E?€¥S§§fif€§fl£fé?flL MED GR‘GW‘TH REGULATOR EFFECTS ON WE MGRPHOLUGY 0F SQUBLE FLGWERENG PETWEA HYBREEA HOW. Thesis for the Degree of M. S. MiCHEGAN STATE UNIVERSITY HELEN NiEN-CHUEN SBU 1969 v , 9"..‘3‘7-5 LIBRAR. 1’ Michigan Sgt: University -4 -————. ‘r' y \ V BINDING av "' "BAG & SflNS’ ABSTRACT ENVIRONMENTAL AND GROWTH REGULATOR EFFECTS ON THE MORPHOLOGY OF DOUBLE FLOWERING PETUNIA HYBRIDA HORT. By Helen Nien-chuen Siu Double flowering in Petunia hybrida Hort, is monogenic dominant to the single flower type. The double genotypes Q and Ed; are generally female sterile whereas single types Q are always fertile. Natarella (1968) has shown that the sterility in the double genotypes is a result of physical hindrance of carpel development by the numerous petal and stamen initials which occur on the receptacle during initiation and early differentiation. It is desirable to restore female fertility in double genotypes for breeding and genetic purposes and doubleness is also a character which can be analyzed for gene action in the control of floral morphology. Thus, these studies were conducted to deter- mine the influence of growth regulator substances and environmental factors on the 2 gene which determines floral doubleness. Preliminary tests were conducted with triiodobenzoic acid (T IBA), tri- chlorobenzoic acid (T CBA), indoleacetic acid (LAA), naphthaleneacetic acid (NAA), estrogen, S-fluorouracil, phenylboric acid and ethrel. Further studies were conducted mainly with TIBA and 1AA; the former at 80, 100, 120 or 140 ppm was found to produce single flower phenocopies in the heterozygous double _D_d_ Helen Nien-chuen Siu but complete reversion and restoration of female fertility in the homozygous 92 was not observed. Indoleacetic acid at 25 ppm and above inhibited growth and flowering and when growth resumed no change in flower morphology was observed. The effect of temperature on pistil formation was studied both in the green- house and in growth chambers. In the summer months, the percent of _D_I_)_ and _D2 flowers with reduced and/or malformed pistils was significantly increased. The presence of pistillate structures in 22 was markedly influenced by the tem- perature in the growth chamber study; again, high temperature favored pistil development. Several possible modes of action of TIBA and auxin interrelation- ships on the l_3__ gene are presented. ENVIRONMENTAL AND GROWTH REGULATOR EFFECTS ON THE MORPHOLOGY OF DOUBLE FLOWERING PETUNIA HYBRIDA HORT. By Helen Nien-chuen Siu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1969 3 C5 C“) | _ _ ‘3 \3‘ " \ s ACKNOWLEDGEMENTS The author wishes to thank Dr. Kenneth C. Sink under whose direction this research was conducted. Acknowledgement and appre- ciation is also extended to the members of the committee: Dr. M. J. Bukovac and Dr. M. W. Adams. I am also grateful to Mr. N. Natarella for his helpful suggestions and encouragement, and Mrs. G. Burldiardt for typing the final thesis. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS. . . . . . ................................. . . . ii LIST OF TABLES. ........................ . ............ . ........ iv LIST OF FIGURES. ...................... ...... v I. INTRODUCTION..... ....................... ....... 1 II. LITERATURE REVIEW. ............... . .................. .. 3 III. MATERIALS AND METHODS ............ . .................. . 9 A. Plant WteriaIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... 9 B. Cultural Procedures .............. ..... 9 C. Experimental DeSIgn ......... 10 D. Growth Regulator Studies 10 E. Greenhouse Environment Study 14 F. Controlled Environment Study 14 G. Low Temperature Study 15 IV. RESULTS..... ..... ............ ..... 16 A. Growth Regulator Studies................. ..... ....... 16 Triiodobenzoic Acid...... ....... ....... 16 'I‘richlorobenzoic Acid..... ..... 23 Indoleacetic Acid.. ..... . ............... 24 Naphthaleneacetic Ac1d 26 l, 3, 5(10)-Estratrien-3ol-17-one. . ...... . . . . . . . . .‘ ........ . . 26 2-Chloroethylphosphonic Acid. . . . ....... . . . . .............. 26 Other Growth Regulators................................. 26 B. Greenhouse Environment Study ............ 27 C. Controlled Environment Study... .......... ...... .. 40 D. Low Temperature Study 41 V. DISCUSSION ........................... . .................... 44 VI. CONCLUSIONS AND SUMMARY .............................. 51 VII. BIBLIOGRAPHY ............................................. 52 iii Tables 1 LIST OF TABLES A summary of the material and methods of the growth regulatorstudies.................. The effect of TIBA treatments on flowering and pistil formation of the MSU-SOO single-flowered and MSU- 499 and MSU-503 double-flowered Petunia . . . . . . Effect of TIBA treatment on pistil formation of MSU-499 homozygous. double-flowered Petunia . . . . . . . . . The effect of IAA treatment on flowering and pistil formation of MSU-SOO single-flowered and MSU-499 and MSU-503 double-flowered Petunia . . . . . . . . The weekly total number of flowers collected and the corresponding weekly mean high temperature in the greenhouse environment study, 1968 . . . . . . . . The weekly total number of flowers collected and the corresponding weekly mean high temperature in the greenhouse environment study, 1969 . . . . . . . . The effect of controlled environment temperature on pistil formation of double-flowered Petunia . . . . . . . . iv Page 13 17 23 25 36 39 4O Figure LIST OF FIGURES Pistil categories and the effect of chloroethylphosphonic acid on flowering of MSU-499 homozygous double- floweredPetunia.................... 11 The effect of TIBA treatment on flower morphology of Petmalybridatbrt.................. 18 Flowers from plants of MSU-499 treated with TIBA . . . . . 21 The influence of greenhouse environment on pistil forma- tion in homozygous double-flowered Petunia, MSU-499, showing percent of flowers with reduced and/or malformed pistils from April to August 1968 . . . . . . 28 The influence of greenhouse environment on pistil forma- tion in heterozygous double-flowered Petunia, LIEU-503, showing percent of flowers with reduced and/or malformed or normal appearing pistils from April to August 1968 . 30 The influence of greenhouse enviromnent on pistil forma- tion in homozygous double-flowered Petunia, MSU-499, showing percent of flowers with reduced and/or mal- formed pistils from April to August 1969. . . . . . . . 32 The influence of greenhouse environment on pistil forma- tion in heterozygous double-flowered Petunia, NBU-503, showing percent of flowers with reduced and/or mal- formed pistils from March to September 1969 . . . . . 34 The effect of controlled environment temperature on pistil formation of homozygous double -flowered Petunia, MSU- 499, showing percent of flowers with reduced and/or malformed pistils over a period of 9 weeks . . . . . . 42 I. INTRODUCTION Petunia hybrida Hort. is an economically important member of the Solanaceae. The double-flowered character in this species is monogenically inherited, 92 and _D_d_, and is dominant to the single-flowered type dd. The morphogenetic effect of this gene has been studied by Natarella (1968). He observed that in the single-flowered genotype 9.3 the initiation of floral parts is acropetal, whereas in the double genotypes _D_g and 92, the initiation pattern is altered by a proliferation of primordia which differentiate into petals and stamens. In the case of the heterozygous 2d, a few normal pistfls are observed in some flowers but none are found in the homozygous PE flowers. Induction of fertile pistils in the female sterile homozygous double geno- type would be a method of maintaining homozygous double inbreds from seed. This is desirable since this genotype, the only one which can be used as a pollen parent for hybrid seed production, is currently maintained by asexual means and infection by tobacco mosiac virus (T MV) leads to a degeneration and loss of valuable pollen production stock. Modifacation of sex-expression in flowering plants by environmental and hormonal agencies has become a common approach to solve these problems of development of floral organs. It was the purpose of this