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Induction of male flowers on female plants of Cannabis sativa by gibberellin and its inhibition by abscisic acid

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Hassan Ram at King Abdulaziz University
Hassan Ram
V S Jaiswal
V S Jaiswal
V S Jaiswal
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Abstract and Figures

Gibberellins (GA3, GA4+7, GA7 and GA9) induce male flowers on female plants of Cannabis sativa. This is, depending on concentration, partially or fully inhibited by abscisic acid (ABA). The ABA effect can in turn be partially overcome by increasing the concentration of GA3.
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Planta (Berl.) 105, 263--266 (1972)
9 by Springer-Verlag 1972
Short Communications
Induction of Male Flowers on Female Plants
of
Cannabis sativa
by Gibberellins and Its Inhibition
by Abscisic Acid
It. u Mohan Ram and V. S. Jaiswal
Department of Botany, University of Delhi, India
Received February 16, 1972
Summary. Gibberellins (GA3, GAt+~, GA 7 and GAg) induce male flowers on
female plants of Cannabis sativa. This is, depending on concentration, partially or
fully inhibited by abscisie acid (ABA). The ABA effect can in turn be partially
overcome by increasing the concentration of GA a .
We reported the induction of female (pistillate) flowers and femini-
zation of flowers in male plants of Cannabis sativa L. by treatment with
2-ehloroethanephosphonie acid (Mohan Ram and Jaiswal, 1970) and a
morphactin (Mohan Ram and Jaiswal, 1971). Earlier, Herich (1960) had
shown that soaking of seeds of C. sativa in gibberellin stimulated the
development of a larger number of female individuals while Heslop-
Harrison and Heslop-Harrison (1961) had observed that gibberellie acid
(GAs) treatment of the plants had no effect on primary sex differentiation
in the same species. We have noted marked stem elongation and produc-
tion of male (staminate) flowers in gibberellin (GA)-treated female plants
of Cannabis. Various GA-induced responses, including shoot elongation,
seed germination, senescence, and production of hydrolases and synthesis
of endoplasmic reticulum in barley aleurone cells, can be counteracted or
inhibited by abseisie acid (ABA) (Addieott and Lyon, 1969; Evins and
Varner, 1971). This communication describes the effects of gibberellins and
ABA on the production of male flowers in female plants of Cannabis sativa.
Seedlings of C. sativa were raised and their sex was determined after flower
initiation. Only femaIe plants were selected for study because male plants showed
no change in sex expression when treated with GAs. Ten plants were used for each
treatment. GAs and ABA (RS form), separately or in combination, were applied in
cotton wicks to the shoot apices of the plants; control plants received distilled water.
The GAs were first dissolved in ethanol and ABA in 1 N Na0H; both were sub-
sequently diluted with distilled water. The number of nodes bearing male flowers
and the average number of male flowers in each treated plant were recorded.
Plants treated with GA 3 on 10 consecutive days, to a total of 50 ~g/
plant (Table 1, Experiment 1) show two types of response : marked inter-
264 H.Y.M. Ram and V. S. Jaiswal:
Table 1. Interaction between gibberellins and ABA on male flower formation in
female plants of
Cannabis sativa
Treatments a Average no. Average no. of
of nodes/plant c~ flowers/plant
with c~ flowers
Experiment 1
Control 0 0
GA 3 50 3.6 33.8
ABA 25 0 0
ABA 50 0 0
ABA 75 0 0
ABA 100 0 0
GA 3 50 + ABA 25 0.5 6.8
GA 3 50 + ABA 50 0 0
GAa 50 + ABA 75 0 0
GA s 50 A- ABA 100 0 0
GA 3 100 ~- ABA 50 1.1 23.5
Experiment 2
Control 0 0
ABA 50 0 0
GA~ 50 3.8 35.0
ABA 50 after 5 days GA 3 50 1.2 23.8
GA 3 50 after 5 days ABA 50 1.0 19.0
Experiment 3
Control 0 0
ABA 5O 0 0
GA4+ 7 50 1.2 17.0
GA~+7 50 -]- ABA 50 0 0
GA7 50 2.3 25.0
GA 7 50 ~- ABA 50 0 0
GA9 50 0.6 7.5
GA9 50 + ABA 50 0 0
a Total amount of chemicals applied in vg per plant.
