Morphogenesis of floral buds of cucumber cultured in vitro

DEVELOPMENTAL

BIOLOGY,

Morphogenesis

6, 370-387

of Floral Buds of Cucumber in Vitro1

ESRA GALUN,’ Division

( 1963)

Cultured

YVONNE JUNG, AND ANTON LANG

of Biology, Ca&fosniu Institute Pasadena, California Accepted

February

of Technology,

18, 1963

INTRODUCTION

Sex expression of flowering plants was studied quite extensively in the past, and a considerable amount of information both on the genetic basis of sex determination (see review by Westergaard, 1958) and on its modification by environmental conditions and by chemical agents, mainly plant growth regulators, has accumulated 1957; Lang, 1961)) but we know (see reviews by Heslop-Harrison, little about the underlying physiological mechanisms. This state of affairs is clearly exemplified in cucurbits, where we know that temperature and photoperiod modify sex expression (Nitsch et al., 1952; Ito et al., 1954) and that applied growth regulators have similar effects (Laibach and Kribben, 1950; Galun, 1959a,b; Peterson and Anhder, 1960; Mitchell and Wittwer, 1962), but we do not know whether there is any interaction between the effects of environment and those of the applied regulators, nor whether natural hormones, similar in action to the applied chemicals, act directly on the floral buds or indirectly, by causing some changes in the plant as a whole. With respect to the last problem, it is especially desirable to isolate the floral bud from the rest of the plant in order to study its reaction to chemicals supplied directly, through the culture medium. The cucumber floral bud seems to be well adapted to such a study as it is bisexual up to a certain, early stage of its ontogeny (Atsmon and Galun, 1960) and its future sex can be controlled by chemical treat’ This work was in part supported by grants G-16408 and G-17483 from the National Science Foundation. *Present address: Plant Genetics Section, The Weizmann Institute of Science, Rehovoth, Israel. 370

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ment of the whole plant, given at precise stages in the development of the young bud (Galun, 1961a). Moreover, in this species there exist genetic lines of monoecious, gynoecious (purely female), and hermaphrodite plants (Galun, 1961b). When grown under appropriate environmental conditions, the monoecious types will produce on their main stem only male (staminate) flowers, at least for a certain period of time. The gynoecious types produce only female (pistillate) flowers; the hermaphrodite produce bisexual flowers in certain positions. One can therefore predict, even at the earliest, bisexual bud stages, what the sex of a bud will be if the latter is left attached to the plant. On the basis of these considerations, we have studied the morphogenesis of cucumber Aoral buds and its control by certain growth substances in an in vitro system. This study had to be preceded by the establishment of conditions suitable for the growth and development of the buds on sterile media, as previous experience in this field was lacking. The present paper will give information on a method for growing floral buds of cucumber on a culture medium; on the morphogenetic effects of indoleacetic acid (IAA) and gibberellie acid (GA,,) on potentially male, potentially female, and potentially hermaphrodite buds; and on the effect of stage of development of the excised bud on its development and its reaction to auxin. A short note on part of this study was published (Galun et al., 1962). MATERIAL

AND

METHODS

Plant muterid. Three monoecious varieties of cucumber (Cucumis sativus L.) were used: Long Marketer, Beit-Alpha, and Yorkstate Pickling (YSP); the first two served for preliminary tests only, and most of the work reported here was done with an inbred line of YSP. The female plants were all of an inbred, homozygous gynoecious line of YSP (reproduced with the aid of GA,), and the hermaphrodite plants were of our own inbred stock. Plants were grown in the Earhart and Campbell Plant Research Laboratories, the hermaphrodite ones under short-day conditions (8 hours of natural light daily, 8 A.M. to 4 P.M.), the monoecious and female ones under long-day conditions (16 or 18 hours of light: natural light extended with incandescent or fluorescent light of approximately 20 foot-candles; total light period 4 A.M. to S and 10 P.M., respectively). The daily temperature regime was in either case