study to investigate environmental and hormonal effects on pistil development and flower morphology of the homozygous and heterozygous double, and the single flowered Petunia hybrida Hort. The objectives were to observe changes in flower morphology and pistil formation as affected by greenhouse environment, controlled temperatures, and various growth regulators including triiodobenzoic acid, trichlorobenzoic acid, indoleacetic acid, naphthalaleneacetic acid and 2-chloroethylphosphonic acid, 5-fluorouracil and phenylboric acid. II. LITERATURE REVIEW Heslop-Harrison (1963a) proposed a scheme for the ontogeny of a hermaphrodite flower which consists of four stages: (1) (u) , (iii) (iv) Apex Sequential initiation Determination of Outgrowth of vegetative of primordium lateral primordium lateral organs Stages (ii) and (iv), which are the objects of this study, involve the actual initiation and mtogeny of the individual floral organs. It is this stage (iv) of flower development that may be influenced by exogenously applied growth substances such as auxins. He stated that two stages, (1) to (ii) and (iv), of flowering are affected by auxin level. A high auxin level is required for (i), the onset of floral initiation and secondly the auxin concentration promoting stamen growth is lower than that for pistil growth. In some plants this level is susceptible to chemical and enviromnental control through the auxin balance of the plant. Both IAA and NAA, applied exogenously, are effective in promoting pistil development (Heslop-Harrison, 1957). He also suggested that: in hermaphroditic species, unisexual flower bearing rudiments of the missing sex, pistil, are a result of differential sup- pression (stage iv) by the intrusion of the additional stamen primordia before the pistil primordia. Therefore, such unisexual flowers are not committed to a predetermined path of development but may be made to develop characteristics of both staminate and/or pistillate flowers. The action of IAA on sex-expression in cucumber was studied by Galun, 1962; Ito and Saito, 1956; Laibach and Kribben, 1950; Nitsch et a1. , 1952; and many others. Laibach and Kribben (1950) and Nitsch et al. (1952) found that IAA and NAA can induce the formation of pistillate flowers and suppress male flowers in cucumber whereas NAA was more effective than 1AA. Ito and Saito (1956) gave a similar report. Galun (1962) reported that floral buds, according to the position on the plant, should be male, but when detached and cultured in the presence of IAA, they developed into female flowers. Smith (1967) reported that auxin induces femaleness in Carex. Leopold (1961) stated that the most remarkable feature of auxin transport in plants is its polarity. There are many other physiological implications of auxin transport, such as, tropistic movements, apical dominance, and root initiation. These can be useful tools for studying the effect of auxin on the genetics and physiology of flowering and flower morphology. Growth inhibitors and auxin transport inhibitors thus can be the major agents for studying the control of flower development. These effects can be correlated with physiological responses which characterize auxin transport. Triiodobenzoic acid (T IBA) is readily mobile in plant tissue, accumulates both in the growing tips of roots and shoots and is extremely stable in plants. Almost all the morphological responses of vegetative plants to TIBA, such as loss of apical dominance (Kuse, 1953), also of polarity of tissue (Keitt, 1966; Niedergang and Skoog, 1956), seem to indicate that auxin levels are altered in the plant (Galston, 1947). T IBA inhibits the secretion of 1AA out of the tissue (Hertel and Leopold, 1963; Keitt and Baker, 1967). Kays (1968) applied 100 ppm TIBA weekly on the single-flowered, grandiflora petlmia White Cascade and observed a random deletion of stamens, elongation and fusion of petal lobes and elongated pistils. Pollination with pollen from un- treated plants resulted in good seed set and these seeds germinated and grew normally. It is suggested that TIBA behaves like a weak auxin, probably by its inhibition of transport of endogenous or added auxin (Keitt and Baker, 1966; Kuse, 1953; Niedergang and Skoog, 1956). The growth regulating effect of 2,3,6-trichlorobenzoic acid (T CBA) closely resembles TIBA. It is reported that TCBA treatment causes epinasty, malformed and reduced size leaves, distortion of inflorescence, small but regularly shaped fruits and overall reduction in growth of plants (Way, 1964; Cock, Taylor and Jubb, 1965). 2-chloroethylphosphonic acid (Ethrel) has been actively used as a growth regulating chemical in cucumber and many other plants. Its ethylene releasing property in plant tissue has been confirmed (Cooke and Randall, 1968; Warner and Leopold, 1969). Promotion of femaleness in Ethrel treated cucumber plants has recently been reported by Miller, Lower and McMurray, 1969; Putnam, 1969; Robinson, Shannon and Manuel, 1969; Rudich et al. , 1969; and Sims, 1969. Other Ethrel responses reported are reduced internode (number and lengths) and permanent or temporary stunting, (Miller, Lower and McMurray, 1969; Robinson et al. , 1969), induction of flowering (Cooke and Randall, 1968) and flower bud abortion at high Ethrel levels (Robinson et a1. , 1969). Opposing effects of gibberellin and ethylene on plant growth were re- ported by Bukovac and Wittwer, (1961); Mitchell and Wittwer, (1962); Scott and Leopold, (1967). Gibberellins are known as promoter of maleness in flowering plants (Bukovac and Wittwer, 1961; Peterson et al. , 1960). Burg, 1962 and Burg and Burg, 1966, reported that the ethylene responses in plant growth and development are strikingly similar to auxin responses and they suggested that auxin effects may be due to auxin induced ethylene production (Zimmerman and Wilcoxon, 1935). Galun, et al. (1965) reported that auxin content was higher in hermaphroditic cucumber plants than in andromonecious plants, whereas Atsom, et a1. (1968) determined that GA is higher in monoecious plants. 5-fluorouracil is a nucleic acid synthesis inhibitor which has been re- ported by Zeevaart (1962) to affect floral differentiation by blocking DNA mul- tiplication. Galston et a1. (1969) suggested that S-fluorouracil curtailed RNA synthesis without affecting auxin induced growth. Phenylboric acid has been used as a tool for studying the action of the lanceolate gene in tomato (Mathan, 1965). This chemical stimulates the gene action through induction of an increase in oxidative enzyme activities. Haccius and Messfeller (1961) found that phenylboric acid caused a reduction of petals in Kalanchoe blossfeldiana. TIBA enhanced the rate of IAA uptake, presumably by inhibiting basipetal transport (Keitt, 1967). Atsom et a1. (1967) reported that TIBA inhibits the synthesis of gibberellin (GA). Love and Love (1945), by applying mammalian female hormones, the estrogen group, in lanolin paste to the leaf axils of male plants, found that this hormone caused a partial suppression of the stamens and promotion of pistil formation. Unfortunately, further investigations along this line were discontinued. The mode of action of auxin is not known; it seems that the enzymatic controlling mechanism is through a general increase of production rather than a triggering of the production of a particular protein (Galston et a1, 1969). One evidence is that TIBA, a sulfhydryl inhibitor, might act as an inhibitor of the enzymatic destruction of IAA (Pilet, 1963). Auxin inhibition is more specific than through the general respiratory scheme (Neidergang and Leopold, 1967). According to Stebbins and Yagil (1966), morphological expression may be the result of a complex physiological chain reactions triggered by a single gene. They studied the awned and hooded lemma in barley which is known to be controlled by a single gene, hood K which is temperature sensitive. This gene gives rise to a hooded lemma which bears one or two extra florets on the lemma, the proximal one being inverted morphologically. Probably the hooded gene initiated another differentiation cycle by altering the mitotic rhythm. The first accountable evidence of gene action was the accelerated synthesis of DNA and RNA during the earliest deve10pment of the hood. They postulated that the synthesized nucleic acids speed up mitosis, causing decreased cell size and resulting in a change in the orientation of the spindles from lemma axis -para11el to a three dimensional direction. Inverted polarity, they believe, is a hormonal induced action which occurs at the later stage of hood develop- ment, and could be the primary action of the hooded genes. Cold temperature (4. 