nodal elongation accompanied by certain formative changes in the vegeta-
tive parts, and production of male flowers. Only the latter response will
be considered in this note. 2-3 weeks after treatment the plants start
bearing male flowers in the newly formed 3-6 nodes (Fig. 1A, C). At the
end of the following week the plants begin to form also female flowers
(Fig. 1 B). In a few instances the terminal node, in addition to male
flowers, may also bear flowers with organs of both sexes. The induced
male flowers have normal stamens with viable pollen grains.
ABA at 25, 50, 75, or 100 ~g per plant, applied alone did not cause
any significant change in extension growth. It caused abscission of leaves,
Induction of Male Flowers by Gibberellins 265
~.~
9
~'~ ~
.Q
~.~#x
9 ~ xd
r
~~ ~
~o~
senescence of portions of younger leaves, and slight injury to the apex,
but there was no effect on flowers.
When 25 ag of ABA and GA a were applied together there was marked
reduction in the number of nodes showing male flowers (Table 1). Plants
receiving 50 ag of GA s along with 50, 75 or 100 ag of ABA showed com-
plete inhibition of the formation of male flowers. If the GAs concentra-
tion was increased from 50 ag to 100 ag/plant and the ABA concentra-
tion was kept at 50 ag/plant, the GA 3 effect dominated over the inhibi-
tory effect of ABA.
18 Plant~ (Berl.), Bd. 105
266 H.Y.M. Ram and V. S. Jaiswal: Induction of Male Flowers by Gibberellins
In another experiment (Table 1, Experiment 2) it was found that sub-
sequent app]ication of 50 [zg of GA s to plants which had received 50 tzg
of ABA permitted, on the average, 1.2 nodes to bear male flowers.
Thus GA a could exert its influence after an initial ABA treatment. If
GA 3 was supplied first and ABA later on, the latter was able to decrease
the extent of formation of male flowers but not totally suppress it.
It was of interest to know whether GAs other than GA 3 elicited the
induction of male flowers in female
C. sativa.
At equal concentrations,
GA~ was found to be most effective, followed by GAy, a mixture of GA 4
and GAv (Ca. 77 and 23%, respectively), and GA 9. ABA was able to
overcome the effect of all the GAs when applied simultaneously (Table 1).
The results clearly demonstrate that all the GAs used in our experi-
ments (GA3, GA4+7, GAy, GAo) stimulate the formation of male flowers
on female plants
of C. sativa.
The GA-induced formation of male flowers
can be inhibited by ABA either totally (at 50 ~zg) or partially (at 25 ~zg)
and this inhibition can be overcome to some extent by increasing the
concentration of GA 3 (100 [zg). We know of only one report (Abdel-
Gawad and Ketellapper, 1969) in which ABA has been shown to stimu-
late the initiation of female flowers.
We are thankful to F. Hoffmann-La Roche & Co., Ltd., Basle, Switzerland, for
supplying a gift sample of abscisic acid.
References
Abdel-Gawad, H. A., Ketellapper, H. J. : Regulation of growth, flowering and senes-
cence of squash plants. II. Effects of 2-ehloroethanephosphonie acid (Ethrel) and
abscisic acid. Plant Physiol. 44, Suppl., 15 (1969).
Addieott, F. T., Lyon, J. L.: Physiology of abscisie acid and related substances.
Ann. Rev. Plant Physiol. 20, 139-169 (1969).
Evins, W. H., Varner, J. E. : Hormone-controlled synthesis of endoplasmic reticulum
in barley aleurone cells. Proc. nat. Acad. Sci. (Wash.) 68, 1631-1633 (1971).
Herich, R. : Gibberellin and sex differentiation of flowering plants. Nature (Lond.)
188, 599-600 (1960).