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23°C from 8 A.M. to 5 P.M. and 19” from 5 P.M. to 8 A.M. When cucumber plants are grown under these conditions, the monoecious plants bear only male flowers on the first 20 nodes, the gynoecious plants produce only female flowers, and at least the first flower in each leaf axil of a hermaphrodite plant is hermaphrodite. The plants were grown in 200-ml plastic cups filled with a mixture of equal volumes of gravel and vermiculite; they were watered once daily with half-strength Hoagland nutrient solution, and in addition with deionized water as needed. Seedlings were harvested 3 weeks after planting. All leaves larger than 1 cm were removed, the stems were placed in test tubes half filled with sterile water, and the plants were taken to the transfer room. Culture medium. The composition of the standard (St) culture medium was developed empirically-starting with White’s basal medium as given by Henderson et al. (1952)-through the addition of organic compounds that proved to be of advantage. As these tests were not made systematically with all possible combinations, the medium should not be considered as optimal. The growth rate was inferior to that in situ. However, the medium does support growth of cucumber floral buds isolated at early, bisexual stages, when potentially male, female, and hermaphrodite buds are morphologically exactly alike, to a stage when their sex is clearly determined. In 20 days’ culture, the buds reached a size of up to approximately 1 cm. It may be mentioned that pale buds seemed to develop less well than pigmented ones, and this paling was prevented by the addition of kinetin. This kinetin effect may be comparable to that found by Richmond and Lang (1957) in detached cocklebur leaves. The stock solutions and the amounts thereof used for the St medium are listed in Table 1. The St medium included also 150 ml of coconut milk, 20 gm of saccharose, and 8 gm of agar per liter. In preparing the medium, filter sterilization in a way similar to that of Ball ( 1961) was used (autoclaved coconut milk seemed to promote callus rather than organized growth). Solutions I through VII as well as the coconut milk were passed through a type S-l, Seitz filter and glass-distilled water was added to make one-fourth of the total volume of the medium. Agar and sugar were dissolved in glass-distilled water to make up the remaining three-fourths volume of the medium and were autoclaved. For making the St medium the autoclaved and the filtered parts were mixed when the former had cooled to about 50°C

MORPHOGENESIS

OF

CUCUMBER

TABLE COMPOSITION OF THE STANDBRD OF ISOLATED CUCUMBER

Components ~-~__~ Ca(NO&4 Hz0 KNO3 NaH,P04.H& Na2S04 MgSO,.i H,O KC1 Ferric citrate MnSOa4 H,O ZnSOa.7 IT,0 HaBOa Glycinc~ Niacin Pyridoxinc Thiamine Tryptophan Kin&in Casein hydrolysate Coconut milk Saccharoso Agar

Stock solution

I I II II III III IV IV IV IV v v v v V VI VII

FLOWERS

W

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1 PVI~~DIUM FOR CIILTURI~ FLORAL Buns

concentratKm

Concentration

~___-

8.0 gm/l 3.2 gm/l 0. i gm/l 8.0 gm/l 14 4 gm/l 2.6 gm/l 3.0 gm/l 4.5 gm/l 1.5 gm/l 1.5 gm/l 0.30 gm/lOO 0.05 gm/lOO 0.01 gm/lOO 0.01 gm/lOO 0.50 gm/lOO 0.01 gm/lOO lOc,o

per liter

_~..- ~~ 25 25 25

1

ml ml ml ml ml ml

I

1 2

200 mg 80 mg 17.5 mg 200 mg 360 mg 65 mg 2.0 mg 1.5 mg 1.5mg 1.5 mg 3.0 mg 0.5 mg 0.1 mg 0.1 mg 5.0 mg 0.1 mg 0.02y~ 150 ml 20 gm 8w

