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Use of growth regulating substances as an aid in hybridization of Phaseolus

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Title:
Use of growth regulating substances as an aid in hybridization of Phaseolus
Creator:
Jyotishi, Rameshwer Prasad, 1920-
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English
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iv, 123 leaves : illustrations ; 28 cm

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Subjects / Keywords:
Abscission ( jstor )
Chemicals ( jstor )
Fertilization ( jstor )
Germination ( jstor )
Hormones ( jstor )
Pollen ( jstor )
Pollen tubes ( jstor )
Seed pods ( jstor )
Seedlings ( jstor )
Species ( jstor )
Beans ( fast )
Hormones ( fast )
Plant hybridization ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 116-121).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Rameshwer Prasad Jyotishi.

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University of Florida
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This item is presumed in the public domain according to the terms of the Retrospective Dissertation Scanning (RDS) policy, which may be viewed at http://ufdc.ufl.edu/AA00007596/00001. The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
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36433391 ( OCLC )
ocm36433391
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LD1780 1950 .J991 ( lcc )

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Use of Growth Regulating Substances as an Aid

In Hybridization of Phaseolus












By
RAMESHWER PRASAD JYOTISHI


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY












UNIVERSITY OF FLORIDA

Septemler, 1950











ACKNOVLEDGENTS


The author wishes to acknowledge with deep gratitude the kindly advice of Dr. A. P, Lorz, under whose guidance and constant help this study was conducted.

He wishes to express his thanks and sincere appreciation to the members of his committee as well as to the members of the staff of the University of Florida whose assistance and encouragement were a constant source of inspiration.

He particularly acknowledges the kindly advice and criticisms of Dr H. S. Wolfe, Head Professor of Horticulture and Mr. L. W. Ziegler, Assistant Professor of Horticulture who went through the manuscript and Mr. W. D. Hanson, Assistant Professor of Agronomy who gave valuable suggestions regarding the statistical analysis of the data.










TABLE OF CONTENTS


INTRODUCTION ........................................ 1

REVIEW OF LITERATURE ...............,...��...... 4

General .4...� ..... .....�.....����.... 4

Hormones as an Aid to Fruit-Set ..................... 13

( Hormones as an Aid in Hybridization by Checking Incompatibility .................................... 15

Use of Hormones in Increasing Yield of Beans .�����. 18

Effect of Hormones on Germination of Seed and
Growth of Seedling 20...��**.��............... 20

EXPERIMENTAL MATERIAL AND METHODS .........��.���..�� 24

A. General ....g................................ 24
"B. The Technique of Emasculation and Pollination .��� 25 C. Preliminary Studies .............................. 28

1) Especially Designed Micrometer Pipette for
Hormone Application ........................... 37

2) Trials on Phaseolus coccineus (Scarlet-runner
bean) ��.�.�����.�..�...�.��.���.�.��.... 41

3) Place of Application of Hormone ............� 43

4) Trials with Hormone in Glycerine-Karo Mixture � 46

D. Final Experiments ............................. 48

V Experiment 1. Interspecific Crosses .............. 51

Experiment 2. Effect on Fertilization and Ovule
Development ............. ...................... 54

Experiment 3. Effective Range of 2,4-D Concentration ..��..�....... . .��.�������. 54


iii











Experiment 4. Germination Tests .................. 56

Experiment 5. Effect of Hormone Treatment on the
Rate of Development of Pods ...................... 56

RESULTS ............*............. 58

Experiment 1. Interspecific Crosses ................. 58

Experiment 2. Effect on Fertilization and Ovule
Development ........o..........................o.. 70

Experiment 3. Effective Range of 2,4-D Concentration ....�................. 74

Experiment 4. Germination Tests ................... 78

Experiment 5. Effect of Hormone Treatment on the
Rate of Development of Pods ....................�.. 81

DISCUSSION .......................................... 92

General ........................................ 92

Effect on Seed-Set .................................. 97

Medium and Method of Hormone Application ......... 101 CONCLUSION ......... 5*5O .. ........... * .......�..... 108

SUMMARY ......................*.........0... 110

APPENDIX ....�.....o.... .............. ..�.. 113

LITERATURE CITED .................,.................. 116

BIOGRAPHICAL ITEMS .,.....,...,.........,...... 122










INTRODUCTION


Beans are an important truck crop of Florida, Which is also of considerable commercial value to the agricultural economy of the United States. Numerous varieties of beans have been evolved during recent years and a very high standard with regard to the horticultural characteristics has been achieved in the existing varieties grown commercially in Florida. There is, however, never a limit to improvements and perfection has not been reached. Improvements towards still better combinations of characters by hybridization are possible and researches in that direction are in progress. Resistance to diseases, such as downy-mildew, to cold, to drought, and to such insects as Mexican-bean-beetle are some of the desirable qualities that might te possible through further improvements in beans by interspecific crossing.

While hybridization is one of the most effective tools
of the plant breeder working towards crop improvements, there are certain limitations in its' practice. Incompatibilities among species and varieties and often a very low percentage of pod-set are great handicaps in bean breeding work. Incompatibility may be due to various causes and be of various types It may result from a failure of the fusion between male and female gametes, that is, failure of the fertilization proper, or it may be due to any of a number of other

1










causes such as improper germination of the pollen, too slow growth and penetration of the pollen tube,. lack of stimulation to development of ovary and surrounding tissues, abscission of style before fertilization, or the abscission of the whole flower before pod and seed set. For success and ease in hybridization a plant breeder is desirous of obtaining a high percentage of success in crossing resulting in healthy and well-developed seed of good germination capacity. In bean breeding both types of difficulties are met. Many species have not been successfully crossed with the common improved types or the percentage of set obtained has been very low, necessitating pollination of numerous flowers. Furthermore, unfavorable environmental conditions such as extremes of light and heat also result in high abscission rate. The exact causes and nature of incompatibility between certain species are not definitely known and may be many and various.

Hormones have assumed great importance in horticultural research during the recent past and have been used in many ways to stimulate growth and other physiological processes in plants. Checking the abscission of fruits and supplementing normal fruit-set have been the two most important uses of hormones with apples and tomatoes. Although hormones have been generally reported as inhibiting fertilization, by regulating the amount and method of application they may encourage development of seed and fruit, reduce abscission










of the flowers before fertilization and thus overcome the above mentioned difficulties without inhibiting fertilization. Very little work has been done with regard to the use of hormones in this respect but there is evidence in the literature which suggests that hormones may be used with advantage in hand-pollination to obtain a greater number of fruits set and healthier seeds,
The results of investigations and trials with hormones on beans with a view to obtaining various interspecific crosses are reported in the following pages. The aim of the study has been threefold. In the first place, as many species as possible have been used as the staminate parent in attempted crosses with the standard varieties of snapbean in an effort to obtain hybrids which may show desirable combinations of characters or may at least prove helpful in bridging the gulf between them so that they may be later used for still further crossing as one of the parents. Secondly, an attempt has been made to study the effects of different hormone treatments, their concentration and method of application on hand-pollinated snapbean flowers with a view to working out an effective treatment which would overcome some of the incompatibilities and difficulties involved in obtaining good pod-set. Thirdly, the aim of this study has also been to evolve a technique for applying the hormone treatment which would permit measurement and regulation of the quantity applied in order that the procedure might be repeated.










REVIEW OF LITERATURE


.General


Although hormone research is comparatively a recent

venture in the field of horticulture, the literature on the subject is voluminous, It is neither possible nor desirable to attempt a complete review of this literature here, As is well known, hormones have been demonstrated to be useful in favorably affecting the physiological activities of plants in various ways. The most common uses of economic importance are: the stimulation of the rooting of cuttings, the control of fruit-drop by reducing abscission, the thinning of blossoms and budsj the production of parthenocarpic fruits, the inhibition of sprouting, and the killing of weeds, While considerable work has been done on the above mentioned aspects, the literature on the use of hormones for stimulating fruit-set and viable seed production is very meager. In fact there is evidence to the effect that hormones actually inhibit fertilization (37). An attempt is made here

to review the pertinent literature on hormone research regarding their use as an aid to fruit-set, fertilization, overcoming incompatibility and any other physiological activity that directly or indirectly helps in self or cross fertilized fruit development.

Boysen Jensen in 1910 showed that some chemical substance










is translocated and brings about certain growth responses within the plant body (8, 9). This provided an explanation to Darwin's earlier observation that shoots of grass failed to bend towards light if their tips were out oft (4). In 1928 Vent demonstrated the bending of decapitated oat coleoptiles to one side when agar jelly containing some unknown hormones was applied. This method is still the most widely used and probably the best method for measuring concentrations of unknown hormones (50). By 1934 auxintriolic acid (auxin-a), auxinolonic acid (auxin-b) and Indole-acetic acid (heteroauxin) were isolated from plants. Indole-acetle acid is known to occur in higher plants and has been obtained from leaves, endosperm of seeds, etc. (4). Zimmerman and Vilcoxon (57, 58, 59) and later others, found that many other chemical compounds such as indole-butyric, indole-propionic, naphthaleneaoetic, phenylacetic, and indolepyruvic acids

acted as stimulants in various growth processes and so may be called synthetic hormones. Many other compounds have since then been added to the list but the chlorophenoxy acids and their derivatives have been most widely used.

The plant breeder, desirous of crop improvement by
hybridization, is primarily interested in a higher percentage of fruit-set with his crossing, and in production of viable seeds. The use of hormones to accomplish this end is thus or interest and should be considered from two points of view: 1) their use as directly helping the actual pro-










cess of fertilization, stimulation and development of ovary and the fertilized ovules, and creating the necessary favorable condition for such activity, 2) their use as indirectly helping to obtain greater number of fruits, and therefore seeds, by checking abscission of flowers and young fruits. Not much work has so far been done with regard to the first, at least with beans, but there is quite a considerable amount of evidence concerning the usefulness of hormones in checking abscission. Control of fruit drop of apples with the help of hormones has become an established commercial practice.

That hormones play an important part in the process of fruit development is also well known. Van Overbeek et al.

(36) have shown that hormones control the growth of ovules. On injecting 1% solution or emulsion of the ammonium salt of naphthaleneacetic acid into the young ovaries of 4-n e they produced parthenocarpic fruits whose ovules had enlarged and developed seed coats but they contained no embryo. When the emulsion was applied to the outside of the ovary, the ovary was stimulated but not the ovules. They suggested a mechanism of fruit and seed development which consists of
stimulation of ovaries, ovules and placentae, and the necessary stimulation to set in motion the division of polar nuclei and egg cell, which may take place with or without fertilization. They suggest three stages of fertilization.

The first one is the stimulation of the ovary, placentae and










ovules and checking abscission which prepares conditions favorable for embryo development. Actual fertilization, i.e. fusion, is not necessary for these conditions. The second stage is the division of the polar nuclei and, rarely, of the egg cell, which are not always a direct result of fertilization. Instances are known where the polar nuclei fuse to form an endosperm nucleus and this divides many times before the sperm nuclei leave the pollen tube and fusion takes place. The presence of pollen tube in the embryo-sac, however, seems to be necessary for initiating divisions of the endosperm. The last step is the actual fusion of the sperm nuclei with the egg cell and polar nuclei. It is, however, important to note that actual fusion, although indispensible for hybridization, is not all that is needed. For a complete process the other two stages, which are probably hormone induced, may be of equal importance. This fact is evidenced by earlier work of Gustafson (19) who showed that except for the absence of ovule development, the fruit development by chemical treatment is in no way different from that resulting from pollination in the tomato. The matter of food supply is important in both cases and its mobilization seems to be influenced by hormones.

It was formerly believed that the initiation of growth of the ovary is caused by the hormone secreted by the pollen tube (19). It has now been demonstrated that the pollen tube











may only activate the hormone already present in the ovary as precursor (36, 37). Van Overbeek has summarized the literature on the growth-regulating substances in plants

(37). Evidence has been furnished that the hormone activity is brought about by an active auxin complex in the plants which is released from the precursor or bound auxin already present. The pollen or pollen tube, thus, may not contribute the necessary hormone but may bring only a substance which activates the bound, inactive auxin in the ovary. Muir (32) made direct measurements of diffusable growth-hormones in the style and ovaries of the flowers of tobacco. He found much higher amounts of auxin in pollinated flowers than in unpollinated ones. Also the extent of penetration of the pollen tube was found to be closely related to the hormone concentration. The pollen or pollen tube extracts did not show the presence of hormones and Muir suggested that the pollen tubes probably secrete an enzyme which can liberate the growth-hormone from the inactive state in the style and the ovary. A similar phenomenon is also noted in the galls and nodules produced by various species of bacteria in plants which show a high active-auxin content. These organisms probably, much in the same way as pollen tube, only activate the native bound auxin (37).

Reporting on the work of Went (51) and Skoog et al.

(41) Van Overbeek in the above review points out the interaction between auxins and chemically-related substances. It










has been observed that the activity of auxins is greatly in-. creased by the action of certain substances chemically related to them. Went (51) found that the split pea stems shoved a much larger auxin response when they were first soaked in phenylbutyric acid or cyclohexaneacetic acid. Auxin alone or phenybuteric acid alone did not show as much growth response. This is suggestive of a better effect that may be expected with hormone mixtures than single hormones, an effect known as synergesis.

The growth and germination of the pollen tube also

seems to be related to auxins. Smith (42) has studied the growth of pollen with respect to temperature, auxins, colchicine, and vitamin-B in the culture medium. Additions of auxins (3-indole-acetic acid, 3-ndole-butyric acid and naphthaleneacetic acid) showed "a moderate stimulation" with Snapdragon and a *much greater stimulation of both germination and tube elongation" in Bryophyllum. A temperature of 250 C. showed optimum tube growth and also showed greatest auxin effect. Concentrations of auxins higher than 1 : 50,000 were found to be toxic resulting in "decreased germination, bursting and distortion of tubes".

Addieott (1) studied pollen germination and tube growth
of Mill& biflora and TkooaeoluM majus and found that the germination of pollen grains and the growth of tubes are not necessarily related. Both can be independently stimulated.










Vitamins, hormones, pyramidines and purines showed stimulating effects. He tried 33 different growth-promoting substances, of which 16 showed significant increases in germination or tube growth. But the growth of pollen with these substances was not comparable to that in stigma exudate. In the latter case the tubes had an average length of 16 diameters as compared with from 3 to 6 diameters in various other media.
Chen (23), however, could not find any favorable

effect on germination and tube growth with auxins on Prunux armerL*aca which showed favorable responses to additions of micro-nutrient elements to the culture medium.

The work of Kuehlwein (24) throws further light on the
activity and stimulating power of growth-promoting substances on pollen and stigma. His results are of especial interest because they suggest the possibility that the pollen tube growth of certain species might be favorably stimulated with certain hormone treatments. By adding the pollen and stigma

extracts of various species to the culture media and observing the growth of pollen tubes of various species in different media, he found that the growth-promoting substances of pollen and stigma are 'species or organ specific"; some producing a more favorable increase in growth or germination with the pollen of certain species than with the pollen of others.

Lewis (28) studied the problem of incompatibility and










parthenocarpy of cherries, plums and Oenothera. He says that incompatibility is a genetically controlled character. It may be because of the failure of the pollen tube to reach the ovary before abscission of the flower or style. Fertilization thus cannot take place and ovules do not develop. Using alpha-naphthaleneacetamide he studied its effect on pollen tubes, the mechanism of abscission of the flowers and style, and the development of fruits and seeds with and without pollen. In case of Prunus avium the application of an aqueous solution of alpha-naphthaleneacetamide to flowers resulted in delayed abscission of the style. This species is self-incompatible and untreated flowers, both unpollinated and self-pollinated, lost their styles two days earlier than the treated ones. The growth of pollen tubes of compatible pollen was, however, not affected by the hormone treatment. With Oenothera the aqueous solution showed no effect, but application of 1% lanolin emulsion to the lower part of the ovary delayed abscission. Time of treatment, from four days before up to the time of flower opening, shoved no significant difference in effectiveness. Incompatible tubes shoved many abnormalities; with 1% emulsion causing swelling or even bursting. The compatible tubes shoved a more remarkable effect in stopping growth at 4 to 5 mm. and their ends bursting instead of reaching the 20 M. length

of untreated ones.

Lewis thus found that alpha-naphthaleneaoetamide treat-










ment without compatible pollen did not stimulate development of fruits in cherries, plums or pear. In .Oenothera, however, smear treatment of the ovary resulted in parthenocarpie fruit development. The ovules grew to normal size but had no embryos. He suggests that for fruit formation the necessary stimulation is provided by the pollen tubes as they enter the ovary as well as by the developing ovules. The pollen tubes probably give a greater stimulus than the ovules in manyseeded fruits. Therefore parthenocarpic fruit development with chemical treatment is more successful in many-seeded fruits like cucumbers than in fruits like plums. Lewis also showed that hormone treatment checks that abscission which is due to the change in the walls of the existing cells and not that which is due to rapid cell division. The abscission of the style and of mature fruits is of the former type while the premature flower and fruit drop is abscission of the latter type.

Besides the evidences quoted above, there are numerous instances where hormones have been demonstrated to check abscission of fruits. LaRue (25) found that the fall of Coleus leaves can be considerably reduced by hormone applications. Date flowers also show the same reaction, (35).

As early as 1939-40 the use of indole-acetic acid, indole-butyrio acid, indole-propionic acid and napbthaleneacetic acid vas demonstrated to be effective in check-










ing the preharvest drop of apples by Gardner et al. (17, 18). Treatment with various chlorophenoxy compounds has been shown by Zimmerman and Hitchcock (59) to result in the tomato flowers staying longer by 10 to 30 days.
Concentrated applications of naphthaleneacetie acid as an aerosol or as dust have been shown to be as effective as sprays applied with a hydraulic sprayer in checking preharvest drop of McIntosh apple by Mitchell et al. (29). Batjer and Thompson (6) have compared the effectiveness 2,4-D and naphthaleneacetic acid in reducing the drop of pears. They find that 2,4-D may probably be a more effective chemical than naphthaleneacetic acid even with a weaker

concentration. The injury caused with 2.4-D seems to be related to the concentrations used; all concentrations above

215 ppm. appear to be injurious.


Hormoneus as an Aid tg Fruit-Set


The part hormones play in fruit development has already been emphasized in the literature reviewed above. The recent work on the use of hormones to encourage fruit-set by supplementing normal pollination, especially with regard to tomato,

is of especial Interest for the study undertaken. The normal fruit-set of tomato has long been known to be low under greenhouse conditions in the north. Howlett (22) reported that the use of hormone treatment in supplementing the natural










setting of tomatoes in the greenhouse helped in overcoming the reduced fruit-set resulting from cold weather. Using indole-butyric acid, he found that an emulsion at 0.3% concentration was effective as a paste. The cross-pollinated flowers with viable pollen, on treatment, developed fruits superior to the untreated. In ease of self-pollinated flowers, only 60% of the untreated flowers set fruits while the percentage was 100% with treated flowers. Treated flowers had a poorer seed development than untreated flowers. The vapors of naphthoxyacetic acid have also been shown to produce a high percentage of seedless fruits (50 to 98%) in greenhouse tomatoes (30). Randhawa and Thompson (39) used various other hormones, viz., beta-naphthoxyacetic acid (50 ppm.), alpha-0-chlorophenoxyproplonic acid (50 ppm.), p-ohlorophenoxyacetic acid (25 ppm.), 2,4-5-trichlorophenoxyacetic acid (10 ppm.) and 2,5-diehlorobenzoic acid (100 ppm.). Spraying the flowers, either when they were half open or when all were open, gave a higher set of fruits than untreated controls. That the effectiveness of different hormones varies according to species is evident from the work of Crane (13) who found that the aqueous sprays of indole-aoetic acid (1500 to 2670 ppm.) gave parthenocarpic fruit-set of figs and the fruits were not inferior to those caprified. Naphthoxyaeetic acid and 2,4-D were not effective. Stewart (44), however, has obtained seedless figs with










2,4-D at the concentration of 250 ppm.
Hormone treatment has not always shown increased fruit set. Evidences of a negative effect are also met within the literature, eg., with greenhouse potatoes (11), Washington navel oranges and Marsh grapefruit (38). There are many more references in the literature which report effectiveness of hormones in causing increased fruit-set with various other crops. These, however, in general, also report that hormoneinduced fruits, though normally pollinated, have a lower seed content. For example, drench spraying with aqueous solution of naphthoxyacetic acid (20 ppm.) applied to strawberries when in full flower, has been reported by Swarbrick (45) to result in a 17% increase in yield. He reports, however, that the fruits did not have the normal number of seeds and that the increase in yield was due to increase in size rather than the number. Burrel and Whitaker (10) used 1% indoleacetic acid in a lanolin paste with success in obtaining an increased fruit-set with muskmelons. The low fruit-set in muskmelons, due to heavy flower drop, is checked by the application of hormone to one lobe of the stigma. They suggest that this method may be useful to the plant-breeders who are interested in a larger number of fruits by handPollination.


-Hormones as an Aid in Hybridiatjon by CheckjU& Inomati-













The earliest record of the effective use of hormone

treatment to overcome incompatibility and obtain fruit-and seed-set with successful fertilization is found in the work of Eyster (15). A strain of Golden-rose petunia was found to be self-sterile, a genetic character behaving as a simple recessive, The incompatibility was caused by a very slow rate of growth of the pollen tube and formation of the abscission layer much earlier than usual. On the basis of Yasuda's findings (5&i) it was stated that the placenta of Petunia violacea secretes a special substance which diffuses into the style causing inhibition of growth of pollen. Spraying the flowers with 10 ppm. aqueous solution of alphanaphthaleneacetamide immediately after or before pollination resulted in development of capsules and viable seeds. The explanation of this is found in the work of Lewis (26) reported earlier. This instance gives reason to believe that the hormone treatment may prove to be useful in overcoming self-or crosssterility of other plants as well.

A highly significant increase in fruit-sot of handPollinated flowers of �is meig obtained by Whitaker and Prior (54) with hormone treatment deserves special mention here. Although self- or cross-sterility is not a problem
with melons, about one third of the hand-pollinated flowers do not set fruits, which is a great handicap to breeding










work. These authors used various hormones and also ether extract of cantaloupe pollen. The hormones were applied in lanolin paste, where and in most cases 10 to 15 ag. per gram of lanolin was the optimum concentration range. 2,4-D and 4-chlorophenoxyacetic acid were found to be most effective and gave very highly significant results. An increase in set of 27% has been reported with the use of hormone treatment.
Emsweller and Stuart (14) successfully overcame the

self-incompatibility of Creole, Craft and Ace varieties of

Easter lily (Lillum joniflorum) with hormone treatment of flowers. They also tried these treatments with interspecific crossing. Various chemicals were used after dissolving them in lanolin at concentrations from 0.1 to 1%. The paste was applied in a wound produced by removing one

petal and also at the base of the style. Effectiveness was found to be the same in both cases. While untreated, handpollinated flowers did not set a single fruit, 1% naphthaleneacetamide treatment resulted in production of viable seeds although they were weak in germination. Treatment

with the hormone caused a swelling of pedicel and delay in abscission by several weeks. Although hormone treatment was not very effective in species and varietal crossing, the authors say that it can help in making some difficult crosses Possible. Chemical analysis of young developing fruits after treatment showed a higher sugar content which










is suggestive of greater mobilization of food material caused by the hormones. The authors believe that this may be responsible for the delay in abseission and may also check rapid degeneration of embryo sacs, keeping the egg cell viable for a longer period.
There appears to be only one instance in the literature where hormones have been used with beans with favorable responses in controlled cross-pollination. Wester and Narth

(53) used 1% concentration of a mixture of indole-butyric acid and p-chlorophenoxyacetic acid in proportion of 4 : 1 and applied it in three ways to cross-pollinated flowers of lima beans. As an aqueous solution, the mixture was sprayed on the stigma and as an emulsion in lanolin, it was applied on the pedicel, with and without scratching the tissue. When applied in a scratch, the treatment resulted in 28.8% of successful sets as against 18.7% for the control. The degree of success depended also on parental oombination$. The most interesting feature of the trials was an increase in the average number of seeds per pod from 1.95 to 2.43.


Use O Hormones IS Lnoreasins Yield of Beans:

Hormones have been definitely shown to be effective in

increasing yield of beans. It would be desirable here to review briefly the literature concerning this aspect.

Fisher et al. (2, 16) conducted extensive studies and










used several chemicals. They demonstrated that bud, blossom and pod drops of string beans are reduced by hormone treatment. These bud, blossom and fruit drops were said to be due to hot, dry weather and insects such as the tarnished plantbugs and the potato leaf-hopper. They induced abseission by higher temperature, by use of illuminating gas and by caging

insects over the plants. Alpha-naphthaleneacetic acid and 2,4-D were found to be most effective, giving best results as a dust (40 to 80 ppm.). An increase of 40% in yield of

wax beans was obtained, resulting from a *greater number of small, high grade beans rather than larger beans'.

Murneek et al. (34), working with snap beans., also

obtained similar increases in yield with hormone treatment but found that the increase in yield was obtained only when the weather was hot. In the fall crop, hormones actually decreased the yield. The authors believed that the yield Increase, when obtained, was due to increase in the chlorophyll content of leaves and a stimulation of carpelgrowth brought about by the hormone treatment. They report

that, at least under certain environmental conditions, the number of pods per plant and their rate of development can be increased by hormone treatment. The same workers further continued the trials using various other chemicals, e.g.,

the substituted phenoxy-bensoie acids and chlorine-substituted phenoxy aclds, and in 1947 (55) reported most con-










sistent yield increases with p-chlorophenoxyacetic acid.
With a summer crop of string beans, spraying the flowers at weekly intervals with 100 to 150 ppm, aqueous solution of
hormone gave as much as 300% yield increase. Dusts were found to be as effective as sprays. The yield increase was found more pronounced when the temperature was above 90 to 950 F. It was also reported that the increase in yield was due only to stimulation of pod tissue. There was a depressing effect on seed development.
Wester and Marth (52), on the other hand, did not find any significant increase in the total yield of lima beans. Alpha-naphthaleneacetic acid as a dust at 50 ppm. showed no significant yield increases with 13 varieties of lima beans tried. The insignificant results were explained to be due to an exceptionally fine weather for seed and pod setting. Clore (12), using alpha-naphthaleneacetic acid on three varieties of bush lima beans, actually found a reduction in yield due to treatment. Concentrations of 50, 100 and 1000 ppm, applied at rates of 75 to 100 gallons per acre resulted In retarded growth and in foliage epinasty. The 5 ppm. conceitration failed to show any favorable or unfavorable effects.


Effect Of Horumesg on Garmination of Seed and Growth of PAa461wg










Tullis and Davis (48) have shown that there is a considerable persistence of the effect of 2,4-D in plants treated with the chemical, Bean plants, when sprayed with 2,4-D during pod development, produced seed which, when sown, gave seedlings exhibiting malformations. Any hormone treatment of flower buds, therefore, has the possibility of affecting the viability of seed and growth of seedlings.

There is a mass of information in the literature on the effect of hormone treatment of seeds and soil on the germination percentage and development of seedlings. The results, on the whole, are conflicting. The majority of

the evidence seems to show that hormone treatment has a deleterious effect on germination of most seeds, or that, at least, it does not increase the germination percentage.

The results of various studies so far made indicate that seed treatment with hormones may increase the germination capacity of seeds in case of some field and vegetable crops, while with others it may be retarded.
Avery and Johnson (5) have summarised the literature On the subject of hormone treatment of seeds. Among the

workers reported to have obtained increased germination of Seed and development of seedlings, and subsequent increase in yield, two may be mentioned here. Amlong and Naundorf

(3), using various concentrations of a potassium salt of indle-aetic acid, obtained increase n the yield of alfalfa,











corn, spring wheat and sugarbeet. Sewartley and Chadwick
(4), using naphthaleneacetamide and indole-butyric acid with 41 garden perennials, obtained significant increase in percentage of germination with 11.
The property of retarding the germination and growth of seedlings exhibited by some chemicals suggested the possibility of using them effectively for weed control. 2#4-D had shown such effect with various species.
Hamner et al. (20) used 2.4-D. applying it both to the soil and seeds, to study its- effect on germination of various kinds of seeds. With 1 gi. of the chemical per pot of the soil, they found complete retardation of germination of clover and cabbage seeds. The germination of wheat seed obtained, however, was 85%. No effect on germination was observed with 0.001 gm. of the acid per pot. Application of the chemical to the seeds (soaked for 4 hours) at various concentrations from 1 to 100 ppm. also showed effect on germination and growth of the seedlings. Bean seeds treated
with 1 ppm. of 2.4-D in aqueous solution showed *the characterlstle formative effects and 'virus' like symptoms* in the seedlings. At 10 ppm. the seedlings were *severely checked* and exhibited swelling in the hypocotyl, while 100 PPU. of 2,4-D completely checked growth. The authors concluded that soil treatment with 204-D at one ppm. adversely affects seed germination and growth of seedlings In many










cases. Grass seeds are more resistant than the seeds of many of the vegetables.
Tukey et al. (47) studied the histological changes in

bindweed and sowthistle on treatment with 2,4-D at 1000 ppm. as spray. Among the effects produced were plasmolysis of

pollen grains, checking of chlorophyll development, plasmolysis of cells of leaves, increase in cell division in all cambial zones, enlargement and rupture of cortical cells and disappearence of starch from all parts of the flower. The chemical also checked starch hydrolysis.

Mullison et al. (33) treated the seeds of various field and vegetable crops using the vapors of 2,4-D and its: derivatives. Bean seeds subjected to vapor treatment with 2,4-D derivatives did not show much reduction in germination, although the methyl ester caused a reduction in germination by 12% as compared to the control, Isopropyl 2.4-Dichlorophenoryaeetate vapor treatment of lima bean seeds resulted in germination of 44% as against 68% with untreated controls.

Without going further into the voluminous literature

on weed control by hormones, it will be evident that any use

of hormones for effecting greater pod and seed-set may affect the viability of seed and the nature of development of seedlings. This fact, therefore, needs to be borne in mind while studying the effects of hormone treatment of flower buds with a view to obtaining a greater set of fruit.










EXPRIMNTAL MATERIAL AND METHODS

A. General

As pointed out before, the aim of this study was threefold. The primary object was to find out if hormone treatment could be used effectively in controlled cross-pollination of beans. Since the most effective method of hormone application for treatment of individual flowers so far used and recommended is the lanolin paste method, a secondary objective was to work out some other equally effective method which would permit a greater control of the quantity of hormone used with each flower. This precision is desirable so that the treatment may be repeated with consistent results. The third objective was to cross as many species of Phaseolus as possible within the limitations of time, etc., with a standard variety of snap bean, Cherokee Wax (P. vulzarls), so as to obtain hybrids which could be tested later.

Except for some of the preliminary trials, all the work was done in the greenhouse at the Florida Agricultural Experiment Station, Gainesville, from August, 1949 to August, 1950, In all preliminary trials lima bean was also used as one of the parents, but later on it was found desirable to confine efforts to one variety of P. ZuJrJ , which was the Cherokee Wax. The reason for selecting and using this variety was that its flowers are very convenient for hand-










pollination, it is quite prolific, and it is a representative type of the commerially grown varieties with many good horticultural characteristics.
The two most serious greenhouse pests were mildew and red spider-mite. The former was controlled by dusting with sulphur and the latter through the use of the never miticides especially .PNo miticide, generously provided by the Station Department of Entomology. The R.P.N. miticide was applied as a spray at the rate of 1 lb./lO0 gals. of water. The soil at first was sterilized with formalin or D-D. and later on

was better sterilized with steam to eliminate nematodes. When the plants were established, a tablespoonful of 4-7-5 fertilizer mixture was applied to each plant at monthly intervals.

Inasmuch as the success in any hybridization work depends

mainly on the proper technique of emasculation and pollination, the technique found to be very satisfactory during the course

of this work needs to be described in some detail.


B. The Technique of Emasculation and Pollination


The largest flower buds obtainable about one day previous to anthesis are selected for emasculation (Fig. 1 A). The suture which closes the banner around the other flower Parts is opened with extreme care in order not to tear the










margin of the left (observer's left) half, which in everted and held down with the thumb of the left hand. The left wing of the corolla i pushed slightly aside exposing the

distal end of the coiled keel (Fig. 1 B). Vith a sharp, slightly flattened, pointed pair of forceps the upper side

of the distal portion of the keel is removed with the points of the forceps inserted as indicated by the arrow in Fig,

1.B The anthers are then removed taking care to disarrange the other flower parts as little as possible.

The stigmatic surface is then pollinated as follows:
The pollen-laden, distal portion of the pistil of a freshly

opened flower from the selected staminate parent is rubbed against the stigma of the emasculated flower of the selected pistillate parent. Usually the pollen-laden distal

portion of the pistil can be made to protrude from the opening in the end of the keel by the downward pull of the left wing of the corolla with relation to the other flower parts. After pollination, the left side of the banner is returned to its normal position and the other slightly disarranged

flower parts are carefully tucked underneath so that the bud is completely closed, In most eases only a slight separation of the margins of the right and left halves of the banner give any indication that the flower has been worked with. The suture then may be sealed with a quick drying latex suspension applied with a camel's hair brush.


















































The Successive Stages of Emasculation
and Pollination of Bean Flowers.
(Explanation given In the text)


Fi 0 1.










A proprietary name for such a latex suspension is Goodrite VL 100, manufactured by B. F. Goodrich Chemical Company, Cleveland, Ohio. In most oases the use of latex is unnecessary if the flower parts are only slightly disarranged
during the emasculation process and the bud Is returned to its original closed condition after pollination. Where closure is difficult or conditions of low atmospheric humidity prevail, the use of latex is more essential in

order to prevent the withering of the style before the pollen tubes have had a chance to grow through it. The successive steps in the process of emasculation and pollination of bean flowers are illustrated in Fig. 1.


C. Preliminary Studies


Instead of arbitrarily selecting hormones and concentrations for treatments or using the chemicals reported to be effective, it was thought better to find out through several trials which chemicals and concentrations showed some kind of stimulation without causing any undesirable effect. It was therefore necessary to make numerous preliminary trials which consisted of treatment of several hundred flowers both in the field and in the greenhouse.

As the method of sealing the pollinated flowers with
latex suspension is very effective and convenient, the first










attempt was to see if the chemicals could be effectively used with latex. Stock solutions of the following hormones were made in latex at 2000 ppm.

1. Indole-propionie acid

2. Beta-naphthoxyacetic acid

3. 2,4-Dichlorophenoxyacetic acid

4. Potassium salt of alpha-naphthaleneacetic acid

5 o Alpha-naphthaleneacetamide

6. 8Q6 Phalinil (A commercial preparation of n-phenyl phthalimideo )

7. Alpha-naphthaleneacetic acid

8. Methyl ester of alpha-naphthaleneacetie acid

The following two hormones were later included in the trials.