Heslop-Harrison, J., Heslop-I-Iarr'mon~ Y. : Studies on flowering plant growth and
organogenesis. IV. Effects of gibberellic acid on flowering and the secondary
sexual difference in stature in
Cannabis sativa.
Proc. roy. Irish Aead. B 61,
219-231 (1961).
Mohan Ram, H. Y., Jaiswal, V. S. : Induction of female flowers on male plants of
Cannabis sativa L.
by 2-chloroethanephosphonie acid. Experientia (Basel) 26,
214-216 (1970).
Mohan Ram, H.Y., Jaiswal, V. S.: Feminization of male flowers of
Cannabis
sativa
L. by a morphactin. Naturwissenschaften 58, 149-150 (1971).
H. Y. Mohan Ram
Department of Botany
University of Delhi
Delhi 7
India
... Male flowers do not form when plants are treated with abscisic acid (ABA), which inhibits the action of GA, so plants treated with GA followed by ABA do not form male flowers (Ram & Jaiswal, 1972). Cytokinin induces feminization in genetically male C. sativa individuals (Gerashchenkov & Rozhnova, 2013). ...
... Cytokinin induces feminization in genetically male C. sativa individuals (Gerashchenkov & Rozhnova, 2013). Ethephon has been used to produce female flowers on male C. sativa plants by releasing ethylene and manipulating auxin levels, with treated male plants forming female flowers that resemble natural female flowers and produce seeds after pollination (Ram & Jaiswal, 1972, 1970. Early ethephon treatment, shortly after primordia formation, maximizes the effect on female flower formation (Moon et al., 2020). ...
... Converted males or females treated with different chemicals or hormones may show differences in their phenotypes compared to genetically male or female individuals. For example, although male plants showed no change in sex expression when treated with GA, female plants showed internodal elongation besides the production of male flowers, and the induced male flowers had normal stamens (Ram & Jaiswal, 1972). Treating the plants with either silver thiosulfate, colloidal silver, or GA had no effect on plant height (Flajšman et al., 2021). ...
A review of sexual strategies in Cannabis sativa L. under genomic and environmental controls
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Core Ideas The understanding of sex determination mechanisms in the dioecious species Cannabis sativa L. is critical for cultivators, considering the high value of female inflorescences in high cannabinoid production, the need of seeds for hemp‐grain production, and the variation of fiber quality between sexes in fiber hemp. Comprehending mechanisms of sex determination is also useful to enhance breeding programs aimed at developing new varieties with desirable traits. Sex expression in C. sativa appears to be primarily controlled by genetic mechanisms through an XY chromosome system. Environmental factors that influence plant hormones contribute to sex expression. Although genetic mechanisms governing sex determination have not been fully elucidated, there are useful molecular tools available for sex identification during the early stages of plant development. A variety of chemical treatments that influence plant hormones have been developed to force females to produce male flowers or males to produce female flowers. These various chemical treatments that induce female plants to produce pollen enables these plants to fertilize themselves or other females and generate seeds devoid of a Y chromosome, known as feminized seeds. While monoecious individuals appear to have XX chromosomes, the differences between those monoecious X chromosomes that have not yet been assembled, and those from females, remain unknown. The Y chromosome, which is the largest in the genome possibly due to repetitive content and rearrangements, remains to be assembled; thus the genes it harbors remain unknown. The genetic factors that determine sexual strategy and expression in C. sativa are yet undetermined.
View
... In one of the experiment reported by Mohanam and Sett (1982) (35) applied 50, 100, and 150 mg of silver nitrate and 25, 50, and 100 mg of silver thiosulfate (STS) to shoot tips of female cannabis plants [37,[56][57][58][59][60][61][62][63][64]. Both silver compounds successfully evoked the formation of male flowers, but STS was more effective than AgNO3 [37,[56][57][58][59][60][61][62][63][64]. 100 mg of STS caused the highest number of fully altered male flowers, which was significantly higher than the number of reduced male, intersexual, and female flowers [37-56-64]. ...