50C) and short days applied prior and during the earliest stage of hood differentiation weakened or cancelled the hood expression. The fact that high temperature lowers the effectiveness of apical dominance may be a result of inhibited auxin transport (Gregory and Hancock, 1955). This evidence may fit into the scheme hypothesized by Heslop-Harrison (1963a), that flower development may be influenced by auxins through environmental factors. III. MATERIAL AND METHODS A. Plant Material Three genetic lines were selected from the fourth inbred generation obtained by self-pollinating a heterozygote in each generation starting with the double-flowering multifora variety Cherry Tart. These lines are MSU-SOO single-flowered genotype dd, MSU-499 homozygous double-flowered genotype 22, and MSU—503 heterozygous double-flowered genotype _D_c_l_. Plants for research purposes were obtained by vegetative propagation of cuttings from stock plants of these three genotypes. B. Cultural Procedures Stem cuttings were treated with Horrnodin No. 2 rooting compound, then placed in sterilized sand and watered twice daily. A glass cover and/or bottom heat were used to encourage rooting during the winter months; the cuttings usually were well rooted in two to three weeks. The rooted cuttings were then transplanted into a 1:1:1 mixture of soil, peat and sand in 2 1/4 inch square peat pots. In two weeks the root system was well developed and the plants were then potted into 4-inch clay pots with the same soil moisture and were automatically watered. ‘ The fertility was maintained by weekly application of N-P-K 20-20-20 fertilizer and 12% EDTA at 5 ppm. Greenhouse temperatures were a minimum of 70°F day and 65°F night; they varied with the prevailing outdoor temperature. During the winter months, supplemental illumination by 100 watt incandescent bulbs 3 feet apart and 3 feet above the plants was provided from 10:00 p.m. 9 to 2:00 a. m. to maintain flowering. C. Experimental Design Experiments were designed either as a randomized block design or a completely randomized design, of three or four replications and were con- ducted either in the greenhouse or in growth chambers. Flowers at anthesis were collected starting four weeks after the first growth regulator application, and the morphology of the pistils was examined. The pistils were classified as follows: (1) normal appearing, (2) malformed and/or reduced, and (3) pistil not present; and were recorded on a per treat- ment per replication basis (Figure 1A). D. Growth Regulators Eight growth regulators were used in different experiments (Table 1). These included 2,3, 5-triiodobenzoic acid (TIBA) from Eastman Organic Chemicals; Floratone, the commercial product of the salt form containing TIBA active in- gredient from Amchem Products, Inc.; 2,3,6-trichlorobenzoic acid (TCBA); 3,indoleacetic acid (IAA) and 1-naphthaleneacetic acid (NAA) from Eastman Organic Chemicals; 1,3,5(10)-Estratrien-301-l7-one (Estrogen) from Mann Re- search Laboratories; 2-chloroethylphosphonic acid (Ethrel) from Amchem Products, Inc.; 5-fluorouracil from Calbiochem; and phenylboric acid from Aldrich. Growth regulators were applied either by foliar spray or by droplet application. Air pressure sprayers were used for the foliar spray. The 10 Figure 1. Pistil categories and the effect of chloroethylphosphonic acid on flowering of MSU-499 homozygous double-flowered Petunia. A. Pistil completely absent in a flower of MSU-499. B. A pistil of reduced size in a flower of MSU-499. C. A malformed pistil in a flower of MSU-503. D. A normal appearing pistil in a flower of MSU-SOO. E. Effect of 200 and 1,000 ppm 2-chloroethylphosphonic acid (Ethrel) on flowering plants of MSU-499 and an untreated plant. 11 12 82 .823 B8 -....-..-.:.- w m :3? $3003 wfizwumm ooedoN mow ofloflmfifia soon .o~\m 8882 .oN\v- -8? .583 N m N}. 88 m .58 5883 818.2 88 8888.68 8818? a m 881218 .28 mafia... 88.188 8% 85m 8832 .oH\~H wooa .m\NH- -25 .583 N m is 88 m 388 5888 2.8 88 888m 8032 .mn\w .......... N m -2). .583 5.58 818.2 88.88.38 .22 8882 .ma\w .......... N m -23 .583 5.33... 818 .2 8888.38 3: mean .o~\m 8882 .o~\~ -32 .583 N N -2: .583 5.5% 8.8.2 88.88.83 «S 8082 .oH\NH moon .ON\HH -25 .583 N m -018 .583 5388 818 88 4mg 83 .28 83 .2), 82 838805 -2}. .583 N m -28 .583 5388 818188 3% 4m:- 3832 .oN\m mama .o~\2 -32 .583 2 m -2: .583 mafia... 818188 8888.38 45. mesh .oH\~H moan .o~\HH -25 .583 N m -813 .583 538% 818 88.8.8... .8:- Uouoofioo non mama woman @0522 Ema RES-mg Hobie-a 36303 \353 ”ENE 5380 .3826 Snag-won 53on 9: Ho mvofiofi one HERB-3h 05 mo hogs-Sm < .H End-H. 13 entire plant was thoroughly and evenly sprayed until the run-off point was reached. Or, alternatively, three drops of the growth regulator solution were placed on the meristem. E. Greenhouse Environment Study This study was conducted to determine the influence of temperature, and aging on pistil formation. The study was made from March through August, 1968 and was re- peated again in 1969. All three genotypes, MSU-SOO, MSU-499 and LIEU-503, were examined. A randomized block design with 4 replications of 4 plants each was used each year. F. Controlled Environment Study The temperatures used in these studies were 80°F DT, 75°F NT and 62°F DT, 57°F NT in two growth chambers, respectively. Fourteen hours of light was maintained in both chambers. Twenty-five foot candle (980 erg/sq cm) light was maintained by 8 incandescent lights, 25 watts each and 6 cool white-influorescent light tubes. There were six plants each of MSU-499 and MSU-503 placed at random in each growth chamber, and they were watered daily and fertilized weekly with 600 ppm N using 20-20-20. The experiment was conducted from November 26, 1968 until February 26, 1969. Later, it was found that growth of the plants in the high temperature growth chamber was suppressed due to rapid transpiration. Therefore, starting on February 5, 14 the temperatures were modified to 75°F DT, 70°F NT and 60°F DT, 55°F NT, respectively. Also, the plants in the high temperature chamber were watered twice daily. As a result, growth of the plants in the high temperature cham- ber returned to normal. Another temperature study was conducted with further modifications. Only one growth chamber was used, with ten MSU-499 plants. Throughout the 9-week experimental period, the plants were provided with fourteen hours of light and the light intensity was the same as with the previous study. In the first 3 weeks, the plants were grown under 85°F DT and 700 NT; the second 3 weeks, 65°F DT and 50°F NT; and the last 3 weeks, 85°F DT and 70°F NT, again. G. Low Temperature Study A 35°F temperature environment was provided to the MSU-499 and MSU-503 plants to observe the effect of low temperature on pistil formation. There were three replications of two plants of each double-flowered genotype in each treatment. The plants were kept in the 35°F growth chamber during the treatment period and then returned to the greenhouse. The experiment was commenced on February 28, 1969, and was repeated in April with the duration of the low temperature treatment being modified to 5 days and 10 days, respectively. 