9. Indole-butyric acid

10. Para-chlorophenoxyaoetic acid

Several flowers of snap beans (P. vulxaris) and lima beans (P. lunatus) were emasculated and sealed with the hormone mixture in latex as usual without pollination. A
little of the mixture was also applied at the base of the ovary. The object was to study the effect and kind of response of the flower to the hormone without the stimulation supplied by the pollen as is the case in pollinated flowers. Various dilutions wore made from the stock solution to arrive at a concentration where a response could be Induced without any toxicity. The 200 ppm. mixtures of Nos. 2. 3#










4, and 5 gave responses in the majority of flowers treated, The flowers treated with these lasted for more than four days while the others as well as the controls (treated with latex but without hormone) dropped within that time, Although, no definite growth stimulation or swelling was noticed, the peduncle did appear to be straight and stiff. When the trials were repeated, however, the responses were not consistent. Inasmuch as none of the hormones excepting Nos. 3 and 4 are soluble in water or the latex suspension, they could hardly be expected to be effective consistently, because the quantity applied in each ease was not definite and uniform. Alcohol coagulated the latex; therefore, it could not be used to dissolve the hormones before mixing with the latex suspension.

Of all the hormones used only 2,4-D and potassium salt of naphthaleneaoetio acid are appreciably soluble in water. To study the nature of the effect of these two, saturated solutions of each were made in water. A number of emasculated flower buds of snap bean were sealed with latex and a little of the water solution of each was applied by dipping a needle in the solution and making a small scratch at the base of the ovary with it. Controls were maintained. The effect was spectacular. All treated flowers stayed long after the ontrols dropped. Many parthenocarpic pods developed, the 2,4-D sho ing greater effect. Abscission was










definitely delayed or in some cases completely prevented.
Since many of the hormones are soluble in glycerine,

those which shoved response in latex mixture were dissolved in glycerine to saturation. After emasculating the flowers

in the field, a very minute drop was applied on the peduncle at the point of junction with the receptacle of the flower. Controls were maintained. The treatment also was used with flowers emasculated and pollinated with P. lathyrides pollen. In all cases the treated flowers dropped, the peduncle drying at the point of application. No response due to the hormone was seen. However, it was observed that glycerine itself was toxic and killed the tissues at the point of application, thereby causing the flower to wither.

As 2,4-D showed greatest response in thickening of the peduncle, as also reported by Wester and Marth (53) and checking of abscission even of emasculated flowers, further trials were made with 2,4-D latex and lanolin paste. One percent solutions were used and snap bean flowers were

treated after emasculating and pollinating them as summarized in Table I

The results definitely shoved the effect of 2,4-D treatment in delaying the absoission of flowers as is evident from Table I. The results also show a definite timulating effect of pollination even though seed development failed. 2,4-D was thus the most effective and merited





TABLE I


Results of Treatment of the Flower Buds of & XgjzIs with 2,4-D


No. of flowers Growth-regulator Medium Pollination No. of flowers No. of emasculated used used drojujed within parthenocarpic
I to 4 4 to 9 pods dedays days veloped


10 2,4-D 1% Lanolin Not 4 .2 4
pollinated

10 2,4-D 1% Latex Not 10 pollinated

10 Untreated Latex Not 10 Control pollinated


10 2,4-D 1% Lanolin Pollinated - 2 8 vith &

10 2,4-D 1% Latex U 2 5 3

10 Untreated _ 10 - Control










further consideration In the matter of establishing definite dosages which would be effective in preventing abscission without producing an over-stimulation which would be detrimental to seed development. It is not soluble in the common vegetable fats which are fluid enough for ease in application, but it is soluble in Karo, a commercial preparation of corn-syrup which appeared to be very convenient for application. Instead of weighing out the chemical and using different measured concentrations it was easier to make a saturated solution with excess of 2,4-D in Karo which could be separated out by centrifuging or filtration with the aid of heat. This was used as a basis for various dilutions in the viscous media of the Kare type.

The effective range of 2,4-D was found to be very

narrow during the course of further trials. Para-chlorophenoxyacetic acid, which has been reported to be effective with lima beans by Vester and Marth (53), was also included in the trials hereafter. It was also saturated in Karo and

the excess separated out by centrifuging. The treatments then tested were:

1. Saturated 2,4-1D in Karo,

2. 1% 2,4-D In lanolin,

3. Saturated para-ohlorophenoxymeetic sold In Karo and
4. Control, (Karo only).
These were used with both snap bean and lima bean flowers










after emasculating and pollinating them with pollen from various species. Saturated 2,4-D being toxic, the strengths were gradually reduced to 1/2 saturation, and then to 1/4 saturation. The 1/4 saturation appeared to be the safer concentration to use. 2,4-D in lanolin was applied in a scratch by a needle on the calyx at the base of the ovary. 2,4..D and pars-�hlorophenoxyacetic acid in Karo were applied at the same place with and without a scratch, Ordinary glass tubing with one end drawn out to a thin capillary tube and with a piece of a rubber tubing attached to the other end was used at this stage as applicator for the Karo mixture. It was helpful in releasing a minute drop of the chemical in Karo with uniformity within certain limits.

The number and kind of crosses made and the number of sets obtained are given In Table 2.

None of the numerous crosses between lima bean (a Fordhook selection) and a wild type of lima bean and also between snap bean and P. lath=2rodes set any pods when 2.4-D was used, either saturated in Karo or 1% in lanolin. Several crosses between the two types of lima bean (P. t) with reduced strength of 2,4-D were also made. These have been sunmarised in Table 3.

Although these preliminary trials did not show results
Buggestive of a very favorable effect of the hormone application, they at least gave sufficient indication that 2,4-D




TABLE 2


Results of Attempted Crosses between p and . 2o31ne1


Kind of cross Treatment No. of flowers No, of treated pods set


P. vULzaris.

x
P. Zognlau


1. 2,4-D 1/2 saturation in
Karo with puncture

2. 2,94-D 1/2 saturation in
Karo without puncture

3. 2,4-D) 1/4 saturation in
Kara without puncture

4. 2,4-) 0.5% in lanolin in
a scratch

5. Para-ehlorophenoxyacetic
acid saturated in Karo

6. Control, Kara alone


35 12





TABLE 3


Result of Attempted Crosses betveen E.lun&t and a Wild Lima Bean


Kind of oross Treatment No. of crosses No. of sets
made obtained


Lima bean (Q. , unat )


Wild lima


1. 2,4-D 1/2 saturation in
Karo with puncture

2. 204-D 1/2 saturation in
Karo without puncture

3. 2,4--D I/4 saturation in
Karo without puncture

4. 2,4-D 0.5% in lanolin in
a scratch

5. Para-hlorophenoxyaeetic said
saturated n Karo


6. Control, Karo alone 37










was showing response and was not too drastic at 1/4 saturation. The precise quantities however were not controlled in these trials and such control is undoubtedly an important factor on which the effectiveness or inhibitory action depends. A need for a device for measuring a tiny drop with

accuracy and facility was apparent. Para-ehlorophenoxyacetic acid saturated in Karo appeared to be too low in concentration. Even then, it did show a response in slight thickening of petiole and delay in abscission. It was evidently safer to use while 2,4-D showed a narrow limit within which the right effect could be expected.


1) Egwalally Psi ed Migrometer Pipette f= Hormon

Apil, Iation

For further studies with regard to the use of the

growth-regulators in hybridization of beans It became necessary to develop a device for dispensing volumetrically very minute quantities of these highly effective substances. Standard mieropipettes were found to be unsatisfactory for handling viscous media and the necessity for frequent filling because of their low capacities made their use cumbersome. They were also unable to dispense small enough quantities with a sufficiently high degree of accuracy. Accordingly a special pipette was devised to suit the needs of the work. It was found that very minute droplets










of consistent size could be dispensed from this micrometer pipette, which, although developed especially for this work, may have a wider application in the field of microchemical technique. It was found to be highly satisfactory and as such may be described and illustrated here.

The pipette employs the micrometer principle and the amount of fluid medium dispensed is in direct relation to the displacement capacity of a calibrated screw which is provided with a scale in order to make possible accurate measurements of the amounts dispensed by each revolution or fraction thereof. The micrometer pipette illustrated in Fig. 3 was designed so that rotation of the arm (fixed to the main screw) through one centimeter on the circular scale would result in the delivery (from the end of the 27gauge hypodermic needle fragment) of a droplet of water at 40 C. weighing 0.97 mg., having the volumetric equivalent to 0.0010 ml.* with a constant correction factor of 3% to be added where extreme accuracy is required. Hovement of the arm through 1 mm. on the circular scale will result in a droplet barely visible to the naked eye, having a volume of 0.0001 ml. less 3%, for which no correction was made for practical reasons in dealing with such small quantities. The accessory screw activates a worm gear mechanism of fine adjustment which makes possible a slow rotation speed with more positive control. The accessory screw is easily removed by counterclockwise rotation when rapid turning of






















rr




















Fig, 2. Longitudinal Section of the Micrometer Pipette.
(Detailed explanation given in text.)







Ij


Fig. 3. Photograph of the Micrometer Pipette vith
a few Refinements.


in










the main screw in desired. All parts except the hypodermic needle and the screws are fabricated from polysterene plastic resin, In the growth regulator studies it was not necessary to use quantities smaller than 0.0002 .l., which were large enough for the practical purposes of this work.


2) Trials on PhaseolUS coccineup (Scarlet-runner bean)


A few plants of Scarlet-runner bean (E. ggejcieus)

were growing vigorously in the greenhouse and flowered profusely and continuously during fall and spring. All flowers, however, abscissed without producing any pods. Evidently there was some incompatibility resulting from some peculiarity of the environmental condition within the greenhouse because the same material growing outside in a frost-protected location was capable of pod development. It was an excellent plant material for trials and tests to study the nature of the effect of 2,4-D, para-chlorophenoxyacetio acid and also other hormone mixtures. The pollen was definitely viable because sets were obtained easily on JP vuhJgis with P. oooineus pollen.

In order to study the effect of 1/4 saturation of 2,4-D in Karo and saturated para-chlorophenoxyacettc acid in Kero, and simultaneously to see if the self-incompatibility of PL. 9 b could be overcome with hormone treatment, several flower buds were treated with the hormone and sealed. Also










a number of crosses with pollen of various species, with and without the hormone treatment, were made.

Out of the 10 buds treated with 2,4-D at 1/4 saturation,

6 developed pods which were later on found to be parthenocarpic. Those treated with para-ablorophenoxyacetic acid did not develop pods, but the flowers did not absciss for about another 3-4 days beyond the controls. Slight straightening and thickening of the peduncle was also noticed In many cases. Incompatibility of JP.1 occneux with many other species was also present, as the crosses with many species, as listed below in Table 4, set no pods.


TABLE 4
Attempted Inter-specific Crosses with P.o.coineus as Pistillate Parent

No. of crosses attempted with completely Name of species negative results used as pollen Para... . ... .
Para-.chloro2p4-D, 1/4 satu- phenoxyaoetic Control
ration in Karo acid saturated Unin Kare treated


J.P& jgth2odes 13 13 13 Pl 6 6 6 P ( lga al ) 7 7
(Cherokee Wax)
PL troaurnreue 5 5 5 L1I tug 3 3 3
(Fordhoo selection) ..










Two pods developed with t Wzgjurls (Cherokee Vax) pollen from para-chlorophenoxyacetic acid treatment. One pod with seed also developed from an untreated bud pollinated with FL .gIziar8 pollen but this was very near another flower treated with hormone and might possibly have obtained some effect from translocation. All other treated flowers stayed from 3 days to an indefinite period after all other controls dropped. Study of the causes and exact nature of incompatibility in P. cocoineus could have been further studied and presented a problem in itself. But as the hormone treatment did not seem to show very encouraging results in overcoming incompatibility, further investigations were not pursued.



3) flaX1e g ADVlioation o Hormone

Trials were also made to see if the place where the

hormone was applied made any difference in the effect produced. Two places .- 1) on the lower part of the calyx, and 2) at the point of union of the peduncle and the receptacle -- were tested. The effect was first studied on the & 0ocineg flowers which were crossed with & vulzarig pollen and were treated with 2,4-D, 1/4 saturation in Karo, para-chlorophenoxyacetic acid saturated in Karo and with Xaro alone. The hormones were applied at each of the two places in sets of 10 with each treatment. Effects similar










to those already reported were observed and abscission was delayed in both cases. This showed that applications at both places were equally effective.

Having observed the effectiveness to be the same

whether the hormone was applied on the abscission zone or on the calyx, with P cooineus flowers, the method had to be further tested with snap bean flowers using the usual pollination technique. A small preliminary trial using 66 flowers with snap bean as pistillate parent and P, cocoineus as staminate was made. Two places of application as mentioned before were tried. The treatments and number of pollinations made and sets obtained are given in Table 5. The hormones were applied without making any puncture.

These trials showed that application of a hormone drop on the abscission zone was as good on the peduncle as on the ovary and probably was safer as less inhibitory effect on the ovule development could be expected. The best point of application was decided to be the point where the receptacle joins the peduncle. Besides giving information regarding the place of application this trial also suggested the possibility that the hormone treatment may prove useful in obtaining a greater number of sets when the abscission rate is high. During the course of these trials the enironmental conditions seemed ideal for pod setting and a very low percentage of abscission was manifest with untreated flowers.





TABLE 5

Effect of the Place of Hormone Application on Pod-et from Crosses & yulzaris and




On calyx On abscission xone Treatment
Buds No of seas Buds No* or sops treated S treated P


2p4-D 1/4 saturation in Karo 11 1 3 11 2 4 Para-chlorophenoxyacetic acid saturated In Karo 11 1 5 11 1 3 Control, aro alone 11 - 11 1 5


parthenocarpic

seed-containing










4) als with fHormone I& Givcerine-Karo Mixture

The slow-drying property of glycerine, as well as the greater solubility of certain hormones in it, makes it a desirable reagent if possible to use. It was found that 1 part of glycerine to 4 parts of syrup provided a slow-drying medium in which the glycerine was not sufficiently concentrated to be toxic. This mixture was also of a desirable consistency and viscosity for ease of handling. It was decided to test once again with glycerine and Karo other

hormones which could easily be dissolved in glycerine. Saturated solutions of all hormones tested before were again made in warm glycerine and the glycerine so saturated was
mixed with 4 times of its volume of Karo.

A number of buds of P, cogcineus were treated with
hormone mixtures by applying a tiny drop on the peduncle at the abscission zone. An equal number of controls was maintained. Para-chlorophenoxyaoetic acid, methyl ester of naphthaleneacetio acid, beta-naphthoxyacetic acid and indolepropionic acid showed response by delaying abscission by several days and causing slight thickening of the peduncle. A mixture of para-chlorophenoxyacetic acid and indolebutyrio acid was also found to be effective. It was decided to use the mixture of all these hormones in glycerine-Karo mixture also containing 2,4-D. On the basis of these trials the following treatments were chosen for further final analysis.










1. PCPA: Para-chlorophenoxyacetic acid saturated In warm

glycerine and the mixture diluted with four
times Its volume of Karo (corn syrup)

2. PCPA &. IA: Para-chlorophenoxyacetio acid and indolebutyric acid saturated in warm glycerine and
the mixture diluted with four times its volume

of Karo.

3. H.M.: Hormone-mixture made by saturating indolepropionic acid, beta-naphthoxyacetic acid,
para-ohlorophenoxyacetic acid and methyl ester

of naphthaleneacetic acid each in 2 cc. of glycerine and then mixing this with 4 times

its volume of 1/8 saturation of 2,4-D in laro.
Only a small drop of the methyl ester was

sufficient to give a turbid emulsion in

glycerine.
4. KNA: 0.5% solution of potassium salt of naphthaleneacetic acid In glycerine-Karo mixture (1:4).

5. Control: A mixture of glycerine and Karo (1:4) without any hormone,
In all further experiments the above mentioned treatments were used, In all crosses attempted the amount of the chemical used was within the range of 0,0004 - 0.0006 Ml. of the mixture applied with the pipette made for this purpose. These treatments were then tested with PL zHUI1










as pistillate parent with pollen from a number of species. The number of crosses made with different species and the number of sets obtained are given in Table 6. Parthenocarpic pods more than 1 inch in length only have been recorded.

These results did not give a precise information with

respect to individual treatments but definitely shoved that hormone treatment produced some kind of effect without being toxic because 46.43% of the attempted pollinations with hormone application produced either parthenocarpic or seed containing pods in contrast with 5.8% for the control. These results may be regarded as highly significant whether the resulting seed represents valid crosses or not, because the same technique and conditions obtained for the flowers treated as for the control. The glycerine-Karo mixture, instead of Karo alone, increased the ease of application and preciseness of the sise of the drop because of the lover viscosity. The amount used, however, was not as carefully controlled as in the later experiments made.


D. Final Experiments


The preliminary experiments gave enough information
required for the final experiments which were conducted during spring and summer of 1950. The hormone treatments used were: I) PCPA, 2) PCPA & IRA, 3) H.M., 4) KNA, and 5) Control. The methods of their preparation have al-











TABLE 6 Results of Attempted Crosses on P with Hormone Treatments



Kind of cross Treatment No. of No. of sets obtained buds
treated


Parthenocarpic


PL vulZMrill
x


Seed containing


PCPA

PCPA & IBA

MH. KNA

Control


P 3Mu!arig PCPA 12 5 2

X PCPA & 1BA 12 4 a trovurpureus H.M. 7 5 1 KNA 11 2 2 Control 12 1


P Weals PCPA 2 2

X PCPA & IBA - P1.L caloaratug, HIM.

KNA 2 1 Control 2 -


i










ready been given. The different species of Phaseolus and their sources, used in this study were as follows:

A. Phase Olus yulgaris (Cherokee Wax). B. Phaseolus coccineus (Scarlet-runner) P. I. 165421.
Collected by Ware and Manning in Mexico.

C. asegluo coeolneus var Rlbus from 0. f. Parson
(Bartel, 'bush lima').

D. haseolus atropurureus M557 from 0. W. Norwell. E. P j riliformis 51-2 op from 0. W. Norwell. F. 1gna.cljgraa (Korean rice-bean, long day
form), from E. M. Meader, New Hampshire.
G. Phaseolus lathyroides M 248-1 op from 0. V. Morwell. H. M500eoX lunatus A Fordhook lima selection. I. PhaseolUs a utifolius 81 op - 1 op from 0. W. Norwell. J. h acutifolius (white seeded) from Rex Thomas

Beltsville, U. S. Department of Agriculture.

K. !Phasgegl, acutifolius 257 from 0. V. Morwell. L. .Phaseolus anrulari P. I. 174907. M. Phaseolus anular:js (Adzuki) from Rex Thomas, Beltsville.
N. Phaseolus - from E. C. Stair.
0. Phageolus aureus from E. C. StaiT.

In all crosses attempted the amount of hormone preparation used was within a range of 0.0004 - 0.0006 MI., applied with the special pipette. The chemicals were










applied at the point of union of the peduncle and the receptacle without making any scratch. In all 1272 snap

bean flower buds were emasculated and treated with hormones after pollinating them with pollen from different species. 410 buds were allowed to be self-pollinated and then were treated with hormone mixtures or used as controls. As reported by workers in the past (53, 54), for any reliable inferences, a large number of buds should be treated. The percentage of sets being so low, and the variability being so high, this evidently is imperative. he time required for emasculating and treating individual flowers and the limitation set by availability of uniform sized buds restricted the planning of the entire experiment on a statistical basis. The variability In such work is large and less under the control of the experimenter. Size. age, position of the buds on the plant, location, age and stage of growth of the plant, time of emasculation and pollination, all may be expected to affect the sets. A proper design for statistical analysis was, however, used with some interspecific crosses.


erient I. ntrersueific crosses


In the experiment designed for statistical analysis three species were used as pollen parents: ?. aggnuP.L LU1rrmU and Z. njr2!ymt uRM. The number of flowers










pollinated and treated with each were 250,250 and 200 respectively. Bach replication consisted of 10 buds with each treatment, i.e., a total of 50 buds. The treatments were replicated 5 times with the first two while with the third there were only 4 replications. Unlike field experiments in which soil heterogeneity is the main factor causing variability, the replications were made with respect to time as well as position in the greenhouse bench. The first

formed buds may be different from those formed in the middle or latter part of the plant's life. Therefore, a 4-day

period was considered to be the block during which all buds
(50) under each replication were treated. The complete experiment with 5 replications each with P� coccineus and P. f :IfornLi was completed in 40 days. With & atroIRorureus due to the shortage of pollen material and irregularity in buds appearing, each replication consisted of 8 days. While treating and pollinating buds, care was taken to select buds from similar positions on the plant and of uniform vigor and size. Five buds, one with each material, were treated

Simultaneously.

Besides the three species mentioned above, a number of Other species of Phaseolus were also used as pollen parents On snap bean. As it was not possible to repeat similar experiments with each species, as many flowers were pollinated with each as was possible within limitations of time,

Space and availability of staminate and pistillate material.










With some species the treatment KNA was dropped as it did not appear to be promising. The names of various species used as pollen parents besides the three mentioned above, along with the number of crosses attempted with each, are given in Table 7.

TABLE 7

Table Showing the Pollen Material Used and the Number of Crosses Attempted with each on P vularis (Cherokee Wax) as Pistillate Parent,

Pollen parent Treatments and number of attempted crosse s

PCPA PCPA&ThA H.H. KNA Control Total

.aujjjfojU-I 13 13 13 13 13 65 P--. �ouflu- 18 16 16 16 16 80 P&eut2ol - 11 11 11 11 11 55 J aPL, -L 10 10 10 - 10 40 & -N 4 4 4 - 4 le JPL calcaratus -F 20 20 20 20 20 100 . ljh hLgjo A-G 20 20 20 - 20 80 P.- IuAtus -H 15 15 15 - 15 60 P -M 4 4 4 - 4 16 PL -0 10 10 10 - 10 40 ntnM ~aR -C 5 5 5 - 5 20
Total 128 128 128 60 128 572










xueriMnt 2. Effect on Fertilization and uW2 DevelobMent


There is evidence in the literature that hormone treatment Inhibits growth of ovules and their fertilization. Whether this tendency can be overcome by regulating the amount and using proper chemicals was important to investigate. An experiment with snap bean, the parent used for all crosses attempted in this study, was conducted in which the buds were allowed to be self-fertilized. Ten buds of uniform size were subjected to each of the treatments, and each treatment was replicated 4 times for statistical analysis. The amount and method of application were the same as with the species crosses. The results of this experinmnt are reported in Tables 14 and 15. The entire experiment consisted of treatment of 200 buds,



Sueml 2 J. Jffective Rae of 2.A-D Concentration


Although the preliminary studies showed that 2,4-D,
vhich caused quick responses, was likely to be too drastic In effects it was decided to work out the effective ranges With respect to both amount applied in each application and strength of the concentration used. With the help of the special pipette it was possible to control the size of the drop with a higher degree of accuracy than is obtained










by any other known technique of the same facility in use. The aim was to find out whether small amounts of 2,4-D solutions in high concentration were as effective as larger amounts of 2,4-D solutions in lover concentration where equivalent actual amounts of 2,4-D were concerned.
Four different strengths of 2,4-D in the glycerineKaro mixture were used as follows:

1. 2,4-D, 3/4 saturation in a glyeerine-Karo mixture (1:4)

Amounts used: 0.0002 ml., 0.0003 ml., 0.0004 ml.,
0.0006 M., and 0.0008 ml.
2. 2,4-9, 1/2 saturation in glycerine-Karo mixture (1:4)

Amounts used: 0.0002 ml., 0.0004 ml., 0.0006 ml.,

0.0008 m., and 0.0010 ml,
3. 2,4-D, 1/4 saturation in glycerine-laro mixture (1:4)

Amounts used: 0.0002 al., 0.0004 ml., 0*0006 ml.,

0.0008 ml., and 0.0010 ml.
4. 2,4-D 1/4 saturation in glycerine-Karo mixture (1:4)
Amounts used: 0.0004 ml., 0.0006 ml., 0.0008 ml.,

0.0010 ml., and 0.0012 ml.

There were thus 4 different concentrations and 5 different amounts used as treatments. Each time, with these treatments, a control was maintained which consisted of treatment with the glycerine-Karo mixture alone. In all, 10 flower buds were treated with each and with the COntrol and were allowed to be self-pollinated; the total










number of buds treated thus was 210. Observations with regard to response were regularly made and the number of buds which dried out from time to time was recorded. The data

regarding this experiment are reported in Table 16.


Exieriment 4. Cermination Tests


Hormone treatment has been known to inhibit develop. ment of embryo. It was therefore necessary to find out if the seed-set with these treatments produced viable seeds or not, and whether the seeds subjected to hormone treatment during early development were affected with regard to their germination capacity. Germination tests were, therefore, run with the seed obtained from the crosses between P., vulgar.i (A) and P_, coccneux (B) with different hormone treatments and control. 20 seeds were selected at random out of eaoh lot and germinated in sterilized soil in flats. Also similar tests with seeds obtained as a result of selling snap bean (.L a gu i) flowers with various treatments were conducted. The data regarding these tests have been reported in Tables 17, 18, and 19.


Rx~eri ,nt 5. fM;t ,of Hormone Treatment on the Rate at

Develeument of Pods


To ascertain if the stimulation provided by the growth-










regulator mixtures on treatment of buds causes any effect on the rate of development of podsO a subsidiary experiment was run using PL vulgari material. A number of buds of uniform sise were allowed to be self-pollinated and treated with PCPA and 2,4-D (1/4 saturation) and also with control. At each application 0.0006 ml. of PPA and 0.0004 ml. of 2,4-D mixture were used. Measurements of 4 pods from each treatment were taken daily for a period of 8 days, and the experiment was replicated 3 times. Usually the pods reach their maximum sir. In about 8 to 10 days after they have started developing. The aim was to compare the rate of development of pods with and without hormone treatment to determine if there was any initial or continued effect of hormones on the developing pods. The data regarding this study are recorded in

Table 20.










RESULTS


Experiment 1. Ilntersecific Cr0ose


It has been pointed out that the planning of the experiment with crosses between P jaga (Cherokee Wax) as pistillate parent and various other species of Ehseolue as staminate parents was done on a statistical basis only with

three species, viz., P. coccineus. _ a trODurDLs and P& f[liformis. The number of sets obtained with P, atro urous as staminate parent was exceedingly low, making any statistical interpretation impractical, and therefore the results with this species are grouped with those of others. The results of crosses with P. coccineus and PL filiformis have, however, been statistically analysed.

The results of attempted crosses with . ogineW_ (B) as staminate parent on PL vulgarls (A) as pistillate are given in Tables 8 and 9. PCPA and PCPA & MhA treatments gave highly significant increases in pod-set and PCPA & IBA gave a similar increase in the number of seeds obtained as Oompared with control. Without the hormone treatment 38%

of the pollinated flowers set pods containing seeds, while treatments PCPA and PCPA & IBA gave pod-sets of 52% and 64% respectively. As is clear from the table 8, the average number of pods obtained per 10 flower buds treated was 5.2










and 6.4 with PCPA and PCPA & IBA respectively as against

3.8 with control. The least significant difference was calculated as 1.21, so that both treatments gave significant increase in set.


TABLE 8

Results of Attempted Crosses of P. vuLarls X P. coocineus

(B) Using Various Treatments.


Replication Number of pods containing seeds obtained from treating 10 flower buds,


PCPA PCPA&IBA H.M. KNA Control


I 6 7 5 4 2 II 5 6 6 4 6


III 4 6 3 3 3

IV 5 7 5 4 5 V 6 6 5 3 3 Total 26 32 24 18 19 Average 5.2 6.4 4.8 3.6 3.8 % Pod-set 52 64 48 36 38 L.S.D. 1.21
(Analysis of varianoe given in Appendix Table 23)










Since the total number of seeds obtained as a result

of a cross is important rather than the number of pods set, the data regarding the number of seeds obtained with various treatments are given in Table 9.


TABLE 9

Number of Seeds Obtained as a Result of Attempted Crosses
between P ,ulgarls and P.. coccneus (B)
Using Various Treatments.


Replications


Seeds obtained per 10 flowers treated

PCPA PCPA&IBA HI.M. KNA Control


I

II III

IV

V


Total Average


72 106 71 54 59 14.4 21.2 14.2 10.8 11.6


Average number of seeds per pod


L.S.D.

(Analysis of variance given


4.07

in Appendix Table 24)


The data presented in Table 9 show that the treatment


2.77


3.31


2.96


3.0


3.1










with PCPA & IDA gave a significant increase in the total number of seeds obtained from crosses between P., vulKaris and P.. cooginEus. The average number of seeds per 10 flowers pollinated and treated with this hormone mixture was found to be 21.2 as compared with 11.8 for the control. The L.S.D. was calculated to be 4.07. PCPA, which gave significantly higher pod-set than control, did not give an average number of seeds exceeding the number obtained with control by the L.S.D., 4.07. The difference, which is 2.6, however, may not be regarded as definitely insignificant, and an experiment designed with greater number of flowers used in each replication might show a significant difference between seeds obtained with treatment PCPA and with the control.

The data, however, show that treatment with PCPA and PCPA & IRA give. a higher percentage of pod-set in crosses between F. vulcaris and P cogcineue (B) than the control. The number of seeds obtained is significantly increased by the treatment PCPA & IBA, by as much as 79% over the control. Hormone treatment in the amounts used does not seem to affect significantly the number of seeds per pod as is evident from Table 9.

Out of the 50 attempted crosses between P.L vx a and L .QralI (E) with each treatment, only one pod with seed was obtained with PCPA and 3 pods with seeds with INA. The Pod-set with these treatments was thus 2% and 6% respectively,










as against no pod-net with control and other remaining treatments, The effect of hormone treatment was, however, significant inasmuch as a large number of parthenocarpic pods was obtained with PCPA, PCPA & IRA, and H.N, The data, in which the number of parthenocarpic and seed containing pods have been combined, are reported in Table 10. Parthenocarpic

pods above one inch in length only have been included.

The table shows that the average number of pod-set

(parthenocarpic and seed-containing) was 3.2, 4.4 and 4.2 respectively with PCPA, PCPA & IBA and R.N., as against 1.0 with control. Pod-set with these treatments was, therefore, significantly higher than that without hormone treatment.

The total number of parthenocarpic pods and those containing seeds from crosses between P. vjlarjg and P. filifoMg (B), along with the average length of pods with various treatments, have been summarized in Table 11.

As is evident from this table, the percentage of sets with seeds is very low being 2% with PCPA and 6% with KNA. Such a low pod-set may be considered to be inadequate to draw conclusions regarding the effectiveness of the hormone treatments in aiding fruitful pod production. However, the fact that 3 sets have been obtained with lNA and none with the control cannot be ignored and considered as only accidental. Further study of these sets by planting the Seeds and studying the plant characteristics will, however,










TABLE 10

Results of the Crosses P. vulgars X ZL (N) with Different Treatments. (The numbers represent the number of parthenocarpic and seed-containing pods obtained out of 10 flowers pollinated and treated in each case.)



Pods set out of 10 flowers treated R e p l i c a t i o n .... .. .. ... .. . . . ..... .................. . ....

PCPA PCPA&IBA H.M. KNA Control


I 1 1 1 - 1


I1 5 6 4 1 2

III 3 5 5 1

IV 5 5 8 5 1 V 2 5 3 2 1



Total 16 22 21 9 5 Average per
10 buds treated 3.2 4,4 4.2 1.8 1 L.S.D. 1.79

(Analysis of variance given in Appendix Table 25)





TABLE 11

Total Number of Pods Set from Attempted Crosses between P vuja1g and

P. ft-Ilorals (9) with Various Treatments, and the Average Length of Pods Obtained.


No. of flowers No. of pods No. of parth- Average Treatment treated with seeds set enocarpic length of pods-set pods in
am*


PCPA 50 1 15 5.7 PCPA&IBA 50 - 22 6.1 R.M. 50 - 21 6.7 KNA 50 3 6 6.1 Control 50 - 5 5.7










be necessary to establish the validity of the crosses. The effect of hormone treatments is very evident in giving an increased number of parthenocarpic sets with PCPAV PCPA&IBA and H1.. The slightly greater average length of pods following treatment with H.M. suggests that this mixture has a greater stimulation to growth than any other treatment.


Other Interayecific Crosses


The results of the attempted crosses between jP8. ,ulrar (Cherokee Wax) as pistillate parent and different species of

Phaseolu and their varieties as staminate parents are reported in Tables 12 and 13. The number of pods containing seeds and parthenocarpic pods (more than one inch in length) have been separately recorded and also the average lengths of pods set with different treatments have been given.

Table 12 clearly shows that hormone treatment aids in obtaining increased set of pods containing seeds, at least with E calcaratus (F). Vith this species KNA and PCPA&IBA again gave increased pod-set of 20% as against 5% with the control. H.M. and PCPA also gave an increased number of pods

ontaining seeds. the percentage of set being 15% and 10% respectively. In order to get more precise information, Several hundred flowers should be treated. The hormone treatments, except INA, also gave a higher number of










parthenocarpic pods of comparatively greater average length than the control. Pods containing seeds have also been obtained with hormone treatments from crosses with FL atronzrureus (D), P& acutlfolius (J) and P. acutifoltug (1) as staminate parent on P. vu!zaris (A) as pistillate, but the percentages were so low that no definite or conclusive statements regarding the effectiveness of hormones in these cases can be made.
Considering the overall effects of treatments on attempted crosses with five different species and varieties grouped in Table 12p It is seen that out of a total of 110 flowers pollinated and treated, PCPA&IBA, U.M. and KNA gave a higher number of pods containing seeds than did the control. Also
hormone treatments in general stimulated a greater pod-set, whether the pods were seed-containing or parthenocarpic. The total number of pods obtained out of 110 attempts in each case was 47 with PCPA&IBA, 36 with PCPA, 32 with H.M., 27 with INA and 18 with control.

The results of attempted crosses between & 31a-ri
(A) and 7 other species of a and their varieties, with treatment KNA omitted, are summarized in Table 13. This table shows that successful crossing is possible only With two species, PL Lathyroides (G), and P. gggging (C). Out of 20 flowers pollinated with P. aJUode pollen, 2 pods containing seeds were obtained with U.N. treatment and










one each with PCPA and the control. The total number of

pods set (parthenocarpic and those containing seeds) was greatest with N.M., being 8, followed by PCPA with 7, while PCPAUBA and the control each had 5. J.& goineux (C) also seems to cross successfully with P& vuiir&. since one pod containing seed was obtained when treatment N.M. was used on five flowers.

The overall effects of hormone treatment with these species in Table 13 are not as significant as those in Table 12, partly because of Insufficient data due to the extremely low percentage of sets. But all hormone treatments have given a higher pod-set than the control, In general, the average length of pods with hormone treatments

appears to be increased when compared with the control. On the basis of all the inter-specific crosses attempted and reported in the foregoing tables, the fact seems apparent

that the effectiveness of different treatments varies with different species. With P coccineus (B) PCPA and PCPA&IBA appear to result in increased pod-set. With P& C11fo rms

(9) as staminate parent PCPA&IBA and N.H. gave a greater number of total pods set but more seed-containing pods were obtained with KNA, which did not give a large number of parthenocarpl sets. A similar set of seed-containing pods was obtained with PL 9alEaktuj (F) when KNA was used, but a greater number of parthenocarpic and seed-containing pods was obtained with treatments PCPA&IBA and N.M. Con-





TABLE 12
Results of Attempted Crosses betveen J. vulg"Is (A) and Various Other Species,
Number of Sets and Average Length of Pods (in cmt) Obtained with Various Treatments.