... In one of the experiment reported by Mohanam and Sett (1982) (35) applied 50, 100, and 150 mg of silver nitrate and 25, 50, and 100 mg of silver thiosulfate (STS) to shoot tips of female cannabis plants [37,[56][57][58][59][60][61][62][63][64]. Both silver compounds successfully evoked the formation of male flowers, but STS was more effective than AgNO3 [37,[56][57][58][59][60][61][62][63][64]. 100 mg of STS caused the highest number of fully altered male flowers, which was significantly higher than the number of reduced male, intersexual, and female flowers [37-56-64]. On the other hand, the treatment of shoot tip with 100 mg of AgNO3 resulted in more than half the lower number of male flowers, with the highest amount of AgNO3 (150 mg) being ineffective in altering sex expression [37-56-64]. ...
... On the other hand, the treatment of shoot tip with 100 mg of AgNO3 resulted in more than half the lower number of male flowers, with the highest amount of AgNO3 (150 mg) being ineffective in altering sex expression [37-56-64]. Furthermore, pollen from all induced male flowers was viable in vitro and also successfully induced seed set [37,[56][57][58][59][60][61][62][63][64]. More recently, two other studies have also successfully used silver thiosulfate (STS) to induce male flowers [37,[56][57][58][59][60][61][62][63][64]. ...
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Cannabis sativa L. is a dioecious wind-pollinated, although monoecious plants (male and female flowers on same plant) can occur in some population or developed by breeding efforts. Monoecious species have many advantages in the agricultural and industrial sectors due to the self-pollination and minimum variation in cannabinoid content from generation to generation. Monoecious species of cannabis also play an important role in the production of feminized seed production. Production of all female seed requires induction of female plants to develop male flowers that produce genetically female pollen. The aim of the cannabis commercial growers would like to access the feminized seed to produce all-female crops. Exogenous application of plant growth regulators can modify or reverse sex morphology in plants. There are a number of solutions that can be sprayed on female plants to create male pollen sacs: benzothiadiazole, gibberellic acid, silver thiosulphate, silver nitrate, and colloidal silver. Silver thiosulfate (STS) is the most effective compound for inducing sex reversal and feminizing seeds in high-THC cannabis cultivars. A hermaphrodite flower is one that has both staminate (male) and pistillate (female) floral structures on the same plant. A hermaphrodite flower is in the form of banana-shaped male anthers that emerge from the female flowers in small bunches, changing from green to a pale yellow as they develop. However, plants grown from feminized seeds are more likely to become hermaphrodites. The development of male anthers within the female inflorescence is known as hermaphrodite. Hermaphroditism is due to physical or chemical stress.
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... It has been shown that low levels of light exposure during the dark period can affect floral development in C. sativa (Kusuma, Westmoreland, Zhen, & Bugbee, 2021), but no study can confirm that hermaphroditism is one of the effects. Some studies suggest that the plant growth hormone gibberellin causes hermaphroditism in C. sativa (Galoch, 1978;Mohan Ram & Jaiswal, 1972;Sarath & Mohan Ram, 1979). ...
... Gibberellins have been known to alter sex ratios in favour of males in monecious cucurbits (Heslop-Harrison, 1972;Gupta & Chakrabarty, 2013), and several studies have demonstrated that exogenous gibberellin application induces hermaphroditism in female C. sativa (Galoch, 1978;Mohan Ram & Jaiswal, 1972;Sarath & Mohan Ram, 1979). Galoch (1978) found that the application of either ethrel or kinetin induced female flowering on genetically male C. sativa and reduced hermaphroditism on female C. sativa caused by gibberellin application. ...
... Galoch (1978) found that the application of either ethrel or kinetin induced female flowering on genetically male C. sativa and reduced hermaphroditism on female C. sativa caused by gibberellin application. Mohan Ram & Jaiswal (1972) found abscisic acid to have similar feminizing effects, with the addition of gibberellins reversing them. Further research is required to determine whether these results are due to the antagonistic relationship between gibberellins and "feminizing" growth hormones documented in Galoch (1977) and Mohan Ram & Jaiswal (1972). ...
