Short Communication

Reversal of Ethephon-Induced Feminization in Male Plants

of Cannabis sativa by Ethylene Antagonists
H. Y. MOHAN RAM and RINA SETT

Department of Botany, University of Delhi, Delhi 110007, India

Received January 4,1982· Accepted April 25, 1982

Summary
Interaction between ethephon and the two ethylene antagonists - silver nitrate and cobalt
chloride - applied exogenously to the male Cannabis sativa plants was studied. The feminiza-
tion effect caused by two sprays of 960 mg .1- 1 of ethephon could be overcome by 100 J.'g of
AgN0 3 or CoCh. However, neither of the antagonists could suppress the feminization effect
of 1920 mg .1- 1 of ethephon sprayed twice.
Key words: Cannabis sativa; cobalt ion; ethephon; sex expression; silver ion.

Introduction
Ethylene regulates several plant processes and has been recognized as a plant
hormone (Abeles, 1973). Application of ethephon, an ethylene releasing compound
enhances female flowers in monoecious plants (see Abeles, 1973; Mohan Ram, 1980
and the references cited therein). In the male plants of Cannabis sativa ethephon
spray causes the formation of female and intersexual flowers (Mohan Ram and
Jaiswal, 1970). Beyer (1976 a) reported that silver ion [Ag(I)] acts as a potent inhibitor
of ethylene action. Lau and Yang (1976) and Yu and Yang (1979) demonstrated inhi-
bition of ethylene synthesis from methionine by cobalt ion [Co(I)] and by ami-
noethoxyvinyl glycine. Silver ion (applied as silver nitrate) stimulates the formation
of fertile male flowers in gynoecious lines of cucumber (Beyer, 1976 b; Atsmon and
Tabbak, 1979). Recently Sarath and Mohan Ram (1979), Mohan Ram and Sett (1979,
1982) reported the production of fertile male flowers on the female plants of Can-
nabis sativa by silver nitrate, cobalt chloride and aminoethoxyvinyl glycine respec-
tively. These compounds also induced fertile male flowers in the primary terminal
inflorescence of a strictly pistillate line of Ricinus communis (Mohan Ram and Sett,
1980).
The present investigation was taken up to ascertain whether or not the effect of
ethephon on male plants of Cannabis can be reversed by Ag(I) and Co(I).

Z. Pjlanzenphysiol. Ed. 107. S. 85-89. 1982.

86 H. Y. MOHAN RAM and RINA SETT

Material and Methods

Seedlings of Cannabis sativa growing naturally in the Botanical Garden of the Department were
transplanted at the 3- or 4-leaf stage to 25 cm wide earthenware pots filled with garden soil. As the
vegetative plants of either sex are indistinguishable, it became necessary to wait until the first flowers
appeared before separating the pots containing male and female plants. Male plants of nearly uni-
form height (24-30 cm) bearing 5 or 6 flowering nodes were selected for each experiment.
Freshly prepared aqueous solution of either 1920 or 960 mg .1- 1 ethephon; 2-chloroethyl-
phosphonic acid (Agromore Ltd, India) was applied by means of a sprayer on the foliage till the
point of run off. An aqueous solution (50 or 100 pg/plant) of either silver nitrate or cobalt
chloride was applied to the growing shoot tips of the ethephon-treated plants. A 10 pI drop of
the test compound was applied onto a wick of cotton placed on the shoot tip each day for 5
days to make up the total amount. Tween-80 (polyoxyethylene mono-oleate sorbitane, Sigma
Chemical Company, USA) at 0.01 % was used as surfactant.
In the present work 50 male plants were given a foliar spray of an aqueous solution of
ethephon and this treatment was repeated after seven days. One set of 10 treated plants served as
ethephon-treated controls besides the 10 untreated control plants which received only 0.01 % of
the surfactant solution. The remaining 40 ethephon treated plants were given apical application
of either AgN03 (50 and 100 pg per plant) or CoCh (50 and 100 pg per plant) one day after the
second spray of ethephon.