PCPA PCPA & IRA H.m. KNA Control No. of No. of Av No:o Av. No.oF Av, No.of rAv. NR;oF A.'. Kind of cross flowers Sfsts 1. Sets 1. Seta 1. s 1W j ,.1 1, treated of of of of of P S pod P S pod P S pod P 8 pod P S pod


riauzi&l (A)
X .... 20 8 2 7.4 11 4 7.3 9 3 7.0 4 4 7.9 5 1 58 .. pal Scutum (F)

XMIX"Ig (A)
) 50 8 - 5e4 11 - 5,7 8 2 68 3 -4.1 2 1 6,7 j. a o=iaMia (D)
. eSri (A)
X 11 4 - 6.3 1 - 511 - 3.5 1 -3.7 3 -4.7 Z. utjrgjg (K)

. zIuMlg1 (A)
X 16 10 - 4.37 10 - 5.22 3 - 5.1 6 1 5.0 4 -3*2 I. aau.iros (J)

~.XMIiazna (A)
x 13 6 - 6.9 10 - 5.55 1 8.0 8 -5.0 2 -495 z. aeutIfollum (1)
Total 110 34 2 - 43 4 - 26 6 - 22 5 - 16 2 -


aS - Seed-containing


*p . Parthenoearpic




TABLE 13


Results of Attempted Crosses between P. vulgaris (A) and Number of' Pods set and Average Length of Pods (in om.) Treatments.


Various Other Species, Obtained with Various


PA co
No. of. No.of Av, No.of Av. No.of Av No~of Av, Kind of cross flowers pods 1. pods 10 pods 1 pods 1 treated set of of " of t of . l a (A) X 20 6 1 5*6 5 - 6.5 6 2 7.9 4 1 6 4 . (A)X 10 6 - 5.0 3 - 6.1 2- 50 3 - 4,8


() 15 4 - 4.4 8 -6.5 7- 57 4 - 39 j: nI )Ig (A) X 4 1 - 5.2 2 1 7.6 -- - 1- 5,7 �. J (A) 10 1 - 4.1 1 - 40 1 -4,2 -:(C) 5 1 - 8.5 2 - 3.5 12,p 8 1- 4,6
)4 - - - 1 - 7.1 -- - 1- 52

Total a8 19 1 22 1 16 3 14 1
.... - Parthenooarpo 9 - �eed-ontainTs~










sidering other species, PCPA gave a larger number of

parthenocarple sets when L. aLOitiol1us (K), X. agutjilius

(J), P. zath jdu (G) and P. #IMM (0) were used as staminate parents. PCPA & IBA gave increased sets with R. calcaratus (F), P. atromwriureus (D), j. acutifolius (J), X. ,.utifolIgg (I) and F. lunatus (H). An appreciably high percentage of parthenocarpic pod-set was obtained with H.N. au compared with the control when the staminate parents used were F. calaratus (F), Z. aPtroj=uru !eu (D), j. &euall (I) X. l1a e deg (G) and F. lunatus (H).


Mj2rMnt. . Effect on FErtLization and vle Develoiment


The results of an experiment in which 200 flower buds
of P. M (A) were allowed to be self-pollinated and were subjected to various treatments are reported in Tables 14 and 15. It is clear from Table 14 that treatment with PCPA gave a significantly greater number of pods as compared with the oontrol. The differences between the number of pods obtained with other treatments and control are not significant statistically. The average number of pods obtained with PCPA treatment per 10 flowers treated was 7.25, as against 5.25 with the control. The difference needed for sisnifieance at the 5% level has been calculated as 1.12. The difference between the pod-set obtained with










PCPA&BA and that with control, although not more than the LoS.D., is very nearly equal to it. Since this treatment has PCPA as its active agent and has also given an increas-. ed set of pods and seeds with the interspecific crosses between X. Wzaris.and P. 9o06nHU& it should not be regarded as merely aceidental.

Table 15 shows that the treatment PCPA also gave a significantly higher number of seeds as compared with control. The average number of seeds obtained per 10 flowers treated in the experiment was 29.75 with PCPA and 17,50 with the control. The difference is much higher than the 7.16 needed for significance at the 5% level. PCPAUA also seems to have a favorable effect on seed- and pod-set as compared to the control, although the difference is not quite significant when compared with the L.S.D. This experiment thus definitely shoved that the hormone treatment, in the amounts used, has no inhibitory effect on pod or seed development. On the contrary PCPA treatment of flower buds very significantly increased set of both pods and seeds. From Table 15 it is also noticed that hormone
treatment did not decrease the number of seeds per pod. There was, In fact, a small increase in the number of seeds per pod with PCPA and also with PCPA&IBA and KNA. The difference, however, Is not so significant as in the numbers of pod and seed set. Unless shown to be statistically Significant it cannot be considered to be a definite ad-










vantage. Among the various treatments used only H.M, gave a slightly smaller number of seeds per pod than the control. Since all other treatments have given at least some increase in the number of seeds per pod, it can very definitely be inferred that at least the hormones other than N.M, in the concentrations used in this study do not inhibit fertilization and seed development.



TABLE 14

Number of Seed-containing Pods Set from i0 Flowers Allowed to be Self-pollinated and Subjected to Various Hormone Treatments.

No. of pods set out of 10 flowers treated Replioation
PCA WPA&WA N.M. INA Control Total


1 5 6 4 5 4 24
1I 7 5 5 4 5 26

111 9 8 6 7 7 37

IV 8 6 4 a 5 29 Total 29 25 19 22 21 116 Average 7.25 6.25 4.75 5.5 5.25


L.S.D. : 1.12


(Analysis of variance given in Appendix Table 26)







73

TABL 15

Number of Seed Obtained from 10 Flowers of . u (A)

Allowed to Be Self-pollinated and Given Various Hormone Treatments.


Number of seeds obtained and Replication Uthe treatments used
PCPA PCPA H.N.M. KNA Control Total



I 19 21 14 25 16 95 II 30 15 10 10 11 76

III 37 36 21 26 28 148 I' 33 19 12 21 15 100




Total 119 91 57 82 70 419



Average 29.75 22.75 14.25 20.5 17.5



No. of eeds
per pod 4.1 3.4 3.16 3.73 3.33 L.S.D. : 7.16
(Analysis of varlanee given in Appendix Table 27)










Exgremnt 2. Effective Ranzs o- 24-D Concentration


The number of pods and seeds obtained with different amounts of various 2,4-D concentrations in glycerine and Kare mixture are reported in Table 16 and the data are presented graphically in Fig. 4. A study of the table indicates that the concentration of 2,4-D in glycerine-Karo mixture is comparatively more important than the amount of the mixture used in each application. With l/8 saturation the results were quite comparable to those obtained in the control, while higher concentration of 2.4-D decreased the number of pods set. Apparently these higher concentrations are in some way toxic or adversely affect pod development. On the basis of these data and considering the 1/8 saturation of 2,4-D which seems safe to use, there is no appreciable difference in effectiveness when different amounts varying from 0.0004 ml. to 0.0010 ml, of the mixture are used in each application. With higher concentrations, however, there is some suggestion that the

toxicity is increased as the amount used is increased. The fact that 0.0002 ml. of 2,4-D at 3/4 saturation did not give a single set while 0.0003 ml. of the same gave 3 sets and 0.0004 ml. and 0.0006 ml. also gave 2 sets each suggests that there is a considerable amount of variability and factors other than the hormone treatment also affect the Percentage of pod-set. For more precise information, there-










fore, this experiment should be repeated and replicated 4 or 5 times to reduce the variability and bring out the differences in the effects produced by hormone treatment more precisely.
During the course of the experiment the daily observations shoved that flowers treated with 3/4 saturation of 2,4-D and also with amounts between 0.0004 and 0.0010 al. of 1/2 saturation of 2,4-D in glycerine-Karo mixture, in most oases, developed swelling and distortion of the peduncle and receptacle. This effect was more pronounced when higher amounts of these concentrations were used. The fact that 0.0008 ml. and 0.0012 ml. of 1/8 saturation of 2,4-D gave 7 and 8 pods respectively while 0.0004 ml. and 0,0006 ml. of 1/4 saturation gave only 2 and 3 respectively$, Indicates that large amounts of a lower concentration and equivalent smaller amounts of a higher concentration of 2,4-D are not similar in effectiveness. If the sets obtained from flower buds treated with various amounts of the concentrations other than the 1/8 saturation are examined in Table 16, it is observed that the amount of the hormone mixture may be Important in determining the effect on podset. However, the concentration of 2,4-b In the medium seems to be omparatively more important than the amount of the mixture used in each application. Treatment of the buds with 1/8 saturation of this chemical in glycerine-Karo







76

TANLN 16

Effect of Different Coneentrations and Amounts of 2,4-D Mixture on
Pod and Seed-et of o z aaJIE& Self-pollinatedo


. ... ntrations of 2.4-D in Lvoerine-Kro mIXtur Amount
of the 3/4 Saturation 1/2 Saturation 1/4 Satura tion 1/8 Saturation No 2.4-V Control Mixture Used Flowers Novers Flwers P lovers $Ot[lovers .S~t.
treated d S*Ods reated Pods Seeds treated



0.0000 181 - - - 10 7 30 0.0002 M. 10 0 0 10 4 12 10 3 11 .....

0.0003 ml. 10 3 5 ....

0.0004 m. 10 2 7 10 2 8 10 2 10 10 5 22 0.0008 Jl. 10 2 8 10 0 0 10 3 15 10 7 22 0.0008 ml. 10 1 2 10 2 10 10 2 11 10 7 26 0.0010 ml. - - 10 2 6 10 1 2 10 8 18 0.0012M14 lo. ..10 8 24










































Fir.4 ifeat of Different Concentrations and Amounts of 2,4-D in Glycerieharo Mixture on Pod-set of W. j (A).
(Data from Table 16)










mixture, whieh apprently does net seen to be toxic in effeet in any of the different amounts used in this experiment, gave no increased pod-set ever the control. This may possibly be due to the fact that the conditions during the course of this experiment were particularly suitable for pod-set and abscission was at a minimum, which is suggested by the high percentage of pod-set, as much as 70%, without the use of hormone treatment.


Ej~rj~gt 4s, fxton. Tets


The results of germination tests and observations of
growing seedli n have Indicated that the hormone treatment of flowers after pollination does not adversely affect the gernination percentage of seeds obtained from some crosses.. The results of tests with the seed obtained an a result of crosses between E. ZagM!g (A) and E. 2 Leu (B), which are sumarised in Table 17, show that, in this particular Gase, the germination percentage is actually increased.

From Table 17 it is seen that the maximum number of
seedlings was secured in a period of 11 days after sowing. Seeds obtained from flower buds treated with PCPA gave the highest germination percentage which was 65. Hormones also increased the germination percentage of seeds as compared to the control, with which it was the lowest. These hybrid seeds were found to be low in germination capacity as com-







79

TABlLB 17

Germination of Seeds Resulting from Crosses between

Z.. xBIu3M11 and L. ggg (B) With
Various Hormone Treatmonts.


No. of germinated seeds and groving seedlings out of 20 sown on 8/9/50. Date vhen
observed
POPA PCPA&UBA IM. INA Control 6/13/50 2 1 1 1 1 6/14/50 3 2 1 3 1 8/15/50 5 2 1 3 1 6/16/50 9 4 3 4 1 8/17/50 10 4 3 7 a 6/19/50 13 6 8 10 6 8/20/50 13 7 8 11 a Germination
pereentago 65 35 40 55 30 8/29/50 8 6 5 7 2










pared to those obtained as a result of self ing X. MALuWIo and also took longer time to emerge. Observations of the growing seedlings showed that the seeds obtained from the attempted crosses between P. 3Ul&aris (A) and . oeineun

(B) resulted from valid crosses. The elevation of the cotyledons of the hybrid seedlings was, in general, intermediate between the two parental extremes but in no case was it as high as in the J. Mv l pistillate parent. There were many abnormalities noticed In the seedlings such as the curvature of the stem, difficulty in the tip breaking through the soil and the cotyledons, and upside down germination of seeds with roots coming up. The seedlings were weak and delicate and, as is evident from the observations made on 6/29/50 in Table 17, many of them died early. The growth of seedlings from seeds with hormone treatment of flover buds before fertilization was more accelerated in the beginning than that of the control but this effect gradually beosame less noticeable later on. This test suggested that the seed obtained as a result of this cross should be germinated in a soft medium such as vermiculite vhtch does not offer much resistance to the emerging tip.

The germination tests of the seed obtained as a result of selfting . ALg ll (A) and treating the flowers with hormone mixtures, reported in Table 18, conclusively show that hormone treatment of flowers does not have a dele-










terlous effect on germination capacity or the resulting seeds but may possibly be responsible for weakness in the stem of the seedlings. The germination percentage of seeds from PCPA-treated flowers was found to be 95, the same as for the control. The germination percentages of seed from flowers treated with PCPA&IBA, fL.M., and RNA were found to be 90, 90, and 85 respectively. A very marked difference in the rate of growth of seedlings from hormone treatments was observed in the early stages of growth. Seeds obtained from flowers treated with hormones gave seedlings which grew faster in the beginning than did these from the control. This Is evident from Table 19 in which the average heights of seedlings measured on the seventh and eighth day after sowing are recorded. Figure 5, which is a photograph of the seedlings rowing in a flat taken on the ninth day after sowing, also clearly shows that the hormone treatment resulted in increased growth rate of seedlings in the initial stages,

which was more pronounced with PCPA and PCPA&IBA.


Effet- of Hormone TreItmnt on the Rate of
,.Dvelomnat of Pods


As has been described earlier, daily measurements of
pod length were taken after treating the flowers with 2,4-Dp PCPA and control. Each day 4 pods were measured from each of










TAM 18

Germination of Seeds from Flowers of X. Mlaris (A) Self-pollinated and Given Various Hormone Treatments.


Date of 6/13/5o 8/15/5o

65/5o 6/1o


No. of *"ds gerinated out


12 10 15 18 17 18

19 17 18 19 18 18


of 20 sown on INA .Control 14 15 17 19 18 19 17 19


Germination %


TABL 19

Average Belot (in inches) of Seedlings from Seeds of . Z VI Selfed and Treated with Various Hormones.


Tim when PCPA PCPA&A E*M. KNA Control observed

7 days
after soWing 3.98 3.58 2.56 2.55 2.1

8 days
after mo ing 6.2 5.8 4.9 4.2 3.6


rl


1.








































Fig. 5
Photograph of the Groving Seedlings Obtained from F. ] Plovers Self-pollinated and Treated with Various Hormones and Control, (The photograph was taken on the ninth day after moving.)










three rplications of each treatment, In the analysis of the data used here it was not necessary to consider the data separately in each replication, therefore an average of pod length from three replications is considered to be the average length of pods on different days. In Table 20, in

which the data regarding the pod lengths on days have been recorded, the pod lengths, thus, are averages of 12 pods from each treatment.

The relationship between pod length and days, as may well be expected, is not a straight .line relationship but follows the pattern of a growth curve. Treating it as a straight line relationship. the regression of pod lengths on days has been calculated with each treatment and the regression coefficients have been used to compare the rate of development of pods resulting from the three treatments. The regression of pod length on days and the regression coeffiieonts (b) with the three treatments are as follows

2,4-D ..... b : 1.3469
A
Y : 1.349 X - 2.0358


PCPA ..... b : 1.1541

Y : 1.1541 X - 1.5938

Control ..... b : 1.2579

S 1.2579 X - 2.1889








85


TABLE 20 Avorage Pod-length (in our.) on Different Days


Treatment 1.
2,4-D


Days Length x y 3 2.06 5 4.57 6 5.97 7 7.30 8 9.00 9 10.40 10 11.10


Tre


Days


3 5 6 7 8




10


atment 2. PCPA


Length
y


frel Das


Days
x


3 5 6 7 8 9 10


2.07 3.87 5.27


6.40 7,83 9.03 9.77


atment 3.
control


Length
Y


1.63 3.67 5.63

6.67 7.97 9.57 9.93


48 50.40 48 44.24 48 45.07


" _._ L--










































Fion 6
Rep"*egj0on of Pod-1.n~gth on Days with 2,4-I), PCPA and Control.










The average pod lengths on days and the regression of pod length on days have been plotted in Fig. 6. In order to determine if the three regression coefficients of average pod length on days are significantly different from each

other, the analysis of eovariance is given in tables 21 and 22.

ased on the average of replications (Table 20), the total am of squares for the averages of pod length have been divided into variation between treatment means and the variation of averages within treatments. This latter sum of squares has been divided into that which can be attributed to the average regression of the pod length on days for

one degree of freedom and that which cannot be attributed to the regression, or the errors of estimate for 17 degrees of freedom. This sum of squares is the variation of averages from the average regression after the variations of treatments have been removed.

The variations of the averages from the specific regressions of pod length on days within each treatment also have been computed each for 5 degrees of freedom.

The total error of the estimate for 15 degrees of

freedom is the estimate of the true variation of pod length where the knowledge of the relationship of pod length and days within each treatment has been employed.

The difference of the sum of squares of errors of

estimate for twe degrees of freedom is an estimate of the

variation of the regression coefficient (Snedicor, 43).





TABLE 21

Analysis of Covarianee for Data Presented in Table 20


Errore of Estimat
Source D.F. D.F. 8.8. H.S.
.... 82 x 82


Total

Treatments Average In Treatments


20 104.5714

2 0.0


18 104.5714


131.0286

0.0


131.0286


169.2784 3.1925


166.0859


19 5.0988


- F lz 15 0 =14 .23 9
17 1,9083 0.1 1121
2 3.1925 1.59021 611


Treatment I Treatment 2 Treatment 3


34.8572 34.8572 34.8572


46.95

40.23 43.8486


63.5494 46.7006 55.8359


0.3114 0.2697 0.6766


15 1.2577 0.0838) F24 3243 = 3.8699
2 0.6486 0.3243 F. 838 368
15 7.05 a .6





TABLE 22

Analysis of Covariance Considering PCPA and Control


Errors of Estimate
Souroe D,F. D.F, SS. M.S.


Total Treatment


69.7143

0.0


84.0786 102.5857 0.0 0.0492


Average in
Treatment 12 69.7143 84.0786 102.5365 11 1.1339


Treatment 2 6 34.8572 40.23 46.7006 5 0.2697 Treatment 3 6 34.8572 43.8486 55.8359 5 0.6766


10 09463 1 0.1876


0.0946 F =187- 1.98

01876 F.:05- 4.96













The first F test in Table 21, which gives a high significance, shows that if the average pod lengths from different treatments are adjusted to a common value of X, I.e. days, there is a highly significant difference among the average pod lengths with various treatments. The observed values of pod length on a common value of X are, however, not significantly different among the three treatments. The simple analysis of variance, which was run using the pod lengths on 6th and 10th day with three replications showed that the treatments had no significant effect on the observed values of T (pod length).

In the second F test in Table 21, in which the variance due to the regression coefficients Is compared with the error variance, shows that there is a significant difference in the regression coefficients and that the hormone treatment affects the rate of development of pods significantly.

In order to determine which of the three regression coefficients differ from each other significantly, a similar analysis of covarianee considering treatments PCPA and Control is made in Table 22. Sines a significant difference in the

three regression coefficients is indicated in Table 21, and the widest difference between the regression coefficients Is seen In case of 2,4-D and PCPA, the two other treatments compared are PCPA and Control which have the next widest differ-







91

once in their regression coefficients. The analysis in Table 22 shows that these two treatments, PCPA and Control. do not differ significantly in their regression coefficients. It is thus seen that 2,4-D increased the rate of development of pods significantly over PCPA but there was no significant difference between either 2,4-D and control or between PCPA and control.










DISCUSSION


ineral


The results of the trials with various growth-regulating chemicals in different amounts and concentrations used in this study show that hormone treatment in right dosages results in responses favorable for greater pod-set. It is, however, evident that the use of hormones mainly stimulates the ovary and other tissues chiefly responsible for pod development and it is not very definite whether the effect is extended to the developing ovules in a similarly favorable manner. The results of various nterspecific crosses reported with P. vulgajrls as pistillate parent show, in in general, that with some species at least, a greater podand seed-set can be obtained with the use of treatments PCPA, PCPA&IBA and KNA. With many other species which have not shown increased set of seed-containing pods, hormone treatment has given a greater number of parthenocarpic pods

set. The greater number of pods obtained may be explained as resulting from:

1. Reduced absoission rate,
2. Stimulation provided by the hormone supplementing any

that may have resulted from the pollen tubes to cause development of the ovary and other tissues concerned

with pod development,










3, Stimulation provided by the hormone supplementing any

that may have resulted from the pollen tube in causing

greater ovule development.
During the course of the preliminary trials as well as in the final experiments, the effective part the chemicals play in checking abscission has been definitely observed.
Reduced yield of beans due to high temperature conditions resulting in a high abscission rate has been reported to be a problem by Fisher et al. (16) and ittwer and Hurneek (55).

The reason for obtaining increased yields of beans with hormone treatment has been explained to be its property of checking abscission. The findings of this study are in agreement with the results of these workers. In hand pollination with beans this tendency of the flowers to abseiss is Increased even more, probably, due to handling of buds and the unavoidable injury caused to the flower parts durIng emasculation. In Florida the weather conditions are often favorable for high abscission rate due to high temperatures and bright sunlight. The principal causes of a low percentage of sets obtained from hand-pollinated flowers thus can be said to be the high rate of abscission of flowers before pod-set or following pod-set if adverse conditions prevail later. Whether the abscission of the style also is a cause of Incompatibility and failure of pod-set can not be said with certainty, but this appears

unlikely.










Levis (26) has reported on the effectiveness of

naphthaleneacetamide treatment of Prung aMIM buds which resulted in prolonging the period before abseission of style and flowers. He explained that the hormone treatment checks abscission resulting from changes taking place in the existing cells consisting of the accumulation of starch below the abscission line resulting in starvation of the tissues above. Exactly how the abscission of bean flowers

takes place was not determined in this study but it is definite that hormone treatment prevents this to a considerable extent. It is quite possible that this is achieved by preventing changes in cells of the type described above.

Van Overbeek et al. (36) have reported that the

hormone released in the ovary by the presence of pollen tubes extends its effect to the peduncle and checks abscission of the flower. The transmission of this

hormone effect to the peduncle is probably Interfered with under unfavorable environmental conditions or in case of incompatible species the pollen tube fails to cause the desired activation of the inactive hormone present in the ovary. This probably results in insufficient stimulation to initiate the development of the ovary and other tissues connected with pod development and also causes abscission of the flower before fertilization is achieved.










An increased number of parthenocarpic sets obtained with hormone treatments may be thus explained.

Many parthenocarpic pods were obtained with treatment RM, with various species used as pollen parents during the course of this study. Some of these were almost similar in size to normal seed-containing pods but had no seeds at all. Without hormone treatment and with the stimulation from the pollen tube alone, both the number and lengths of parthenocarpic pods were comparatively much smaller. The reason for the development of fewer and smaller parthenocarpic pods in crosses between supposedly incompatible species without use of hormones may be attributed to the lack of the stimulation necessary to initiate the development of ovarian and other tissues and also to the lack of stimulation provided by developing ovules as in normal pods, In an attempted cross, if the egg cell and the sperm cell are not inoompatible the hormone application should be able to overcome any lack of stimulation normally provided by the hormone naturally occurring in the ovary which is activated by the pollen tube.

A few sets of seed-containing pods have been obtained from P., galaratu . P, P athroideg and . flf3 pollen with the use of hormone treatment. This shows that the sperm cells of these and the egg cell of F. Ymjgaj are probably not incompatible and the reason for failure to obtain sets from attempted crosses between these may possibly










be due to improper growth of pollen tubes, the lack of stimulation resulting from an insufficient number of themo the degeneration of embryo sac before fertilization, or some other causes not as yet studied. These causes are to some degree controlled by the hormone treatment and seed-containing pods are obtained.

ith many other species which have not given any seedcontaining pods and have given significantly large numbers of parthenocarpic pods, it may be postulated that the cause of incompatibility in such cases possibly is the incompatibility between the sperm and egg cells themselves# which cannot be overcome by hormone treatment. Hormone treatment thus cannot substitute for or cause fertilization directly, but it can certainly help in providing the necessary conditions for it and can also supplement any inadequate natural stimulation for initial development of pods.

It seems definite that with the help of PCPA and PCPA & IBA a greater percentage of pod-set with seed can be obtained from attempted crosses between . yzva. and E. coccineus than without this treatment. The data on these crosses and on other attempted crosses reported In Tables 12 and 13 suggest that ordinarily the species which cross without the use of hormone treatment give increased set of pods and seeds if treated with PCPA or PCPA&IBA in the amounts used in this study. Wittwer and Hurneek (55) have also reported consistent Yield increases of snap beans with para-chlorephonoxyacetic




Full Text
96
be due to improper growth of pollen tubes, the lack of
stimulation resulting from an insufficient number of them,
the degeneration of embryo sae before fertilization, or some
other causes not as yet studied. These causes are to some
degree controlled by the hormone treatment and seed-contain
ing pods are obtained.
With many other species whieh have not given any seed-
containing pods and have given significantly large numbers
of parthenocarpic pods, it may be postulated that the cause
of incompatibility in such cases possibly is the incompati
bility between the sperm and egg cells themselves, which
cannot be overcome by hormone treatment. Hormone treatment
thus cannot substitute for or cause fertilization directly,
but it can certainly help in providing the necessary con
ditions for it and can also supplement any inadequate natural
stimulation for initial development of pods.
It seems definite that with the help of PCPA and PCPA &
IBA a greater percentage of pod-set with seed can be obtained
from attempted crosses between P. vulgaris and P. coccineus
than without this treatment. The data on these crosses and
on other attempted crosses reported in Tables 12 and 13 sug
gest that ordinarily the species which cross without the use
of hormone treatment give increased set of pods and seeds if
treated with PCPA or PCPA&IBA in the amounts used in this
study. Wittwer and Hurneek (55) have also reported consistent
yield increases of snap beans with para-chlorophenoxyacetic


31
definitely delayed or in some cases completely prevented.
Since many of the hormones are soluble in glycerine,
those which showed response in latex mixture were dissolved
in glycerine to saturation. After emasculating the flowers
in the field, a very minute drop was applied on the peduncle
at the point of junction with the receptacle of the flower.
Controls were maintained. The treatment also was used with
flowers emasculated and pollinated with P. lathvroides
pollen. In all cases the treated flowers dropped, the
peduncle drying at the point of application. No response
due to the hormone was seen. However, it was observed that
glycerine itself was toxic and killed the tissues at the
point of application, thereby causing the flower to wither.
As 2,4-D showed greatest response in thickening of the
peduncle, as also reported by Hester and Marth (53) and
checking of abscission even of emasculated flowers, further
trials were made with 2,4-D latex and lanolin paste. One
percent solutions were used and snap bean flowers were
treated after emasculating and pollinating them as summa
rized in Table I.
The results definitely showed the effect of 2,4-D
treatment in delaying the abscission of flowers as is
evident from Table I. The results also show a definite
stimulating effect of pollination even though seed develop
ment failed. 2,4-D was thus the most effective and merited


99
unlikely. This might have resulted in a slight depressing
effect on fertilization of egg and development of fertilized
ovules.
In the experiment to study the effective range of 2,4-D
on self-pollinated flowers of P. vulgaris. a few pods were
obtained with higher concentrations of 2,4-D (3/4 saturation
and l/2 saturation) in which seeds had not developed. In
some the number of seeds obtained was much smaller. Even if
hormones inhibit the fertilization of ovules and development
of seeds, the results of these experiments support the view
that this depressing effect can be overcome by regulating the
amount and concentrations of the chemicals used. A fairly
precise mechanism to control the amount of the chemical
applied is therefore imperative for obtaining the desired
effects
Literature on the effect of hormone treatment on germi
nation of seeds has already been reviewed. Direct seed
treatment with chemicals, or treatment of soil, can be
expected to affect the germination of seeds and growth of
seedlings. The treatment of flowers with a very small
quantity of very dilute mixture as used in this study cannot
be expected to cause any deleterious effect on the germination
capacity of seeds obtained. It could be argued that any per
sistence of effect of the chemicals in the seed to the extent
that it can cause undesirable effects on germination and
growth of seedling would in the first place be severe enough


67
one each with PCPA and the control. The total number of
pods set (parthenocarpic and those containing seeds) was
greatest with H.M., being 8, followed by PCPA with 7, while
PCPA&IBA and the control each had 5. coccineus (C) also
seems to cross successfully with P^ vulgaris. since one pod
containing seed was obtained when treatment H.M. was used
on five flowers.
The overall effects of hormone treatment with these
species in Table 13 are not as significant as those in
Table 12, partly because of insufficient data due to the
extremely low percentage of sets. But all hormone treat
ments have given a higher pod-set than the control. In
general, the average length of pods with hormone treatments
appears to be increased when compared with the control. On
the basis of all the inter-specific crosses attempted and
reported in the foregoing tables, the fact seems apparent
that the effectiveness of different treatments varies with
different species. With P*. coccineus (B) PCPA and PCPA&IBA
appear to result in increased pod-set. With P*. filiformis
(E) as staminate parent PCPA&IBA and H.M. gave a greater
number of total pods set but more seed-containing pods
were obtained with SNA, which did not give a large number
of parthenocarpic sets. A similar set of seed-containing
pods was obtained with calcaratus (P) when KNA was used,
But a greater number of parthenocarpic and seed-containing
pods was obtained with treatments PCPA&IBA and H.M. Con-


66
parthenocarpic pods of comparatively greater average length
than the control Pods containing seeds have also been
obtained with hormone treatments from crosses with P.
atropuronreus (D), acutifolius (J) and P^ acutifolius (I)
as staminate parent on P*. vulgaris (A) as pistillate, but
the percentages were so low that no definite or conclusive
statements regarding the effectiveness of hormones in these
cases can be made.
Considering the overall effects of treatments on attempt
ed crosses with five different species and varieties grouped
in Table 12, it is seen that out of a total of 110 flowers
pollinated and treated, PCPA&IBA, H.M. and KNA gave a higher
number of pods containing seeds than did the control. Also
hormone treatments in general stimulated a greater pod-set,
whether the pods were seed-containing or parthenocarpic.
The total number of pods obtained out of 110 attempts in
each case was 47 with PCPA&IBA, 36 with PCPA, 32 with H.M.,
27 with KNA and 18 with control.
The results of attempted crosses between P^ vulgaris
(A) and 7 other species of Phaseolus and their varieties,
with treatment KNA omitted, are summarized in Table 13.
This table shows that successful crossing is possible only
with two species, P^ lathvroides (G), and P*. coccineus (C).
Out of 20 flowers pollinated with P,. lathyroides pollen, 2
pods containing seeds were obtained with H.M. treatment and


29
attempt was to see If the chemicals could be effectively
used with latex. Stock solutions of the following hormones
were made in latex at 2000 ppm.
1. Indole-propionic acid
2. Beta-naphthoxyacetic acid
3. 2,4-Dichlorophenoxyacetic acid
4. Potassium salt of alpha-naphthaleneacetlc acid
5. Alpha-naphthaleneacetamide
6. 8Q6 Phalinil (A commercial preparation of
n-phenyl phthalimide.)
7. Alpha-naphthaleneacetic acid
8. Methyl ester of alpha-naphthaleneacetic acid
The following two hormones were later included in the trials.
9. Indole-butyric acid
10.Para-chlorophenoxyacetic acid
Several flowers of snap beans (P. vulgaris) and lima
beans (P. lunatus) were emasculated and sealed with the
hormone mixture in latex as usual without pollination. A
little of the mixture was also applied at the base of the
ovary. The object was to study the effect and kind of
response of the flower to the hormone without the stimulation
supplied by the pollen as is the case in pollinated flowers.
Various dilutions were made from the stock solution to
arrive at a concentration where a response could be induced
without any toxicity. The 200 ppm. mixtures of Nos. 2, 3,


IN CM.
86
Pig. 6
Regressions of Pod-length on Days with 2,4-D, PCPA and Control.


87
The average pod lengths on days and the regression of
pod length on days have been plotted in Pig. 6. In order
to determine if the three regression coefficients of average
pod length on days are significantly different from each
other, the analysis of covariance is given in tables 21 and
22.
Based on the average of replications (Table 20), the
total sum of squares for the averages of pod length have
been divided into variation between treatment means and the
variation of averages within treatments. This latter sum
of squares has been divided into that which can be attribut
ed to the average regression of the pod length on days for
one degree of freedom and that which cannot be attributed
to the regression, or the errors of estimate for 17 degrees
of freedom* This sura of squares is the variation of aver
ages from the average regression after the variations of
treatments have been removed.
The variations of the averages from the specific re
gressions of pod length on days within each treatment also
have been computed each for 5 degrees of freedom.
Hie total error of the estimate for 15 degrees of
freedom is the estimate of the true variation of pod length
where the knowledge of the relationship of pod length and
days within each treatment has been employed.
The difference of the sura of squares of errors of
estimate for two degrees of freedom is an estimate of the
variation of the regression coefficient (Snedicor, 43).


TABLE 21
Analysis of Covariance for Data Presented in Table 20
Source
D.F.
s*2
Sxv
Sy2
Errors of Estimate
D.F. S.S. M.S.
Total
20
104.5714
131.0286
169.2784
19
5.0988
Treatments
2
0.0
0.0
3.1925
Average in
Treatments
18
104.5714
131.0286
166.0859
*7
1.9063
0,1121|
F-i = 15962 =14.239
1 1121
2
3.1925
1.5962)
F#0i 6.11
Treatment 1
6
34.8572
46.95
63.5494
5
0.3114
)
Treatment 2
6
34.8572
40.23
46.7006
5
0.2697
Treatment 3
6
34.8572
43.8486
55.8359
5
0.6766
15
2
1.2577
0.6486
0.0838)
0.3243j
F2* 3243 = 3,8699
A 838
P,05 3-68


Experiment 4. Germination Tests 56
Experiment 5. Effect of Hormone Treatment on the
Rate of Development of Pods 56
RESULTS 58
Experiment 1. Interspecific Crosses 58
Experiment 2. Effect on Fertilization and Ovule
Development . 70
Experiment 3. Effective Range of 2,4-D Concen
tration 74
Experiment 4. Germination Tests 78
Experiment 5. Effect of Hormone Treatment on the
Rate of Development of Pods ... 81
DISCUSSION 92
General 92
Effect on Seed-Set 97
Medium and Method of Hormone Application 101
CONCLUSION 108
SUMMARY 110
APPENDIX 113
LITERATURE CITED 116
BIOGRAPHICAL ITEMS 122
iv


75
fore, this experiment should he repeated and replleated 4
or 5 times to reduce the variability and bring out the
differences in the effects produced by hormone treatment
more precisely.
During the course of the experiment the daily obser
vations showed that flowers treated with 3/4 saturation of
2,4-D and also with amounts between 0.0004 and 0.0010 ral.
of l/2 saturation of 2,4-D in glycerine-Karo mixture, in
most eases, developed swelling and distortion of the pe
duncle and receptacle. This effect was more pronounced
when higher amounts of these concentrations were used. The
fact that 0.0008 ml. and 0.0012 ml. of l/8 saturation of
2,4-D gave 7 and 8 pods respectively while 0.0004 ml. and
0,0006 ml. of 1/4 saturation gave only 2 and 3 respective
ly, indicates that large amounts of a lower concentration
and equivalent smaller amounts of a higher concentration of
2,4-D are not similar in effectiveness. If the sets obtain
ed from flower: buds treated with various amounts of the
concentrations other than the l/s saturation are examined
in Table 16, it is observed that the amount of the hormone
mixture may be important in determining the effect on pod-
set. However, the concentration of 2,4-D in the medium
seems to be comparatively more important than the amount
of the mixture used in each application. Treatment of the
buds with l/8 saturation of this chemical in glycerine-Karo


46
4) Trials with Hormone in Glycerine-Karo Mixture
The slow-drying property of glycerine, as well as the
greater solubility of certain hormones in it, makes it a
desirable reagent if possible to use. It was found that 1
part of glycerine to 4 parts of syrup provided a slow-drying
medium in which the glycerine was not sufficiently concen
trated to be toxic. This mixture was also of a desirable
consistency and viscosity for ease of handling. It was
decided to test once again with glycerine and Karo other
hormones which could easily be dissolved in glycerine.
Saturated solutions of all hormones tested before were again
made in warm glycerine and the glycerine so saturated was
mixed with 4 times of its volume of Karo.
A number of buds of P,. coccineus were treated with
hormone mixtures by applying a tiny drop on the peduncle at
the abscission zone. An equal number of controls was main
tained. Para-chlorophenoxyacetic acid, methyl ester of
naphthaleneacetic acid, beta-naphthoxyacetic acid and indole-
propionic acid showed response by delaying abscission by
several days and causing slight thickening of the peduncle.
A mixture of para-chlorophenoxyacetic acid and indole-
butyrie acid was also found to be effective. It was decided
to use the mixture of all these hormones in glycerine-Karo
mixture also containing 2,4-D. On the basis of these trials
tiie following treatments were chosen for further final
analysis.