and were poured into the culture vials. Au toclaving of GA, reduced its biological activity to less than one-fifth, as judged by a pea seedling bioassay developed in this laboratory (E. Reinhard and A. Lang, unpublished data). Therefore stock solutions of 100 mg/l GA, and IAA were filter sterilized. The supplemented media were then prepared by pipetting the GA, and/or IAA solutions into the filtered part of the nutrient solution and then adding the appropriate amount of warm agar-sugar solution, to make the same final concentrations as in the St medium. Culture and staining methods. Transfer work was performed in a room sterilized by an ultraviolet germicidal tube. Contamination of the buds was eliminated by immersing the upper part of the seedling for 4 minutes in 1% sodium hypochlorite, followed by dipping in ethanol and several rinsings with sterile water. The buds were then dissected out under 20 or 40 times magnification, and planted in

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pairs ( buds of similar size) in the slanted agar ( -10 ml) in screwcap 2.5 X 6-cm vials. During incubation, the cultures were kept at 25”-26”C, 12 hours’ light (white fluorescent tubes, intensity about 160 foot-candles) for 20 days, At the termination of culturing, the buds were either dissected and the results recorded visually, or the buds were fixed in Randolph’s modified Navashin fluid, dehydrated by the “short” Randolph (1935) method, and stained by Ehrlich’s hematoxylin-erythrosin method, RESULTS

Potentially

Male Buds

As pointed out above, one can predict into which flower type a sexually undetermined embryonic bud will develop, according to the genotype of the plant, the environmental conditions under which the plant is grown, and the site (node) from which the bud is taken. The embryonic buds of the first 20 nodes of long-day grown monoecious plants will thus be further designated as potentially male buds. Potentially male buds were isolated from the 6th to 10th node of monoecious plants. Usually two buds of succeeding nodes were isolated from each seedling. One bud 0.6-0.7 mm in diameter (Fig. 1) was taken from the lower node, and another bud of 0.5 mm or less in diameter from the node above it (Fig. 9). The first type of bud will be referred to as “Large” ( L), the second as “Small” ( S ) . The buds were cultured either in St medium with O-0.1-0.3-1.0 mg/l IAA, or with O-0.3-1.0-3.0 mg/l GA,, or with the two growth substances combined in all possible combinations, i.e., altogether 16 different treatments. The cultures were started with 12-16 L and S buds each, per treatment. Lower concentrations of both GA, and IAA were also used, but as these treatments did not cause visible effects, compared to St alone, they will not be mentioned further. The results are summarized in Table 2. They are based on photographs of microslides prepared at the termination of culturing. It will be noted that the data are not based on the full number of buds cultured initially. This loss of buds was due to contamination during the isolation and culturing and to damage during microslide preDaration. The L type floral buds may be considered first. From Fig. 1, it can

FIGS. l-16. Potentially male buds of stages L or S. All enlarged x23. C, calyx (sepal) primordia; P, petal primordin; S, stamen primordia; 0, incipient ovary ( pistil ) . FIGS. l-8. Stage L. Fig. 1, at beginning of culturing; h’igs. 2-8, after 20 days of culturing in St medium (Fig. 2 ), in 0.3 m~/l GA3 (Fig. 3 ), in 1.0 mg/l GA., (Fig. 4), in 3.0 mg/l GA, (Fig. 5), in 0.1 mg/l IAA (Fig. B), in 0.3 mg/l IAA (Fig. 7), and in 1.0 mg/l IAA (Fig. 8). FIGS. 9-16. Stage S. Fig. 9, at beginning of culturing; Figs. lo-16 aftcl 20 days of culturing in St medium ( Fig. lo), in 0.3 mg/l GA, (Fig. 11)) in 1.0 mg/l GA., (Fig. 12), in 3.0 mg/l GA, (Fig. 13), in 0.1 q/l IAA (Fig. 14), in 0.3 mgil IAA ( Fiz. 15), and in 1 .O mg/l IAA (Fig. 16 ). 375

376

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OVARY

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AND

TABLE

2

LANG

FORMATION IN POTENTIALLY MALE CUCUMBER FLORAL AND CULTURED AT Two STAGES OF DEVELOPMENT DIFFERENT CONCENTRATIONS OF IAA AND GA, Floral buds of stage IJ Concentration of IAA (mg/l)