15 IV. RESULTS A. Growth Regulator Studies Triiodobenzoic Acid: Preliminary studies with triiodobenzoic acid (T IBA) were conducted in the fall of 1968. In all three genotypes, vegetative growth was retarded and flower size reduced. Lateral branching and flower number were increased as shown in Table 2. Plant height and leaf size were decreased. In some flowers, the inner whorls of petals were modified to petalloid anthers, either in original color or green. In other flowers, the corolla completely resembled that of the MSU-SOO single genotype and in the extreme case, only portions of the single whorl of petals appeared (Figure 2A). No concentration effect was observed. A reduction in the number of stamens was also observed for all concentrations of TIBA-treated MSU-499 and MSU-503. Each control flower always had about twenty stamens, whereas in many TBA treated plants, most of the flowers had only aifew stamens in each; some of the stamens in NBU-503 were shortened. In some flowers of MSU-499, the stamens were completely deleted and the central part of the flowers were clustered with greenish petaloid structures. It was observed that the complete or partial disappearance of the stamens was always correlated with a reduction of the petals. Flowers of distorted shape were occasionally found in the T IBA treated plants (Figure 2B). Some of the TIBA treated MSU-SOO bore flowers of distorted shape, or had 16 Table 2. The effect of TIBA treatments on flowering and pistil formation of the MSU-SOO single-flowered and MSU-499 and MSU-503 double-flowered Penmia. % of flowers with Total no. reduced and/or % of flowers with Treatment of flowers malformed pistils normal appearing pistils a. Control 134 1.6 ~- 34} 100 ppm 205 8.4 ~- 5 120 ppm 170 12.5 -- 5 140 ppm 103 6.0 -- .0 Control 52 15. 0 -- 8 100 ppm 103 54.0 3.0 :2 120 ppm 110 74.5 5. 5 E 140 ppm 126 43.0 23.0 o Control 108 -- 100. O 8 100 ppm 182 2.7 97.3 5 120 ppm 233 5.0 95.0 g 140 ppm 132 0.7 99.3 folded corollas (Figure 2A). Table 2 shows the total number of flowers, percent of flowers with reduced and/or malformed pistils or with normal appearing pistils. In all three TIBA concentrations applied, the highest percent of reduced and/or mal- formed pistils was found in the plants of MSU-499 and MSU-503 treated with 120 ppm TIBA. In the MSU-503 plants, the greatest promotion of normal appearing pistil development was achieved with the 140 ppm TIBA treatment. 17 Figure 2. The effect of TIBA treatment on flower morphology of Petunia hybrida Hort. A. A flower from an untreated plant and 3 flowers from 100 ppm TIBA treated plants of MSU-SOO showing distortion of flower shape and a reduction in flower size. A flower from an untreated plant and 3 flowers from 100 ppm TIBA treated plants of MSU-499 showing distortion of flower shape, reduction in flower size and floral doubleness. A flower from an untreated plant and 3 flowers from 100 ppm TIBA treated plants of MSU-503 showing dis- tortion of flower shape, reduction in flower size and floral doubleness. A flower from an untreated plant, without pistil, and a flower from 140 ppm TIBA treated plant of MSU-499 showing a thick, short, malformed pistil. A flower from an untreated plant, without pistil, and a flower from 140 ppm TIBA treated plant of MSU-499 showing a reduced size pistil. 18 19 Twenty-three percent of the flowers developed normal appearing pistils, whereas only 5. 5 and 3.0 percent in the 120 and 100 ppm treatments, respectively. In MSU-SOO, development of normal appearing pistils was slightly inhibited in the TIBA treatments as compared with the control with 100 percent normal appearing pistils. Again, the greatest effect was found in the 120 ppm TIBA treatment in which only 95 percent of the flowers had normal appearing pistils, whereas there were 97.3 and 99.3 percent in the 100 and 140 ppm T IBA treatments, respectively. In the second TIBA treatment experiment with MSU-499 homozygous double, a change from double to single flower form was observed as it was in the preliminary study (Figure 3). In some flowers, the number of stamens was reduced and they were modified as such with long, thin and straight filaments instead of the typical short, malformed type commonly observed in control flowers of MSU-499. In the 200 ppm TIBA treatment, growth and flowering were completely inhibited in replications II and 111. All flower buds were aborted. However, flowering was only partially inhibited in the first replication of which the plant material was older than the other two replications. The percent of flowers with reduced size or malformed pistils in this replication was almost identical with the control plants. Table 3 shows that there appeared to be a concentration effect on pistil formation in the 120 and 140 ppm TIBA treatment. There were 5. 5 and 20 20 Figure 3. Flowers from plants of MSU-499 treated with TIBA. A. A flower from an untreated plant and two flowers from 100 ppm T IBA treated plants showing a reduction in flower size and floral doubleness. B. Two flowers from 120 ppm TIBA treated plants showing a reduction in flower size and floral doubleness, and partial deletion of stamens as compared to a flower from an untreated plant. C. A flower from 140 ppm TIBA treated plant showing a reduction in flower size and floral doubleness as compared to a flower from an untreated plant. 21 22 Table 3. Effect of TIBA treatment on pistil formation of MSU-499 homozygous double flowered Petunia. Number of flowers % of flowers Rep. 1* Rep. 11 Rep. 111 Total showing reduced and/or Treatment no* * P no P no P no P malformed pistils Control 125 5 72 6 46 l 243 12 4. 7 100 ppm 155 3 40 2 24 1 249 6 3. 7 120 ppm 93 5 32 1 29 3 154 9 5. 5 140 ppm 58 19 14 0 9 1 81 20 20. 0 200 ppm 73 3 -- - -- - 73 3 4. O * Plants in the first replication were older. ** no = without pistils percent of flowers developed reduced and/or malformed pistils in these two treatments, respectively. However, the 100 and 200 ppm TIBA treatment showed no effect on pistil formation when compared with the control. Trichlo robenzoic Acid: The effects observed in trichlorobenzoic acid (T CBA) treated MSU-499 and MSU-503 plants mere similar to the TIBA treatments. Lateral branching and flower number were increased; petal doubleness, flower size and plant height were reduced. 23 Indoleacetic Acid: In all three genotypes observed, flowering was suppressed in the 40 ppm and higher 1AA treatments. Flowering in plants treated with 20 ppm 1AA was delayed three weeks. In LIEU-499, 7 percent of the flowers had reduced and/or malformed pistils at 20 ppm, and also at 40 ppm 1AA, which was high when compared with the control, 1.6 percent. Plants treated at 10 ppm 1AA resulted in only 2 percent of flowers with reduced and/or malformed pistils. In MSU-503, there were 39.4, 39.6 and 16.8 percent of reduced and/or malformed pistils in the plants treated at 10, 20 and 40 ppm 1AA, respectively; a significant increase compared to the control, 15 percent. A higher percen- tage of normal appearing pistils developed in the plants treated at 20 and 40 ppm 1AA; 3.4 and 7.2 percent, respectively, compared to the 10 ppm treat- ment. No normal appearing pistils were observed in the control. Pistil development in all 1AA treated MSU-SOO plants was not affected. In the second study with 25, 50 and 100 ppm 1AA, flowering was severely inhibited in 25 ppm, and was completely inhibited in the 50 and 100 ppm treatments. Flowering was resumed at a period of four weeks after the last application of IAA. There was no observable morphological change in those flowers . 24 Table 4. The effect of IAA treatment on flowering and pistil formation of MSU-500 single-flowered and MSU-499 and MSU-503 double- flowered Petunia. % of flowers with % of flowers Total no. reduced and/or with normal Treatment of flowers malformed pistils appearing pistils Control 134 1. 6 - - § 10 ppm 167 2.0 -- g 20 ppm 160 7. 0 -- 40 ppm 115 7. 0 -- Control 52 15. 0 -- 8 10 ppm 87 39. 4 0.6 m a 20 ppm 73 39.6 3.4 40 ppm 52 16. 8 ' 7. 2 Control 108 - - 100. 0 o 10 ppm 247 -- . 100.0 E 20 ppm 113 -- 100.0 a 40 ppm 121 -- 100.0 25 Naphthaleneacetic Ac id: Normal vegetative growth was severely suppressed in the plants treated at 25, 50 and 100 ppm naphthaleneacetic acid (NAA) and anthesis was completely inhibited. Leaves were large and thick and the plants were dwarfed. Vegetative growth gradually resumed after the NAA treatment was stopped, and anthesis was observed in four-weeks time. The observed growth suppressing effect in NAA treated plants was greater than in the IAA treated plants. 1, 3, 5(lO)-Estratrien-3 ol-l7-one: In the preliminary study of 1,3,5(10)-Estratrien-301-l7-one (Estrogen), there was no significant morphological change observed in the treated plants. 