74
Experiment 3. Effective Range of 2.4-D Concentration
Hie number of pods and seeds obtained with different
amounts of various 2,4-D concentrations in glycerine and
Karo mixture are reported in Table 16 and the data are pre
sented graphically in Pig 4. A study of the table
indicates that the concentration of 2,4-D in glycerine-Karo
mixture is comparatively more important than the amount of
the mixture used in each application With l/8 saturation
the results were quite comparable to those obtained in the
control, while higher concentration of 2,4-D decreased the
number of pods set Apparently these higher concentrations
are in some way toxic or adversely affect pod development.
On the basis of these data and considering the l/8 satu
ration of 2,4-D which seems safe to use, there is no
appreciable difference in effectiveness when different
amounts varying from 0.0004 ml. to 0.0010 ml, of the mix
ture are used in each application With higher concen
trations, however, there is some suggestion that the
toxicity is increased as the amount used is increased. The
fact that 0.0002 ml, of 2,4-D at 3/4 saturation did not give
a single set while 0.0003 ml. of the same gave 3 sets and
0.0004 ml. and 0.0006 ml. also gave 2 sets each, suggests
that there is a considerable amount of variability and
factors other than the hormone treatment also affect the
Percentage of pod-set. For more precise information, there-


7
ovules and checking abscission which prepares conditions
favorable for embryo development. Actinal frtilization,
i.e. fusion, is not necessary for these conditions. The
second stage is the division of the polar nuclei and, rarely,
of the egg cell, which are not always a direct result of
fertilization. Instances are known where the polar nuclei
fuse to form an endosperm nucleus and this divides many
times before the sperm nuclei leave the pollen tube and
fusion takes place. The presence of pollen tube in the
embryo-sac, however, seems to be necessary for initiating
divisions of the endosperm. The last step is the actual
fusion of the sperm nuclei with the egg cell and polar
nuclei. It is, however, important to note that actual
fusion, although indispensible for hybridization, is not
all that is needed. For a complete process the other two
stages, which are probably hormone induced, may be of equal
importance. This fact is evidenced by earlier work of
Gustafson (19) who showed that except for the absence of
ovule development, the fruit development by chemical treat
ment is in no way different from that resulting from
pollination in the tomato. The matter of food supply is
important in both cases and its mobilization seems to be
influenced by hormones.
It was formerly believed that the initiation of growth
of the ovary is caused by the hormone secreted by the pollen
tube (19). It has now been demonstrated that the pollen tube
is-


21
Tulls and Davis (48) have shown that there is a con
siderable persistence of the effect of 2,4-D in plants
treated with the chemical* Bean plants, when sprayed with
2,4-D during pod development, produced seed which, when
sown, gave seedlings exhibiting malformations* Any hormone
treatment of flower buds, therefore, has the possibility of
affecting the viability of seed and growth of seedlings*
There is a mass of information in the literature on
the effect of hormone treatment of seeds and soil on the
germination percentage and development of seedlings. The
results, on the whole, are conflicting. The majority of
the evidence seems to show that hormone treatment has a
deleterious effect on germination of most seeds, or that,
at least, it does not increase the germination percentage.
The results of various studies so far made Indicate that
seed treatment with hormones may increase the germination
capacity of seeds in case of some field and vegetable crops,
while with others it may be retarded.
Avery and Johnson (5) have summarised the literature
on the subject of hormone treatment of seeds* Among the
workers reported to have obtained increased germination of
seed and development of seedlings, and subsequent increase
in yield, two may be mentioned here* Amlong and Naundorf
(3), using various concentrations of a potassium salt of
indole-acetic acid, obtained increase in the yield of alfalfa.


INTRODUCTION
Beans are an important truck crop of Florida, which is
also of considerable commercial value to the agricultural
economy of the United States. Numerous varieties of beans
have been evolved during recent years and a very high standard
with regard to the horticultural characteristics has been
achieved in the existing varieties grown commercially in
Florida. There is, however, never a limit to improvements
and perfection has not been reached. Improvements towards
still better combinations of characters by hybridisation are
possible and researches in that direction are in progress.
Resistance to diseases, such as downy-mildew, to cold, to
drought, and to such insects as Mexican-bean-beetle are some j
of the desirable qualities that might be possible through
further improvements in beans by interspecific crossing.
While hybridization is one of the most effective tools
of the plant breeder working towards crop improvements, there
are certain limitations in its practice. Incompatibilities
among species and varieties and often a very low percentage
of pod-set are great handicaps in bean breeding work. In
compatibility may be due to various causes and be of various
types. It may result from a failure of the fusion between
male and female gametes, that is, failure of the fertili
sation proper, or it may be due to any of a number of other
1


42
a number of crosses with pollen of various species, with
and without the hormone treatment, were made.
Out of the 10 buds treated with 2,4-D at l/4 saturation,
6 developed pods which were later on found to be partheno-
carpic. Those treated with para-chlorophenoxyacetic acid
did not develop pods, but the flowers did not absciss for
about another 3-4 days beyond the controls. Slight
straightening and thickening of the peduncle was also
noticed in many cases. Incompatibility of P^ coccineus
with many other speeies was also present, as the crosses
with many species, as listed below in Table 4, set no pods.
TABLE 4
Attempted Inter-specific Crosses with P^ coccineus as
Pistillate Parent
No. of crosses attempted with completely
Name of species negative results
used as pollen
Para-chloro-
2,4-D, l/4 satu- phenoxyacetic Control
ration in Karo acid saturated Un-
in Karo treated
latftyroldes
13
13
13
£*. liUfgrgAS
6
6
6
P,- vulgaris
(Cherokee Wax)
7
7
7
P* atronurnureus
5
5
5
P. lunatus
(Fordhook selection)
3
3
3


26
margin of the left (observers left) half, which is everted
and held down with the thumb of the left hand. The left
wing of the corolla is pushed slightly aside exposing the
distal end of the coiled keel (Fig. IB). With a sharp,
slightly flattened, pointed pair of forceps the upper side
of the distal portion of the keel is removed with the points
of the forceps inserted as indicated by the arrow in Fig.
IB. The anthers are then removed taking care to disarrange
the other flower parts as little as possible.
The stigmatic surface is then pollinated as follows:
The pollen-laden, distal portion of the pistil of a freshly
opened flower from the selected staminate parent is rubbed
against the stigma of the emasculated flower of the select
ed pistillate parent. Usually the pollen-laden distal
portion of the pistil can be made to protrude from the open
ing in the end of the keel by the downward pull of the left
wing of the corolla with relation to the other flower parts.
After pollination, the left side of the banner is returned
to its normal position and the other slightly disarranged
flower parts are carefully tucked underneath so that the
bud is completely closed. In most eases only a slight
separation of the margins of the right and left halves of
the banner give any indication that the flower has been
worked with. The suture then may be sealed with a quick
drying latex suspension applied with a camels hair brush.


TABLE OF CONTENTS
INTRODUCTION 1
REVIEW OF LITERATURE 4
General 4
Hormones as an Aid to Fruit-Set 13
Hormones as an Aid in Hybridization by Checking
Incompatibility 15
Use of Hormones in Increasing Yield of Beans 18
Effect of Hormones on Germination of Seed and
Growth of Seedling 20
EXPERIMENTAL MATERIAL AND METHODS 24
A. General 24
r B. Hie Technique of Emasculation and Pollination .... 25
C. Preliminary Studies 28
1) Especially Designed Micrometer Pipette for
Hormone Application 37
2) Trials on Phaseolus coecineus (Scarlet-runner
bean) 41
3) Place of Application of Hormone 43
4) Trials with Hormone in Glycerine-Karo Mixture 46
D. Final Experiments 48
v Experiment 1. Interspecific Crosses 51
Experiment 2. Effect on Fertilization and Ovule
Development 54
Experiment 3. Effective Range of 2,4-D Concen
tration 54
iii


TABLE 12
Results of Attempted Crosses between P. vulgaris (A) and Various Other Species.
Number of Sets and Average Length of Pods (in cmf) Obtained with Various Treatments,
PCPA
PCPA &
IBA
H,M.
KNA
Control
No. of
No, of
Av.
No.of
Av,
No.of Av,
No.of
Av,
No.of Av,
Kind of cross flowers
Sets
1.
Sets
1.
Sets 1.
Sets
1.
Sets 1,
treated
P S
of
pod
P S
of
pod
of
P S pod
P S
of
pod
of
P S pod
£ vulgaris (A)
X
£ calcaratus (F)
20
6
2
7.4
11
4
7.3
9
3
7.0
4
4
7.9
5
1
5.8
£ na&a.rig (A)
X 50
£ SLteBflrPMrgMff
8
*
5.4
11
-
5.7
8
2
6.8
3
-
4,1
2
1
6*7
£ vulgaris (A)
X
P. acutifolius (K)
11
4
-
6.3
1
-
5.1
1
-
3.5
1
3.7
3
-
4.7
£. yuJLftarjs (A)
X
£ aoutltg.llus (J)
16
10
mm
4.37
10
-
5.22
3
mm
5.1
6
1
5.0
4
I
3,2
£ yigarjLp (a)
X
£. aouttfollus (I)
13
6
mm
6.9
10
-
5.5
5
1
8.0
8
-
5,0
2
-
4,5
Total
110
34
2
-
43
4
-
26
6
mm
22
5
-
16
2
mm
*P Parthenooarpic
*S Seed-containing


91
ence In their regression coefficients. The analysis in Table
22 shows that these two treatments, PCPA and Control, do not
differ significantly in their regression coefficients. It
is thus seen that 2,4-D increased the rate of development of
pods significantly over PCPA but there was no significant
difference between either 2,4-D and control or between PCPA
and control.


13
ing the preharvest drop of apples by Gardner et al. (17, 18),
Treatment with various chlorophenoxy compounds has been shown
by Zimmerman and Hitchcock (59) to result in the tomato
flowers staying longer by 10 to 30 days.
Concentrated applications of naphthaleneacetic acid as
an aerosol or as dust have been shown to be as effective as
sprays applied with a hydraulic sprayer in checking pre-
harvest drop of McIntosh apple by Mitchell et al, (29),
Batjer and Thompson (6) have compared the effectiveness
2,4-D and naphthaleneacetic acid in reducing the drop of
pears. They find that 2,4-D may probably be a more effective
chemical than naphthaleneacetic acid even with a weaker
concentration. The injury caused with 2,4-D seems to be
related to the concentrations used; all concentrations above
215 ppm. appear to be injurious.
Hormones as an Aid to Fruit-Set
The part hormones play in fruit development has already
been emphasized in the literature reviewed above. The recent
work on the use of hormones to encourage fruit-set by supple
menting normal pollination, especially with regard to tomato,
is of especial interest for the study undertaken. The normal
fruit-set of tomato has long been known to be low under green
house conditions in the north. Howlett (22) reported that
the use of hormone treatment in supplementing the natural


102
the exact amount of the emulsion used in each case vas not
accurately measured. Linder et al. (28) have, however,
adopted a technique to use precisely measured quantities of
radio-active 2,4-dlehloro-5-iodo-phenoxyacetic acid and its
derivatives on leaves of Leans. The method consisted of
stamping a ring of lanolin on the upper surface of the leaf
on the midrib enclosing 0.5 cm2, area and then applying
0.01 ml. of the mixture of hormone in alcohol and Tween 20
as carrier with the help of a 0,1 ml. pipette. The chemical
thus was not only measured in amount but was also restricted
to a specific area on the leaf. This technique may be
i
satisfactory for treating relatively small numbers of leaves
in experiments of this kind, but certainly is very cumber
some to use as a standard method in hybridization work,
where numerous flowers have to be treated. Also the small
est amount that can be applied with a 0.1 ml. pipette is
much larger than the amounts used in this study and found
effective.
Glycerine employed as a solvent for the ehemicals
used in this study and Karo as a viscous medium have been
found to be very effective and have many advantages.
Saturated solutions of various growth-regulating substances
in 1 part glycerine and 4 parts Karo provide concentrations
which are strong enough to cause the desired effects. Also
the glycerine in such dilutions does not cause any withering
of flower parts as observed when it is used undiluted by Karo.


39
Fig. 2. Longitudinal Section of the Micrometer Pipette.
(Detailed explanation given in text.)


98
made. However, it can be said that the hormone treatments in
the dosage used did not cause any inhibition of fertilization
and ovule development. There are many instances reported in
literature when hormones caused reduction of the number of
seeds although they increased fruit-set and its development.
Van Overbeek has mentioned such an effect in his review of
literature on hormones (37). Swarbrick (45), who reported an
increase in yield of strawberries with naphthoxyacetic acid,
also has stated that the fruits obtained os a result of
hormone treatment, although larger in size, had a lower seed
content. Wester and Marth (53), however, obtained an
appreciable increase in the number of seeds per pod in lima
beans with para-ehlorophenoxyacetic acid and indole-butyric
acid treatment. The slight increase in number of seeds per
pod from the same treatment in this study, therefore, is
in agreement with Wester and Marth. With P. vulgaris
(Cherokee Wax) flowers self-pollinated and treated with H.M.,
the number of seeds per pod was found to be less than the
control. The difference may possibly be due to chance only,
but it is also possible that this mixture caused a small
inhibition of fertilization or ovule development. Of all
the four hormone treatments used in the final experiments,
H.M. definitely appeared to be strongest in effect.
Since this hormone mixture has been made by using a number
of different chemicals saturated in glycerine and since it
also contains 2,4-D, a stronger effect on the tissues is not


20
sistent yield increases with p-chlorophenoxyacetic acid.
With a summer crop of string ¡beans, spraying the flowers at
weekly intervals with 100 to 150 ppm. aqueous solution of
hormone gave as much as 300$ yield increase. Dusts were
found to he as effective as sprays. *01 yield Increase was
found more pronounced when the temperature was above 90 to
95 F. It was also reported that the increase in yield was
due only to stimulation of pod tissue. There was a depress
ing effect on seed development.
Wester and Marth (52), on the other hand, did not find
any significant increase in the total yield of lima beans.
Alpha-naphthaleneaeetie acid as a dust at 50 ppm. showed no
significant yield increases with 13 varieties of lima beans
tried. The insignificant results were explained to be due
to an exceptionally fine weather for seed and pod setting.
Clore (12), using alpha-naphthaleneacetic acid on three
varieties of bush lima beans, actually found a reduction in
yield due to treatment. Concentrations of 50, 100 and 1000
ppm. applied at rates of 75 to 100 gallons per acre resulted
in retarded growth and in foliage epinasty. The 5 ppm.
concentration failed to show any favorable or unfavorable
effects.
Effect of Hormones on Germination of Seed and Growth of
Seedling


103
Both media are slow-drying and this property is of special
advantage in better absorption of the chemicals over a
longer period* The viscosity and fluid consistency of the
glycerine-Karo mixture make it possible to control the
volumetric measurements with considerable precision with
the special pipette designed for this purpose. The aqueous
solubility of both the components of the medium may also
facilitate the transfer of the dissolved hormone into the
cell sap*
Translocation of 2,4-D in plants has been reported
to parallel carbohydrate movement (27, 31, 40). It has
been reported that the movement of 2,4-D is far less from
leaves low in carbohydrates at the time of application than
from leaves high in carbohydrates* How far the syrup as a
medium indirectly affects translocation of the chemical will
depend upon whether any minute amounts of the soluble syrup
are also absorbed or not. But it is quite possible that
minute amounts of the Karo (soluble carbohydrate) thus
absorbed with the chemical might favor its translocation
and thus increase the effectiveness of the hormone.
During the course of these experiments care was taken
to select buds for various treatments as distant as
possible from each other. They were generally from differ
ent branches of the plant to avoid any confounding of re
sults due to translocation effects* Batjer and Thompson
(7) have, however, shown that when the spurs of apple were


112
hormone mixture used in each application in producing an
adequate effect. l/8 saturation of the chemical in glycerine-
Karo mixture was found to he the safest concentration tried.
Hormone treatment with para-chlorophenoxyacetic acid or
2,4-D, in the dosages found to he effective, did not cause
a significant difference in the rate of development of pods
as compared to that with the untreated controls.
This study gave sufficient indication of an effective
use of growth-regulating substances as an aid in obtaining
increased set of pods from hand-pollinated flowers in beans.
The technique developed may prove useful for other
crop plants as well.


6
cess of fertilization, stimulation and development of ovary
and the fertilized ovules, and creating the necessary favor
able condition for such activity, 2) their use as indirectly
helping to obtain greater number of fruits, and therefore
seeds, by checking abscission of flowers and young fruits.
Not much work has so far been done with regard to the first,
at least with beans, but there is quite a considerable amount
of evidence concerning the usefulness of hormones in checking
abscission. Control of fruit drop of apples with the help
of hormones has become an established commercial practice.
That hormones play an important part in the process of
fruit development is also well known. Van Overbeek et al.
(36) have shown that hormones control the growth of ovules.
On injecting 1$ solution or emulsion of the ammonium salt of
naphthalene&cetic acid into the young ovaries of 4-n Meladrium.
they produced parthenocarpic fruits whose ovules had enlarged
and developed seed coats but they contained no embryo. When
the emulsion was applied to the outside of the ovary, the
ovary was stimulated but not the ovules. They suggested a
mechanism of fruit and seed development which consists of
stimulation of ovaries, ovules and placentae, and the
necessary stimulation to set in motion the division of polar
nuclei and egg cell, which may take place with or without
fertilization. They suggest three stages of fertilization.
Hie first one is the stimulation of the ovary, placentae and
M- Is


108
CONCLUSION
Hormone treatment of flower bud, after emasculation
and pollination results in increased pod-set without
inhibiting seed development from certain attempted inter
specific crosses. In case of the species in which the
egg cell and the sperm are incompatible, hormone treatment
stimulates the ovary and checks abscission which results in
a higher percentage of parthenocarpic pods. The seeds
obtained from the hormone treated flowers are not adversely
affected in germination capacity. PCPA and PCPA&IBA, in
the concentrations and dosages used, appear to give the
desired responses without producing any undesirable effects
with most of the possible interspecific crosses. With some
species KNA 0.5$, gives a higher number of fruitful sets.
Glycerine-Karo mixture (1:4) is a very suitable medium
for hormone application and is particularly convenient to
use with the micrometer pipette designed for the purpose.
Both the amount and the concentration of the chemicals in
the medium are important in obtaining the desired stimulus.
Desired effects are obtained when a minute drop varying from
0.0004 to 0.0006 ml. of the hormone mixture is applied on
the abscission zone without any scratch. In proper dosage,
with some species, the hormone treatment also increases the
number of seeds per pod.


43
Two pods developed with vulgaris (Cherokee Wax) pollen
from para-chlorophenoxyacetic acid treatment. One pod with
seed also developed from an untreated hud pollinated with
P. vulgaris pollen hut this was very near another flower
treated with hormone and might possibly have obtained some
effect from translocation. All other treated flowers stayed
from 3 days to an indefinite period after all other controls
dropped. Study of the causes and exact nature of incom
patibility in Pj¡. coecineus could have been further studied
and presented a problem in itself. But as the hormone treat
ment did not seem to show very encouraging results in over
coming incompatibility, further investigations were not pur
sued.
3) Place of Application of Hormone
Trials were also made to see if the place where the
hormone was applied made any difference in the effect pro
duced. Two places l) on the lower part of the calyx,
and 2) at the point of union of the peduncle and the
receptacle were tested. The effect was first studied on
the £*. coecineus flowers which were crossed with P^ vulgaris
pollen and were treated with 2,4-D, l/4 saturation in Karo,
para-chlorophenoxyacetic acid saturated in Karo and with
Karo alone. The hormones were applied at each of the two
places in sets of 10 with each treatment. Effects similar


122
BIOGRAPHICAL ITEMS
Rameshwer Prasad Jyotishl was "born In Raipur,
Madhyapradesh, India on July 1, 1920. He graduated from
the Municipal High School Saugor in 1936 and received his
B.Sc.(Agr) degree from the Nagpur University in 1941.
Prom June 1941 until May 1945 he worked as an Agri
cultural Assistant at the Agriculture College Experimental
Farm, Nagpur. In June 1945 he joined the staff of the
Agriculture College Nagpur as a lecturer in Agriculture.
In August 1948 he was sent to the United States for
advanced studies in Horticulture as a stipendiary depu-
tationist of the Government of Madhyapradesh. He prosecut
ed his studies towards a Ph.D. degree in Horticulture at
the University of Florida at Gainesville.


90
Interpretation
The first P test in Table 21, which gives a high signifi
cance, shows that if the average pod lengths from different
treatments are adjusted to a common value of X, i.e. days,
there is a highly significant difference among the average
pod lengths with various treatments. The observed values of
pod length on a common value of X are, however, not signifi
cantly different among the three treatments. The simple
analysis of variance, which was run using the pod lengths
on 6th and 10th day with three replications showed that the
treatments had no significant effect on the observed values
of Y (pod length).
In the second P test in Table 21, in which the variance
due to the regression coefficients is compared with the error
variance, shows that there is a significant difference in the
regression coefficients and that the hormone treatment affects
the rate of development of pods significantly.
In order to determine which of the three regression co
efficients differ from each other significantly, a similar
analysis of covariance considering treatments PCPA and Control
is made in Table 22. Since a significant difference in the
three regression coefficients is indicated in Table 21, and
the widest difference between the regression coefficients is
seen in case of 2,4-D and PCPA, the two other treatments com
pared are PCPA and Control which have the next widest differ-


106
This may be considered to be an advantage over the lanolin
paste method with which a scratch has been recommended. It
has already been shown that the place of application of the
hormone drop, whether at the point of union of the receptacle
to the peduncle or on the calyx, makes no difference in the
effects obtained. Hie former was considered to be safer and
better place to use because checking of abscission appeared
to be the primary objective and application at this point
was considered to be helpful in checking a direct and
drastic effect, if any, on fertilization and ovule develop
ment. Since glycerine has been included in the mixture at
20$ concentration, consideration should be given to the
possibility of its withering effect on the peduncle as was
observed in the preliminary trials when no Karo was used.
Although 20$ glycerine does not seem to be injurious, it
might be safer to apply it on the calyx where killing of a
few cells, if done, will not cause as much damage to the
flower as the killing of a similar number of cells on the
peduncle would because of its thinness and small number of
cells involved. Destruction of comparatively few cells of
the peduncle can easily result in the withering of the
flower.
The pipette designed for this study has proved to be
very suitable and it should be possible to repeat the
technique used with consistent results. No such device to
dispense such minute quantities of this fluid as are possible


56
number of buds treated thus was 210. Observations with re
gard to response were regularly made and the number of buds
which dried out from time to time was recorded. The data
regarding this experiment are reported in Table 16.
Experiment 4. Germination Tests
Hormone treatment has been known to inhibit develop
ment of embryo. It was therefore necessary to find out if
the seed-set with these treatments produced viable seeds
or not, and whether the seeds subjected to hormone treatment
during early development were affected with regard to their
germination capacity. Germination tests were, therefore,
run with the seed obtained from the crosses between P.
vulffafis (A) and P. coccineus (B) with different hormone
treatments and control. 20 seeds were selected at random
out of eaeh lot and germinated in sterilized soil in flats.
Also similar tests with seeds obtained as a result of
selfing snap bean (P. vulgaris) flowers with various treat
ments were conducted. The data regarding these tests have
been reported in Tables 17, 18, and 19.
Experiment 5. Effect of Hormone Treatment on the Rate of
Development of Pods
L-
To ascertain if the stimulation provided by the growth-


47
1. PCPA: Para-chlorophenoxyacetic acid saturated in warm
glycerine and the mixture diluted with four
times its volume of Karo (corn syrup)
2. PCPA & IBA: Para-chlorophenoxyacetic acid and indole-
butyric acid saturated in warm glycerine and
the mixture diluted with four times its volume
of Karo.
3. H.M.: Hormone-mixture made by saturating indole-
propionic acid, beta-naphthoxyacetic acid,
para-chlorophenoxyacetic acid and methyl ester
of naphthaleneacetic acid each in 2 cc. of
glycerine and then mixing this with 4 times
its volume of l/8 saturation of 2,4-D in Karo.
Only a small drop of the methyl ester was
sufficient to give a turbid emulsion in
glycerine.
4. KNA: 0.5% solution of potassium salt of naphthalene-
acetic acid in glycerine-Karo mixture (1:4).
5. Control: A mixture of glycerine and Karo (1:4) with
out any hormone.
In all further experiments the above mentioned treat
ments were used. In all crosses attempted the amount of
the chemical used was within the range of 0.0004 0.0006
ml. of the mixture applied with the pipette made for this
purpose. These treatments were then tested with P^ vulgaris


70
sidering other speeies, PCPA gave a larger number of
parthenoearpie sets when P. acutifollus (K), P. acutifolius
(J), £* lathvroides (G) and P. aureus (0) were used as
staminate parents PCPA & IBA gave increased sets with P.
calcaratus (P), P. atropurpureus (D), £. acutifolius (J),
P. acutifolius (I) and P. lunatus (H). An appreciably high
percentage of parthenoearpie pod-set was obtained with H.M.
as compared with the control when the staminate parents
used were P. ealcaratus (F), P. atropurpureus (D), P.
acutifolius (I), £. lathvroides (G) and J. lunatus (H).
Experiment 2. Effect on Fertilization and Ovule Development
The results of an experiment in which 200 flower buds
of £ vnlsarig (A) were allowed to be self-pollinated and
were subjected to various treatments are reported in Tables
14 and 15. It is clear from Table 14 that treatment with
PCPA gave a significantly greater number of pods as compared
with the control. The differences between the number of
pods obtained with other treatments and control are not
significant statistically. The average number of pods
obtained with PCPA treatment per 10 flowers treated was
7.25, as against 5.25 with the control. The difference
needed for significance at the 5% level has been calculated
as 1*12. The difference between the pod-set obtained with


115
TABLE 27
Analysis of Variance for Table 15
Source
D.F.
Sum of squares
Mean square
Total
19
1372.95
Treatments
4
550.70
137.675 **
Replications
3
562.95
187.65
Error
12
259.30
21.608
L.S.D.
7.1623
. F i 137.675/21.608 a 6.37


104
sprayed with naphthalenescetic acid, little or no effect of
the hormone was translocated from treated to untreated spurs.
However, Veintraub et al. (49) have demonstrated that not
only the growth-regulatory activity of 2,4-2) is observed on
the other parts of the plants when the chemical is applied
to the primary leaves of the bean seedlings, but also that
the effect so translocated is due to the actural trans
location of the 2,4-B itself rather than auxin-a, auxin-b
or indole-acetic acid or any other organic compound
activated by the 2,4-B. It should be pointed out that 2,4-D,
the most active of all the hormones tried was not used at all
in most of these experiments. Since PCPA, found to be
effective in this study, is related to 2,4-D, but is less
active, a similar effect is less likely. Caution is, however,
necessary in the matter of using buds sufficiently distant
from each other, and if possible on different plants, to
avoid any confounding of the results.
Mention may be made here of the interesting results
obtained and reported by Hamner and Kiang (21) regarding
the use of the "plastic material" to increase the effect
iveness of 2,4-D on bean plants. These workers found an
appreciable increase in the effects produced by 2,4-D used
as a spray when the treated branches were sprayed with 5%
Geon 31X latex. Similar results were obtained when the
chemical was mixed with the latex and a single drop of the
solution was applied at the base of one of the primary leaves.


94
Lewis (26) has reported on the effectiveness of
naphthaleneacetamide treatment of Prturns avium buds which
resulted in prolonging the period before abscission of
style and flowers. He explained that the hormone treatment
checks abscission resulting from changes taking place in
the existing cells consisting of the accumulation of starch
below the abscission line resulting in starvation of the
tissues above. Exactly how the abscission of bean flowers
takes place was not determined in this study but it is
definite that hormone treatment prevents this to a con
siderable extent. It is quite possible that this is
achieved by preventing changes in cells of the type
described above.
Tan verbeek et al. (36) have reported that the
hormone released in the ovary by the presence of pollen
tubes extends its effect to the peduncle and checks
abscission of the flower. The transmission of this
hormone effect to the peduncle is probably interfered
with under unfavorable environmental conditions or in
case of incompatible species the pollen tube fails to
cause the desired activation of the inactive hormone
present in the ovary. This probably results in insufficient
stimulation to initiate the development of the ovary and
other tissues connected with pod development and also causes
abscission of the flower before fertilization is achieved.


61
with PCPA & IBA gave a significant increase in the total
number of seeds obtained from crosses between vulgaris
and Pj. coccineus. The average number of seeds per 10
flowers pollinated and treated with this hormone mixture
was found to be 21,2 as compared with 11.8 for the control.
The L.S.D. was calculated to be 4.07. PCPA, which gave
significantly higher pod-set than control, did not give an
average number of seeds exceeding the number obtained with
control by the L.S.D., 4.07. The difference, which is 2.6,
however, may not be regarded as definitely insignificant,
and an experiment designed with greater number of flowers
used in each replication might show a significant difference
between seeds obtained with treatment PCPA and with the
control.
The data, however, show that treatment with PCPA and
PCPA & IBA gives a higher percentage of pod-set in crosses
between P^ vulgaris and P,. coccineus (B) than the control.
The number of seeds obtained is significantly increased by
the treatment PCPA & IBA, by as much as 79# over the control.
Hormone treatment in the amounts used does not seem to affect
significantly the number of seeds per pod as is evident from
Table 9.
Out of the 50 attempted crosses between Pu vulgaris and
Pjl filiformis (E) with each treatment, only one pod with seed
was obtained with PCPA and 3 pods with seeds with KNA. The
pod-set with these treatments was thus 2# and 6% respectively.


52
pollinated and treated with each were 250,250 and 200
respectively. Bach replication consisted of 10 huds with
each treatment, i.e*, a total of 50 buds. The treatments
were replicated 5 times with the first two while with the
third there were only 4 replications. Unlike field experi
ments in which soil heterogeneity is the main factor causing
variability, the replications were made with respect to time
as well as position in the greenhouse bench. The first
formed buds may be different from those formed in the middle
or latter part of the plants life. Therefore, a 4-day
period was considered to be the block during which all buds
(50) under each replication were treated. The complete
experiment with 5 replications each with P*. coccineus and
P. filiformis was completed in 40 days. With atropurpureus.
due to the shortage of pollen material and irregularity in
buds appearing, each replication consisted of 8 days. While
treating and pollinating buds, care was taken to select buds
from similar positions on the plant and of uniform vigor
and size. Five buds, one with each material, were treated
simultaneously*
Besides the three species mentioned above, a number of
other species of Phaseolus were also used as pollen parents
on snap bean. As it was not possible to repeat similar
experiments with each species, as many flowers were polli
nated with each as was possible within limitations of time,
space and availability of staminate and pistillate material.


63
TABLE 10
Results of the Crosses P^ vulgaris X fillformis (E)
with Different Treatments. (The numbers represent the
number of parthenocarpic and seed-containing pods obtain-
ed out of 10
flowers
pollinated and
treated in
each case.)
Replication
Pods set out of
10 flowers
treated
PCPA
PCPA&IBA
H.M.
KNA
Control
I
1
1
1
-
1
II
5
6
4
1
2
III
3
5
5
1
-
IV
5
5
8
5
1
V
2
5
3
2
1
Total
16
22
21
9
5
Average per
10 buds treated 3.2
4.4
4.2
1.8
1
L.S.D. :
1.79
(Analysis of variance given in Appendix Table 25)


62
as against no pod-set with control and other remaining treat
ments. The effect of hormone treatment was, however, sig
nificant inasmuch as a large number of parthenocarpic pods
was obtained with PCPA, PGPA & IBA, and H.M. The data, in
which the number of parthenocarpic and seed containing pods
have been combined, are reported in Table 10. Parthenocarpic
pods above one inch in length only have been included.
The table shows that the average number of pod-set
(parthenocarpic and seed-containing) was 3.2, 4.4 and 4.2
respectively with PCPA, PCPA & IBA and H.H., as against 1.0
with control. Pod-set with these treatments was, therefore,
significantly higher than that without hormone treatment.
The total number of parthenocarpic pods and those con
taining seeds from crosses between P*. vulgaris and P,
filiformis (E), along with the average length of pods with
various treatments, have been summarized in Table 11.
As is evident from this table, the percentage of sets
with seeds is very low being 2$ with PCPA and 6% with KNA.
Such a low pod-set may be considered to be Inadequate to
draw conclusions regarding the effectiveness of the hormone
treatments in aiding fruitful pod production. However, the
fact that 3 sets have been obtained with KNA and none with
the control cannot be ignored and considered as only
accidental. Further study of these sets by planting the
seeds and studying the plant characteristics will, however.