Concentration of GAP

(WC/l)

NIXE

TQ

0.1

Ob

T

0.3

0

T

T

ISOLATED

Florsl buds of stage S Concentration of IAA (mg/l) I .o

0

BUDS IN

NIXW

0

0.1

0.3

1.0

T

0

T

0

T

0

T

0

9 13

8 5

lti 12

15

8

8

9

9

4 3 2

2

9 6

7 7 5

5

83 10 2

3 2 ---

-

None

11

0.3

L4 8 T-

1.0 3.0

-

13

12

5

8 lc 8 9 5 5-7-j-

* Total number of buds. b Number of buds which developed c Slight development of ovary.

27 1” 9

-

6

-

-

1

7

1

5

2

ovaries.

be seen that at time of isolation such buds have clearly developed sepals and petals as well as spherical stamen primordia, while the future pistil is represented by only a small depression at the inner base of the bud. When such buds were grown on St medium or on any of the GA, media, this depression was not developed further and no ovary development could be observed (see Figs. 2 through 5). But when grown in St with 0.1 mg/l IAA most of the buds developed a well-advanced ovary (Fig. 6). Such ovaries were quite similar to those developing in intact and cultured female buds. Higher concentrations of IAA did not promote ovary formation further; in fact, at 1.0 mg/l IAA, ovary production was found in only one out of 7 cultures (Figs, 7 and 8). Whenever combinations of IAA and GA, were used the effect of IAA in promoting ovary development was completely nullified. The S buds seemed to behave differently when cultured. In buds of this size (Fig. 9), stamen primordia are not yet spherical and there is no depression at the site of the future pistil. In almost all treatments, including unsupplemented St medium, some S buds developed ovaries. The addition of GA, to the medium seemed to reduce the tendency of ovary formation in a way similar to the L buds. However, GA, did not promote stamen formation, and when high GA, concentrations were added to the medium the stamen primordia were changed into long, slim structures without any apparent change in the number of cells (Figs. 12 and 13).

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In both L and S buds it was observed that whenever development of the ovary started, further development of the stamen primordia was completely arrested. But even when the ovary did not develop and stamen primordia started to grow (as on St medium and on low GA, concentrations) stamen development did not reach a very advanced stage. From the comparison of L and S buds it was quite apparent that the stage of development at which the buds are removed from the plant is decisive for their future development in isolated culture. In order to investigate this further, the same range of buds was divided into four groups, I through IV, and the buds were cultured on St medium without IAA or with 0.1 mg/l IAA. Drawings of longitudinal sections of these buds, showing their average diameters, are shown in Fig. 17, and the results of the culture experiments are summarized in Table 3. T:ZBLE 'lb

EFFECT

ON THEIR S$Gr;pf brld

OF

STAGE

AT

DEVEI,~PMENT

With or without 0. ;~p

Total number of budr cultured

WHICH

Ix \-cry slight or no growth

3

POTENTIALLY MALE IA.4 -END ST CCLTURE

Br:~s

WERE

ISOLZTED,

MEDI~~M*

Number of buds with doveloped ovary --~ Slight Fair Adranwd dewlopme~t devrlopment developmalt

Number of bud9 with d2veloq d stamens

* Buds were dissected after 20 days of culturing.

Examining first the results with buds of different size grown on St medium without IAA, it may be observed that stage I buds, the smallest buds with barely developed stamen primordia, never developed stamens. They either showed only slight or no further development of any kind, or started to develop ovaries. Of the stage II buds, which had clearly visible stamen primordia at the start of culturing,

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w J--

0.75 mm ___(

E

0.45 mm +

FIG. 17. Semidiagrammatic used in the test on the effect

k

0.6mm

t0.3mm

StageDI -{

-/

drawings of four stages of potentially of stage at isolation on development

male buds in culture.