2-Chloroethylphosphonic Acid: Preliminary study of 2-chloroethylphosphonic acid (Ethrel) showed that two sprays of 200 or 1,000 ppm Ethrel inhibited growth and flowering com- pletely (Figure IE. Flowering was resumed five weeks after the second treat- ment, no morphological change was observed in the flowers. Other Growth Regulators: No morphological response was observed in the preliminary studies on 5-fluorouracil and phenylboric ac id. 26 8. Greenhouse Environment Study The influence of environment on pistil formation of the three genotypes of Petunia hybrida Hort. was studied from March through August in 1968 and 1969. The results were plotted as the weekly occurrence in percent for each category of pistil structure of all flowers: with reduced and/or malformed, or normal appearing pistils. The number of flowers without pistil structure was also recorded. No pistils of category 1 were observed in flowers from MSU-499 (Figure 1). The weekly average of the local daily maximum temperature at ten-day intervals before anthesis was also plotted to show the relationship of temperature with pistil formation (Figures 4-7). The weekly total number of flowers collected and the corres- ponding weekly mean high temperature are listed in Table 5. In the 1968 study, the percent of all flowers showing reduced and/or malformed pistils in MSU-499 showed a tendency to increase from March to July and then to level off in August. In Figure 4, it can be observed that pistil development was closely related with the charted weekly mean high temperature ten days prior to anthesis. There were four noticeable peaks at which pistil development was expressed as percent of flowers with reduced and/or malformed pistils: 5 percent, 17 percent, 30 percent and 57 percent on May 7, May 28, June 18 and July 16, respectively, and this corresponds to when the temperature ten days before anthesis was also high. The Figure 4. The influence of greenhouse environment on pistil formation in homozygous double-flowered Petunia, MSU-499, showing percent of flowers with reduced and/or malformed pistils from April to August 1968. 28 — 'l. REDUCED AND/OI MAI-FORMED PISYILS -—- YIMPEIATUII ID DAYS IIFOIE ANYHISIS io IIDLVUIJWIL O 3 v. 1 1 ~60 ,Pso I I I l t O O O O ‘ 0 N l- n S1ILSI‘ OMOflVWIO/ONVOJDDDII ONIMOHS SllMO‘H 11V ‘0 INIDlld 29 ‘-- ~ ~------ ~~~ a. 20 13 f JUN JULY 1. 25 I 2. T 14 MAY r 30 a n (N I968 DAY! Figure 5. The influence of greenhouse environment on pistil formation in heterozygous double-flowered Petunia, MSU-503, showing percent of flowers with reduced and/or malformed or normal appearing pistils from April to August 1968. 30 Jo IIDLVUIdWI-I. ON 2 0 on an: 00-. 00+ an on o N .00— b an Op — p Q A L. i u b ( O in a '— b h — \/\\. . s I s I s e . S I x 4 23M a.m.:hz¢ .8050. 0>(fl 0— H's-h‘lut‘ub -"' nd.hn: O.‘CO$.-(‘ 8032‘ OUUDOUI 8 io nd-hn.£ d‘:.°z 3 III 51”!“ DNIMOHS SIIMO‘H 11' IO lNlDlld [GD— 31 Figure 6. The influence of greenhouse environment on pistil formation in homozygous double-flowered petunia, MSU-499, showing percent of flowers with reduced and/or malformed pistils from April to August 1969. 32 — % REDUCED AND/OI MAI-FORMED PISYILS YEMPEIAYUIE ID DAYS IEEOIE ANTNESIS 4. annivudwn O 0 fl ~70 l l O O ' fl 20- S'IILSIG GIWIOJ'IVW IOIDNV 033003. DNIMOHS SIIMO‘IJ 11' ” INJDIJII 33 -SD - 40 an J 5: s 91 1 28 1 AUG 5 I2 I JULY 23 30 IO 16 1969 1 JUN 2 26 1 1 MAY 5 12 M API 7 MAI 3| DATE Figure 7. The influence of greenhouse environment on pistil formation in heterozygous double-flowered Petunia, MSU-503, showing percent of flowers with reduced and/or malformed, or normal pistils from March to September 1969. 34 IUDIVIIJWIJ 03‘ Q— hen... On nu 000— wh¢£ a_nu8h2( ulOuu- n>(n O— ulflh‘dutzuh 3.5T out-0...:- -o\92¢ 32::- s all-o nd-hn-‘ d“.°z 3 All 10 IN! 3‘31! 11' S1l1$ld 9NIMOHS SIIMO1I 35 Table 5. The weekly total number of flowers collected and the corres- ponding weekly mean high temperature in the greenhouse environment study, 1968. Weekly mean Date high temperature MSU-500 MSU-499 MSU-503 April 2 62 295 131 158 9 60 141 90 74 16 65 196 163 184 23 64 254 125 151 30 60 230 103 98 May 7 58 238 195 164 14 69 200 111 129 21 67 200 134 133 28 65 196 194 153 June 4 70 180 151 136 11 87 112 91 74 18 72 168 163 139 25 80 210 248 232 July 2 75 206 99 55 9 79 170 71 16 16 86 208 167 180 23 84 280 115 20 30 78 300 142 180 Aug. 6 81 --- --- --- 13 81 170 97 95 20 84 113 118 101 36 heterozygous double LIEU-503, Figure 3, had an increase in the percent of flowers with reduced and/or malformed pistils from March to August, whereas the percent of normal appearing pistils (Category 2) remained low, within the 12 percent, throughout the experimental period, and showed a decline after July. There was an average of 64 percent reduced and/or malformed pistils and 5 percent normal appearing pistils found throughout the period of study. From April to June, the rise and decline of the reduced and/or malformed pistils and of temperature were roughly parallel. In Figure 5 there are three major peaks of reduced and/or malformed pistils: 62, 60 and 69 percent on April 23, May 28 and June 11, respectively. The corresponding temperature ten days before was correlated with these major peaks except for June 11. In July and August the percentage of reduced and/or malformed pistils was high and relatively constant at about 90-95 percent. The weekly mean high temperature of these two months was between 75-870F, which could be correlated with the high percentage of reduced and/or malformed pistils. Throughout the experimental period, NEU-503 had a higher percent of flowers with reduced and/or malformed pistils when compared with MSU-499. There was no observed change in the floral morphology of MSU-SOO during these studies. The weekly increase and decrease in flower number of all three genotypes seemed to be parallel with each other. MSU-SOO always had a higher flower 37 number than MSU-499 and MSU-503. Flowering markedly declined from July 30 on in NBU-499 and in MSU-500, whereas the decline of flowering in NBU-503 was more gradual. The total number of flowers of the three genotypes seemed to be in contrast with the rise and fall of the temperature at or a few days before anthesis (Table 5). In the 1969 study, in WU-499, the percent of flowers with reduced pistils in general was higher in the later months, but not as great as in 1968. Pistil development again was associated with the daily maximum tem- perature ten days before anthesis, showing four peaks at April 21, June 9, July 7-14 and September 9, successively (Figure 6). In MSU-503, 40 percent of the flowers had reduced and/or malformed pistils and 10 percent had normal appearing pistils. The former figure was lower than 1968, and the latter higher. However, as with LIEU-499, the increase in percent of flowers with pistils was not as sharp as in 1968. the highest being 78 percent, but still appeared to parallel the rise and fall of temperature. Development of normal appearing pistils was steady and little affected by temperature (Figure 7). Similar to the 1968 study, the weekly increase and decrease in flower number of all three genotypes seemed to be parallel with each other. From July 21 on, flower number markedly declined in the two double types while the single type remained steady throughout the period of study. Again, flower abortion and reduction in flower size was observed in July and August when temperature was high (Table 6). 