Results
Characteristic responses to the two sprays of 1920 mg .1- 1 of ethephon appeared
two days after the final spray in all the treated plants. The leaves which were present
at the time of treatment abscised after yellowing, and exhibited marked epinasty. The
degree of epinasty was inversely proportional to the age of the leaf. The newly for-
med 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 the treated con-
trols as compared to the untreated controls (Table 1). The male flowers (Fig. 1 A)
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 primary lateral
branches already present at the time of ethephon spray increased in length, giving a
bushy appearance to the plants. However, no new lateral branches developed imme-
diately below the growing shoot tip. The untreated control plants continued to
produce new nodes bearing male flowers (Fig. 1 B). In the ethephon-treated control
plants (Fig. 1 C) the first few nodes on the main axis bore intersexual flowers with
varied morphology (Fig. 1 D); the subsequently formed nodes, however, produced
only female flowers (Fig. 1 C).
The shoot tip of the plants which received either Ag(I) or Co(I), did not grow fur-
ther and dried up (Fig. 1 E). The slight increase in shoot length in these plants was
caused by internodal elongation (Table 1). At least four primary lateral branches were
present in all the treated plants from which the data on flower number were coll-
ected. These branches bore male, intersexual and fully altered female flowers (Fig.
1 E, F). The percentage of altered flowers (these include intersexual and female flow-
ers) along each primary lateral branch was higher in plants treated with 50 p.g of Ag(I)

Z. Pjlanzenphysiol. Bd. 107. S. 85-89. 1982.

 Reversal of ethephon-induced feminization in Cannabis sativa 87

Fig. 1 A-F: A. Male flower showing 5 tepals and 5 stamens. - B. Terminal portion of a control
male plant. - C. Apical part of a treated male plant (1920 mg .1- 1 ethephon) bearing flowers of
altered sex [intersexual (if) and fully altered female flower (ff) on the main axis; photographed
35 days after treatment]. - D. An intersexual flower bearing a bract (br) typical of the female
flower and stamens (st). - E. Apical region of a male plant treated with ethephon (1920 mg ·I-I)
+ AgN03 (100 I-'g) [arrow indicates dried shoot tip]. - F. A portion enlarged from Fig. 1 E to
show the female flowers.

or Co(I) as compared to the plants treated with 100 Jtg of these chemicals (Table 1).
This clearly indicated that Ag(I) and Co(I} were not able to completely overcome the
effect of ethephon (1920 mg .1- 1; 2 sprays). Presuming that the dosage of ethephon
was too high, in a subsequent experiment two sprays of 960 mg .1- 1 of ethephon
were given and these were followed by apical application of either AgN03 (50 or
100 Jtg per plant) or CoCh (50 or 100 J.tg per plant).
As compared to the situation in which 1920 mg .1- 1 of ethephon was applied, the
extent of epinasty and yellowing of the leaves in the treated plants was. reduced and
the plants produced intersexual, female and male flowers at the newly formed nodes
on the main axis. The treated plants reverted to the production of flowers of the orig-
inal sex after bearing a few female flowers.
Apical treatment with Ag(I} and Co(I} following two sprays of ethephon arrested
the growth of the main shoot meristem, the effect being greater at 100 Jtg than at
50 Jtg per plant. Interestingly, interaction of each of these compounds was able to
inhibit the formation of fully altered female flowers. In response to treatment with
50 Jtg of either AgN03 or CoCh, the primary lateral branches bore flowers of altered
sex (Table 2). However, in response to treatment with 100 Jtg of either of the com-
pounds, only male flowers were formed on the primary lateral branches and the
production of altered flowers was totally inhibited (Table 2). This experiment show-

Z. Pjlanzenphysiol. Bd. 107. S. 85-89. 1982.

Table 1: Effect of two sprays of ethephon (1920 mg .1- 1) and their interaction with AgN03 and
CoCh on the growth of the main shoot and flower sex expression in the male plants of Can·
nabis sativa').
Treated Ethephon + Ethephon + Ethephon+ Ethephon +
control AgN0 3 (50 J.Lg) AgN0 3
(100 J.Lg) CoCh (50J.Lg) CoCh (100 J.Lg)
x CI x CI x CI x CI x CI
Increment in 17.82 3.88**) 5.52 2.17**) 7.00 3.13**) 7.87 1.12**) 7.87 1.12**)
height (em)
Increment in 11.18 1.19**) D D D D
node number