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... Phytohormone manipulation has also been documented in C. sativa, with the use of the ethylene-blocking chemical, silver thiosulfate to initiate the generation of male flowers producing genetically female pollen [13]. Similarly, the phytohormone gibberellic acid (GA) has also been demonstrated to modify sex expression in female C. sativa plants, with abscisic acid (ABA) ameliorating the effects of GA in a dosage-dependant manner [14]. Concentrations of the main psychoactive cannabinoid in C. sativa, ∆-9-tetrahydrocannabinol (THC), along with several upstream precursors and secondary metabolites, were modulated by the application of exogenous ABA [15]. ...
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... For example, application of GA 3 can either inhibit or promote flowering depending on the plant species, as well as impacting sex determination of flowers in some monoecious and dioecious plants [27]. In the case of cannabis, GA 3 has been found to induce male flower development in genetically female plants [28]. Further, the GA 3 biosynthesis inhibitor PBZ, has been found in illicit cannabis samples where it is used to promote earlier and heavier flowering [29]. ...
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... The concentration of GA solution was 50 μg/l. Female plants were sprayed three times before f lowering and the whole treatment lasted 10 days [36] . GA solutions were applied to the F 1 female plants on the first, fifth, and tenth days, after which the plants began producing male f lowers at the newly formed nodes. ...
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... Additionally, dioecious cannabis genotypes have been known to display hermaphroditism (Monthony et al. 2021c), reversion from a sexual to vegetative state (Monthony et al. 2021a), and sometimes complete changes in sex expression in response to certain environmental factors and chemical Communicated by Lars Ostergaard . reagents (Schaffner 1923;Heslop-Harrison 1956;Ram and Jaiswal 1972;Mohan Ram and Sett 1982;Lubell and Brand 2018;Moon et al. 2020a). Taken together, these observations suggest that cannabis sex is plastic and not strictly controlled by its XX or XY chromosomal complement. ...
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Background: Spinach (Spinacia oleracea L.) is an important leafy vegetable with dioecious and occasional monoecious plants. Monoecious lines are more suitable for hybrid production than dioecious lines due to their extended flowering period. However, genetic research on the sex determination of monoecism remains limited. Methods: In this study, RNA-seq analysis of monoecious and female spinach plants was performed at two distinct flowering stages. In total, we identified 4586 differentially expressed genes (DEGs), which were primarily involved in biological processes such as hormone signaling, cell wall biosynthesis, photosynthesis, and flower development, based on Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results: Among these DEGs, 354 transcription factors, including 27 genes associated with the ABCDE gene, were discovered. Furthermore, a co-expression gene regulatory network was built, identifying nine key genes that play important roles in regulating sex differentiation between female and monoecious plants. Conclusions: Our findings provide crucial molecular insights into the mechanisms of monoecism in spinach and offer a scientific basis for future spinach breeding.
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Ethephon's effects on sexual expression and cannabinoid production in monoecious and dioecious varieties of hemp (Cannabis sativa L.): A field trial
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  • Feb 2024
  • IND CROP PROD
In pharmaceutical cannabis (Cannabis sativa L.) crops sex distribution plays a pivotal role in influencing the yield and quality of inflorescences. Male plants or inflorescences do not produce sufficient concentrations of key bioactive compounds, including cannabinoids and terpenes; moreover, pollination significantly reduces the quality of female inflorescences. Existing studies suggest that ethephon may be effective in feminizing hemp crops and altering their canna-binoid content. However, comprehensive research is essential to fully understand ethephon's specific effects on hemp. This study, incorporating a two-year field trial, was conducted to assess the effects of ethylene-releasing ethephon on sex expression and the concentrations of main cannabinoids in monoecious, dioecious, cannabi-diolic, and cannabigerolic hemp varieties. Aqueous ethephon solutions of 420, 840, 1240, and 1660 µM m − 1 were administered biweekly via foliar spraying during the flowering period. The trial incorporated three varieties of hemp: Felina 32 (monoecious, cannabidiol dominant chemotype), Santhica 27 (monoecious, cannabigerol dominant chemotype), and Kompolti (dioecious, cannabidiol dominant chemotype). All treatments significantly (p < 0.05) increased the number of fully female phenotypes and reduced the number of monoecious phenotypes in monoecious varieties Felina 32 and Santhica 27. Almost all but a few phenotypes turned into 100% female flower phenotypes. However, these treatments did not produce feminizing effects on either 100% male phenotypes in monoecious varieties or male plants in the dioecious variety Kompolti. Almost all concentrations of ethephon significantly (p < 0.05) altered the content of dominant cannabinoids in the inflorescences of Felina 32, Kompolti, and Santhica 27 varieties. However, the effect of the 840 µM treatment on the Santhica 27 variety was not statistically significant. The alteration in cannabinoid production did not correlate with the increased number of female plants. All treatments significantly (p < 0.05) decreased the height of plants in all varieties.