123
This dissertation was prepared under the direction of
the Chairman of the candidate*s Supervisory Committee and
has heen approved by all members of the committee. It was
submitted to the Graduate Council and was approved as a
partial fulfilment of the requirements for the degree of
Doctor of Philosophy.
Dean
SUPERVISORY COMMITTEE:


48
as pistillate parent with pollen from a number of species.
The number of crosses made with different species and the
number of sets obtained are given in Table 6. Parthenocarpic
pods more than 1 inch in length only have been recorded.
These results did not give a precise information with
respect to individual treatments but definitely showed that
hormone treatment produced some kind of effect without being
toxic because 46.43$ of the attempted pollinations with hor
mone application produced either parthenocarpic or seed con
taining pods in contrast with 5.8$ for the control. These
results may be regarded as highly significant whether the
resulting seed represents valid crosses or not, because the
same technique and conditions obtained for the flowers
treated as for the control. The glycerine-Karo mixture,
instead of Karo alone, increased the ease of application
and preciseness of the size of the drop because of the low
er viscosity. The amount used, however, was not as care
fully controlled as in the later experiments made.
D. Final Experiments
The preliminary experiments gave enough information
required for the final experiments which were conducted
during spring and summer of 1950. The hormone treatments
used were: 1) PCPA, 2) PCPA & IBA, 3) H.M., 4) KNA,
and 5) Control. The methods of their preparation have al-


105
Hi authors have not given any satisfactory explanation of
this effect produced hy latex but have suggested that
possibly the plastic material, due to its low moisture-
vapor transmission coefficient, increases the action of
2,4-D by sealing in its vapors. Since the same plastic
material has been used for sealing the emasculated and
pollinated buds in this study, consideration of this
property of the latex should be made* However, since the
plastic material has been used in this work only for seal
ing the eorolla and the hormone mixture drop has only been
applied on the abscission zone, any sealing affect as sug
gested by the above mentioned workers is out of the question*
In case there is some other way, not yet known, in which the
latex increases the effectiveness of the hormone, then, when
it is not necessary to seal the corolla with latex, the
dosage found effective in this study might prove to be in
adequate for the desired results.
With regard to the method of application, it seems
quite definite that the hormone need not be applied in a
scratch as has been done by most of the workers (22, 14, 53)
who have used a lanolin base* Desirable effects are shown
by the organs needing stimulation when correct amounts of
the hormone in glycerine-Karo mixture are applied on the
abscission zone without making any scratch, and even over
stimulation (conspicuous distortion of the developing pod
which eventually withers) may result from excessive dosages.


TABLE I
Results of Treatment of the Flower Buds of jP*. vulgaris with 2,4-D
No* of flowers
emasculated
Growth-regulator
used
Medium
used
Pollination
No* of flowers
dropped within
1 to 4 4 to 9
days days
No. of
parthen-
ocarpic
pods de
veloped
10
2,4-D 1%
Lanolin
Not
pollinated
4
2
4
10
2,4-D 1%
Latex
Not
pollinated
10
-
-
10
Untreated
Control
Latex
Not
pollinated
10
-

10
2,4-D 1#
Lanolin
Pollinated
with P*
lathvroides
2
8
10
2,4-D 1%
Latex
n
if
2
5
3
10
Untreated
Control
-
n
if
10
-
-


9
has been observed that the activity of auxins is greatly in
creased by the action of certain substances chemically re
lated to them. Went (51) found that the split pea stems
showed a much larger auxin response when they were first
soaked in phenylbutyric acid or cyclohexaneacetic acid.
Auxin alone or phenybuteric acid alone did not show as much
growth response. This is suggestive of a better effect that
may be expected with hormone mixtures than single hormones,
an effect known as synergesis.
The growth and germination of the pollen tube also
seems to be related to auxins. Smith (42) has studied the
growth of pollen with respect to temperature, auxins,
colchicine, and vitamin-B in the culture medium. Additions
of auxins (3-indole-acetic acid, 3-indole-butyric acid and
naphthaleneacetic acid) showed "a moderate stimulation" with
Snapdragon and a "much greater stimulation of both germi
nation and tube elongation" in Bryophyllum. A temperature
of 25 C. showed optimum tube growth and also showed great
est auxin effect. Concentrations of auxins higher than 1 :
50,000 were found to be toxic resulting in "decreased
germination, bursting and distortion of tubes".
Addicott (1) studied pollen germination and tube growth
of Milla biflora and Tropaeolum maius and found that the
germination of pollen grains and the growth of tubes are not
necessarily related. Both can be independently stimulated.


8
may only activate the hormone already present in the ovary
as precursor (36, 37). Van Overbeek has summarized the
literature on the growth-regulating substances in plants
(37), Evidence has been furnished that the hormone activity
is brought about by an active auxin complex in the plants
which is released from the precursor or bound auxin already
present. The pollen or pollen tube, thus, may not contribute
the necessary hormone but may bring only a substance which
activates the bound, inactive auxin in the ovary. Muir (32)
made direct measurements of diffusable growth-hormones in
the style and ovaries of the flowers of tobacco. He found
much higher amounts of auxin in pollinated flowers than in
unpollinated ones. Also the extent of penetration of the
pollen tube was found to be closely related to the hormone
concentration. The pollen or pollen tube extracts did not
show the presence of hormones and Muir suggested that the
pollen tubes probably secrete an enzyme which can liberate
the growth-hormone from the inactive state in the style and
the ovary. A similar phenomenon is also noted in the galls
and nodules produced by various species of bacteria in plants
which show a high active-auxin content. These organisms
probably, much in the same way as pollen tube, only activate
the native bound auxin (37).
Reporting on the work of Went (51) and Skoog et al.
(41) Van Overbeek in the above review points out the inter
action between auxins and chemically-related substances. It


85
TABLE 20
Average Pod-length (in caa .) on Different Days
Treatment 1.
2,4-D
Treatment 2.
PCPA
Treatment 3.
Control
Days
Length
Days
Length
Days
Length
X
Y
X
Y
X
Y
3
2.06
3
2.07
3
1.63
5
4.57
5
3.87
5
3.67
6
5.97
6
5.27
6
5.63
7
7.30
7
6.40
7
6.67
8
9.00
8
7.83
8
7.97
9
10.40
9
9.03
9
9.57
10
11.10
10
9.77
10
9.93
48
50.40
48
44.24
48
45.07


109
The technique developed may also prove useful in
hybridization work with other crop plants in obtaining a
higher percentage of sets from attempted crosses.


TABLE 5
_ t
Effect of the Place of Hormone Application on Pod-set from Crosses P^ vulgaris and
P. coccineus
Treatment
On calyx
On abscission zone
Buds No. of
treated P*
se|s
Buds
treated
No. of
P*
sets
S*
2,4-D 1/4 saturation in Karo
11 1
3
11
2
4
Para-ehlorophenoxyacetic
acid saturated in Karo
11 1
5
11
1
3
Control, Karo alone
11
3
11
1
5
*
P: parthenocarpic
*
S: seed-containing


84
three replications of each treatment. In the analysis of
the data used here it was not necessary to consider the data
separately in each replication, therefore an average of pod
length from three replications is considered to be the
average length of pods on different days. In Table 20, in
whieh the data regarding the pod lengths on days have been
recorded, the pod lengths, thus, are averages of 12 pods
from each treatment.
The relationship between pod length and days, as may
well be expected, is not a straight line relationship but
follows the pattern of a growth curve. Treating it as a
straight line relationship, the regression of pod lengths
on days has been calculated with each treatment and the
regression coefficients have been used to compare the rate
of development of pods resulting from the three treatments.
The regression of pod length on days and the regression
coefficients (b) with the three treatments are as follows:
2,4D
b !
1.3469
Y :
1.3469 X 2.0358
PCPA
b
1.1541
1.1541 X 1.5938
Control
b :
1.2579
1.2579 X 2.1869


Use of Growth Regulating Substances as an Aid
In Hybridization of Phaseolus
By
RAMESHWER PRASAD JYOTISHI
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
September, 1950

ACKNOWLEDGEMENTS
The author wishes to acknowledge with deep gratitude
the kindly advice of Dr, A, P. Lorz, under whose guidance
and constant help this study was conducted.
He wishes to express his thanks and sincere appreci
ation to the members of his committee as well as to the
members of the staff of the University of Florida whose
assistance and encouragement were a constant source of
inspiration.
He particularly acknowledges the kindly advice and
criticisms of Dr* H. S, Wolfe, Head Professor of Horti
culture and Mr, I, W, Ziegler, Assistant Professor of
Horticulture who went through the manuscript and Mr,
W. D, Hanson, Assistant Professor of Agronomy who gave
valuable suggestions regarding the statistical analysis
of the data.
ii

TABLE OF CONTENTS
INTRODUCTION 1
REVIEW OF LITERATURE 4
General 4
Hormones as an Aid to Fruit-Set 13
Hormones as an Aid in Hybridization by Checking
Incompatibility 15
Use of Hormones in Increasing Yield of Beans 18
Effect of Hormones on Germination of Seed and
Growth of Seedling 20
EXPERIMENTAL MATERIAL AND METHODS 24
A. General 24
r B. Hie Technique of Emasculation and Pollination .... 25
C. Preliminary Studies 28
1) Especially Designed Micrometer Pipette for
Hormone Application 37
2) Trials on Phaseolus coecineus (Scarlet-runner
bean) 41
3) Place of Application of Hormone 43
4) Trials with Hormone in Glycerine-Karo Mixture 46
D. Final Experiments 48
v Experiment 1. Interspecific Crosses 51
Experiment 2. Effect on Fertilization and Ovule
Development 54
Experiment 3. Effective Range of 2,4-D Concen
tration 54
iii

Experiment 4. Germination Tests 56
Experiment 5. Effect of Hormone Treatment on the
Rate of Development of Pods 56
RESULTS 58
Experiment 1. Interspecific Crosses 58
Experiment 2. Effect on Fertilization and Ovule
Development . 70
Experiment 3. Effective Range of 2,4-D Concen
tration 74
Experiment 4. Germination Tests 78
Experiment 5. Effect of Hormone Treatment on the
Rate of Development of Pods ... 81
DISCUSSION 92
General 92
Effect on Seed-Set 97
Medium and Method of Hormone Application 101
CONCLUSION 108
SUMMARY 110
APPENDIX 113
LITERATURE CITED 116
BIOGRAPHICAL ITEMS 122
iv

INTRODUCTION
Beans are an important truck crop of Florida, which is
also of considerable commercial value to the agricultural
economy of the United States. Numerous varieties of beans
have been evolved during recent years and a very high standard
with regard to the horticultural characteristics has been
achieved in the existing varieties grown commercially in
Florida. There is, however, never a limit to improvements
and perfection has not been reached. Improvements towards
still better combinations of characters by hybridisation are
possible and researches in that direction are in progress.
Resistance to diseases, such as downy-mildew, to cold, to
drought, and to such insects as Mexican-bean-beetle are some j
of the desirable qualities that might be possible through
further improvements in beans by interspecific crossing.
While hybridization is one of the most effective tools
of the plant breeder working towards crop improvements, there
are certain limitations in its practice. Incompatibilities
among species and varieties and often a very low percentage
of pod-set are great handicaps in bean breeding work. In
compatibility may be due to various causes and be of various
types. It may result from a failure of the fusion between
male and female gametes, that is, failure of the fertili
sation proper, or it may be due to any of a number of other
1

2
causes such as improper germination of the pollen, too slow
growth and penetration of the pollen tube, lack of stimu
lation to development of ovary and surrounding tissues,
abscission of style before fertilization, or the abscission
of the whole flower before pod and seed set. For success
and ease in hybridization a plant breeder is desirous of
obtaining a high percentage of success in crossing resulting
in healthy and well-developed seed of good germination
capacity. In bean breeding both types of difficulties are
met. Many species have not been successfully crossed with
the common improved types or the percentage of set obtained
has been very low, necessitating pollination of numerous
flowers. Furthermore, unfavorable environmental conditions
such as extremes of light and heat also result in high
abscission rate. The exact causes and nature of incompati
bility between certain species are not definitely known and
may be many and various.
Hormones have assumed great importance in horticultural
research during the recent past and have been used in many
ways to stimulate growth and other physiological processes
in plants. Checking the abseisslon of fruits and supplement
ing normal fruit-set have been the two most important uses
of hormones with apples and tomatoes. Although hormones
have been generally reported as inhibiting fertilization,
by regulating the amount and method of application they may
encourage development of seed and fruit, reduce abscission

3
of the flowers before fertilization and thus overcome the
above mentioned difficulties without inhibiting fertili
zation. Very little work has been done with regard to the
use of hormones in this respect but there is evidence in
the literature which suggests that hormones may be used with
advantage in hand-pollination to obtain a greater number of
fruits set and healthier seeds.
The results of investigations and trials with hormones
on beans with a view to obtaining various interspecific
crosses are reported in the following pages. The aim of
the study has been threefold. In the first place, as many
species as possible have been used as the staminate parent
in attempted crosses with the standard varieties of snapbean
in an effort to obtain hybrids which may show desirable com
binations of characters or may at least prove helpful in
bridging the gulf between them so that they may be later
used for still further crossing as one of the parents.
Secondly, an attempt has been made to study the effects of
different hormone treatments, their concentration and method
of application on hand-pollinated snapbean flowers with a
view to working out an effective treatment which would over
come some of the incompatibilities and difficulties involved
in obtaining good pod-set. Thirdly, the aim of this study
has also been to evolve a technique for applying the hormone
treatment which would permit measurement and regulation of
the quantity applied in order that the procedure might be re
peated.

4
REVIEW OP LITERATURE
General
Although hormone research is comparatively a recent
venture in the field of horticulture, the literature on the
subject is voluminous. It is neither possible nor desirable
to attempt a complete review of this literature here. As is
well known, hormones have been demonstrated to be useful in
favorably affecting the physiological activities of plants
in various ways. The most common uses of economic import
ance are: the stimulation of the rooting of cuttings, the
control of fruit-drop by reducing abscission, the thinning
of blossoms and buds, the production of parthenocarpic
fruits, the inhibition of sprouting, and the killing of
weeds. While considerable work has been done on the above
mentioned aspects, the literature on the use of hormones
for stimulating fruit-set and viable seed production is very
meager. In fact there is evidence to the effect that hormones
actually inhibit fertilization (37), An attempt is made here
to review the pertinent literature on hormone research re
garding their use as an aid to fruit-set, fertilization,
overcoming incompatibility and any other physiological
activity that directly or indirectly helps in self or cross
fertilized fruit development,
Boysen Jensen in 1910 showed that some chemical substance

5
is translocated and brings about certain growth responses
within the plant body (8, 9). This provided an explanation
to Darwins earlier observation that shoots of grass failed
to bend towards light if their tips were cut off (4). In
1928 Went demonstrated the bending of decapitated oat
coleoptiles to one side when agar jelly containing some un
known hormones was applied. This method is still the most
widely used and probably the best method for measuring
concentrations of unknown hormones (50). By 1934 auxintriolic
acid (auxin-a), auxinolonic acid (auxln-b) and indole-acetic
acid (heteroauxin) were isolated from plants. Indole-acetic
acid is known to occur in higher plants and has been obtained
from leaves, endosperm of seeds, etc. (4). Zimmerman and
Wilcoxon (57, 58, 59) and later others, found that many other
chemical compounds such as indole-butyric, indole-propionic,
naphthaleneacetic, phenylacetic, and indolepyruvic acids
acted as stimulants in various growth processes and so may
be called synthetic hormones. Many other compounds have
since then been added to the list but the chlorophenoxy
acids and their derivatives have been most widely used.
The plant breeder, desirous of crop improvement by
hybridization, is primarily interested in a higher percent
age of fruit-set with his crossing, and in production of
viable seeds. The use of hormones to accomplish this end
is thus of Interest and should be considered from two points
of view: l) their use as directly helping the actual pro-

6
cess of fertilization, stimulation and development of ovary
and the fertilized ovules, and creating the necessary favor
able condition for such activity, 2) their use as indirectly
helping to obtain greater number of fruits, and therefore
seeds, by checking abscission of flowers and young fruits.
Not much work has so far been done with regard to the first,
at least with beans, but there is quite a considerable amount
of evidence concerning the usefulness of hormones in checking
abscission. Control of fruit drop of apples with the help
of hormones has become an established commercial practice.
That hormones play an important part in the process of
fruit development is also well known. Van Overbeek et al.
(36) have shown that hormones control the growth of ovules.
On injecting 1$ solution or emulsion of the ammonium salt of
naphthalene&cetic acid into the young ovaries of 4-n Meladrium.
they produced parthenocarpic fruits whose ovules had enlarged
and developed seed coats but they contained no embryo. When
the emulsion was applied to the outside of the ovary, the
ovary was stimulated but not the ovules. They suggested a
mechanism of fruit and seed development which consists of
stimulation of ovaries, ovules and placentae, and the
necessary stimulation to set in motion the division of polar
nuclei and egg cell, which may take place with or without
fertilization. They suggest three stages of fertilization.
Hie first one is the stimulation of the ovary, placentae and
M- Is

7
ovules and checking abscission which prepares conditions
favorable for embryo development. Actinal frtilization,
i.e. fusion, is not necessary for these conditions. The
second stage is the division of the polar nuclei and, rarely,
of the egg cell, which are not always a direct result of
fertilization. Instances are known where the polar nuclei
fuse to form an endosperm nucleus and this divides many
times before the sperm nuclei leave the pollen tube and
fusion takes place. The presence of pollen tube in the
embryo-sac, however, seems to be necessary for initiating
divisions of the endosperm. The last step is the actual
fusion of the sperm nuclei with the egg cell and polar
nuclei. It is, however, important to note that actual
fusion, although indispensible for hybridization, is not
all that is needed. For a complete process the other two
stages, which are probably hormone induced, may be of equal
importance. This fact is evidenced by earlier work of
Gustafson (19) who showed that except for the absence of
ovule development, the fruit development by chemical treat
ment is in no way different from that resulting from
pollination in the tomato. The matter of food supply is
important in both cases and its mobilization seems to be
influenced by hormones.
It was formerly believed that the initiation of growth
of the ovary is caused by the hormone secreted by the pollen
tube (19). It has now been demonstrated that the pollen tube
is-

8
may only activate the hormone already present in the ovary
as precursor (36, 37). Van Overbeek has summarized the
literature on the growth-regulating substances in plants
(37), Evidence has been furnished that the hormone activity
is brought about by an active auxin complex in the plants
which is released from the precursor or bound auxin already
present. The pollen or pollen tube, thus, may not contribute
the necessary hormone but may bring only a substance which
activates the bound, inactive auxin in the ovary. Muir (32)
made direct measurements of diffusable growth-hormones in
the style and ovaries of the flowers of tobacco. He found
much higher amounts of auxin in pollinated flowers than in
unpollinated ones. Also the extent of penetration of the
pollen tube was found to be closely related to the hormone
concentration. The pollen or pollen tube extracts did not
show the presence of hormones and Muir suggested that the
pollen tubes probably secrete an enzyme which can liberate
the growth-hormone from the inactive state in the style and
the ovary. A similar phenomenon is also noted in the galls
and nodules produced by various species of bacteria in plants
which show a high active-auxin content. These organisms
probably, much in the same way as pollen tube, only activate
the native bound auxin (37).
Reporting on the work of Went (51) and Skoog et al.
(41) Van Overbeek in the above review points out the inter
action between auxins and chemically-related substances. It

9
has been observed that the activity of auxins is greatly in
creased by the action of certain substances chemically re
lated to them. Went (51) found that the split pea stems
showed a much larger auxin response when they were first
soaked in phenylbutyric acid or cyclohexaneacetic acid.
Auxin alone or phenybuteric acid alone did not show as much
growth response. This is suggestive of a better effect that
may be expected with hormone mixtures than single hormones,
an effect known as synergesis.
The growth and germination of the pollen tube also
seems to be related to auxins. Smith (42) has studied the
growth of pollen with respect to temperature, auxins,
colchicine, and vitamin-B in the culture medium. Additions
of auxins (3-indole-acetic acid, 3-indole-butyric acid and
naphthaleneacetic acid) showed "a moderate stimulation" with
Snapdragon and a "much greater stimulation of both germi
nation and tube elongation" in Bryophyllum. A temperature
of 25 C. showed optimum tube growth and also showed great
est auxin effect. Concentrations of auxins higher than 1 :
50,000 were found to be toxic resulting in "decreased
germination, bursting and distortion of tubes".
Addicott (1) studied pollen germination and tube growth
of Milla biflora and Tropaeolum maius and found that the
germination of pollen grains and the growth of tubes are not
necessarily related. Both can be independently stimulated.

10
Vitamins, hormones, pyramidines and purines shoved stimu
lating effects. He tried 33 different growth-promoting
substances, of which 16 showed significant increases in
germination or tube growth. But the growth of pollen with
these substances was not comparable to that in stigma
exudate. In the latter case the tubes had an average
length of 16 diameters as compared with from 3 to 6 diame
ters in various other media,
Chen (23), however, could not find any favorable
effect on germination and tube growth with auxins on Prunus
armeniaca which showed favorable responses to additions of
micro-nutrient elements to the culture medium.
The work of Kuehlwein (24) throws further light on the
activity and stimulating power of growth-promoting substances
on pollen and stigma. His results are of especial interest
because they suggest the possibility that the pollen tube
growth of certain species might be favorably stimulated with
certain hormone treatments. By adding the pollen and stigma
extracts of various species to the culture media and observ
ing the growth of pollen tubes of various species in differ
ent media, he found that the growth-promoting substances of
pollen and stigma are "species or organ specific"; some pro
ducing a more favorable increase in growth or germination
with the pollen of certain species than with the pollen of
others.
Lewis (26) studied the problem of incompatibility and

11
parthenocarpy of cherries, plums and Oenothera. He says
that incompatibility is a genetically controlled character.
It may be because of the failure of the pollen tube to reach
the ovary before abscission of the flower or style. Fertili
zation thus cannot take place and ovules do not develop.
Using alpha-naphthaleneacetamide he studied its effect on
pollen tubes, the mechanism of abscission of the flowers
and style, and the development of fruits and seeds with and
without pollen. In case of Prunus avium the application of
an aqueous solution of alpha-naphthaleneacetamide to flowers
resulted in delayed abscission of the style. This species
is self-incompatible and untreated flowers, both unpolli
nated and self-pollinated, lost their styles two days earlier
than the treated ones. The growth of pollen tubes of com
patible pollen was, however, not affected by the hormone
treatment. With Oenothera the aqueous solution showed no
effect, but application of 1% lanolin emulsion to the lower
part of the ovary delayed abscission. Time of treatment,
from four days before up to the time of flower opening, show
ed no significant difference in effectiveness. Incompatible
tubes showed many abnormalities; with 1% emulsion causing
swelling or even bursting. The compatible tubes showed a
more remarkable effect in stopping growth at 4 to 5 mm* and
their ends bursting instead of reaching the 20 ram. length
of untreated ones.
Lewis thus found that alpha-naphthaleneacetamide treat-

12
ment without compatible pollen did not stimulate development
of fruits in cherries, plums or pear. In Oenothera, however,
smear treatment of the ovary resulted in parthenocarpic fruit
development. The ovules grew to normal size but had no
embryos. He suggests that for fruit formation the necessary
stimulation is provided by the pollen tubes as they enter the
ovary as well as by the developing ovules. The pollen tubes
probably give a greater stimulus than the ovules in many-
seeded fruits. Therefore parthenocarpic fruit development
with chemical treatment is more successful in many-seeded
fruits like cucumbers than in fruits like plums. Lewis also
shoved that hormone treatment checks that abscission which
is due to the change in the walls of the existing cells and
not that which is due to rapid cell division. The abscission
of the style and of mature fruits is of the former type while
the premature flower and fruit drop is abscission of the
latter type.
Besides the evidences quoted above, there are numerous
instances where hormones have been demonstrated to check
abscission of fruits. LaRue (25) found that the fall of
Coleus leaves can be considerably reduced by hormone appli
cations. Date flowers also show the same reaction, (35).
As early as 1939-40 the use of indole-acetic acid,
indole-butyric acid, indole-propionic acid and naphtha-
leneacetic acid was demonstrated to be effective in check-

13
ing the preharvest drop of apples by Gardner et al. (17, 18),
Treatment with various chlorophenoxy compounds has been shown
by Zimmerman and Hitchcock (59) to result in the tomato
flowers staying longer by 10 to 30 days.
Concentrated applications of naphthaleneacetic acid as
an aerosol or as dust have been shown to be as effective as
sprays applied with a hydraulic sprayer in checking pre-
harvest drop of McIntosh apple by Mitchell et al, (29),
Batjer and Thompson (6) have compared the effectiveness
2,4-D and naphthaleneacetic acid in reducing the drop of
pears. They find that 2,4-D may probably be a more effective
chemical than naphthaleneacetic acid even with a weaker
concentration. The injury caused with 2,4-D seems to be
related to the concentrations used; all concentrations above
215 ppm. appear to be injurious.
Hormones as an Aid to Fruit-Set
The part hormones play in fruit development has already
been emphasized in the literature reviewed above. The recent
work on the use of hormones to encourage fruit-set by supple
menting normal pollination, especially with regard to tomato,
is of especial interest for the study undertaken. The normal
fruit-set of tomato has long been known to be low under green
house conditions in the north. Howlett (22) reported that
the use of hormone treatment in supplementing the natural

14
setting of tomatoes In the greenhouse helped in overcoming
the reduced fruit-set resulting from cold weather. Using
indole-butyric acid, he found that an emulsion at 0.3$
concentration was effective as a paste. The cross-polli
nated flowers with viable pollen, on treatment, developed
fruits superior to the untreated. In case of self-polli
nated flowers, only 60$ of the untreated flowers set fruits
while the percentage was 100$ with treated flowers. Treated
flowers had a poorer seed development than untreated flowers.
The vapors of naphthoxyacetic acid have also been shown to
produce a high percentage of seedless fruits (50 to 98$) in
greenhouse tomatoes (30). Randhawa and Thompson (39) used
various other hormones, viz., beta-naphthoxyacetie acid
(50 ppm.), alpha-O-chlorophenoxypropionic acid (50 ppm.),
p-chlorophenoxyacetic acid (25 ppm.), 2,4-5-trichloro-
phenoxyacetic acid (10 ppm.) and 2,5-dichlorobenzoie acid
(100 ppm.). Spraying the flowers, either when they were
half open or when all were open, gave a higher set of fruits
than untreated controls. That the effectiveness of differ
ent hormones varies according to species is evident from the
work of Crane (13) who found that the aqueous sprays of
indole-acetic acid (1500 to 2670 ppm.) gave parthenocarpic
fruit-set of figs and the fruits were not inferior to those
caprified. Naphthoxyacetic acid and 2,4-D were not effect
ive. Stewart (44), however, has obtained seedless figs with

15
2,4-D at the concentration of 250 ppm.
Hormone treatment has not always shown increased fruit
set. Evidences of a negative effect are also met witliin the
literature, eg., with greenhouse potatoes (11), Washington
navel oranges and Marsh grapefruit (38). There are many more
references in the literature which report effectiveness of
hormones in causing increased fruit-set with various other
crops. These, however, in general, also report that hormone-
induced fruits, though normally pollinated, have a lower seed
content. For example, drench spraying with aqueous solution
of naphthoxyacetic acid (20 ppm.) applied to strawberries
when in full flower, has been reported by Swarbrick (45) to
result in a 17$ increase in yield. He reports, however,
that the fruits did not have the normal number of seeds and
that the increase in yield was due to increase in size rather
than the number. Burrel and Whitaker (10) used 1$ indole-
acetic acid in a lanolin paste with success in obtaining an
increased fruit-set with muskmelons. The low fruit-set in
muskmelons, due to heavy flower drop, is checked by the
application of hormone to one lobe of the stigma. They
suggest that this method may be useful to the plant-breeders
who are interested in a larger number of fruits by hand-
pollination.
Hormones as an Aid in Hybridization by Checking Incompatj-

16
frility.:
The earliest reeord of the effective use of hormone
treatment to overcome Incompatibility and obtain fruit- and
seed-set with successful fertilization is found in the work
of Eyster (15), A strain of Golden-rose petunia was found
to be self-sterile, a genetic character behaving as a simple
recessive. The incompatibility was caused by a very slow
rate of growth of the pollen tube and formation of the
abscission layer much earlier than usual. On the basis of
Yasuda*s findings (5fr) it was stated that the placenta of
Petunia violcea secretes a special substance which diffuses
into the style causing inhibition of growth of pollen. Spray
ing the flowers with 10 ppm. aqueous solution of alphanaphtha-
leneacetamide immediately after or before pollination resulted
in development of capsules and viable seeds. The explanation
of this is found in the work of Lewis (26) reported earlier.
This instance gives reason to believe that the hormone treat
ment may prove to be useful in overcoming self-or cross
sterility of other plants as well.
A highly significant increase in fruit-set of hand-
pollinated flowers of Cucumis melo obtained by Whitaker and
Prior (54) with hormone treatment deserves special mention
here. Although self- or cross-sterility is not a problem
with melons, about one third of the hand-pollinated flowers
do not set fruits, which is a great handicap to breeding

17
work. These authors used various hormones and also ether
extract of cantaloupe pollen, Hie hormones were applied In
lanolin paste, where and in most cases 10 to 15 mg. per
gram of lanolin was the optimum concentration range. 2,4-D
and 4-ehlorophenoxyacetic acid were found to he most effect
ive and gave very highly significant results. An increase
in set of 27$ has been reported with the use of hormone
treatment.
Emsweller and Stuart (14) successfully overcame the
self-incompatibility of Creole, Craft and Ace varieties of
Easter lily (Lilium longiflorum) with hormone treatment of
flowers. They also tried these treatments with inter
specific crossing. Various chemicals were used after dis
solving them in lanolin at concentrations from 0.1 to 1$.
The paste was applied in a wound produced by removing one
petal and also at the base of the style. Effectiveness was
found to be the same in both cases. While untreated, hand-
pollinated flowers did not set a single fruit, 1$ naphtha-
leneacetamide treatment resulted in production of viable
seeds although they were weak in germination. Treatment
with the hormone caused a swelling of pedicel and delay in
abscission by several weeks. Although hormone treatment
was not very effective in species and varietal crossing,
the authors say that it can help in making some difficult
crosses possible. Chemical analysis of young developing
fruits after treatment showed a higher sugar content which

18
is suggestive of greater mobilization of food material caus
ed by the hormones. The authors believe that this may be
responsible for the delay in abscission and may also check
rapid degeneration of embryo sacs, keeping the egg cell
viable for a longer period.
There appears to be only one instance in the literature
where hormones have been used with beans with favorable
responses in controlled cross-pollination. Wester and Marth
(53) used 1$ concentration of a mixture of indole-butyric
acid and p-chlorophenoxyaeetic acid in proportion of 4 : 1
and applied it in three ways to cross-pollinated flowers of
lima beans. As an aqueous solution, the mixture was sprayed
on the stigma and as an emulsion in lanolin, it was applied
on the pedicel, with and without scratching the tissue.
When applied in a scratch, the treatment resulted in 28.8$
of successful sets as against 18.7$ for the control. The
degree of success depended also on parental combinations.
The most interesting feature of the trials was an increase
in the average number of seeds per pod from 1.95 to 2.43.
Pse of Hormone in Increasing Yield of Beans:
Hormones have been definitely shown to be effective in
increasing yield of beans. It would be desirable here to
review briefly the literature concerning this aspect.
Fisher et al. (2, 16) conducted extensive studies and

19
used several chemicals. They demonstrated that bud, blossom
and pod drops of string beans are reduced by hormone treat
ment. These bud, blossom and fruit drops were said to be due
to hot, dry weather and insects such as the tarnished plant-
bugs and the potato leaf-hopper. They induced abscission by
higher temperature, by use of illuminating gas and by caging
insects over the plants. Alpha-naphthaleneacetle acid and
2,4-D were found to be most effective, giving best results
as a dust (40 to 80 ppm.). An increase of 40$ in yield of
wax beans was obtained, resulting from a "greater number of
small, high grade beans rather than larger beans".
Murneek et al. (34), working with snap beans, also
obtained similar increases in yield with hormone treatment
but found that the increase in yield was obtained only when
the weather was hot. In the fall crop, hormones actually
decreased the yield. The authors believed that the yield
increase, when obtained, was due to increase in the
chlorophyll content of leaves and a stimulation of carpel-
growth brought about by the hormone treatment. They report
that, at least under certain environmental conditions, the
number of pods per plant and their rate of development can
be increased by hormone treatment. The same workers further
continued the trials using various other chemicals, e.g.,
the substituted phenoxy-benzoic acids and chlorine-substi
tuted phenoxy acids, and in 1947 (55) reported most con-

20
sistent yield increases with p-chlorophenoxyacetic acid.
With a summer crop of string ¡beans, spraying the flowers at
weekly intervals with 100 to 150 ppm. aqueous solution of
hormone gave as much as 300$ yield increase. Dusts were
found to he as effective as sprays. *01 yield Increase was
found more pronounced when the temperature was above 90 to
95 F. It was also reported that the increase in yield was
due only to stimulation of pod tissue. There was a depress
ing effect on seed development.
Wester and Marth (52), on the other hand, did not find
any significant increase in the total yield of lima beans.
Alpha-naphthaleneaeetie acid as a dust at 50 ppm. showed no
significant yield increases with 13 varieties of lima beans
tried. The insignificant results were explained to be due
to an exceptionally fine weather for seed and pod setting.
Clore (12), using alpha-naphthaleneacetic acid on three
varieties of bush lima beans, actually found a reduction in
yield due to treatment. Concentrations of 50, 100 and 1000
ppm. applied at rates of 75 to 100 gallons per acre resulted
in retarded growth and in foliage epinasty. The 5 ppm.
concentration failed to show any favorable or unfavorable
effects.
Effect of Hormones on Germination of Seed and Growth of
Seedling

21
Tulls and Davis (48) have shown that there is a con
siderable persistence of the effect of 2,4-D in plants
treated with the chemical* Bean plants, when sprayed with
2,4-D during pod development, produced seed which, when
sown, gave seedlings exhibiting malformations* Any hormone
treatment of flower buds, therefore, has the possibility of
affecting the viability of seed and growth of seedlings*
There is a mass of information in the literature on
the effect of hormone treatment of seeds and soil on the
germination percentage and development of seedlings. The
results, on the whole, are conflicting. The majority of
the evidence seems to show that hormone treatment has a
deleterious effect on germination of most seeds, or that,
at least, it does not increase the germination percentage.
The results of various studies so far made Indicate that
seed treatment with hormones may increase the germination
capacity of seeds in case of some field and vegetable crops,
while with others it may be retarded.
Avery and Johnson (5) have summarised the literature
on the subject of hormone treatment of seeds* Among the
workers reported to have obtained increased germination of
seed and development of seedlings, and subsequent increase
in yield, two may be mentioned here* Amlong and Naundorf
(3), using various concentrations of a potassium salt of
indole-acetic acid, obtained increase in the yield of alfalfa.