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some produced stamens, but most of them developed ovaries. When buds of a still more advanced stage (111) were cultured on St medium, only 3 out of 16 showed slight development of ovaries while the rest developed stamens. Buds of the most advanced siagc’ (WI all developed stamens and no ovaries. The addition of 0.1 mg/l IAA to the St medium does not seem to change the future development of stage IV buds-all but one produced stamens, thus behaving like stage IV bucls grown on unsupplemented medium. The two youngest stages, I and II, also did not show a very pronounced reaction to the addition of IAA, although the data may indicate a slight further shift toward female development. On the other hand, when buds of stage 111 were grown on IAA-supplemented medium they showed a clear change toward ovary development as compared to stage III buds on ausin-fret> medium. These entire results are in line with those of the first experiment, showing that smaller buds have a tendencv to develop ovaries on unsupplemented media, and that IAA may change the sex of an isolated potentially male bud of a certain stage. The second experiment shows furthermore that the earlier the stage at which potentially male buds are isolated and cultured on St medium the greater is their tendency to develop ovaries. It may also be pointed out that there was never a simultaneous development of both ovary and stamens in the same floral bud, even when IAA, GA,, or a combination of both were added to the medium. Potentially

Female Buds

Such buds were dissected out of gynoecious (female) plants which, unless treated with gibberellin, produce only female flowers. The procedure of isolation and culturing was as in the first experiment with potentially male buds and sections of L and S buds at the beginning of cultivation are shown in Figs. 18 and 26, respectively. No difference between potentially male and potentially female buds could be detected at these stages. Twelve buds of each stage were cultured on St medium and on any of the 15 media with IAA, GA,, or a combination of the two. After 20 days’ culture, the buds were fixed and permanent slides were prepared. In general no treatment caused a basic change in the future development of the buds. Buds cultured on St medium or on one of the IAA media all showed normal development as in intact buds. The addition of GA, caused some distortions and sometimes breakdown of the ovary tissues.

FIGS. 18-33. Potentially female buds of stage L or S. FIGS. 18-25. Fig. 18, at beginning of culturing; Figs. 19-25, after 20 days of culturing in St medium (Fig. 19), in 0.3 mg/l CA3 (Fig. 20), in 1.0 mg/l GA, (Fig. 21), in 3.0 mg/l GA, (Fig. 22), in 0.1 mg/l IAA (Fig. 23), in 0.3 mg/l IAA (Fig. 24)) and in 1.0 mg/l IAA ( Fig. 25). FIGS. 26-33. Stage S. Fig. 26, at beginning of culturing; Figs. 27-33, after 20 days of culturing in St medium (Fig. 27), in 0.3 q/l GAa (Fig. 28), in 1.0 mg/l G& (Fig. 29), in 3.0 m&l CA, (Fig. 30), in 0.1 mg/l IAA (Fig. 31), in 0.3 mg/l IAA (Fig. 32), and in 1.0 mg/l IAA (Fig. 33). 380

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The main results are illustrated by sections of the cultured buds in Figs. 18 through 33. Variability among buds of the same stage and treatment was very small, and the figures are a good representation of the results. Combinations of IAA and GA, did not give additional information; the results were similar to those with addition of GA,, alone to the medium. Potentially