38 Table 6. The weekly total number of flowers collected and the corresponding weekly mean high temperature in the greenhouse environment study, 1969. Weekly mean Date high temperature NBU-SOO MSU-499 NBU-503 March 31 -- 186 80 77 April 7 40 264 135 102 14 46 256 138 117 2 l 63 2 13 45 126 28 62 264 2 18 138 May 5 61 375 209 111 12 69 -- 142 187 19 65 507 284 125 26 68 425 170 240 June 2 63 552 186 335 9 80 715 327 3 19 16 62 715 327 3 19 23 76 621 280 306 30 72 820 324 389 July 7 80 782 153 2 12 14 79 11 13 651 703 21 81 946 2 17 291 28 83 1008 345 119 Aug 5 82 1136 494 133 12 81 1016 144 122 18 82 1120 237 74 28 85 12 10 376 190 Sept 4 85 1130 95 236 39 C. Controlled Environment Study MSU-499 plants in the 60°F growth chamber had 6.2% flowers with reduced and/or malformed pistils, whereas those in the 75°F DT chamber had 12.4 (Table 7). The percentage of flowers of MSU-503 with reduced and/or malformed pistils in the 60°F DT chamber and 75oF chamber were 25% and 57%, respectively. There were 4.3% of the MSU-503 flowers with normal appearing pistils in the 60°F DT chamber and 10.9% in the 75°F DT chamber. The results indicated that in 75°F DT and 70°F NT, pistil develop- ment was increased twofold When compared to the 60°F DT and 55°F NT chamber. Table 7. The effect of controlled environment temperature on pistil forma- tion of double-flowered Petunia. Total % of flowers with % of flowers no. of reduced and/or with normal Temperature flowers malformed pistils appearing pistils g“ 0 0 1,. 60 F DT, 55 F NT 113 6.2 -- l g 75°F DT, 70°F NT 161 12.4 -- § 60°F DT, 55°F NT 140 25.0 4.3 I :9) 75°F DT, 70°F NT . 73 57.0 10.9 In the second growth chamber temperature study, flower collecting was started 10 days after the first high temperature treatment and then at the end 40 of the third week. Flowers were collected three times during the second three weeks (low temperature). In the remainder of the study period, flowers were collected at five day intervals. The results are expressed as percent of flowers with reduced and/or malformed pistils and are shown in Figure 8. It shows that pistil development was enhanced starting from the tenth day of high temperature treatment and markedly decreased at the low temperature period, dropped to zero percent at the end of that treatment, then increased at the third stage when temperature was higi again, starting at the tenth day of high temperature treatment and increased. D. Low Temperature Study The effect of temperature, below the range for optimal growth, on pistil development of MSU-499 and MSU-503 was studied. There was no observable change in flower morphology in this study. 41 Figure 8. The effect of controlled environment temperature on pistil formation in homozygous double-flowered Petunia, MSU-499, showing percent of flowers with reduced and/or malformed pistils over a period of 9 weeks. 42 0N an n— hz neck .2. moan 000p ub‘b 'N J S. .83. .5 :3 '— »! a can .hfl mono an >a3n¢ l I! l 0 us 583M011 so 1N3)!“ 1 - - 1 2 8 1‘ S1ll-Sld DIWUOJ'IVW IO 033003! DNIMO 41 0 fl 1 n 0 43 V. DISCUSS ION Numerous biochemical and morphological changes occur in the transition of a vegetative apex to a mature flower. Heslop-Harrison (1963a) in a review of sex expression in plants, has defined four stages of develop- ment in floral morphogenesis. (i) (ii) (iii) (1V) Apex, vegetative Sequential Determination Outgrowth of "‘—"" initiation of—""""" of lateral -—"""lateral organs primordia primordia In step (i) the transition from vegetative to floral initiation occurs most likely through some hormonal agent which has not yet been identified. In stage (ii) the apex passes through an orderly sequential initiation of primordia. Stage (iii) is included for diclinous flowers in which unisexuality occurs. In step (iv) the actual ontogeny of the floral organs including differentiation of stamens, petals and carpel occurs. Petunia flowers are hermaphroditic and therefore in this discussion stage (iii) as proposed by Heslop-Harrison (1963a) will not be included. The double -flowering gene _D_ in Petunia has been mor- phogenetically studied by Natarella (1968). He observed that in the double- flowering genotypes DR and 21 carpel development was physically hindered by the petal and stamen growth that _D_ affects on the receptacle. Since the carpel is centrally located it is actually encased by the petals and stamens during 44 differentiation. Rarely, a normal pistil capable of seed set is observed in pg genotype flowers, but one has not been observed in 2L) types. Thus, the production of pistil organs in Penmia is actually not a type of sex expression in the classical sense that the plant is a hermaphrodite, dioecious or monoecious. Rather, a gene _D_ is active during initiation and it has a secondary influence on pistil presence and function. Control of floral ontogeny by environment and growth substances is usually operative at stage (ii) or (iv) and as Heslop-Harrison (1957, 1963a, 1963b) mentions, with regard to the former stage, the initiation will proceed unless the "treatment is so severe as to arrest growth altogether." Stage (iv) is also readily influenced by growth substances and environmental conditions. There are numerous reports of auxin influences on floral ontogeny and sex-expression (Galun, 1962; Heslop-Harrison, 1957, 1963b; Ito and Saito, 1956; Laibach, 1952; Nitsch et a1. , 1952: and Smith, 1967). In this study, of all the growth substances tested, the auxin transport inhibitor 2,3, 5-triiodobenzoic acid (T IBA) and 236-trichlorobenzoic acid (TCBA) were the only ones that had an effect on flower morphology and pistil development. The former compound was used more extensively since TCBA was male phytotoxic to petunia plants when applied as a foliar spray. TIBA can act as a weak auxin (Niedergang and Leopold, 1957); an SH reagent. will overcome apical dominance (Galston, 1947; Galston et al. , 1968), reduce auxin concentration and polarity in callus formation and influence auxin movement in isolated sections, and reduce the IAA content in pea roots (Hertel and Leopold, 1963; Keitt and Baker, 1966; Kuse, 1953, Niedergang and Leopold, 1957; Neidergang and Skoog, 1956; 45 Thimann et a1. , 1948; and Winter, 1967). Kiermayer (1961) reported that TIBA simulated auxins in supressing stamen development while not affecting pistil growth in Solanaceous plants and the action was greater when NAA was included. The exact mode of action of TIBA does not at this time appear to be precisely known but a reduction in 1AA, increased ratio of immobile to mobile IAA and decreased polar transport all may apply to the results of this study. Certainly the appearance of TIBA treated plants indicated an auxin response. There was a loss of apical dominance, and increase in the number of flowers and an obvious retardation of plant growth. There was also a differential in genotypic response to TIBA. Single flower phenocopies were observed on heterozygous E plants but 92 plants did not express a complete reversion to single flowers. The TIBA did not exhibit specific action on double flower morphogenesis. All degrees of flower doubleness from complete expression to single phenocopies were observed and malformed and distorted flowers occurred on single dd genotype plants. Thus, TIBA appeared to act on both stage (ii) and (iv) in floral morphogenesis of Petunia, and possibly only stage (iv) was involved since anatomical examina- tion was not done to confirm if initiation, stage (ii), was inhibited. This finding supports the suggestion that TIBA may act as an auxin transport inhibitor since auxin is known to influence both stages in floral morphogenesis (Heslop- Harrison, 1957). It is noteworthy that TIBA will effectively inhibit the action of Q in some cases without causing an inhibition of carpel initiation and develop- ment which occur later on in the differentiating receptacle. 46 A preliminary experiment applying TIBA at 100 ppm and including 1AA at 40 ppm in the same solution gave a typical TIBA response. This may be evidence for polar transport inhibition of IAA (Galston et a1, 1969; Heslop-Harrison, 1967; Kuse, 1953; Hertel and Leopold, 1963; Thimann and Bonner, 1948; Winter, 1967) but extensive studies need to be undertaken to verify this supposition. The IAA and NAA studies did not result in a change in flower morphology mainly because vegetative growth and flowering were completely inhibited by the exogenous auxin treatments employed. Here the problem may be one of absorption and/or concentration and frequency in applying the exogenous auxin. A lower concentration and higher frequency of application might be desirable. Plants with 5-fluorouracil showed no morphological response, probably because this RNA synthesis inhibitor does not interact with the auxin affected system (Galston et al. , 1969 and Zeevaart, 1962). Phenylboric acid has been reported (Mathan, 1965) to induce an increase in activity of oxidative enzymes which induce a change in leaf form to resemble the effect of the lanceolate gene in tomato. This chemical did not affect the morphological expression of the 2 gene. Perhaps the sequence of biochemical events that _D_ affects were not altered by phenylboric acid, oxidative enzymes were not involved and also the 2 gene is active only during differentiation , whereas the lanceolate characte1 is one that is manifest in total plant growth and development. 