22
corn, spring wheat and sugarbeet, Swartley and Chadwick
(46), using naphthaleneacetamide and indole-butyric acid
with 41 garden perennials, obtained significant increase in
percentage of germination with 11,
The property of retarding the germination and growth
of seedlings exhibited by some chemicals suggested the
possibility of using them effectively for weed control,
2,4-D had shown such effect with various species,
Hamner et al, (20) used 2,4-D, applying it both to the
soil and seeds, to study its effect on germination of
various kinds of seeds, With 1 gm, of the chemical per pot
of the soil, they found complete retardation of germination
of clover and cabbage seeds. The germination of wheat seed
obtained, however, was 85$. No effect on germination was
observed with 0,001 gm, of the acid per pot. Application
of the chemical to the seeds (soaked for 4 hours) at various
concentrations from 1 to 100 ppm, also showed effect on
germination and growth of the seedlings. Bean seeds treated
with 1 ppm, of 2,4-D in aqueous solution showed "the
characteristic formative effects and virus' like symptoms0
in the seedlings. At 10 ppm, the seedlings were "severely
checked* and exhibited swelling in the hypocotyl, while 100
Ppm, of 2,4-D completely checked growth. The authors con
cluded that soil treatment with 2,4-D at one ppm, adversely
affects seed germination and growth of seedlings in many

23
cases. Grass seeds are more resistant than the seeds of
many of the vegetables,
Tukey et al, (47) studied the histological changes in
bindweed and sowthistle on treatment with 2,4-D at 1000 ppm,
as spray. Among the effects produced were plasraolysis of
pollen grains, checking of chlorophyll development,
plasmolysis of cells of leaves, increase in cell division
in all cambial zones, enlargement and rupture of cortical
cells and disappearence of starch from all parts of the
flower* The chemical also cheeked starch hydrolysis,
Mullison et al, (33) treated the seeds of various field
and vegetable crops using the vapors of 2,4-D and its
derivatives. Bean seeds subjected to vapor treatment with
2,4-D derivatives did not show much reduction in germination,
although the methyl ester caused a reduction in germination
by 12$ as compared to the control. Isopropyl 2,4-Dichloro-
phenoxyacetate vapor treatment of lima bean seeds resulted
in germination of 44$ as against 68$ with untreated controls.
Without going further into the voluminous literature
on weed control by hormones, it will be evident that any use
of hormones for effecting greater pod and seed-set may affect
the viability of seed and the nature of development of
seedlings. This fact, therefore, needs to be borne in mind
while studying the effects of hormone treatment of flower
buds with a view to obtaining a greater set of fruit.

24
EXPERIMENTAL MATERIAL AND METHODS
A* General
As pointed out before, the aim of this study was three
fold. The primary object was to find out if hormone treat
ment could be used effectively in controlled cross-polli
nation of beans. Since the most effective method of hormone
application for treatment of individual flowers so far used
and recommended is the lanolin paste method, a secondary
objective was to work out some other equally effective method
which would permit a greater control of the quantity of
hormone used with each flower. This precision is desirable
so that the treatment may be repeated with consistent results.
The third objective was to cross as many species of Phaseolus
as possible within the limitations of time, etc., with a
standard variety of snap bean, Cherokee Wax (P. vulgaris),
so as to obtain hybrids which could be tested later.
Except for some of the preliminary trials, all the work
was done in the greenhouse at the Florida Agricultural Ex
periment Station, Gainesville, from August, 1949 to August,
1950. In all preliminary trials lima bean was also used as
one of the parents, but later on it was found desirable to
confine efforts to one variety of P. vulgaris, which was the
Cherokee Wax. The reason for selecting and using this
variety was that its flowers are very convenient for hand-

25
pollination, it is quite prolific, and it is a representative
type of the commercially grown varieties with many good
horticultural characteristics.
The two most serious greenhouse pests were mildew and
red spider-mite. The former was controlled by dusting with
sulphur and the latter through the use of the newer miticides
especially E.P.N. miticide, generously provided by the Station
Department of Entomology. The E.P.N. miticide was applied
as a spray at the rate of 1 lb./lOO gals, of water. The soil
at first was sterilized with formalin or D-D, and later on
was better sterilized with steam to eliminate nematodes.
When the plants were established, a tablespoonful of 4-7-5
fertilizer mixture was applied to each plant at monthly
intervals.
Inasmuch as the success in any hybridization work depends
mainly on the proper technique of emasculation and pollination,
the technique found to be very satisfactory during the course
of this work needs to be described in some detail.
B. The Technique of Emasculation and Pollination
The largest flower buds obtainable about one day previ
ous to anthesis are selected for emasculation (Pig. 1 A).
The suture which closes the banner around the other flower
parts is opened with extreme care in order not to tear the

26
margin of the left (observers left) half, which is everted
and held down with the thumb of the left hand. The left
wing of the corolla is pushed slightly aside exposing the
distal end of the coiled keel (Fig. IB). With a sharp,
slightly flattened, pointed pair of forceps the upper side
of the distal portion of the keel is removed with the points
of the forceps inserted as indicated by the arrow in Fig.
IB. The anthers are then removed taking care to disarrange
the other flower parts as little as possible.
The stigmatic surface is then pollinated as follows:
The pollen-laden, distal portion of the pistil of a freshly
opened flower from the selected staminate parent is rubbed
against the stigma of the emasculated flower of the select
ed pistillate parent. Usually the pollen-laden distal
portion of the pistil can be made to protrude from the open
ing in the end of the keel by the downward pull of the left
wing of the corolla with relation to the other flower parts.
After pollination, the left side of the banner is returned
to its normal position and the other slightly disarranged
flower parts are carefully tucked underneath so that the
bud is completely closed. In most eases only a slight
separation of the margins of the right and left halves of
the banner give any indication that the flower has been
worked with. The suture then may be sealed with a quick
drying latex suspension applied with a camels hair brush.

Fig. 1. The Successive Stages of Emasculation
and Pollination of Bean Flowers.
(Explanation given in the text)

28
A proprietary name for such a latex suspension is Goodrite
VL 100, manufactured by B. F. Goodrich Chemical Company,
Cleveland, Ohio. In most cases the use of latex is un
necessary if the flover parts are only slightly disarranged
during the emasculation process and the bud is returned to
its original closed condition after pollination. Where
closure is difficult or conditions of low atmospheric
humidity prevail, the use of latex is more essential in
order to prevent the withering of the style before the
pollen tubes have had a chance to grow through it. The
successive steps in the process of emasculation and polli
nation of bean flowers are illustrated in Fig. 1.
C. Preliminary Studies
Instead of arbitrarily selecting hormones and concen
trations for treatments or using the chemicals reported to
be effective, it was thought better to find out through
several trials which chemicals and concentrations showed
some kind of stimulation without causing any undesirable
effect. It was therefore necessary to make numerous pre
liminary trials which consisted of treatment of several
hundred flowers both in the field and in the greenhouse.
As the method of sealing the pollinated flowers with
latex suspension is very effective and convenient, the first

29
attempt was to see If the chemicals could be effectively
used with latex. Stock solutions of the following hormones
were made in latex at 2000 ppm.
1. Indole-propionic acid
2. Beta-naphthoxyacetic acid
3. 2,4-Dichlorophenoxyacetic acid
4. Potassium salt of alpha-naphthaleneacetlc acid
5. Alpha-naphthaleneacetamide
6. 8Q6 Phalinil (A commercial preparation of
n-phenyl phthalimide.)
7. Alpha-naphthaleneacetic acid
8. Methyl ester of alpha-naphthaleneacetic acid
The following two hormones were later included in the trials.
9. Indole-butyric acid
10.Para-chlorophenoxyacetic acid
Several flowers of snap beans (P. vulgaris) and lima
beans (P. lunatus) were emasculated and sealed with the
hormone mixture in latex as usual without pollination. A
little of the mixture was also applied at the base of the
ovary. The object was to study the effect and kind of
response of the flower to the hormone without the stimulation
supplied by the pollen as is the case in pollinated flowers.
Various dilutions were made from the stock solution to
arrive at a concentration where a response could be induced
without any toxicity. The 200 ppm. mixtures of Nos. 2, 3,

30
4, and 5 gave responses in the majority of flowers treated.
The flowers treated with these lasted for more than four
days while the others as well as the controls (treated with
latex hut without hormone) dropped within that time. Al
though no definite growth stimulation or swelling was noticed,
the pedunele did appear to he straight and stiff. When the
trials were repeated, however, the responses were not con
sistent. Inasmuch as none of the hormones excepting Nos. 3
and 4 are soluble in water or the latex suspension, they
could hardly he expected to he effective consistently, be
cause the quantity applied in each ease was not definite and
uniform. Alcohol coagulated the latex; therefore, it could
not he used to dissolve the hormones before mixing with the
latex suspension.
Of all the hormones used only 2,4-D and potassium salt
of naphthaleneacetic acid are appreciably soluble in water.
To study the nature of the effect of these two, saturated
solutions of each were made in water. A number of emascu
lated flower buds of snap bean were sealed with latex and a
little of the water solution of each was applied by dipping
a needle in the solution and making a small scratch at the
base of the ovary with it. Controls were maintained. The
effect was spectacular. All treated flowers stayed long
after the controls dropped. Many parthenocarpic pods de
veloped, the 2,4-D showing greater effect. Abscission was

31
definitely delayed or in some cases completely prevented.
Since many of the hormones are soluble in glycerine,
those which showed response in latex mixture were dissolved
in glycerine to saturation. After emasculating the flowers
in the field, a very minute drop was applied on the peduncle
at the point of junction with the receptacle of the flower.
Controls were maintained. The treatment also was used with
flowers emasculated and pollinated with P. lathvroides
pollen. In all cases the treated flowers dropped, the
peduncle drying at the point of application. No response
due to the hormone was seen. However, it was observed that
glycerine itself was toxic and killed the tissues at the
point of application, thereby causing the flower to wither.
As 2,4-D showed greatest response in thickening of the
peduncle, as also reported by Hester and Marth (53) and
checking of abscission even of emasculated flowers, further
trials were made with 2,4-D latex and lanolin paste. One
percent solutions were used and snap bean flowers were
treated after emasculating and pollinating them as summa
rized in Table I.
The results definitely showed the effect of 2,4-D
treatment in delaying the abscission of flowers as is
evident from Table I. The results also show a definite
stimulating effect of pollination even though seed develop
ment failed. 2,4-D was thus the most effective and merited

TABLE I
Results of Treatment of the Flower Buds of jP*. vulgaris with 2,4-D
No* of flowers
emasculated
Growth-regulator
used
Medium
used
Pollination
No* of flowers
dropped within
1 to 4 4 to 9
days days
No. of
parthen-
ocarpic
pods de
veloped
10
2,4-D 1%
Lanolin
Not
pollinated
4
2
4
10
2,4-D 1%
Latex
Not
pollinated
10
-
-
10
Untreated
Control
Latex
Not
pollinated
10
-

10
2,4-D 1#
Lanolin
Pollinated
with P*
lathvroides
2
8
10
2,4-D 1%
Latex
n
if
2
5
3
10
Untreated
Control
-
n
if
10
-
-

33
further consideration in the matter of establishing definite
dosages which would be effective in preventing abscission
without producing an over-stimulation which would be detri
mental to seed development. It is not soluble in the common
vegetable fats which are fluid enough for ease in appli
cation, but it is soluble in Karo, a commercial preparation
of corn-syrup which appeared to be very convenient for
application. Instead of weighing out the chemical and using
different measured concentrations it was easier to make a
saturated solution with excess of 2,4-D in Karo which could
be separated out by centrifuging or filtration with the aid
of heat* This was used as a basis for various dilutions in
the viscous media of the Karo type.
The effective range of 2,4-D was found to be very
narrow during the course of further trials. Para-ehloro-
phenoxyacetic acid, which has been reported to be effective
with lima beans by Wester and Marth (53), was also included
in the trials hereafter* It was also saturated in Karo and
the excess separated out by centrifuging. The treatments
then tested were:
1. Saturated 2,4-D in Karo,
2. Vfo 2,4-D in lanolin,
3. Saturated para-chlorophenoxyacetic acid in Karo and
4. Control, (Karo only).
These were used with both snap bean and lima bean flowers

34
after emasculating and pollinating them with pollen from
various species. Saturated 2,4-D being toxic, the strengths
were gradually reduced to l/2 saturation, and then to l/4
saturation. The l/4 saturation appeared to be the safer
concentration to use. 2,4-D in lanolin was applied in a
scratch by a needle on the calyx at the base of the ovary.
2,4-D and para-chlorophenoxyacetic acid in Karo were applied
at the same place with and without a scratch. Ordinary
glass tubing with one end drawn out to a thin capillary tube
and with a piece of a rubber tubing attached to the other
end was used at this stage as applicator for the Karo mix
ture. It was helpful in releasing a minute drop of the
chemical in Karo with uniformity within certain limits.
The number and kind of crosses made and the number of
sets obtained are given in Table 2.
None of the numerous crosses between lima bean (a Ford-
hook selection) and a wild type of lima bean and also be
tween snap bean and P. lathvroides set any pods when 2,4-D
was used, either saturated in Karo or 1% in lanolin.
Several crosses between the two types of lima bean (P.
lunatus) with reduced strength of 2,4-D were also made.
These have been summarized in Table 3.
Although these preliminary trials did not show results
suggestive of a very favorable effect of the hormone appli
cation, they at least gave sufficient indication that 2,4-D

TABLE 2
Results of Attempted Crosses between P,. vulgaris and coccineus
Kind of cross
Treatment
No, of flowers
treated
No, of
pods set
P, vulgaris
1.
2,4-D l/2 saturation in
Karo with puncture
6
X
P. coccineus
2.
2,4-D l/2 saturation in
Karo without puncture
19
6
3,
2,4-D 1/4 saturation in
Karo without puncture
10
6
4*
2,4-D 0,5% in lanolin in
a scratch
35
16
5.
Para-chlorophenoxyacetic
acid saturated in Karo
19
9
6.
Control, Karo alone
35
12

TABLE 3
Result of Attempted Crosses between lunatus and a Wild Lima Bean
Kind of oross
Treatment No.
of crosses
made
No. of sets
obtained
Lima bean (P.
lunatus)
1.
2,4-D l/2 saturation in
Karo with puncture
10
-
X
2.
2,4-D l/2 saturation in
Karo without puncture
23
2
Wild lima
3.
2,4-D 1/4 saturation in
Karo without puncture
4
2
4.
2,4-D 0.5$ in lanolin in
a scratch
32
4
5.
Para-chlorophenoxyacetic acid
saturated in Karo
4
1
6.
Control, Karo alone
37
6

37
was showing response and was not too drastic at l/4 satu
ration* The precise quantities however were not controlled
in these trials and such control is undoubtedly an important
factor on which the effectiveness or inhibitory action de
pends. A need for a device for measuring a tiny drop with
accuracy and facility was apparent. Para-chlorophenoxyaeetie
acid saturated in Karo appeared to be too low in concen
tration. Even then, it did show a response in slight thick
ening of petiole and delay in abscission. It was evidently
safer to use while 2,4-D showed a narrow limit within which
the right effect could be expected.
1) Especially Designed Micrometer Pipette for Hpjrjpone
Application
For further studies with regard to the use of the
growth-regulators in hybridization of beans it became
necessary to develop a device for dispensing volumetrically
very minute quantities of these highly effective substances.
Standard micropipettes were found to be unsatisfactory for
handling viscous media and the necessity for frequent fill
ing because of their low capacities made their use cumber
some. They were also unable to dispense small enough
quantities with a sufficiently high degree of accuracy.
Accordingly a special pipette was devised to suit the
needs of the work. It was found that very minute droplets

38
of consistent size could be dispensed from this micrometer
pipette, which, although developed especially for this work,
may have a wider application in the field of microchemical
technique. It was found to be highly satisfactory and as
such may be described and illustrated here.
The pipette employs the micrometer principle and the
amount of fluid medium dispensed is in direct relation to
the displacement capacity of a calibrated screw which is
provided with a scale in order to make possible accurate
measurements of the amounts dispensed by each revolution or
fraction thereof. The micrometer pipette illustrated in
Pig, 3 was designed so that rotation of the arm (fixed to
the main screw) through one centimeter on the circular
scale would result in the delivery (from the end of the 27-
gauge hypodermic needle fragment) of a droplet of water at
4 C, weighing 0,97 mg., having the volumetric equivalent
to 0,0010 ml., with a constant correction factor of 3$ to
be added where extreme accuracy is required. Movement of
the arm through 1 mm. on the circular scale will result in
a droplet barely visible to the naked eye, having a volume
of 0.0001 ml. less 3$, for which no correction was made
for practical reasons in dealing with such small quantities.
The accessory screw activates a worm gear mechanism of fine
adjustment which makes possible a slow rotation speed with
more positive control. The accessory screw is easily re
moved by counterclockwise rotation when rapid turning of

39
Fig. 2. Longitudinal Section of the Micrometer Pipette.
(Detailed explanation given in text.)

40
Pig. 3. Photograph of the Micrometer Pipette with
a few Refinements.

41
the main screw is desired. All parts except the hypodermic
needle and the screws are fabricated from polysterene
plastic resin. In the growth regulator studies it was not
necessary to use quantities smaller than 0.0002 ml., which
were large enough for the practical purposes of this work.
2) Trials on Phaseolus coccineus (Scarlet-runner bean)
A few plants of Scarlet-runner bean (P*. coccineus)
were growing vigorously in the greenhouse and flowered pro
fusely and continuously during fall and spring. All flowers,
however, abscissed without producing any pods. Evidently
there was some incompatibility resulting from some peculiarity
of the environmental condition within the greenhouse because
the same material growing outside in a frost-protected
location was capable of pod development. It was an excell
ent plant material for trials and tests to study the nature
of the effect of 2,4-D, para-chlorophenoxyacetic acid and
also other hormone mixtures. The pollen was definitely
viable because sets were obtained easily on P*. vulgaris with
P. coccineus pollen.
In order to study the effect of l/4 saturation of 2,4-D
in Karo and saturated para-chlorophenoxyacetic acid in Kero,
and simultaneously to see if the self-incompatibility of P.
coccineus could be overcome with hormone treatment, several
flower buds were treated with the hormone and sealed. Also

42
a number of crosses with pollen of various species, with
and without the hormone treatment, were made.
Out of the 10 buds treated with 2,4-D at l/4 saturation,
6 developed pods which were later on found to be partheno-
carpic. Those treated with para-chlorophenoxyacetic acid
did not develop pods, but the flowers did not absciss for
about another 3-4 days beyond the controls. Slight
straightening and thickening of the peduncle was also
noticed in many cases. Incompatibility of P^ coccineus
with many other speeies was also present, as the crosses
with many species, as listed below in Table 4, set no pods.
TABLE 4
Attempted Inter-specific Crosses with P^ coccineus as
Pistillate Parent
No. of crosses attempted with completely
Name of species negative results
used as pollen
Para-chloro-
2,4-D, l/4 satu- phenoxyacetic Control
ration in Karo acid saturated Un-
in Karo treated
latftyroldes
13
13
13
£*. liUfgrgAS
6
6
6
P,- vulgaris
(Cherokee Wax)
7
7
7
P* atronurnureus
5
5
5
P. lunatus
(Fordhook selection)
3
3
3

43
Two pods developed with vulgaris (Cherokee Wax) pollen
from para-chlorophenoxyacetic acid treatment. One pod with
seed also developed from an untreated hud pollinated with
P. vulgaris pollen hut this was very near another flower
treated with hormone and might possibly have obtained some
effect from translocation. All other treated flowers stayed
from 3 days to an indefinite period after all other controls
dropped. Study of the causes and exact nature of incom
patibility in Pj¡. coecineus could have been further studied
and presented a problem in itself. But as the hormone treat
ment did not seem to show very encouraging results in over
coming incompatibility, further investigations were not pur
sued.
3) Place of Application of Hormone
Trials were also made to see if the place where the
hormone was applied made any difference in the effect pro
duced. Two places l) on the lower part of the calyx,
and 2) at the point of union of the peduncle and the
receptacle were tested. The effect was first studied on
the £*. coecineus flowers which were crossed with P^ vulgaris
pollen and were treated with 2,4-D, l/4 saturation in Karo,
para-chlorophenoxyacetic acid saturated in Karo and with
Karo alone. The hormones were applied at each of the two
places in sets of 10 with each treatment. Effects similar

44
to those already reported were observed and abscission was
delayed In both cases. This showed that applications at
both places were equally effective.
Having observed the effectiveness to be the same
whether the hormone was applied on the abscission zone or
on the calyx, with Pg. coccineus flowers, the method had to
be further tested with snap bean flowers using the usual
pollination technique. A small preliminary trial using 66
flowers with snap bean as pistillate parent and P*. coccineus
as staminate was made. Two places of application as mention
ed before were tried. The treatments and number of polli
nations made and sets obtained are given in Table 5. The
hormones were applied without making any puncture.
These trials showed that application of a hormone drop
on the abscission zone was as good on the peduncle as on
the ovary and probably was safer as less inhibitory effect
on the ovule development could be expected. The best point
of application was decided to be the point where the
receptacle joins the peduncle. Besides giving information
regarding the place of application this trial also suggested
the possibility that the hormone treatment may prove useful
in obtaining a greater number of sets when the abscission
rate is high. During the course of these trials the
environmental conditions seemed ideal for pod setting and a
very low percentage of abscission was manifest with untreat
ed flowers.

TABLE 5
_ t
Effect of the Place of Hormone Application on Pod-set from Crosses P^ vulgaris and
P. coccineus
Treatment
On calyx
On abscission zone
Buds No. of
treated P*
se|s
Buds
treated
No. of
P*
sets
S*
2,4-D 1/4 saturation in Karo
11 1
3
11
2
4
Para-ehlorophenoxyacetic
acid saturated in Karo
11 1
5
11
1
3
Control, Karo alone
11
3
11
1
5
*
P: parthenocarpic
*
S: seed-containing

46
4) Trials with Hormone in Glycerine-Karo Mixture
The slow-drying property of glycerine, as well as the
greater solubility of certain hormones in it, makes it a
desirable reagent if possible to use. It was found that 1
part of glycerine to 4 parts of syrup provided a slow-drying
medium in which the glycerine was not sufficiently concen
trated to be toxic. This mixture was also of a desirable
consistency and viscosity for ease of handling. It was
decided to test once again with glycerine and Karo other
hormones which could easily be dissolved in glycerine.
Saturated solutions of all hormones tested before were again
made in warm glycerine and the glycerine so saturated was
mixed with 4 times of its volume of Karo.
A number of buds of P,. coccineus were treated with
hormone mixtures by applying a tiny drop on the peduncle at
the abscission zone. An equal number of controls was main
tained. Para-chlorophenoxyacetic acid, methyl ester of
naphthaleneacetic acid, beta-naphthoxyacetic acid and indole-
propionic acid showed response by delaying abscission by
several days and causing slight thickening of the peduncle.
A mixture of para-chlorophenoxyacetic acid and indole-
butyrie acid was also found to be effective. It was decided
to use the mixture of all these hormones in glycerine-Karo
mixture also containing 2,4-D. On the basis of these trials
tiie following treatments were chosen for further final
analysis.

47
1. PCPA: Para-chlorophenoxyacetic acid saturated in warm
glycerine and the mixture diluted with four
times its volume of Karo (corn syrup)
2. PCPA & IBA: Para-chlorophenoxyacetic acid and indole-
butyric acid saturated in warm glycerine and
the mixture diluted with four times its volume
of Karo.
3. H.M.: Hormone-mixture made by saturating indole-
propionic acid, beta-naphthoxyacetic acid,
para-chlorophenoxyacetic acid and methyl ester
of naphthaleneacetic acid each in 2 cc. of
glycerine and then mixing this with 4 times
its volume of l/8 saturation of 2,4-D in Karo.
Only a small drop of the methyl ester was
sufficient to give a turbid emulsion in
glycerine.
4. KNA: 0.5% solution of potassium salt of naphthalene-
acetic acid in glycerine-Karo mixture (1:4).
5. Control: A mixture of glycerine and Karo (1:4) with
out any hormone.
In all further experiments the above mentioned treat
ments were used. In all crosses attempted the amount of
the chemical used was within the range of 0.0004 0.0006
ml. of the mixture applied with the pipette made for this
purpose. These treatments were then tested with P^ vulgaris

48
as pistillate parent with pollen from a number of species.
The number of crosses made with different species and the
number of sets obtained are given in Table 6. Parthenocarpic
pods more than 1 inch in length only have been recorded.
These results did not give a precise information with
respect to individual treatments but definitely showed that
hormone treatment produced some kind of effect without being
toxic because 46.43$ of the attempted pollinations with hor
mone application produced either parthenocarpic or seed con
taining pods in contrast with 5.8$ for the control. These
results may be regarded as highly significant whether the
resulting seed represents valid crosses or not, because the
same technique and conditions obtained for the flowers
treated as for the control. The glycerine-Karo mixture,
instead of Karo alone, increased the ease of application
and preciseness of the size of the drop because of the low
er viscosity. The amount used, however, was not as care
fully controlled as in the later experiments made.
D. Final Experiments
The preliminary experiments gave enough information
required for the final experiments which were conducted
during spring and summer of 1950. The hormone treatments
used were: 1) PCPA, 2) PCPA & IBA, 3) H.M., 4) KNA,
and 5) Control. The methods of their preparation have al-

49
TABLE 6
Results of Attempted Crosses on P*. vulgaris with
Hormone Treatments
Kind of cross
Treatment
No. of
Buds
treated
No. of sets
obtained
Partheno-
Seed con-
carpic
taining
p*. ratorJjg
PCPA
2
,
_
X
POPA & IBA
2
1
-
P. coecineus
H.H.
2
-
1
KNA
2
-
-
Control
2
-
-
ZM.lfiaris
PCPA
12
5
2
X
PCPA & IBA
12
4
-
P. atroournureus
H.M.
7
5
1
KNA
11
2
2
Control
12
1
-
P. vulgaris
PCPA
2
2
-
X
PCPA & IBA
-
-
-
Et ealcaratus
H.M.
-
-
-
KNA
2
1
-
Control
2
-
m

50
ready been given. The different species of Phaseolus and
their sources, used in this study were as follows:
A. Phaseolus vulgaris (Cherokee Wax).
B. Phaseolus coccineus (Scarlet-runner) P. I. 165421.
Collected by Ware and Manning in Mexico.
C. Phaseolus coccineus var albus from 0. H. Parson
Hs
(Bartel "bush lima").
D. Phaseolus atropurnureus M557 from 0. W. Horwell,
E. Phaseolus filiformis 51-2 op from 0. W. Morvell.
F. Phaseolus calcaratus (Korean rice-bean, long day
form), from E. M. Meader, New Hampshire.
G. Phaseolus lathvroides M 248-1 op from 0. W. Morvell.
II. Phaseolus lunatus A Fordhook lima selection.
I. Phaseolus acutifolius 81 op 1 op from 0. W. Morvell.
J. Phaseolus acutifolius (white seeded) from Rex Thomas
Beltsville, U. S. Department of Agriculture.
K. Phaseolus acutifolius 257 from 0. W. Morvell.
Ii. Phaseolus angularis P. I. 174907.
M. Phaseolus angularis (Adzuki) from Rex Thomas, Belts
ville.
N. Phaseolus mungo from E. C. Stair.
O. Phaseolus aureus from E. C. Stair.
In all crosses attempted the amount of hormone prepa
ration used was within a range of 0.0004 0.0006 ml.,
applied with the special pipette. The chemicals were

51
applied at the point of union of the peduncle and the
receptacle without making any scratch. In all 1272 snap
bean flower buds were emasculated and treated with hormones
after pollinating them with pollen from different species.
410 buds were allowed to be self-pollinated and then were
treated with hormone mixtures or used as controls. As re
ported by workers in the past (53, 54), for any reliable
inferences, a large number of buds should be treated. The
percentage of sets being so low, and the variability being
so high, this evidently is imperative. The time required
for emasculating and treating individual flowers and the
limitation set by availability of uniform sized buds re
stricted the planning of the entire experiment on a
statistical basis. The variability in such work is large
and less under the control of the experimenter. Size, age,
position of the buds on the plant, location, age and stage
of growth of the plant, time of emasculation and pollination,
all may be expected to affect the sets. A proper design for
statistical analysis was, however, used with some inter
specific crosses.
Experiment 1. Interspecific Crosses
In the experiment designed for statistical analysis
three species were used as pollen parents: P*. coccineus.
E*. fillformis and atropurpureus. The number of flowers

52
pollinated and treated with each were 250,250 and 200
respectively. Bach replication consisted of 10 huds with
each treatment, i.e*, a total of 50 buds. The treatments
were replicated 5 times with the first two while with the
third there were only 4 replications. Unlike field experi
ments in which soil heterogeneity is the main factor causing
variability, the replications were made with respect to time
as well as position in the greenhouse bench. The first
formed buds may be different from those formed in the middle
or latter part of the plants life. Therefore, a 4-day
period was considered to be the block during which all buds
(50) under each replication were treated. The complete
experiment with 5 replications each with P*. coccineus and
P. filiformis was completed in 40 days. With atropurpureus.
due to the shortage of pollen material and irregularity in
buds appearing, each replication consisted of 8 days. While
treating and pollinating buds, care was taken to select buds
from similar positions on the plant and of uniform vigor
and size. Five buds, one with each material, were treated
simultaneously*
Besides the three species mentioned above, a number of
other species of Phaseolus were also used as pollen parents
on snap bean. As it was not possible to repeat similar
experiments with each species, as many flowers were polli
nated with each as was possible within limitations of time,
space and availability of staminate and pistillate material.

53
With some species the treatment KNA vas dropped as it did
not appear to be promising. The names of various species
used as pollen parents besides the three mentioned above,
along with the number of crosses attempted with each, are
given in Table 7.
TABLE 7
Table Showing the Pollen Material Used and the Number of
Crosses Attempted with each on 3P*. vulgaris (Cherokee Vax)
as Pistillate Parent.
Pollen parent Treatments and number of attempted
crosses
PCPA
PCPA&IBA
H.M.
KNA
Control
Total
Ll ££utlfolius-I
13
13
13
13
13
65
Is. .aptttifolius-J
16
16
16
16
16
80
P, aeutifolius-E
11
11
11
11
11
55
Ei. angularis
-L
10
10
10
-
10
40
P. mungo
-N
4
4
4
-
4
16
P. calcaratus
-F
20
20
20
20
20
100
P. lathvroides-G
20
20
20
-
20
80
P. lunatus
-H
15
15
15
-
15
60
£*. angularis
-M
4
4
4
-
4
16
P^. aureus
-0
10
10
10
-
10
40
E* ooccineus
-C
5
5
5
-
5
20
Total
128
128
128
60
128
572

54
Experiment 2. Effeet on Fertilisation and Ovule Development
There is evidence in the literature that hormone treat
ment inhibits growth of ovules and their fertilisation.
Whether this tendency can be overcon by regulating the
amount and using proper chemicals was important to investi
gate. An experiment with snap bean, the parent used for
all crosses attempted in this study, was conducted in which
the buds were allowed to be self-fertilised. Ten buds of
uniform sise were subjected to each of the treatments, and
each treatment was replicated 4 times for statistical
analysis. The amount and method of application were the
same as with the species crosses. Hie results of this
experiment are reported in Tables 14 and 15. Hie entire
experiment consisted of treatment of 200 buds.
Experiment 3. Effective Range of 2.4-P Concentration
Although the preliminary studies showed that 2,4-D,
which caused quick responses, was likely to be too drastic
in effect, it was decided to work out the effective ranges
with respect to both amount applied in each application
and strength of the concentration used. With the help of
the special pipette it was possible to control the sise of
the drop with a higher degree of accuracy than is obtained

55
by any other known technique of the same facility In use.
The aim was to find out whether small amounts of 2,4-D
solutions in high concentration were as effective as larger
amounts of 2,4-D solutions in lower concentration where
equivalent actual amounts of 2,4-D were concerned.
Pour different strengths of 2,4-D in the glycerine-
Karo mixture were used as follows:
1, 2,4-D, 3/4 saturation in a glycerine-Karo mixture (1:4)
Amounts used: 0,0002 ml,, 0,0003 ml,, 0,0004 ml.,
0,0006 ml,, and 0,0008 ml,
2, 2,4-D, l/2 saturation in glycerine-Karo mixture (1:4)
Amounts used: 0,0002 ml,, 0,0004 ml., 0.0006 ml.,
0,0008 ml., and 0,0010 ml,
3, 2,4-D, l/4 saturation in glycerine-Karo mixture (1:4)
Amounts used: 0,0002 ml., 0,0004 ml,, 0.0006 ml.,
0,0008 ml., and 0,0010 ml,
4, 2,4-D, 1/4 saturation in glycerine-Karo mixture (1:4)
Amounts used: 0,0004 ml., 0.0006 ml., 0.0008 ml.,
0,0010 ml,, and 0,0012 ml.
There were thus 4 different concentrations and 5
different amounts used as treatments. Each time, with
these treatments, a control was maintained which consisted
of treatment with the glycerine-Karo mixture alone. In
all, 10 flower buds were treated with each and with the
control and were allowed to be self-pollinated; the total

56
number of buds treated thus was 210. Observations with re
gard to response were regularly made and the number of buds
which dried out from time to time was recorded. The data
regarding this experiment are reported in Table 16.
Experiment 4. Germination Tests
Hormone treatment has been known to inhibit develop
ment of embryo. It was therefore necessary to find out if
the seed-set with these treatments produced viable seeds
or not, and whether the seeds subjected to hormone treatment
during early development were affected with regard to their
germination capacity. Germination tests were, therefore,
run with the seed obtained from the crosses between P.
vulffafis (A) and P. coccineus (B) with different hormone
treatments and control. 20 seeds were selected at random
out of eaeh lot and germinated in sterilized soil in flats.
Also similar tests with seeds obtained as a result of
selfing snap bean (P. vulgaris) flowers with various treat
ments were conducted. The data regarding these tests have
been reported in Tables 17, 18, and 19.
Experiment 5. Effect of Hormone Treatment on the Rate of
Development of Pods
L-
To ascertain if the stimulation provided by the growth-

57
regulator mixtures on treatment of buds causes any effect on
the rate of development of pods, a subsidiary experiment was
run using vulgaris material. A number of buds of uniform
sise were allowed to be self-pollinated and treated with PCPA
and 2,4-D (l/4 saturation) and also with control. At each
application 0.0006 ml. of PCPA and 0.0004 ml. of 2,4-D mix
ture were used. Measurements of 4 pods from each treatment
were taken daily for a period of 8 days, and the experiment
was replicated 3 times. Usually the pods reach their maximum
size in about 8 to 10 days after they have started develop
ing. The aim was to compare the rate of development of pods
with and without hormone treatment to determine if there was
any initial or continued effect of hormones on the develop
ing pods. The data regarding this study are recorded in
Table 20.

58
RESULTS
Experiment 1. Interspecific Crosses
It has been pointed out that the planning of the experi
ment with crosses between vulgaris (Cherokee Wax) as
pistillate parent and various other species of Phaseolus as
staminate parents was done on a statistical basis only with
three species, viz., P^ coccineus. P. atropurpureus and P.
filiformis. The number of sets obtained with P^ atropurpureus
as staminate parent was exceedingly low, making any statisti
cal interpretation impractical, and therefore the results
with this species are grouped with those of others. The
results of crosses with P*. coccineus and P^ filiformis have,
however, been statistically analysed.
The results of attempted crosses with P^ coccineus (B)
as staminate parent on P*. vulgaris (A) as pistillate are
given in Tables 8 and 9. PCPA and PCPA & IBA treatments
gave highly significant increases in pod-set and PCPA & IBA
gave a similar increase in the number of seeds obtained as
compared with control. Without the hormone treatment 38$
of the pollinated flowers set pods containing seeds, while
treatments PCPA and PCPA & IBA gave pod-sets of 52$ and 64$
respectively. As is clear from the table 8, the average
number of pods obtained per 10 flower buds treated was 5.2

59
and 6.4 with PCPA and PCPA & IBA respectively as against
3.8 with control. The least significant difference was
calculated as 1.21,
increase in set.
so that
both
treatments
gave
significant
TABUS
8
Results of Attempted Crosses of P
(B) Using Various
. vulaaris
Treatments
X

P. coccineus
Replication
Number
of pods containing seeds obtained from
treating 10 flower buds.
PCPA
PC PM IBA
H.M.
KNA
Control
I
6
7
5
4
2
II
5
6
6
4
6
III
4
6
3
3
3
IV
5
7
5
4
5
V
6
6
5
3
3
Total
26
32
24
18
19
Average
5.2
6.4
4.8
3.6
3.8
% Pod-set
52
64
48
36
38
L.S.D.