Position of primary
lateral branches b) Percentage of altered flowers (intersexual and femaleJA
1 85.1 69.2 89.0 79.0
2 + 84.6 67.4 82.3 86.7
3 88.2 69.0 72.0 68.0
4 85.4 60.0 79.0 73.0

') average of 10 plants, 35 days after treatment. b) numbers indicate position from base
upwards.
D dried shoot tip.
A percentage of flowers of altered sex out of the total number of flowers formed.
+ no lateral branches present at equivalent positions; the percentage of altered flowers present
on the main axis is 100.
CI confidence interval at P ~ 0.05. **) highly significant over control at P ~ 0.01.

Table 2: Effect of two sprays of ethephon (960 mg .1- 1) and their interaction with AgN03 and
CoCh on the growth of the main shoot, and flower sex expression in the male plants of Can·
nabis sativa').
Treated Ethephon + Ethephon + Ethephon + Ethephon+
control AgN03 (50 J.lg) AgN0 3
(100J.Lg) CoCh (50J.Lg) CoCh (100 J.Lg)
x CI x CI x CI x CI x CI
Increment in 27.50 5.73 12.38 5.83**) 1.75 0.45**) 13.38 1.41**) 3.13 1.13**)
height (em)
Increment in 9.50 1.18 D D D D
node number

Position of primary
lateral branches b) Percentage of altered flowers (intersexual and femaleJA
1 48.1 0.0 49.4 0.0
2 + 43.5 0.0 41.0 0.0
3 48.0 0.0 36.8 0.0

') average of 10 plants, 35 days after treatment. b) numbers indicate position from base
upwards.
D dried shoot tip.
A percentage of flowers of altered sex out of the total number of flowers formed.
+ no lateral branches develop at equivalent position; the percentage of altered flowers on the
main axis is 72.
CI confidence interval. **) highly significant over control at P ~0.Q1.

Z. Pjlanzenphysiol. Ed. 107. S. 85-89. 1982.

 Reversal of ethephon-induced feminization in Cannabis sativa 89

ed that the effect of two sprays of 960 mg ·1-1 of ethephon can be completely over-
come by treatment with 100 p.g of AgN03 or CoCh.

Discussion
It has been suggested that in Cannabis sativa ethylene and gibberellin function as
female and male hormones respectively and that the expression of sex is controlled by
their relative endogenous balance (Mohan Ram and Jaiswal, 1974). In a study of the
interaction of ethephon with GA3,· it was noted that the effect of former at 960 and
1920 mg .1- 1 could not be completely reversed by GA3 at any of the concentrations
tried, although it caused marked reduction in the feminizing effect of ethephon
(Mohan Ram and Jaiswal, 1974).
In the present study AgN0 3 or CoCh at 100 /tg has been shown to completely
reverse the feminizing effect of 960 mg .1- 1 of ethephon, whereas neither of these
compounds could totally overcome the effect of ethephon at 1920 mg .1- 1. At 50 /tg
AgN03 or CoCh could only partially reverse the effect of both 960 and 1920 mg . I-I
ethephon. Thus the extent to which flower sex can be altered appears to depend on
the dosage of ethephon and ethylene antagonists applied.
It was believed that whereas Ag(I) blocks the action of ethylene (Beyer, 1976 a),
Co(I) inhibits the conversion of methionine to ethylene (Lau and Yang, 1976; Yu and
Yang, 1979). Our studies on the interaction of ethephon and Co(I) provide evidence
that the action of ethylene is also prevented by Co(I).
On the basis of the present knowledge it may be inferred that whatever be the gen-
etic basis of sex determination, it operates through the production of specific
hormonal substances. Exogenous application of any compound that directly or indi-
rectly modifies the endogenous hormonal status can bring about a phenotypic change
in the flower sex expression.

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LAu, O. L. and S. F. YANG: PI. Physiol. 58, 114-117 (1976).
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Z. Pjlanzenphysiol. Ed. 107. S. 85-89. 1982.