47 An increased percentage of reduced and malformed pistils in both double types is found in the summer months of the greenhouse study and high temperature condition of the growth chamber studies. Such high tem- perature effect might be a consequence of cellular division and enlargement, thus increasing growth rate overcomes the physical interference of pistil development. Gregory and Hancock (1955) noted that high temperatures reduced apical dominance and suggested that auxin transport is inhibited. Strasburger (1900) and Schaffner (1920) have generalized from their extensive experiments that environmental conditions which favor flowering also enhance pistil formation. Since the results of this study indicated that summer conditions favor pistil formation in Petunia, in both double genotypes, therefore, pistil formation may be correlated with the rise of auxin level due to high temperature, either increasing auxin synthesis of inhibiting degredation. The mode of auxin action on pistil formation is not known. According to Natarella (1968), there is no morphological difference in the floral initiation of homozygous and heterozygous flower buds. It is thus suggested that floral development, rat her than initiation is sensitive to environmental factors such as temperature through changes in endogenous hormonal levels. It is known that floral initiation begins approximately three weeks before anthesis in Petunia (Cathey, 1969). From the environmental study, the percent of total flowers with malformed and/or reduced, or normal appearing pistils 48 seemed to be positively correlated with the temperature ten days before anthesis. This is therefore an affect on development and not initiation. The controlled temperature studies in the growth chamber further confirmed this relationship. It is reasoned that since a high temperature environment ten days before anthesis was able to enhance pistil development that a stage of development after initiation was influenced. This major step, most probably determination, may not be as easily affected by environment and/or exogenous growth regulators, as the later developmental stage of the pistil. The development of normal pistils in the single-flowered type, Q was not sensitive to exogenously applied T IBA or to temperature. Malformed and distorted flowers resulted but no gross differences in the presence or absence of individual parts were observed. Also, the percent of normal pistils in the heterozygous double type was unaffected by temperature, whereas the percentage of reduced and/or malformed pistils in both double types was greatly enhanced by TIBA treatment or high temperature. This suggests that development of a normal pistil involves a different development mechanism which is not affected by the temperature or growth regulator treatments used in this study. 49 VI. CONCLUSION AND SUNIMARY Growth regulator substances and environmental factors were studied in relation to female sterility which is commonly observed in double-flowered petunias. Of the numerous growth substances tested, triiodobenzoic acid (TIBA) induced single -flowered phenocopies. Complete reversion of double to single types occurred with the heterozygous genotypes _D_d_, whereas it was only partial on the homozygote _D_Q. Intensive studies previously reported indicated that TIBA can overcome apical dominance, influence auxin movement and simu- late auxin suppressing stamen development. The possible action of TIBA is that it acts as an auxin transport inhibitor and thereby causes an increase of auxin level at the synthetic site, the apex. The balance of endogenous auxin level is believed to influence floral organ development; higher auxin levels suppress the action of _D_ in producing double flowers and thus female fertility is restored since the carpel can develop normally. Summer greenhouse conditions and high temperature growth chamber treatment were effective in inducing a higher percentage of flowers with reduced or malformed pistils in the double types, whereas only very few double flowers in the winter months or at a lower temperature in the growth chamber produce pistillate structures. The results of this study further indicate that a high temperature environment ten days before anthesis enhanced pistil development and thus supports the hypothesis that floral organ differentiation 50 is influenced. The high temperature effect may be related to a change of endogenous auxin level since it has been reported that high temperatures lower apical dominance , thus suggesting that auxin transport is inhibited. Further work could be developed in several aspects: first, to obtain more detailed and precise knowledge of the endogenous auxin level in relation to the expression of flower types as conditioned by Q and _d_; secondly, the improve- ment of the methods of applying exogenous growth regulators. A breakdown of each weekly application into seven diluted daily applications is suggested (Cathey, 1969), with the assumption of creating a more steady and homogenous hormonal environment throughout the treatment period. Thirdly, to find out if there are significant interactions between high temperature and TIBA treatment. Fourth, to observe the hormonal control of floral organ morphogenesis by bud culture so as to eliminate at least partly the complicated interaction of the site of floral organ development with hormones from other plant parts, and fifth, to determine whether fertile pollen is produced and seed set is possible on TIBA treated plants. 51 10. ll. BIBLIOGRAPHY Almeida, C. R. M. 1950. De: Corea do transport polar das auxinas. Anais Inst. Sup Agron. 17:261-305. Atsmon, S. M. . A. Lang and E. N. Light. 1968. Contents and re- covery of gibberellins in monoecious and gynoecious cucumber plants. Plant Physiol. 43:806—810. Bendana, F. E., A. W. Galson, R. Kaur-Sauhney and P. J. Penny. 1965. Recovery of labeled Ribonucleic Acid following adminis- tration of labeled auxin to green pea stem sections. Plant Physiol. 40:977-983. Bunning, E. , U. H. 11g. 1954. Polaritatsstorugen bei Pflanzenzellen durch Athylen. Planta 43:472-476. Bukovac, M. J. and S. H. Wittwer. 1961. Gibberellin modification of flower sex expression in Cucumis sativus L. Adv. in Chemistry Series 28:80-88. Burg, S. P. 1962. The physiology of ethylene formation. Ann. Rev. Plant Physiol. 13:235-302. and E. A. Burg. 1966. The interaction between auxin and ethylene and its role in plant growth. Proc. Nat. Acad. Sci. 55: 262-2690 Cathey, M. 1969. Personal Communication. Clor, M. A. 1967. Translocation of Tritium-labeled gibberellic acid in pea stem segments and potato tuber cylinders. Nature 214: 1263-1264. Cock, L. J., R. E. Taylor and S. Jubb. 1965. The effect of TBA residues on compost on the growth of mushrooms and subsequently on tomatoes and cucumbers. Plant Path. l4(3):105-108. Cooke, A. K. and D. I. Randall. 1968. 2-haloethanephosphonic acid as ethylene releasing agents for the induction of flowering in pineapples. Nature 218:974. 52 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. De la Fuente, R. Kde. , and A. C. Leopold. 1966. Kinetics of polar auxin transport. Plant Physiol. 41:1481-1484. Galston, A. W. 1947. The effect of 2,3, 5-Triiodobenzoic Acid on the growth and flowering of soybeans. Amer. Jour. Bot. 31:356-360. . 1969. Hormonal regulation in higher plants. Science 163: 1288- 1297. Galun, E. 1962. Culture and sex modification of male cucumber buds in Vitro. Nature 194:596-598. , S. Izhar and D. Atsmon. 1965. Determination of relative amcin content in hermaphrodite and andromonoecious Cucumis sativus L. Plant Physiol. 