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Gibberellin and Sex Differentiation of Flowering Plants
Article
  • Nov 1960
  • NATURE
THOUGH work on the influence of gibberellin on flowering of plants has been given much attention, up to now very little is known, about the relation of gibberellin to sex differentiation of plants.
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Hormone-Controlled Synthesis of Endoplasmic Reticulum in Barley Aleurone Cells
Article
  • Aug 1971
The rate of synthesis of the endoplasmic reticulum in barley aleurone cells after treatment with gibberellic acid was determined by measurement of [(14)C]-choline incorporation into acid-insoluble material in a semipurified fraction containing the endoplasmic reticulum. 94% of the (14)C incorporated into this fraction is extractable by lipid solvents and only 9% is removed by procedures for nucleic acid extraction. Gibberellic acid increases the rate of synthesis of the endoplasmic reticulum 4- to 8-fold, starting about 4 hr after addition of the hormone (at about the same time as polysome formation). Abscisic acid inhibits this gibberellic acid-enhanced increase in the rate of synthesis of endoplasmic reticulum.
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Induction of female flowers on male plants ofCannabis Sativa L. by 2-chloroethanephos-phonic acid
Article
  • Feb 1970
  • Experientia
Zusammenfassung Ethrel induziert auf männlichen Hanfpflanzen (Cannabis sativa L.) weibliche Blüten, welche nach der Bestäubung Früchte ausbilden.
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Heslop-I-Iarr'mon~ Y. : Studies on flowering plant growth and organogenesis. IV. Effects of gibberellic acid on flowering and the secondary sexual difference in stature in Cannabis sativa
  • Jan 1961
  • 219-231
  • J Heslop-Harrison
Heslop-Harrison, J., Heslop-I-Iarr'mon~ Y. : Studies on flowering plant growth and organogenesis. IV. Effects of gibberellic acid on flowering and the secondary sexual difference in stature in Cannabis sativa. Proc. roy. Irish Aead. B 61, 219-231 (1961).
Regulation of growth, flowering and senescence of squash plants. II. Effects of 2-chloroethanephosphonic acid (Ethrel) and abscisic acid
  • Jan 1969
  • 15
  • H A Abdel-Gawad
  • H J Ketellapper
  • H. A. Abdel-Gawad
Abdel-Gawad, H. A., Ketellapper, H. J. : Regulation of growth, flowering and senescence of squash plants. II. Effects of 2-ehloroethanephosphonie acid (Ethrel) and abscisic acid. Plant Physiol. 44, Suppl., 15 (1969).
Studies on flowering plant growth and organogenesis. IV. Effects of gibberellic acid on flowering and the secondary sexual difference in stature in Cannabis sativa
  • Jan 1961
  • 219
  • J Heslop-Harrison
  • Y Heslop-Harrison
  • J. Heslop-Harrison
Heslop-Harrison, J., Heslop-I-Iarr'mon~ Y. : Studies on flowering plant growth and organogenesis. IV. Effects of gibberellic acid on flowering and the secondary sexual difference in stature in Cannabis sativa. Proc. roy. Irish Aead. B 61, 219-231 (1961).