1.21
(Analysis of variance given in Appendix Table 23)

60
Since the total number of seeds obtained as a result
of a cross is important rather than the number of pods set,
the data regarding the number of seeds obtained with various
treatments are given in Table 9.
TABLE 9
Number of Seeds Obtained as a Result of Attempted Crosses
between P. vulgaris and Pt eoceineus (B)
Using Various Treatments.
Replications
Seeds
obtained per 10
flowers
treated
PCPA
PCPA&IBA
H.M.
KNA
Control
I
16
17
15
15
7
II
16
24
16
13
20
III
9
20
10
5
9
IV
15
28
17
13
15
V
16
17
13
8
8
Total
72
106
71
54
59
Average
14.4
21.2
14.2
10.8
11.6
Average number
of seeds per
pod
2.77
3.31
2.96
3.0
3.1
L.S.D.


4.07
(Analysis of variance given in Appendix Table 24)
The data presented in Table 9 show that the treatment

61
with PCPA & IBA gave a significant increase in the total
number of seeds obtained from crosses between vulgaris
and Pj. coccineus. The average number of seeds per 10
flowers pollinated and treated with this hormone mixture
was found to be 21,2 as compared with 11.8 for the control.
The L.S.D. was calculated to be 4.07. PCPA, which gave
significantly higher pod-set than control, did not give an
average number of seeds exceeding the number obtained with
control by the L.S.D., 4.07. The difference, which is 2.6,
however, may not be regarded as definitely insignificant,
and an experiment designed with greater number of flowers
used in each replication might show a significant difference
between seeds obtained with treatment PCPA and with the
control.
The data, however, show that treatment with PCPA and
PCPA & IBA gives a higher percentage of pod-set in crosses
between P^ vulgaris and P,. coccineus (B) than the control.
The number of seeds obtained is significantly increased by
the treatment PCPA & IBA, by as much as 79# over the control.
Hormone treatment in the amounts used does not seem to affect
significantly the number of seeds per pod as is evident from
Table 9.
Out of the 50 attempted crosses between Pu vulgaris and
Pjl filiformis (E) with each treatment, only one pod with seed
was obtained with PCPA and 3 pods with seeds with KNA. The
pod-set with these treatments was thus 2# and 6% respectively.

62
as against no pod-set with control and other remaining treat
ments. The effect of hormone treatment was, however, sig
nificant inasmuch as a large number of parthenocarpic pods
was obtained with PCPA, PGPA & IBA, and H.M. The data, in
which the number of parthenocarpic and seed containing pods
have been combined, are reported in Table 10. Parthenocarpic
pods above one inch in length only have been included.
The table shows that the average number of pod-set
(parthenocarpic and seed-containing) was 3.2, 4.4 and 4.2
respectively with PCPA, PCPA & IBA and H.H., as against 1.0
with control. Pod-set with these treatments was, therefore,
significantly higher than that without hormone treatment.
The total number of parthenocarpic pods and those con
taining seeds from crosses between P*. vulgaris and P,
filiformis (E), along with the average length of pods with
various treatments, have been summarized in Table 11.
As is evident from this table, the percentage of sets
with seeds is very low being 2$ with PCPA and 6% with KNA.
Such a low pod-set may be considered to be Inadequate to
draw conclusions regarding the effectiveness of the hormone
treatments in aiding fruitful pod production. However, the
fact that 3 sets have been obtained with KNA and none with
the control cannot be ignored and considered as only
accidental. Further study of these sets by planting the
seeds and studying the plant characteristics will, however.

63
TABLE 10
Results of the Crosses P^ vulgaris X fillformis (E)
with Different Treatments. (The numbers represent the
number of parthenocarpic and seed-containing pods obtain-
ed out of 10
flowers
pollinated and
treated in
each case.)
Replication
Pods set out of
10 flowers
treated
PCPA
PCPA&IBA
H.M.
KNA
Control
I
1
1
1
-
1
II
5
6
4
1
2
III
3
5
5
1
-
IV
5
5
8
5
1
V
2
5
3
2
1
Total
16
22
21
9
5
Average per
10 buds treated 3.2
4.4
4.2
1.8
1
L.S.D. :
1.79
(Analysis of variance given in Appendix Table 25)

TABLE 11
Total Number of Pods Set from Attempted Crosses between vulgaris and
P, flliformis (E) with Various Treatments, and the Average Length of
Pods Obtained,
Treatment
No, of flowers
treated
No, of pods
with seeds set
No, of parth-
enocarpic
pods-set
Average
length of
pods in
cm.
PCPA
50
1
15
5.7
PCPA&IBA
50
-
22
6.1
H.M.
50
-
21
6.7
KNA
50
3
6
6.1
Control
50
5
5,7

65
be necessary to establish the validity of the crosses. The
effect of hormone treatments is very evident in giving an
increased number of parthenocarpic sets with PCPA, PCPA&IBA
and H.M. Hie slightly greater average length of pods follow
ing treatment with H.M. suggests that this mixture has a
greater stimulation to growth than any other treatment.
Other Interspecific Crosses
The results of the attempted crosses between P*. vulgaris
(Cherokee Wax) as pistillate parent and different species of
Phaseolus and their varieties as staminate parents are re
ported in Tables 12 and 13* The number of pods containing
seeds and parthenocarpic pods (more than one inch in length)
have been separately recorded and also the average lengths
of pods set with different treatments have been given.
Table 12 clearly shows that hormone treatment aids in
obtaining increased set of pods containing seeds, at least
with calcara tus (F) With this species KNA and PCPA&IBA
again gave increased pod-set of 20$ as against 5$ with the
control. H.M. and PCPA also gave an increased number of pods
containing seeds, the percentage of set being 15$ and 10$
respectively. In order to get more precise information,
several hundred flowers should be treated. The hormone
treatments, except KNA, also gave a higher number of

66
parthenocarpic pods of comparatively greater average length
than the control Pods containing seeds have also been
obtained with hormone treatments from crosses with P.
atropuronreus (D), acutifolius (J) and P^ acutifolius (I)
as staminate parent on P*. vulgaris (A) as pistillate, but
the percentages were so low that no definite or conclusive
statements regarding the effectiveness of hormones in these
cases can be made.
Considering the overall effects of treatments on attempt
ed crosses with five different species and varieties grouped
in Table 12, it is seen that out of a total of 110 flowers
pollinated and treated, PCPA&IBA, H.M. and KNA gave a higher
number of pods containing seeds than did the control. Also
hormone treatments in general stimulated a greater pod-set,
whether the pods were seed-containing or parthenocarpic.
The total number of pods obtained out of 110 attempts in
each case was 47 with PCPA&IBA, 36 with PCPA, 32 with H.M.,
27 with KNA and 18 with control.
The results of attempted crosses between P^ vulgaris
(A) and 7 other species of Phaseolus and their varieties,
with treatment KNA omitted, are summarized in Table 13.
This table shows that successful crossing is possible only
with two species, P^ lathvroides (G), and P*. coccineus (C).
Out of 20 flowers pollinated with P,. lathyroides pollen, 2
pods containing seeds were obtained with H.M. treatment and

67
one each with PCPA and the control. The total number of
pods set (parthenocarpic and those containing seeds) was
greatest with H.M., being 8, followed by PCPA with 7, while
PCPA&IBA and the control each had 5. coccineus (C) also
seems to cross successfully with P^ vulgaris. since one pod
containing seed was obtained when treatment H.M. was used
on five flowers.
The overall effects of hormone treatment with these
species in Table 13 are not as significant as those in
Table 12, partly because of insufficient data due to the
extremely low percentage of sets. But all hormone treat
ments have given a higher pod-set than the control. In
general, the average length of pods with hormone treatments
appears to be increased when compared with the control. On
the basis of all the inter-specific crosses attempted and
reported in the foregoing tables, the fact seems apparent
that the effectiveness of different treatments varies with
different species. With P*. coccineus (B) PCPA and PCPA&IBA
appear to result in increased pod-set. With P*. filiformis
(E) as staminate parent PCPA&IBA and H.M. gave a greater
number of total pods set but more seed-containing pods
were obtained with SNA, which did not give a large number
of parthenocarpic sets. A similar set of seed-containing
pods was obtained with calcaratus (P) when KNA was used,
But a greater number of parthenocarpic and seed-containing
pods was obtained with treatments PCPA&IBA and H.M. Con-

TABLE 12
Results of Attempted Crosses between P. vulgaris (A) and Various Other Species.
Number of Sets and Average Length of Pods (in cmf) Obtained with Various Treatments,
PCPA
PCPA &
IBA
H,M.
KNA
Control
No. of
No, of
Av.
No.of
Av,
No.of Av,
No.of
Av,
No.of Av,
Kind of cross flowers
Sets
1.
Sets
1.
Sets 1.
Sets
1.
Sets 1,
treated
P S
of
pod
P S
of
pod
of
P S pod
P S
of
pod
of
P S pod
£ vulgaris (A)
X
£ calcaratus (F)
20
6
2
7.4
11
4
7.3
9
3
7.0
4
4
7.9
5
1
5.8
£ na&a.rig (A)
X 50
£ SLteBflrPMrgMff
8
*
5.4
11
-
5.7
8
2
6.8
3
-
4,1
2
1
6*7
£ vulgaris (A)
X
P. acutifolius (K)
11
4
-
6.3
1
-
5.1
1
-
3.5
1
3.7
3
-
4.7
£. yuJLftarjs (A)
X
£ aoutltg.llus (J)
16
10
mm
4.37
10
-
5.22
3
mm
5.1
6
1
5.0
4
I
3,2
£ yigarjLp (a)
X
£. aouttfollus (I)
13
6
mm
6.9
10
-
5.5
5
1
8.0
8
-
5,0
2
-
4,5
Total
110
34
2
-
43
4
-
26
6
mm
22
5
-
16
2
mm
*P Parthenooarpic
*S Seed-containing

TABLE 13
Results of Attempted Crosses Between P, vulgaris (A) and Various Other Species.
Number of Pods set and Average Length of Pods (in cm.) Obtained with Various
Treatments,
1,1
pcPK
rMmzm
ts-c
UfP
... Control
No, of
No, of
Av,
No.
of
Av,
No.
,of
Av,
No,
Of
Av,
Kind of cross
flowers
pods
1,
pods
1*
pods
1,
pods
1,
trea ted
Set £
of
set
of
set
of
set
Of
nod
P
s
pod
, Em
S
pod
F
s
PQd
p. xaUagU (a) X
P. lathyroides (6)
20
6 1
5,6
5
-
6.5
6
2
7,9
4
1
6,4
£ mUftKlg U)
P. aureus 10)
X
10
6 -
5.0
3
a
6.1
2
-
5,0
3

4,8
£* IHifiSEiS. £ Aaaa.tus (h)
X
15
4 -
4,4
8
-
6,5
7
mm
5,7
4
-
3,9
p. xaiMEia (a)
P. punco (N)
X
4
1 -
5.2
2
1
7.6
mm
-
-
1
mm
5,7
£. mUaE.§ (a)
£* aasate*8
X
10
1 -
4.1
1
-
4.0
1
mm
4,2
*
-
p* xaU&gl8 (a)
£ coc^ne^s (C)
X
5
1 -
8.5
2
Ml
3.5
N
1
12,6
1
-
4,6
g* matear (a)
P. angularis (M)
X
4
* Ml
-
1
-
7.1
-
-

1
5,2
Total
68
19 1
22
1
16
3
14
1
* P Parthenocarpic
* S Seed-containing

70
sidering other speeies, PCPA gave a larger number of
parthenoearpie sets when P. acutifollus (K), P. acutifolius
(J), £* lathvroides (G) and P. aureus (0) were used as
staminate parents PCPA & IBA gave increased sets with P.
calcaratus (P), P. atropurpureus (D), £. acutifolius (J),
P. acutifolius (I) and P. lunatus (H). An appreciably high
percentage of parthenoearpie pod-set was obtained with H.M.
as compared with the control when the staminate parents
used were P. ealcaratus (F), P. atropurpureus (D), P.
acutifolius (I), £. lathvroides (G) and J. lunatus (H).
Experiment 2. Effect on Fertilization and Ovule Development
The results of an experiment in which 200 flower buds
of £ vnlsarig (A) were allowed to be self-pollinated and
were subjected to various treatments are reported in Tables
14 and 15. It is clear from Table 14 that treatment with
PCPA gave a significantly greater number of pods as compared
with the control. The differences between the number of
pods obtained with other treatments and control are not
significant statistically. The average number of pods
obtained with PCPA treatment per 10 flowers treated was
7.25, as against 5.25 with the control. The difference
needed for significance at the 5% level has been calculated
as 1*12. The difference between the pod-set obtained with

71
PCPA&IBA and that with control, although not more than the
L.S.D., is very nearly equal to it, Sinoe this treatment
has PCPA as its active agent and has also given an increas
ed set of pods and seeds with the interspecific erosses be
tween P. vulgaris and P. coccineus. it should not be regard
ed as merely accidental,
Table 15 shows that the treatment PCPA also gave a
significantly higher number of seeds as compared with
control. The average number of seeds obtained per 10
flowers treated in the experiment was 29.75 with PCPA and
17.50 with the control. The difference is much higher than
the 7.16 needed for significance at the 5$ level. PCPA&IBA
also seems to have a favorable effect on seed- and pod-set
as compared to the control, although the difference is not
quite significant when compared with the L.S.D. This
experiment thus definitely showed that the hormone treat
ment, in the amounts used, has no inhibitory effect on pod
or seed development. On the contrary PCPA treatment of
flower buds very significantly increased set of both pods
and seeds. From Table 15 it is also noticed that hormone
treatment did not decrease the number of seeds per pod.
There was, in fact, a small increase in the number of seeds
per pod with PCPA and also with PCPA&IBA and KNA. The
difference, however, is not so significant as in the numbers
of pod and seed set. Unless shown to be statistically
significant it cannot be considered to be a definite ad-

72
vantage* Among the various treatments used only H*M, gave
a slightly smaller number of seeds per pod than the control.
Since all other treatments have given at least some increase
in the number of seeds per pod, it can very definitely be
inferred that at least the hormones other than H.M. in the
concentrations used in this study do not inhibit fertili
zation and seed development.
TABLE 14
Number of Seed-containing Pods Set from 10 Flowers Allowed
to be Self-pollinated and Subjected to Various Hormone
Treatments.
Replication
No.
of pods set out
of 10
flowers treated
PCPA
PCPA&IBA
H.M.
SNA
Control
Total
I
5
6
4
5
4
24
11
7
5
5
4
5
26
III
9
8
6
7
7
37
IV
8
6
4
6
5
29
Total
29
25
19
22
21
116
Average
7.25
6.25
4.75
5.5
5.25
L.S.D.
: 1.
,12
(Analysis of variance given in Appendix Table 26)

73
TABLE 15
Number of Seed Obtained from 10 Plovers of P. vulgaris (A)
Allowed to Be Self-pollinated and
Given Various Hormone Treatments.
Number of seeds obtained and
Replication the treatments used.
PCPA
PCPA&IBA
H.M.
KNA
Control
Total
I
19
21
14
25
16
95
II
30
15
10
10
11
76
III
37
36
21
26
28
148
IV
33
19
12
21
15
100
Total
119
91
57
82
70
419
Average
29.75
22.75
14.25
20.5
17.5
No. of seeds
per pod
4.1
3.64
3.16
3.73
3.33
L.S.B. :
7.16
(Analysis of variance given in Appendix Table 27)

74
Experiment 3. Effective Range of 2.4-D Concentration
Hie number of pods and seeds obtained with different
amounts of various 2,4-D concentrations in glycerine and
Karo mixture are reported in Table 16 and the data are pre
sented graphically in Pig 4. A study of the table
indicates that the concentration of 2,4-D in glycerine-Karo
mixture is comparatively more important than the amount of
the mixture used in each application With l/8 saturation
the results were quite comparable to those obtained in the
control, while higher concentration of 2,4-D decreased the
number of pods set Apparently these higher concentrations
are in some way toxic or adversely affect pod development.
On the basis of these data and considering the l/8 satu
ration of 2,4-D which seems safe to use, there is no
appreciable difference in effectiveness when different
amounts varying from 0.0004 ml. to 0.0010 ml, of the mix
ture are used in each application With higher concen
trations, however, there is some suggestion that the
toxicity is increased as the amount used is increased. The
fact that 0.0002 ml, of 2,4-D at 3/4 saturation did not give
a single set while 0.0003 ml. of the same gave 3 sets and
0.0004 ml. and 0.0006 ml. also gave 2 sets each, suggests
that there is a considerable amount of variability and
factors other than the hormone treatment also affect the
Percentage of pod-set. For more precise information, there-

75
fore, this experiment should he repeated and replleated 4
or 5 times to reduce the variability and bring out the
differences in the effects produced by hormone treatment
more precisely.
During the course of the experiment the daily obser
vations showed that flowers treated with 3/4 saturation of
2,4-D and also with amounts between 0.0004 and 0.0010 ral.
of l/2 saturation of 2,4-D in glycerine-Karo mixture, in
most eases, developed swelling and distortion of the pe
duncle and receptacle. This effect was more pronounced
when higher amounts of these concentrations were used. The
fact that 0.0008 ml. and 0.0012 ml. of l/8 saturation of
2,4-D gave 7 and 8 pods respectively while 0.0004 ml. and
0,0006 ml. of 1/4 saturation gave only 2 and 3 respective
ly, indicates that large amounts of a lower concentration
and equivalent smaller amounts of a higher concentration of
2,4-D are not similar in effectiveness. If the sets obtain
ed from flower: buds treated with various amounts of the
concentrations other than the l/s saturation are examined
in Table 16, it is observed that the amount of the hormone
mixture may be important in determining the effect on pod-
set. However, the concentration of 2,4-D in the medium
seems to be comparatively more important than the amount
of the mixture used in each application. Treatment of the
buds with l/8 saturation of this chemical in glycerine-Karo

76
TABLE 16
Effect of Different Concentrations and Amounts of 2,4-B Mixture on
Pod and Seed-set of P. vulearis Self-iJollinated.
-
Concentrations of 2.4-D in glyserine-Karo mixture
Amount
of the
Mixture
3/4 Saturation
1/2 Saturation
1/4 Satura
tion
l/8 Saturation
No 2,4-D Control
used
. . (
Flowers
Sets
Flowers
Sets
Flowers
S,.
Flowers
Set*
i .....
Flowers
Sets
treated
Pods Seeds
treated
Pods Seeds
treated
Pods
Seeds
treated
iPods
Seeds
treated
Pods Seeds
0.0000 ml*
-
-
wm
mm
-
-
-
-
-
-
-
-
10
7 30
0*0002 ml*
10
0
0
10
4
\
12
10
3
11
-
-
mm
-
-
0.0003 ml.
10
3
5
-
Wf '
-
-

-
mm
m
-
0.0004 ml*
10
2
7
10
2
8
10
2
10
10
5
22
0.0006 ml*
10
2
8
10
0
0
10
3
15
10
7
22
0.0008 ml*
10
1
2
10
2
10
10
2
11
10
7
26
0.0010 ml*
mm
am-
mm
10
2
6
10
1
2
10
6
18
0.0012 ml.
m,
mm
pa

*
10
8
24

NO. OF POD
77
Effect of Different Concentrations and Amounts of 2,4-D in
Glycerine-Karo Mixture on Pod-set of P. vulgaris (A).
(Data from Table 16)

78
mixture, which apparently does not seem to he toxic in effect
in any of the different amounts used in this experiment, gave
no increased pod-set over the control. This may possibly be
due to the fact that the conditions during the course of this
experiment were particularly suitable for pod-set and ab
scission was at a minimum, which is suggested by the high
percentage of pod-set, as much as 70$, without the use of
hormone treatment.
Experiment 4, Germination Tests
The results of germination tests and observations of
growing seedlings have indicated that the hormone treatment
of flowers after pollination does not adversely affect the
germination percentage of seeds obtained from some crosses.
The results of tests with the seed obtained as a result of
crosses between P. vulgaris (A) and P. coccineus (b), which
are summarized in Table 17, show that, in this particular
case, the germination percentage is actually increased.
From Table 17 it is seen that the maximum number of
seedlings was secured in a period of 11 days after sowing.
Seeds obtained from flower buds treated with PCPA gave the
highest germination percentage which was 65, Hormones also
increased the germination percentage of seeds as compared
to the control, with which it was the lowest. These hybrid
seeds were found to be low in germination capacity as com-

79
TABLE 17
Germination of Seeds Resulting from Crosses between
E* iriUSgJjjjL **d E- (B) with
Various Hormone Treatments,
Ho, of germinated seeds and growing
seedlings out of 20 sown on 6/9/50,
Bate when
observed
POPA
PCPA&IBA
H.M.
KNA
Control
6/13/50
2
1
1
1
1
6/14/50
3
2
1
3
1
6/15/50
5
2
1
3
1
6/16/50
9
4
3
4
1
6/17/50
10
4
3
7
6
6/19/50
13
6
6
10
6
6/20/50
13
7
8
11
6
Germination
percentage
65
35
40
55
30
6/29/50
8
6
5
7
2

80
pared to those obtained as a result of selfing P. vulgaris
and also took longer time to emerge. Observations of the
growing seedlings showed that the seeds obtained from the
attempted crosses between P. vulgaris (A) and P. coccineus
(B) resulted from valid crosses, the elevation of the
cotyledons of the hybrid seedlings was, in general, inter
mediate between the two parental extremes but in no case
was it as high as in the P. vulgaris pistillate parent.
Hiere were many abnormalities noticed in the seedlings such
as the curvature of the stem, difficulty in the tip breaking
through the soil and the cotyledons, and upside down germi
nation of seeds with roots coming up. The seedlings were
weak and delicate and, as is evident from the observations
made on 6/29/50 in Table 17, many of them died early. The
growth of seedlings from seeds with hormone treatment of
flower buds before fertilisation was more accelerated in
the beginning than that of the control but this effect
gradually became less noticeable later on. Ibis test sug
gested that the seed obtained as a result of this cross
should be germinated in a soft medium such as vermiculite
which does not offer much resistance to the emerging tip.
The germination tests of the seed obtained as a result
of selfing P. vulgaris (A) and treating the flowers with
hormone mixtures, reported in Table 18, conclusively show
that hormone treatment of flowers does not have a dele-

81
terious effect on germination capacity of the resulting seeds
hut may possibly be responsible for weakness in the stem of
the seedlings* The germination percentage of seeds from
PCPA-treated flowers was found to be 95, the same as for the
control* The germination percentages of seed from flowers
treated with PCPA&IBA, H.M., and KNA were found to be 90, 90,
and 85 respectively* A very marked difference in the rate
of growth of seedlings from hormone treatments was observed
in the early stages of growth. Seeds obtained from flowers
treated with hormones gave seedlings which grew faster in
the beginning than did those from the control. This is
evident from Table 19 in which the average heights of
seedlings measured on the seventh and eighth day after sowing
are recorded. Figure 5, which is a photograph of the
seedlings growing in a flat taken on the ninth day after sow
ing, also clearly shows that the hormone treatment resulted
in Increased growth rate of seedlings in the initial stages,
which was more pronounced with PCPA and PCPA&IBA.
Experiment 5. Effect of Hormone Treatment on the Rate of
Development of Pods
As has been described earlier, daily measurements of
pod length were taken after treating the flowers with 2,4-D,
PCPA and control. Each day 4 pods were measured from each of

82
TABLE 18
Germination of Seeds from Flowers of P. vulgaris (A)
Self-pollinated and Given Various Hormone Treatments.
Date of
observation
No.
of seeds germinated out of 20
6/9/50
sown on
PCPA PCPA&IBA
H.M.
KNA
Control
6/13/50
12
10
15
14
15
6/15/50
18
17
18
17
19
6/15/50
19
17
18
18
19
6/16/50
19
18
18
17
19
Germination %
95
90
90
85
95
TABLE 19
Average Height (in inches) of Seedlings from Seeds of
P. vulcaris Selfed and Treated with Various Hormones.
Time when
observed
PCPA PCPA&IBA
H.M.
KNA Control
7 days
after sowing
3.98 3.58
2.56
2.55 2.1
8 days
after sowing
6.2 5.8
4.9
4.2 3.6

83
Fig. 5
Photograph of the Growing Seedlings Obtained from P. vulgaris
Flowers Self-pollinated and Treated with Various Hormones and
Control. (The photograph was taken on the ninth day after
sowing.)

84
three replications of each treatment. In the analysis of
the data used here it was not necessary to consider the data
separately in each replication, therefore an average of pod
length from three replications is considered to be the
average length of pods on different days. In Table 20, in
whieh the data regarding the pod lengths on days have been
recorded, the pod lengths, thus, are averages of 12 pods
from each treatment.
The relationship between pod length and days, as may
well be expected, is not a straight line relationship but
follows the pattern of a growth curve. Treating it as a
straight line relationship, the regression of pod lengths
on days has been calculated with each treatment and the
regression coefficients have been used to compare the rate
of development of pods resulting from the three treatments.
The regression of pod length on days and the regression
coefficients (b) with the three treatments are as follows:
2,4D
b !
1.3469
Y :
1.3469 X 2.0358
PCPA
b
1.1541
1.1541 X 1.5938
Control
b :
1.2579
1.2579 X 2.1869

85
TABLE 20
Average Pod-length (in caa .) on Different Days
Treatment 1.
2,4-D
Treatment 2.
PCPA
Treatment 3.
Control
Days
Length
Days
Length
Days
Length
X
Y
X
Y
X
Y
3
2.06
3
2.07
3
1.63
5
4.57
5
3.87
5
3.67
6
5.97
6
5.27
6
5.63
7
7.30
7
6.40
7
6.67
8
9.00
8
7.83
8
7.97
9
10.40
9
9.03
9
9.57
10
11.10
10
9.77
10
9.93
48
50.40
48
44.24
48
45.07

IN CM.
86
Pig. 6
Regressions of Pod-length on Days with 2,4-D, PCPA and Control.

87
The average pod lengths on days and the regression of
pod length on days have been plotted in Pig. 6. In order
to determine if the three regression coefficients of average
pod length on days are significantly different from each
other, the analysis of covariance is given in tables 21 and
22.
Based on the average of replications (Table 20), the
total sum of squares for the averages of pod length have
been divided into variation between treatment means and the
variation of averages within treatments. This latter sum
of squares has been divided into that which can be attribut
ed to the average regression of the pod length on days for
one degree of freedom and that which cannot be attributed
to the regression, or the errors of estimate for 17 degrees
of freedom* This sura of squares is the variation of aver
ages from the average regression after the variations of
treatments have been removed.
The variations of the averages from the specific re
gressions of pod length on days within each treatment also
have been computed each for 5 degrees of freedom.
Hie total error of the estimate for 15 degrees of
freedom is the estimate of the true variation of pod length
where the knowledge of the relationship of pod length and
days within each treatment has been employed.
The difference of the sura of squares of errors of
estimate for two degrees of freedom is an estimate of the
variation of the regression coefficient (Snedicor, 43).

TABLE 21
Analysis of Covariance for Data Presented in Table 20
Source
D.F.
s*2
Sxv
Sy2
Errors of Estimate
D.F. S.S. M.S.
Total
20
104.5714
131.0286
169.2784
19
5.0988
Treatments
2
0.0
0.0
3.1925
Average in
Treatments
18
104.5714
131.0286
166.0859
*7
1.9063
0,1121|
F-i = 15962 =14.239
1 1121
2
3.1925
1.5962)
F#0i 6.11
Treatment 1
6
34.8572
46.95
63.5494
5
0.3114
)
Treatment 2
6
34.8572
40.23
46.7006
5
0.2697
Treatment 3
6
34.8572
43.8486
55.8359
5
0.6766
15
2
1.2577
0.6486
0.0838)
0.3243j
F2* 3243 = 3,8699
A 838
P,05 3-68

TABLE 22
Analysis of Covariance Considering PCPA and Control
Source
B.F.
Sx2
Sxv
Sy2
Errors of Estimate
D* F. S.S. M.S.
Total
13
69,7143
84.0786
102.5857
Treatment
1
0.0
0,0
0.0492
Average in
Treatment
12
69.7143
84.0786
102.5365
11
1.1339
Treatment 2
6
34.8572
40.23
46.7006
5
0.2697
Treatment 3
6
34.8572
43.8486
55.8359
5
0.6766
10
1
0.9463 0.0946)
0.1876 0.1876)
F=lf2f: 1.98
F.05 4*96

90
Interpretation
The first P test in Table 21, which gives a high signifi
cance, shows that if the average pod lengths from different
treatments are adjusted to a common value of X, i.e. days,
there is a highly significant difference among the average
pod lengths with various treatments. The observed values of
pod length on a common value of X are, however, not signifi
cantly different among the three treatments. The simple
analysis of variance, which was run using the pod lengths
on 6th and 10th day with three replications showed that the
treatments had no significant effect on the observed values
of Y (pod length).
In the second P test in Table 21, in which the variance
due to the regression coefficients is compared with the error
variance, shows that there is a significant difference in the
regression coefficients and that the hormone treatment affects
the rate of development of pods significantly.
In order to determine which of the three regression co
efficients differ from each other significantly, a similar
analysis of covariance considering treatments PCPA and Control
is made in Table 22. Since a significant difference in the
three regression coefficients is indicated in Table 21, and
the widest difference between the regression coefficients is
seen in case of 2,4-D and PCPA, the two other treatments com
pared are PCPA and Control which have the next widest differ-

91
ence In their regression coefficients. The analysis in Table
22 shows that these two treatments, PCPA and Control, do not
differ significantly in their regression coefficients. It
is thus seen that 2,4-D increased the rate of development of
pods significantly over PCPA but there was no significant
difference between either 2,4-D and control or between PCPA
and control.

92
DISCUSSION
General
The results of the trials with various growth-regulat
ing chemicals in different amounts and concentrations used
in this study show that hormone treatment in right dosages
results in responses favorable for greater pod-set. It is,
however, evident that the use of hormones mainly stimulates
the ovary and other tissues chiefly responsible for pod
development and it is not very definite whether the effect
is extended to the developing ovules in a similarly favor
able manner* The results of various interspecific crosses
reported with P. vulgaris as pistillate parent show, in
in general, that with some speeles at least, a greater pod-
and seed-set can be obtained with the use of treatments
PCPA, PCPA&IBA and KNA. With many other species which have
not shown increased set of seed-containing pods, hormone
treatment has given a greater number of parthenocarpic pods
set. The greater number of pods obtained may be explained
as resulting from:
1. Reduced abscission rate,
2. Stimulation provided by the hormone supplementing any
that may have resulted from the pollen tubes to cause
development of the ovary and other tissues concerned
with pod development,

93
3 Stimulation provided by the hormone supplementing any
that may have resulted from the pollen tube in causing
greater ovule development.
During the course of the preliminary trials as well as
in the final experiments, the effective part the chemicals
play in checking abscission has been definitely observed.
Reduced yield of beans due to high temperature conditions
resulting in a high abscission rate has been reported to be
a problem by Fisher et al.(16) and Vittwer and Murneek (55)
The reason for obtaining increased yields of beans with
hormone treatment has been explained to be its property of
checking abscission. The findings of this study are in
agreement with the results of these workers. In hand poll!
nation with beans this tendency of the flowers to absciss
is increased even more, probably, due to handling of buds
and the unavoidable injury caused to the flower parts dur
ing emasculation. In Florida the weather conditions are
often favorable for high abscission rate due to high
temperatures and bright sunlight. The principal causes
of a low percentage of sets obtained from hand-pollinated
flowers thus can be said to be the high rate of abscission
of flowers before pod-set or following pod-set if adverse
conditions prevail later. Whether the abscission of the
style also is a cause of incompatibility and failure of
pod-set can not be said with certainty, but this appears
unlikely.

94
Lewis (26) has reported on the effectiveness of
naphthaleneacetamide treatment of Prturns avium buds which
resulted in prolonging the period before abscission of
style and flowers. He explained that the hormone treatment
checks abscission resulting from changes taking place in
the existing cells consisting of the accumulation of starch
below the abscission line resulting in starvation of the
tissues above. Exactly how the abscission of bean flowers
takes place was not determined in this study but it is
definite that hormone treatment prevents this to a con
siderable extent. It is quite possible that this is
achieved by preventing changes in cells of the type
described above.
Tan verbeek et al. (36) have reported that the
hormone released in the ovary by the presence of pollen
tubes extends its effect to the peduncle and checks
abscission of the flower. The transmission of this
hormone effect to the peduncle is probably interfered
with under unfavorable environmental conditions or in
case of incompatible species the pollen tube fails to
cause the desired activation of the inactive hormone
present in the ovary. This probably results in insufficient
stimulation to initiate the development of the ovary and
other tissues connected with pod development and also causes
abscission of the flower before fertilization is achieved.