40:321-326. Gregory, F. G. and C. R. Hancock. 1955. The rate of transport of natural auxin in woody shoots. Ann. Bot. 19:451-465. Haccius, B. and D. Messfeller. 1961. Untersuchimgen Zur Biologischen Aktivitat der phenylborsaure. Planta 56:174-188. Hamilton, T. H. 1968. Control by estrogen of genetic transcription and translation. Science 162:649-660. Hertel, R. and A. C. Leopold. 1963. Versuche zur analyse des Auxin-transports in der koleoptile von leg mays L. Planta 59: 535-5620 Heslop-Harrison, J. 1956. Auxin and sexuality in Cannabis sativa. Physiol. Plantarum 9:588-597. . 1963a. Sex-expression in flowering plants. In Brookhaven Symposia in Biology, Meristems and differentiations. No. 16. :109-125. . 1963b. The control of flower differentiation and sex expression. Regulateurs de la Croissance Vegetale. 649-664. Ito, H. and T. Saito. 1956. Factors responsible for the sex expression of Japanese cucumber. III. The role of auxin on the plant growth and sex expression (1) Jour. of Hort. Association. Japan. 25:101-110. 53 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. Ito, H. 1957. Factors responsible for the sex expression of Japanese cucumber. V. Causal interpretation of the effects of pinching and growth substances on the transformation of the primordia of the staminate flower nodes. Jour. of Hort. Assoc. Japan. 25:213-220. Jones, K. L. 1947. Studies in ambrosa. IV. Effects of short photo- period and temperature on sex expression. Amer. J. Bot. 34:371- 377. Keitt, W. J. and R. A. Baker. 1966. Auxin activity of substituted Benzoic acids and their effect on polar auxin transport. Plant Physiol. 41:1561-1569. . 1967. Acropetal movement of auxin: dependence on tem- perature. Science. 156:1380-1381. Kentzer, T. and E. Libbert. 1961. The blockage of gibberellin trans- port in hypocotyl segment by TIBA. Planta 56:23-27. Kiermayer, O. 1961. Uber die formative Wirksamkeit der 2,3, 5-Tri- iodobenzoesaure (TIBA) im vegetativen under generativen Bereich von solanum. Osterreich. Bot. Zeitschr. 108:102-156. Kline, J. 1967. Personal communication. The Pan American Seed Company. , Paonia, Colorado. Kuse, G. 1953. Effect of 2,3,5-Triiodobenzoic acid on the growth of .lateral bud and on tropism of petiole. Memoirs Coll. Sci. , Univ. Kyoto, Series B. 20:3. Laibach F. and F. J. Kribben. 1950. Der Einfluss von Wuchstoff auf die bildung mannlicher und weibeic her bluten bei einer monoziochen Planze. Ber. Deut. Bot. Ges. 62:53-55. . 1952. Wuchsstoff und blutenbidung. Beitr. Biol. Pfl. 29: 129-141. Leopold, A. C. 1961. The transport of auxin. Encyclopedia of Plant Physiol. 14:671-682. Love, A. and D. Love. 1945. Experiments on the effects of animal sex hormones on dioecious plants. Arkiv. For Botanik. 32A 13:1-60. 54 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. Lower, R. L. and C. H. Miller. 1969. Ethrel, a tool for plant hybridizers. Nature 222:1072-1073. Mathan, D. S. and R. D. Cole. 1964. Comparative biochemical study of two allelic forms of a gene affecting leaf shape in the tomato. Amer. J. Bot. 51(5):560-566. McMurray, A. L. and C. H. Miller. 1969. The effect of 2-Chloro- ethanephosphonic acid, (Ethrel) on the sex expression and yields of Cucumis sativus. J. Amer. Soc. Hort. Sci. 94(4):400-402. Miller. C. H., R. L. Lower and A. L. McMurray. 1969. Some effects of ethrel (2 -chloroethanephosphonic acid) on vegetable crops. Hort. Sci. Vol. 4(3):248-249. Mitchell, W. D. and S. H. Wittwer. 1962. Chemical regulation of sex expression and vegetative growth in Cucumis sativus L. Science 136:880-881. Molliard, M. 1898. De l'hermaphroditism chez 1a Mercuriale et le chanvie. Rev. Gen. Bot. 10:320-324. Natarella, N. J. 1968. The morphogenetic effects of the double flowering gene in Petunia hybrida Hort. M.S. Thesis, Mich. State Univ. 50 pp. Niedergang, K. E. and F. Skoog. 1956. Studies on polarity and auxin transport in plants. I. Modification of polarity and auxin trans- port by Triiodobenzoic acid. Physiol. Plant. 9:60-73. and A. C. Leopold. 1957. Inhibitors of auxin transport. Physiol. Plant. 10:29-35. Nitsch, J. P., E. Durtz, J. Livermaim and F. W. Went. 1952. The development of sex expression in cucurbit flowers. Amer. J. BOto 39:32T43o Patau, K. , N. K. Das and F. Skoog. 1966. Induction of DNA synthesis by kinetin and indoleacetic acid in excised tobacco pith. Physiol. Plant. 10:949-966. Peterson, C. E. and L. D. Anhder. 1960. Induction of staminate flowers on gynoecious cucumbers with gibberllin. Science 131: 1673-1674. 55 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. Pilet, P. E. 1963. Action de lacide 2,3,5-triiodobenzoique et du glutathion sur la morphologia. Physiol. Plant. 16:299-310. . 1967. Auxin content and auxin catabolism in relation to the growth polarity. Physiol. Plant. 20:285-290. . 1968. Basipetal and acropetal auxin transport in relation with temperature. Physiol. Plant. 23:1196-1200. Podesva. J. 1956. Studies on the use of growth substance solution for spraying forced vegetables. Sborn Csl Akad. Yemed Ved Rostl. Vyroba. 29:737-746. ' Putnam. A. R. 1969. Personal Communication. Robinson, R. W., S. Shannon and Manual D. de la G. 1969. Regulation of sex expression in the Cucumber. BioScience 19:141-144. Rudich, J., A. H. Halevy and N. Keda'r. 1969. Increase in femaleness of three cucurbit by treatment with ethrel, an ethylene releasing compound. Planta 86-1:69-76. Sabins, D. D. and L. J. Audus. 1967. Growth substance interactions during uptake by mesocotyl segments of Led may L. Ann. of Bot. (London) 31(122):263-281. Schaffner, J. H. 1918. Influence of environment on sexual expression in hemp. Bot. Gaz. 71:197-219. Scott, R. C. and A. C. Leopold. 1967. Opposing effects of gibberellin and ethylene. Plant Physiol. 42:1021-1022. Shifriss, O. 1961. Sex control in Cucumbers. J. of Heredity. 52:5-12. Sims, W. L. 1969. Ethrel effects on sex expression and flower development. Calif. Agriculture 23:2. Smith, D. L. 1967. The experimental control of influorescence development in Carex. Ann. of Bot. (London) 31:121. Stebbins, G. L. 1965a. Some relationships between mitotic rhythum, nucleic acid synthesis and morphogensis in higher plants. In Brookhaven Symposia in Biology, Genetic control of differentiation. No. 18:204-221. 56 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. Stebbins, G. L. 1965b. From gene to character in higher plants. American Scientist. 53:104-126. and E. A. Yagil. 1966. The morphogenetic effects of the hooded gene in barley. I. The course development in hooded and awned genotypes. Genetics 54:727-741. Strasburger. E. 1900. Versuche mit dioischen pflanzen in Rucksicht auf Geschlecheeverteilung. Biol. Centralbl. 20:657-753. Tepfer, S. S. 1965. The growth and development of flower buds in culture. Proc. Intern. Conf. on Plant Tissue Culture. ec. P. R. White et al.:287-295. , A. J. Karpoff and R. I. Greyson. 1966. Effects of growth substances on excised floral buds of aquilegia. Amer. J. Bot. Thimann, K. V. and W. D. Bonner, Jr. 1948. The action of tri- iodobenzoic acid on growth. Plant Physiol. 23:158-161. Thompson, A. E. 1955. Methods of producing first generation hybrid seed in spinach. Cornell Agr. Exp. Sta. Memoir. 336:1-48. Warner, H. L. and A. C. Leopold. 1969. Ethylene evolution from 2-chloroethancphosphonic acid. Plant Physiol. 44:156-158. Went, I". W. .1928. Wuchsstoff und Wachstum. Rec. Trav. Bot. Necrl. 2521-116. Winter, A. 1967. The promotion of the immobilization of auxin in avena coleoptiles by Triiodobenzoic acid. Physiol. Plant. 20: 330-336. Wittwer, S. H. and I. G. Hillyer. 1954. Chemical induction of male sterility in cucurbitis. Science 120:893-894. Yagil, E. A. 1965. A morphogenetic comparison between awned and hooded genotype of barley. Ph. D. Thesis. , Univ. of Calif. , Davis. pp. 131. 57 75. Zeevaart, J. A. D. 1962. DNA multiplication as a requirement for expression of floral stimulus in Pharbitis nil. Plant Physiol. 37:296-304. 76. Zimmerman, P. W. and F. Wilcox. 1935. Several chemical growth substances which cause initiation of roots and other responses in plants. Contr. Boyce. Thompson Inst. 7:209-229. 58 395 I. I'll I I ’1 I I It ’I I5 I I l 1' 5' I III I I It I! I, [I I! I l! I II III 9 03169 4 IIIIHUHIH.