Hermaphrodite

Buds

Such buds were the first buds in the leaf axils of hermaphrodite plants. The layout of the culture experiment with these buds was like the one of the first experiment with potentially male buds and the experiment with potentially female buds: 12 L and 12 S buds were cultured on unsupplemented St medium and one each of the 15 media with additions of IAA and GA, or both, followed by fixation and preparation of permanent slides. The main results are illustrated by representative photomicrographs (Figs. 34 through 49). As with potentially female buds, variability among the cultured buds was small and the figures are a good representation of the results. Combinations of IAA and GA, added to the medium gave results similar to those of GA,, alone. The L and S buds at the beginning of culturing do not seem to differ from potentially male or potentially female buds of the same stage. No one of the treatments caused any basic change in the morphogenesis of the bud; the buds grew into clearly hermaphrodite buds with well-developed ovaries and stamens. Nevertheless, the result is of interest in several respects. The lack of mutual exclusion of stamens and ovary is maintained in the isolated bud just as in the attached flower. The absence of any inhibition of stamen development by IAA, even at quite high concentration (1.0 ppm) is obvious and may be of interest in considering its effect on the morphogenesis of florai buds in general. Stamen development in cultured potentially hermaphrodite buds -excluding high-GA, media-is more advanced than in the potential male buds, GA, at 1.0 and 3.0 mg/l caused a considerable distortion of the floral bud tissue. This distortion was especially pronounced in buds cultured at a younger stage (S) and the ovary seemed to be affected most strongly, although the anthers were deformed as well. In contrast, IAA (at the concentrations used) caused no visible change in the normal development of the buds, DISCUSSION

4part from a short note on a part of the present study (Galun et al.,

FIGS. 3449. Potentially hermaphrodite buds of stages L or S. Stage L. Fig. 34, at beginning of culturing; Figs. 35-41 after FIGS. 34-41. 20 days of culturing in St medium (Fig. 35), in 0.3 mg/l GA, (Fig. 36), in 1.0 mg/l GA:, (Fig. 37), in 3.0 mg/l GA, (Fig. 38), in 0.1 mg/l IAA (Fig. 39), in 0.3 mg/l IAA (Fig. 40), and in 1.0 mg/l IAA (Fig. 41). FIGS. 42-49. Stage S. Fig. 42, at beginning of culturing; Figs. 4349, after 20 days of culturing in St medium (Fig. 43), in 0.3 mg/l GA, (Fig. 44), in 1.0 mg/l GA, (Fig. 45), in 3.0 mg/l GA,, (Fig. 46), in 0.1 mg/l IAA (Fig. 47). in 0.3 mg/l IAA (Fig. 48), and in 1.0 mg/l IAA (Fig. 49). 382

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1962) and a recent abstract (Tepfer et al., 1962), no previous report on floral bud culture has come to our knowledge. Flowers at anthesis or a later stage, or parts from such flowers, were cultured successfully and in some cases viable seeds were produced in culture (Nits&, 1951; Maheshwari and Lal, 1958). Stem tips of flowering plants were also successfully grown in culture, and under certain conditions formation of flower buds was induced by certain treatments (Butenko, 1960; Chailakhyan ct al., 1961; Raghavan and Jacobs, 1961; Baldev, 1962). In the present studv it was shown that isolated floral buds of cucumber can be grown on agar medium from an early embryonic stage to quite an advanced one, thus providing a new tool for experimental studies on the morphogenesis of flowers. Our results further show that the development of the isolated cucumber floral bud may be determined by the genotype of the plants, the stage at excision, and in certain cases by the addition of growth substances to the culture medium. Floral buds of monoecious and gynoecious cucumber plants, which carry the gene A, although they are initiated as bisexual organs, develop along strictlv alternative pathways, becoming either male or female flowers. These plants contain evidently some kind of trigger mechanism which permits development of either stamens or the pistil (ovary), but no simultaneous development of both. In our experiments with buds of A plants, we obtained typical development of female flowers, and reasonably advanced development (i.e., development to a stage which left no doubt about the sex type) of male flowers from embryonic buds of A plants, hut in no case did we observe development of a hermaphrodite flower, or even a flower tending to the hermaphrodite state. Thus, the trigger mechanism is retained in isolated floral buds of A plants. However, the path of development which an excised bud from an A plant is going to take can to some extent be determined by an external chemical factor, namely, auxin. Hermaphrodite plants which lack the A gene also are lacking in the trigger mechanism, at kdSt in part of their buds, namely, those formed first in a leaf axil under short-day conditions. Floral buds of these plants and from these positions not only form both stamens and pistil, but the absence of the trigger mechanism seems to make them more stable against modifying influences on their development. The same anxin concentrations in the culture medium which forced

