, but their growth was limited and flower bud
production seldom occurred.
The induced male flowers produced viable pollen grains and 69 % of them ger-
minated in vitro within 30-35 minutes. Also, following treatment with fluorescein
diacetate, the viable pollen grains fluoresced when observed under ultraviolet light.

Stages ofMasculinization
The first stage towards «maleness» was the development of a wide range of intersex-
ual flowers which produced both carpel and stamens (Fig. 1 c). A gradual disap-
pearance of the organs associated with the female flower such as bract, ovary, and
stigmatic lobes was observed. This was followed by the appearance of tepals and sta-
mens in the intersexual flowers. The congregation of flowers at each node and the
presence of pedicels represented additional features of the male plants.
From the above experiment it can be concluded that the minimum effective dosage
of A VG is 5 f.lg and the optimal amount is 75 f.lg per plant. At 100 f.lg A VG caused
strong inhibition of growth.

Studies on Male Plants

As ethephon causes the production of female and intersexual flowers on male
plants of Cannabis (Mohan Ram and Jaiswal, 1970) and AVG induces the formation
of male flowers in female plants (present report), it was logical to ask whether or not
the effect of ethephon on male plants can be reversed by the application of AVG.
Ethephon at 1920 mg . 1-1 was selected because Mohan Ram and Jaiswal (1974) had
shown that a maximum number of nodes (5.2) bearing female flowers is formed in
response to this concentration. Although a slight epinasty occurred two days after the
first spray, the characteristic ethephon effects appeared two days after the final spray.
The leaves which were present at the time of treatment showed epinasty, became yel-
low and abscised. The extent of epinasty was inversely proportional to the age of the
leaf. The newly formed leaves were smaller and darker green than those in the untreated
controls. There was a significant decrease in the height and the number of nodes in all
the treated controls as compared with the untreated controls. The male flowers that
were already present abscised. Cessation of apical growth was noted in all the ethephon
treated control plants for a period of 7 -10 days. The PLBs present at the time of
ethephon spray increased in length, giving a bushy appearance to the plants. However,
no new PLBs developed immediately below the growing shoot tip. In the treated
control plants the first few nodes on the main axis bore intersexual flowers with varied
morphology, whereas the nodes formed subsequently produced only female flowers.
The untreated control plants continued to bear only male flowers (Table 2).

Z. Pjlanzenphysiol. Bd. 105. S. 165-172. 1982.

170 H. Y. MOHAN RAM and RrNA SETT

Table 2: Effect of interaction of ethephon and A VG on the growth of the main shoot, length of
primary lateral branches and flower sex expression!).
Treatments
Untreated Treated Ethephon (1920 mg .1- 1) Ethephon (1920 mg·I- ' )
control control + AVG (25 ~g) + AVG (50 ~g)
(ethephon,
1920 mg' -I)
CI x CI x CI x CI
Increment 28.70 4.17 13.90 2.50*) 1.50 0.38*) 2.55 1.02*)
in height (em)
Increment 17.60 2.06 10.60 0.90 D D
in the number
of nodes

Position of Length (em) Flower Percent- Length (em) Flower Percent-

primary number age of number age of
lateral altered altered
branches') x CI Cf 'l' 9 flowers 3) x CI Cf 'l' 9 flowers 3)

+ + 12.20 3.21 9.40 4.1 2.6 41.61 14.10 2.91 12.7 4.8 - 27.00
11.30 1.65 11.30 3.9 2.3 35.43 11.30 2.94 12.2 3.0 - 19.74
11.60 1.48 9.00 3.0 2.1 36.17 10.50 2.29 14.8 5.4 - 26.73

!) Average of 10 plants 35 days after treatment.

2) Numbers indicate position from base upwards.
'3) Percentage of flowers bearing altered sex out of the total number of flowers.
D Denotes damaged shoot tip.
+ No laterals were present at this position.
- Indicates absence.
CI Confidence interval at P ~ 0.05.
*) Highly significant relative to the control at P ~ 0,01.

Plants which received interaction treatments did not produce new nodes on the
main shoot and the small increase in their height resulted from internodal elongation.
PLBs developed after 15 - 20 days of growth inhibition. At least three newly formed
PLBs were present in all the plants that received the interaction treatment. Hence the
data were collected from the first three PLBs only. In response to treatment with
25 J.lg of AVG, the lateral branches bore intersexual, female and male flowers,
although the number of fully altered female flowers was less than that of the normal
male flowers. It took less time for the plants to resume bearing male flowers than the
treated controls. Curiously, no fully altered female flowers were noticed when
SO J.lg of AVG was applied to ethephon-treated plants. A large number of male flow-
ers with a few intersexual flowers were formed on the PLBs (Fig. 5 a, b). Table 2
shows a drastic reduction in the percentage of altered flowers. Thus the feminizing
effect of 1920 mg . 1-1 of ethephon can be markedly curtailed by treatment with
SO J.lg of AVG.

Discussion
The discovery of rhizobitoxine (2-amino-4-2'-amino-3'-hydroxypropoxy-trans-3-
butenoic acid), a phytotoxin isolated from the root-nodule bacterium (Rhizobium
japonicum), and capable of inhibiting ethylene production in fruit and other plant

Z. Pf/anzenphysiol. Bd. 105. S. 165-112. 1982.

 Modification of sex in Cannabis sativa 171

tissues (Owens et al., 1971), prompted investigators to look for its structural ana-
logues. Aminoethoxyvinylglycine, one such analogue, was shown to be an effective
inhibitor of ethylene production in apple tissues, slices of green tomato and avocado
(Lieberman et al., 1974; Baker et al., 1978) and excised leaves of Petunia hybrida
(Gavinlertvatana et al., 1980).
A VG has been used for delaying fruit ripening, reducing pre harvest drop, and for
increasing flesh firmness etc. (Bangerth, 1978; Williams, 1980). However, the effect of
A VG on flower sex expression has been studied in only two cucurbits. Loy et al.
(1979) reported enhanced production of staminate and/or perfect flowers in a
gynoecious muskmelon. In a gynoecious line of Cucumis sativus A VG induced per-
fect flowers (Atsmon and Tabbak, 1979). In the present investigation various degrees
of masculinization were evoked in the female Cannabis plants following A VG
application. Interestingly, no intersexual flowers developed in response to 75 flg
A VG and the lateral branches bore predominantly male flowers. It is speculated that
A VG triggers male sex expression in female plants by preventing ethylene synthesis.
Whereas gibberellic acid causes shoot elongation in addition to inducing maleness in
Cannabis sativa (Mohan Ram and Jaiswal, 1972), AVG (present study) retards shoot
elongation but promotes male sex expression.
It is significant to note that in male Cannabis plants, A VG (50 flg) markedly
counteracts the effect of two sprays of 1920 mg . 1-1 of ethephon and that the newly
formed PLBs fail to form fully altered female flowers. Ness and Romani (1980) have
recently demonstrated the reversal of ethylene-induced ripening of pear fruits by
AVG. Apparently, AVG is able to overcome the effect of ethephon, either by block-
ing its stimulation of ethylene biosynthesis or by some other, as yet unknown means.
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