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isolated potentially male buds from monoecious plants to develop into female buds had no such effect on hermaphrodite buds isolated and treated at the same developmental stage. The development of potentially male buds from monoecious plants depended on the stage at which the bud was isolated. This fact is at first sight quite surprising. These buds might have been expected to develop all into male flowers when grown on the St medium. In fact buds of young stages tended to develop ovaries; moreover, buds of the youngest stage either did not grow at all or developed into female flower buds, This behavior may be explained in one of two ways. Firstly, the St medium may not be “neutral” with respect to ovary induction. Inclusion of the coconut milk or one of the amino acids (tryptophan?) may result in such an induction, or these substances may be converted in the bud into substances (auxin?) which cause induction. If this speculation is right, the effect of added IAA on the more advanced buds has to be explained by assuming that at this stage stamen development has already been determined and can be counteracted only by a stronger induction of ovary formation, and that this can be achieved by the addition of IAA. Secondly, one may assume the existence of a natural stamen-induction factor in which the St medium is deficient. As pointed out above, stamen development in isolated potentially male buds never approached that of intact ones, whereas the media seemed to supply all the needs for normal ovary formation. Therefore, when the bud is removed from the plant before stamen induction has taken place the development of stamens is prevented and the trigger mechanism controlled by the A gene allows development of the ovary. But when the bud is isolated at a more advanced stage, stamen induction has already started and unless ovary development is promoted by added IAA, stamen development will proceed and the trigger mechanism will prevent ovary formation. In any event, the results with potentially male buds seem to support the idea that IAA is involved in the induction of ovary formation. As ovary induction in plants having the gene A prevents future stamen development, this effect of IAA leads to the production of a female flower. This sequence of events may explain the findings of Laibach and Kribben (195(r), namely, that application of auxins to intact monoecious plants increases the prevalence of female flowers. However, although fitting well into our present information, this interpre-

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tation of the IAA effect should be considered only as one possibility and should be checked in further studies. The effect of GA:, cannot be fully evaluated on the basis of the present data. Although the results seem to indicate that GA, has no direct effect in promoting stamen formation, this finding may be misleading. It is therefore only after a medium which will support normal stamen development has been established that this problem can be studied again, and the effect of GA:, on young potentially female and hermaphrodite buds can be reexamined. But the possibility that IAA acts directly in the bud whereas GA,, affects sex expression more indirectly, either by interacting with IAA or through other parts of the plant, should not be excluded. SUMMARY

Young floral buds of cucumber plants were cultured aseptically on media containing mineral salts, sugar and various organic metabolites and the development of anthers and pistil in these buds was studied. Three types of buds were used: potentially male, potentially female, and potentially hermaphrodite, removed from seedlings of monoecious, gynoecious and hermaphrodite plants, respectively, but at the time of inoculation all buds were at the bisexual stage. Auxin (3-indoleacetic acid) added to the culture medium promoted ovary development in potentially male buds; gibberellic acid counteracted this IAA effect but seemed to have no constructive effect of its own, whether alone or in combination with IAA. Very young potentially male buds tended to form ovaries even without addition of IAA. Isolated potentially female and potentially hermaphrodite buds continued in isolation their normal development and were little affected by IAA as well as GA:,, although GA, at high concentrations seemed to have a deforming effect on the bud (mainly on the ovary tissue). The bearing of these results on the understanding of normal morphogenesis of cucumber flower buds is discussed. REFERENCES ATSAION, D., and GALUN, E. ( 1960). A morphogenetic study of staminate, pistillate and hermaphrodite flowers in Cucrcmis ( t ). Phytomorphobgy 10, 110-115. BALDEV, B. ( 1962). In vitro studies of floral induction on stem apices of cuscuta reflexa-a short day plant. Ann. Botuany (London) [N.S.] 26, 173-180. BALL, E. ( 1961). Cell divisions in living shoot apices. Phytomorph&gy 10,

377-396.

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