95
An increased number of parthenocarpic sets obtained with
hormone treatments may be thus explained
Many parthenocarpic pods were obtained with treatment
H.M with various species used as pollen parents during the
course of this study. Some of these were almost similar in
size to normal seed-containing pods but had no seeds at all.
Without hormone treatment and with the stimulation from the
pollen tube alone, both the number and lengths of partheno
carpic pods were comparatively much smaller. The reason
for the development of fewer and smaller parthenocarpic
pods in crosses between supposedly incompatible species
without use of hormones may be attributed to the lack of
the stimulation necessary to initiate the development of
ovarian and other tissues and also to the lack of stimulation
provided by developing ovules as in normal pods. In an
attempted cross, if the egg cell and the sperm cell are not
incompatible the hormone application should be able to over
come any lack of stimulation normally provided by the hormone
naturally occurring in the ovary which is activated by the
pollen tube.
A few sets of seed-containing pods have been obtained
from P. calcaratus. P. lathvroides and P. filiformis pollen
with the use of hormone treatment. This shows that the
sperm cells of these and the egg cell of P. vulgaris are
probably not incompatible and the reason for failure to
obtain sets from attempted crosses between these may possibly

96
be due to improper growth of pollen tubes, the lack of
stimulation resulting from an insufficient number of them,
the degeneration of embryo sae before fertilization, or some
other causes not as yet studied. These causes are to some
degree controlled by the hormone treatment and seed-contain
ing pods are obtained.
With many other species whieh have not given any seed-
containing pods and have given significantly large numbers
of parthenocarpic pods, it may be postulated that the cause
of incompatibility in such cases possibly is the incompati
bility between the sperm and egg cells themselves, which
cannot be overcome by hormone treatment. Hormone treatment
thus cannot substitute for or cause fertilization directly,
but it can certainly help in providing the necessary con
ditions for it and can also supplement any inadequate natural
stimulation for initial development of pods.
It seems definite that with the help of PCPA and PCPA &
IBA a greater percentage of pod-set with seed can be obtained
from attempted crosses between P. vulgaris and P. coccineus
than without this treatment. The data on these crosses and
on other attempted crosses reported in Tables 12 and 13 sug
gest that ordinarily the species which cross without the use
of hormone treatment give increased set of pods and seeds if
treated with PCPA or PCPA&IBA in the amounts used in this
study. Wittwer and Hurneek (55) have also reported consistent
yield increases of snap beans with para-chlorophenoxyacetic

97
acid, Howiett (22) obtained increased fruit-set of
tomatoes from weakly viable pollen with the help of indole-
butyric acid. Wester and Marth (53) have also obtained an
increased pod-set of hand-pollinated lima beans with a mix
ture of para-chlorophenoxyacetic acid and indole-butyric
acid. These hormones have thus repeatedly shown favorable
responses in obtaining higher pod-set. It would certainly
have been interesting to know the effect of indole-butyric
acid separately as well, since it has shown such favorable
response in conjunction with PCPA.
Effect on Seed-set
Hormone treatment which gave a significant increase
in the number of pods and seeds set from crosses between
P. vulgaris and P. coccineus (Tables 8 and 9) did not,
however, increase the number of seeds per pod appreciably.
A small increase over the control was however obtained with
PCPA&IBA (3.3 as compared to 3.1). In case of self-polli
nated P. vulgaris flowers, those treated with PCPA gave an
increased pod-set as well as a higher number of seeds per
pod. PCPA&IBA also gave an appreciable increase in pod-set
(though not statistically significant) and also a small
increase in number of seeds per pod (Table 15). The effect
of hormones on the number of seeds per pod has not been
statistically studied, and so no conclusive statement can be

98
made. However, it can be said that the hormone treatments in
the dosage used did not cause any inhibition of fertilization
and ovule development. There are many instances reported in
literature when hormones caused reduction of the number of
seeds although they increased fruit-set and its development.
Van Overbeek has mentioned such an effect in his review of
literature on hormones (37). Swarbrick (45), who reported an
increase in yield of strawberries with naphthoxyacetic acid,
also has stated that the fruits obtained os a result of
hormone treatment, although larger in size, had a lower seed
content. Wester and Marth (53), however, obtained an
appreciable increase in the number of seeds per pod in lima
beans with para-ehlorophenoxyacetic acid and indole-butyric
acid treatment. The slight increase in number of seeds per
pod from the same treatment in this study, therefore, is
in agreement with Wester and Marth. With P. vulgaris
(Cherokee Wax) flowers self-pollinated and treated with H.M.,
the number of seeds per pod was found to be less than the
control. The difference may possibly be due to chance only,
but it is also possible that this mixture caused a small
inhibition of fertilization or ovule development. Of all
the four hormone treatments used in the final experiments,
H.M. definitely appeared to be strongest in effect.
Since this hormone mixture has been made by using a number
of different chemicals saturated in glycerine and since it
also contains 2,4-D, a stronger effect on the tissues is not

99
unlikely. This might have resulted in a slight depressing
effect on fertilization of egg and development of fertilized
ovules.
In the experiment to study the effective range of 2,4-D
on self-pollinated flowers of P. vulgaris. a few pods were
obtained with higher concentrations of 2,4-D (3/4 saturation
and l/2 saturation) in which seeds had not developed. In
some the number of seeds obtained was much smaller. Even if
hormones inhibit the fertilization of ovules and development
of seeds, the results of these experiments support the view
that this depressing effect can be overcome by regulating the
amount and concentrations of the chemicals used. A fairly
precise mechanism to control the amount of the chemical
applied is therefore imperative for obtaining the desired
effects
Literature on the effect of hormone treatment on germi
nation of seeds has already been reviewed. Direct seed
treatment with chemicals, or treatment of soil, can be
expected to affect the germination of seeds and growth of
seedlings. The treatment of flowers with a very small
quantity of very dilute mixture as used in this study cannot
be expected to cause any deleterious effect on the germination
capacity of seeds obtained. It could be argued that any per
sistence of effect of the chemicals in the seed to the extent
that it can cause undesirable effects on germination and
growth of seedling would in the first place be severe enough

100
to prevent fertilization or ovule development. Since hormone
treatment has given Increased set of pods and seeds in some
cases, it would normally he expected that the seeds so
obtained are not adversely affected by the treatment.
Germination tests conducted with hybrid seeds reported
in Table 17 and those with seeds obtained as a result of
selfing P. vulgaris reported in Tables 18 and 19 are in
keeping with this view. Furthermore, hormone treatment has
shown a favorable effect on the germination percentage and
vigor of seedlings in the early stages of growth in case of
seeds obtained as result of cross between P. vulgaris and
P. coocineus. In case of the seeds obtained as result of
selfing of P. vulgaris. also, this increase in vigor in the
early stages has been noticed although no increase in the
germination percentage has been observed. This increase in
vigor does not seem to be of any special advantage as the
difference seems to be overcome after about 10 to 15 days.
Seedlings exhibiting accelerated development and apparent
increased vigor in the early stages of growth due to hormone
treatment, however, seem to be more delicate and weak in
stem, which calls for greater care during these early stages.
All the workers, who have so far made studies with re
gard to the use of chemicals as an aid in hand-pollination
of flowers, have stressed the need of using many hundred
flowers in order to arrive at any definite conclusions. In
the statistically planned experiments in this study 10

101
flowers were treated in each replication with various hormone
mixtures and the control. Vith species which cross easily
this number may be considered to be the minimum needed, but
in case of species with which the percentage of pod-set is
very low many times this many flowers should be treated in
each replication.
Medium and Method of Hormone Application
The medium and method of hormone application is an
important factor on which the successful use of these
chemicals depends. The medium and method should not only
be effective in producing desirable results but should also
be convenient* Unlike the use of hormones on a large
number of plants in a wide area, treatment of individual
flowers in hybridization work calls for a very precise
control of amount and concentration of the chemical used.
Very minute amounts of l/4 saturation of 2,4-D have been
found to be enough to produce an effect which tends to be
come toxic* Lanolin has been the most common medium used
by most workers so far, and has proved to be effective*
However only a limited control of the amount used in each
application can be achieved with this medium.
Vaster and Marth (53), Whittaker and Prior (54) and
Emsweller and Stuart (14) have used lanolin paste with
various chemicals for treatment of individual flowers, but

102
the exact amount of the emulsion used in each case vas not
accurately measured. Linder et al. (28) have, however,
adopted a technique to use precisely measured quantities of
radio-active 2,4-dlehloro-5-iodo-phenoxyacetic acid and its
derivatives on leaves of Leans. The method consisted of
stamping a ring of lanolin on the upper surface of the leaf
on the midrib enclosing 0.5 cm2, area and then applying
0.01 ml. of the mixture of hormone in alcohol and Tween 20
as carrier with the help of a 0,1 ml. pipette. The chemical
thus was not only measured in amount but was also restricted
to a specific area on the leaf. This technique may be
i
satisfactory for treating relatively small numbers of leaves
in experiments of this kind, but certainly is very cumber
some to use as a standard method in hybridization work,
where numerous flowers have to be treated. Also the small
est amount that can be applied with a 0.1 ml. pipette is
much larger than the amounts used in this study and found
effective.
Glycerine employed as a solvent for the ehemicals
used in this study and Karo as a viscous medium have been
found to be very effective and have many advantages.
Saturated solutions of various growth-regulating substances
in 1 part glycerine and 4 parts Karo provide concentrations
which are strong enough to cause the desired effects. Also
the glycerine in such dilutions does not cause any withering
of flower parts as observed when it is used undiluted by Karo.

103
Both media are slow-drying and this property is of special
advantage in better absorption of the chemicals over a
longer period* The viscosity and fluid consistency of the
glycerine-Karo mixture make it possible to control the
volumetric measurements with considerable precision with
the special pipette designed for this purpose. The aqueous
solubility of both the components of the medium may also
facilitate the transfer of the dissolved hormone into the
cell sap*
Translocation of 2,4-D in plants has been reported
to parallel carbohydrate movement (27, 31, 40). It has
been reported that the movement of 2,4-D is far less from
leaves low in carbohydrates at the time of application than
from leaves high in carbohydrates* How far the syrup as a
medium indirectly affects translocation of the chemical will
depend upon whether any minute amounts of the soluble syrup
are also absorbed or not. But it is quite possible that
minute amounts of the Karo (soluble carbohydrate) thus
absorbed with the chemical might favor its translocation
and thus increase the effectiveness of the hormone.
During the course of these experiments care was taken
to select buds for various treatments as distant as
possible from each other. They were generally from differ
ent branches of the plant to avoid any confounding of re
sults due to translocation effects* Batjer and Thompson
(7) have, however, shown that when the spurs of apple were

104
sprayed with naphthalenescetic acid, little or no effect of
the hormone was translocated from treated to untreated spurs.
However, Veintraub et al. (49) have demonstrated that not
only the growth-regulatory activity of 2,4-2) is observed on
the other parts of the plants when the chemical is applied
to the primary leaves of the bean seedlings, but also that
the effect so translocated is due to the actural trans
location of the 2,4-B itself rather than auxin-a, auxin-b
or indole-acetic acid or any other organic compound
activated by the 2,4-B. It should be pointed out that 2,4-D,
the most active of all the hormones tried was not used at all
in most of these experiments. Since PCPA, found to be
effective in this study, is related to 2,4-D, but is less
active, a similar effect is less likely. Caution is, however,
necessary in the matter of using buds sufficiently distant
from each other, and if possible on different plants, to
avoid any confounding of the results.
Mention may be made here of the interesting results
obtained and reported by Hamner and Kiang (21) regarding
the use of the "plastic material" to increase the effect
iveness of 2,4-D on bean plants. These workers found an
appreciable increase in the effects produced by 2,4-D used
as a spray when the treated branches were sprayed with 5%
Geon 31X latex. Similar results were obtained when the
chemical was mixed with the latex and a single drop of the
solution was applied at the base of one of the primary leaves.

105
Hi authors have not given any satisfactory explanation of
this effect produced hy latex but have suggested that
possibly the plastic material, due to its low moisture-
vapor transmission coefficient, increases the action of
2,4-D by sealing in its vapors. Since the same plastic
material has been used for sealing the emasculated and
pollinated buds in this study, consideration of this
property of the latex should be made* However, since the
plastic material has been used in this work only for seal
ing the eorolla and the hormone mixture drop has only been
applied on the abscission zone, any sealing affect as sug
gested by the above mentioned workers is out of the question*
In case there is some other way, not yet known, in which the
latex increases the effectiveness of the hormone, then, when
it is not necessary to seal the corolla with latex, the
dosage found effective in this study might prove to be in
adequate for the desired results.
With regard to the method of application, it seems
quite definite that the hormone need not be applied in a
scratch as has been done by most of the workers (22, 14, 53)
who have used a lanolin base* Desirable effects are shown
by the organs needing stimulation when correct amounts of
the hormone in glycerine-Karo mixture are applied on the
abscission zone without making any scratch, and even over
stimulation (conspicuous distortion of the developing pod
which eventually withers) may result from excessive dosages.

106
This may be considered to be an advantage over the lanolin
paste method with which a scratch has been recommended. It
has already been shown that the place of application of the
hormone drop, whether at the point of union of the receptacle
to the peduncle or on the calyx, makes no difference in the
effects obtained. Hie former was considered to be safer and
better place to use because checking of abscission appeared
to be the primary objective and application at this point
was considered to be helpful in checking a direct and
drastic effect, if any, on fertilization and ovule develop
ment. Since glycerine has been included in the mixture at
20$ concentration, consideration should be given to the
possibility of its withering effect on the peduncle as was
observed in the preliminary trials when no Karo was used.
Although 20$ glycerine does not seem to be injurious, it
might be safer to apply it on the calyx where killing of a
few cells, if done, will not cause as much damage to the
flower as the killing of a similar number of cells on the
peduncle would because of its thinness and small number of
cells involved. Destruction of comparatively few cells of
the peduncle can easily result in the withering of the
flower.
The pipette designed for this study has proved to be
very suitable and it should be possible to repeat the
technique used with consistent results. No such device to
dispense such minute quantities of this fluid as are possible

107
vith this pipette has previously been described. It is not
only useful for work of this nature but may prove to be
suitable for use in micro-chemistry where minute measure
ments such as this are also necessary.
With regard to the growth-regulating substances used
in this study and found satisfactory in the glycerine-Karo
medium, the effective concentrations have not been designated
in parts per million because it was determined early that,
except for 2,4-D, maximum concentrations attainable were
desirable since the whole plant was not subjected to treat
ment and only extremely small localized areas of application
were involved. It was found desirable rather to regulate
the dosage by regulating the actual volumetrlcally measured
amounts than to determine relative concentrations in terms
of parts per million. The concentration of chemicals
expressed as percentage saturation in glycerine should be
adequate inasmuch as the mixtures of this strength can be
prepared with consistent accuracy.

108
CONCLUSION
Hormone treatment of flower bud, after emasculation
and pollination results in increased pod-set without
inhibiting seed development from certain attempted inter
specific crosses. In case of the species in which the
egg cell and the sperm are incompatible, hormone treatment
stimulates the ovary and checks abscission which results in
a higher percentage of parthenocarpic pods. The seeds
obtained from the hormone treated flowers are not adversely
affected in germination capacity. PCPA and PCPA&IBA, in
the concentrations and dosages used, appear to give the
desired responses without producing any undesirable effects
with most of the possible interspecific crosses. With some
species KNA 0.5$, gives a higher number of fruitful sets.
Glycerine-Karo mixture (1:4) is a very suitable medium
for hormone application and is particularly convenient to
use with the micrometer pipette designed for the purpose.
Both the amount and the concentration of the chemicals in
the medium are important in obtaining the desired stimulus.
Desired effects are obtained when a minute drop varying from
0.0004 to 0.0006 ml. of the hormone mixture is applied on
the abscission zone without any scratch. In proper dosage,
with some species, the hormone treatment also increases the
number of seeds per pod.

109
The technique developed may also prove useful in
hybridization work with other crop plants in obtaining a
higher percentage of sets from attempted crosses.

110
SUMMARY
Incompatibility between certain species and a low per
centage of pod-set from attempted crosses are great handicaps
in bean hybridization. Some workers have reported successful
use of hormones in overcoming incompatibilities and in in
creasing fruit-set of beans, melons and lily.
An attempt was made to study the effectiveness of
hormones in reducing abscission of hand-pollinated bean
\
flowers and in increasing the set of pods with viable seeds
from attempted crosses between P. vulgaris (Cherokee Wax)
and various other species of Phaseolus.
Preliminary trials with several hundred flowers showed
that some of the chemicals were effective in producing
favorable responses such as checking abscission, increasing
pod-set, and production of parthenocarpic pods.
A mixture of glycerine and Karo in the proportion of 1:4
was found to be a very satisfactory medium for hormone
application. Many chemicals which are not appreciably
soluble in water, when saturated in glycerine and then
diluted with 4 times of its volume of Karo, produced desirable
effects without being toxic. These could be applied either
on the calyx or on the peduncle without making any scratch.
A 0.0004 to 0.0006 ml. size droplet of the mixture was the
amount used in each application. It could be easily dispensed
with the help of the micrometer pipette designed for the pur-

Ill
pose.
Treatment of flowers with para-chlorophenoxyacetic
acid and indole-butyric acid mixture in glycerine-Karo
significantly increased the percentage of sets from attempt
ed crosses between £. vulgaris and P. coccineus by 26# over
the control. A significant increase of 14# was obtained
with par-chlorophenoxyacetic acid. The mixture of the two
also resulted in an increase in seed-set from 14.2 with
control to 21.2 with the hormone treatment per 10 flowers
treated.
Pods containing seeds were obtained from attempted
crosses between P. vulgaris as pistillate parent and P.
calcaratus. P. filiformis. P. atropurnureus. P. acutifolius
and P. lathyroldes as staminate parents. It has not yet
been established by planting and observation whether these
are valid crosses. With some species 0.5# potassium salt
of naphthaleneacetic acid in glycerine-Karo mixture gave
increased set of seed-containing pods. With incompatible
species hormone treatment resulted in increased set of
parthenocarpic pods.
Ihese hormones, in the concentrations and amounts used,
did not inhibit fertilization and ovule development nor did
they affect adversely the germination capacity of resulting
seeds.
With 2,4-D it was found that the concentration of the
hormone in the medium was more important than the amount of

112
hormone mixture used in each application in producing an
adequate effect. l/8 saturation of the chemical in glycerine-
Karo mixture was found to he the safest concentration tried.
Hormone treatment with para-chlorophenoxyacetic acid or
2,4-D, in the dosages found to he effective, did not cause
a significant difference in the rate of development of pods
as compared to that with the untreated controls.
This study gave sufficient indication of an effective
use of growth-regulating substances as an aid in obtaining
increased set of pods from hand-pollinated flowers in beans.
The technique developed may prove useful for other
crop plants as well.

113
APPENDIX
TABLE 23
Analysis of Variance for Table 8
Source
D.F.
Sum of squares
Mean square
Total
24
46.56
Treatments
4
25.76
6.44 **
Replications
4
7.76
1.94
Error
16
13.04
0.815
L.S.D*
: 1.21
. F : 6.44/0.815
= 7.9
TABLE 24
Analysis of Variance for Table
9
Source
D.F. Sum
of squares
Mean square
Total
24
680.24
Treatments
4
329.84
82.46 **
Replications
4
201.84
50.46
Error
16
148.56
9.285
L.S.D. S 4.07 F : 82.46/9.285 = 8.88

114
TABLE 25
Analysis of Variance for Table 10
Source
D.F.
Sum
of squares
Mean square
Total
24
115.84
Treatments
4
44.24
11.06 **
Replications
4
43.04
10.76
Error
16
28.56
1.785
L.S.D.
: 1.79
. P
: 11.06/1.785
= 6.19
TABLE 26
Analysis of Variance for Table
14
Source
D.F.
Sum of squares
Mean square
Total
19
41.20
Treatments
4
15.20
3.8 **
Replications
3
19.60
6.53
Error
12
6.40
0.533
L.S.D. :
1.1248
P i 3.8/0.533 s 7.129

115
TABLE 27
Analysis of Variance for Table 15
Source
D.F.
Sum of squares
Mean square
Total
19
1372.95
Treatments
4
550.70
137.675 **
Replications
3
562.95
187.65
Error
12
259.30
21.608
L.S.D.
7.1623
. F i 137.675/21.608 a 6.37

116
LITERATURE CITED
1. Addicott, Fredrick T. 1943. Pollen germination and
pollen tube growth as Influenced by pure
growth substances. Plant Physio. 18 (2): 270-
79.
2. Allen, T. C. and E. H. Fisher. 1943. Plant hormones
increase yield of wax beans. Wis. Agri. Exp.
Sta. Bull. 460:54-55.
3. Amlong, H. U. and G. Naundorf. 1939. Wuchsstoff und
Pflanzenertrag. Forschungsdienst 7:465-482.
4. Avery, George S. and Elizabeth Bindloss Johnson. 1947.
Hormones and horticulture. McGraw Hill Book
Company Inc. pg 8-9.
5. Ibid pg. 186-205.
6. Batjer, L. P. and A. H. Thompson. 1948. A comparison
of naphthaleneacetic acid and 2,4-D sprays for
controlling preharvest drop of Bartletts'
pears. Proc. Amer. Soc. Hort. Sci. 51:71-74.
7. Batjer, L. P. and A. H. Thompson. 1949. The trans
mission effect of naphthaleneacetic acid on
apple drop as determined by localized appli
cation. Proc. Amer. Soc. Hort. Sci. 51:
77-80.
8. Boysen Jenson, P. 1910. Ueber die Leitung des
phototropisehen Reizes in AvenaKeimpflanzen.
Ber. Deut. Botan. Ges. 28:118-120.
9. Boysen Jenson, P. 1911. La transmission de l*irritation
phototropique dans l'Avena. K. Danske Videnskab.
Selskab. Forh. No. 3:1-24.
10. Burrel, P. C. and T. W. Whitaker. 1940. The effect of
indole-acetic acid on fruit setting in musk-
melons. Proc. Amer. Soc. Hort. Sci. 37:829-830.
11. Clark, H. E., W. C. Edmundson and P. M. Lombard. 1941.
Seed setting in potatoes as affected by spraying
alpha-naphthaleneacetamide and by light. Amer.
Potato Jour. 18:273-279.

117
12. Clore, W. 6. 1948. The effect of alpha-naphthalene-
acetic acid on certain varieties of lima beans.
Proc. Amer. Soc. Hort. Sci. 51:475-478.
13. Crane, Julian C. 1949. The use of growth regulating
chemicals to induce parthenocarpic fruits in
the Calimyrna fig. Plant Physio. 24 (l):44-54.
14. Emsweller, S. L. and N. W. Stuart. 1948. Use of growth
regulating substances to overcome incompati
bilities in Lilium. Proc. Amer. Soc. Hort.
Sci. 514581-589.
15. Eyster, V. H. 1941. Hie introduction of fertility in
genetically self-sterile plants. Science 94:
144-145.
16. Fisher, E. H., A. J. Riker and T. C. Allen. 1946. Bud,
blossom and pod drop of canning string beans
reduced by plant-hormones. Phytopathology 36:
504-523.
17. Gardner, F. E., P. C. Marth and L. P. Batjer. 1940.
Spraying with plant growth substances to pre
vent apple fruit dropping. Science 90:208-
209.
18. Gardner, F. E., P. C. Marth and L. P. Batjer. 1940.
Spraying with plant growth substances for
control of preharvest drop of apples. Proc.
Amer. Soc. Hort. Sci. 37:415-428.
19. Gustafson, Felix G. 1940. Parthenocarpic and normal
fruits compared as to percentage of setting
and size. Botan. Gaz. 102:280-286.
20. Hamner, C. L., J. E. Moulton and H. B. Tukey. 1946.
Effect of treating soil and seeds with 2,4-
Dichlorophenoxyacetic acid on germination
and development of seedlings. Botan. Gaz.
107(3):352-361.
21. Hamner, C. L. and Kiang Chi-Kien. 1948. Use of a
plastic material to increase the action of the
sodium salt of 2,4-D. Science 107(2786):572-
573.
22. Howlett, F. S. 1940. Effect of indole-butyric acid
upon tomato fruit set and development. Proc.
Amer. Soc. Hort. Sci. 39:217-227.

118
23 Huang Tsung Chen. 1948. Chemical stimulation in pollen
germination and pollen tube growth. Botan.
Bull. Acad. Sinica. 2(4):282-290.
24. Kuehwein, Hans. 1948. Ueber Keimungsfordernde
substanzen in Pollen and Narben. Planta 35
(516):528-535.
25. LaRue, C. D. 1936. The effect of auxin on the abscission
of petioles. Proc. Nat. Acad. Sci. 22:254-259.
26. Lewis, D. 1946. Chemical control of fruit formation.
Jour. Pomo, and Hort. Sci. 22:175-183.
27. Linder, Paul J., James V. Brown and John V. Mitchell.
1949. Movement of externally applied phenoxy
compounds in bean plants in relation to
conditions favoring carbohydrate translocation.
Botan. Gaz. 110:628-632.
28. Linder, P. J., J. ¥. Mitehelland, J. ¥. ¥ood. 1950.
Effects of 2,4-Dichloro-5-iodo-phenoxyacetic
acid and its* derivatives as plant growth
regulators. Science 111:518-519.
29. Mitchell, A. E., ¥alter Toenjes and C. L. Hamner. 1948.
The use of dusts and concentrate sprays to
prevent preharvest drop of McIntosh apple.
Quart. Bull. Mich. Exp. Sta. 31(2):160-164.
30. Mitchell, John ¥. and Muriel R. ¥hitehead. 1942.
Effects of vaporous naphthoxyacetic acid on
development of tomato fruits with special
reference to their vitamin C contents. Botan.
Gaz. 104(2):362-365.
31. Mitchell, J. ¥. and J. ¥. Brown. 1946. Movement of
2,4-Bichlorophenoxyacetic acid stimulus and
its* relation to translocation of organic
food materials in plants. Botan. Gaz. 107:
393-407.
32. Muir, Robert M. 1942. Growth hormones as related to
the setting and development of fruit of
Nicotiana tabacum. Amer. Jour. Botan. 29:
716-720.
33. Mullison, ¥endell R. and Richard ¥. Hummer. 1949.
Some effects of the vapor of 2,4-D and
derivatives on various field erops and
vegetable seeds. Botan. Gaz. 111:77-85.

119
34. Murneek, A. E., S. H. Wittwer and B. D. Hemphill. 1944.
Hormone sprays on snap beans. Market Growers
Jour. 73:197, 215, 218, 221.
35. Nixon, R. W. and P. E. Gardner, 1939. Effects of certain
growth substances in inflorescences of dates.
Botan. Gaz. 100:868-871.
36. Overbeek, J. Van, M. E. Conklin and A. E. Blakeslee.
1941. Hormone controls growth of ovules.
Amer. Jour. Botan. 28:647-656.
37. Overbeek, J. Van. 1944. Growth regulating substances
in plants. Annual Review of Biochem. 13:
631-666.
38. Pomery, C. S. and V. V. Aldrich* 1943. Set of citrus
fruit in relation to application of certain
growth substances. Proc. Amer. Soc. Iiort.
Sci. 42:146-148.
39. Randhawa, G. S. and H. C. Thompson. 1949. Effect of
application of hormone on yield of tomatoes
grown in the green house. Proc. Amer. Soc.
Hort. Sci. 53:337-344.
40. Rice, E. L. 1948. Absorption and translocation of
ammonium 2,4-Dichlorophenoxyacetic acid by
bean plants. Botan. Gaz. 109:301-314.
41. Skoog, F., C. L. Schneider and P. Malan. 1942. Inter
actions of auxins in growth and inhibition.
Amer. Jour. Botan. 29:568-576.
42. Smith, Paul F. 1942. Studies of the growth of pollen
with respect to temperature, auxins, colchicine
and vitamin-B. Amer. Jour. Botan. 29:56-65.
43. Snedicor, George V. 1948. Statistical Methods. Fourth
edition, The Iowa State College Press, Ames,
Iowa, pages 318-331.
44. Stewart, V. M. S, and Ira J. Condit. 1949. The effect
of 2,4-D and other plant growth regulators on
the Calirayrna fig. Amer. Jour. Botan. 36(4):
332-335.
45. Swarbrick, T. 1943. Progress report on the use of
naphthoxyacetic acid to increase the fruit set
of strawberry variety Tardive de Leopold. Ann.
Report Agr. Hort. Res. Sta. Long Ashton
(Bristol) 1-32.

120
46. Swartley, J. E. and L. C. Chadwick. 1942. Effects of
synthetic growth substances on cuttings, seeds
and transplants. Ohio Agr. Exp. Sta. Bi
monthly Bull. 27(217):125-144.
47. Tukey, H. B., C. L. Hamner and Barbara Imhofe. 1945.
/ Histological changes in bindweed and sowthistle
following applications of 2,4-D in herbicidal
concentrations. Botan. Caz. 107(1):62-73.
48. Tullis, Edgar C. and Wm. C. Davis. 1950. Persistence
of 2,4-D in plant-tissues. Science 3:90.
49. Weintraub, R. L., J. B. Brown and J. N. Yeatman. 1950.
Recovery of growth regulator from plants treat
ed with 2,4-D. Science 3:493-494.
50. Went, F. W. 1928. Wuehstoff and Wachstum. Rec. trav.
botan, neerland. 25:1-116.
51. Went, F. W. 1939. Further analysis of pea test for
auxin. Bull. Torrey Botan. Club. 66:391-410.
52. Wester, R. E. and Paul C. Marth. 1947. Effect of some
growth regulators on yield of bush lima beans.
Proc. Amer. Soc. Hort. Sci. 49:315-319.
53. Wester, R. E. and Paul C. Marth. 1949. Some effects
of a growth regulator mixture in controlled
cross-pollination of lima bean. Proc. Amer.
Soc. Hort. Sci. 53:315-318.
54. Whitaker, T, W. and Dean E. Pryor. 1946. Effect of
plant growth regulators on set of fruits from
hand pollinated flowers in Cucumis melo.
Proc. Amer. Soc. Hort. Sci. 48:417-422.
55. Wittwer, S. H. and A. E. Murneek. 1947. Hormone sprays
and dusts for snap beans. Market Growers Jour.
76:8-36.
56. Yasuda, Sadawo. 1932. Physiological researches on the
fertility of Petunia violcea. Botan. Mag.
(Tokyo)46(548):519-517.
57. Zimmerman, P. W. and F. Wilcoxon. 1935. Several
growth substances which cause initiation of
roots and other processes in plants. Contrib.
Boyce Thompson Inst. 7:209-229.

121
58, Zimmerman, P. W. and A. E, Hitchcock. 1941. Formative
effects induced with h-naphthoxyacetic acid.
Contrih. Boyce Thompson Inst. 12:1-14.
59. Zimmerman, P. V. and A. E. Hitchcock. 1942.
Substituted phenoxy and benzoic acid growth
substances and the relation of structure to
physiological activity. Contrib. Boyce
Thompson Inst. 12:321-343.

122
BIOGRAPHICAL ITEMS
Rameshwer Prasad Jyotishl was "born In Raipur,
Madhyapradesh, India on July 1, 1920. He graduated from
the Municipal High School Saugor in 1936 and received his
B.Sc.(Agr) degree from the Nagpur University in 1941.
Prom June 1941 until May 1945 he worked as an Agri
cultural Assistant at the Agriculture College Experimental
Farm, Nagpur. In June 1945 he joined the staff of the
Agriculture College Nagpur as a lecturer in Agriculture.
In August 1948 he was sent to the United States for
advanced studies in Horticulture as a stipendiary depu-
tationist of the Government of Madhyapradesh. He prosecut
ed his studies towards a Ph.D. degree in Horticulture at
the University of Florida at Gainesville.

123
This dissertation was prepared under the direction of
the Chairman of the candidate*s Supervisory Committee and
has heen approved by all members of the committee. It was
submitted to the Graduate Council and was approved as a
partial fulfilment of the requirements for the degree of
Doctor of Philosophy.
Dean
SUPERVISORY COMMITTEE:



92
DISCUSSION
General
The results of the trials with various growth-regulat
ing chemicals in different amounts and concentrations used
in this study show that hormone treatment in right dosages
results in responses favorable for greater pod-set. It is,
however, evident that the use of hormones mainly stimulates
the ovary and other tissues chiefly responsible for pod
development and it is not very definite whether the effect
is extended to the developing ovules in a similarly favor
able manner* The results of various interspecific crosses
reported with P. vulgaris as pistillate parent show, in
in general, that with some speeles at least, a greater pod-
and seed-set can be obtained with the use of treatments
PCPA, PCPA&IBA and KNA. With many other species which have
not shown increased set of seed-containing pods, hormone
treatment has given a greater number of parthenocarpic pods
set. The greater number of pods obtained may be explained
as resulting from:
1. Reduced abscission rate,
2. Stimulation provided by the hormone supplementing any
that may have resulted from the pollen tubes to cause
development of the ovary and other tissues concerned
with pod development,


100
to prevent fertilization or ovule development. Since hormone
treatment has given Increased set of pods and seeds in some
cases, it would normally he expected that the seeds so
obtained are not adversely affected by the treatment.
Germination tests conducted with hybrid seeds reported
in Table 17 and those with seeds obtained as a result of
selfing P. vulgaris reported in Tables 18 and 19 are in
keeping with this view. Furthermore, hormone treatment has
shown a favorable effect on the germination percentage and
vigor of seedlings in the early stages of growth in case of
seeds obtained as result of cross between P. vulgaris and
P. coocineus. In case of the seeds obtained as result of
selfing of P. vulgaris. also, this increase in vigor in the
early stages has been noticed although no increase in the
germination percentage has been observed. This increase in
vigor does not seem to be of any special advantage as the
difference seems to be overcome after about 10 to 15 days.
Seedlings exhibiting accelerated development and apparent
increased vigor in the early stages of growth due to hormone
treatment, however, seem to be more delicate and weak in
stem, which calls for greater care during these early stages.
All the workers, who have so far made studies with re
gard to the use of chemicals as an aid in hand-pollination
of flowers, have stressed the need of using many hundred
flowers in order to arrive at any definite conclusions. In
the statistically planned experiments in this study 10


114
TABLE 25
Analysis of Variance for Table 10
Source
D.F.
Sum
of squares
Mean square
Total
24
115.84
Treatments
4
44.24
11.06 **
Replications
4
43.04
10.76
Error
16
28.56
1.785
L.S.D.
: 1.79
. P
: 11.06/1.785
= 6.19
TABLE 26
Analysis of Variance for Table
14
Source
D.F.
Sum of squares
Mean square
Total
19
41.20
Treatments
4
15.20
3.8 **
Replications
3
19.60
6.53
Error
12
6.40
0.533
L.S.D. :
1.1248
P i 3.8/0.533 s 7.129


53
With some species the treatment KNA vas dropped as it did
not appear to be promising. The names of various species
used as pollen parents besides the three mentioned above,
along with the number of crosses attempted with each, are
given in Table 7.
TABLE 7
Table Showing the Pollen Material Used and the Number of
Crosses Attempted with each on 3P*. vulgaris (Cherokee Vax)
as Pistillate Parent.
Pollen parent Treatments and number of attempted
crosses
PCPA
PCPA&IBA
H.M.
KNA
Control
Total
Ll ££utlfolius-I
13
13
13
13
13
65
Is. .aptttifolius-J
16
16
16
16
16
80
P, aeutifolius-E
11
11
11
11
11
55
Ei. angularis
-L
10
10
10
-
10
40
P. mungo
-N
4
4
4
-
4
16
P. calcaratus
-F
20
20
20
20
20
100
P. lathvroides-G
20
20
20
-
20
80
P. lunatus
-H
15
15
15
-
15
60
£*. angularis
-M
4
4
4
-
4
16
P^. aureus
-0
10
10
10
-
10
40
E* ooccineus
-C
5
5
5
-
5
20
Total
128
128
128
60
128
572


117
12. Clore, W. 6. 1948. The effect of alpha-naphthalene-
acetic acid on certain varieties of lima beans.
Proc. Amer. Soc. Hort. Sci. 51:475-478.
13. Crane, Julian C. 1949. The use of growth regulating
chemicals to induce parthenocarpic fruits in
the Calimyrna fig. Plant Physio. 24 (l):44-54.
14. Emsweller, S. L. and N. W. Stuart. 1948. Use of growth
regulating substances to overcome incompati
bilities in Lilium. Proc. Amer. Soc. Hort.
Sci. 514581-589.
15. Eyster, V. H. 1941. Hie introduction of fertility in
genetically self-sterile plants. Science 94:
144-145.
16. Fisher, E. H., A. J. Riker and T. C. Allen. 1946. Bud,
blossom and pod drop of canning string beans
reduced by plant-hormones. Phytopathology 36:
504-523.
17. Gardner, F. E., P. C. Marth and L. P. Batjer. 1940.
Spraying with plant growth substances to pre
vent apple fruit dropping. Science 90:208-
209.
18. Gardner, F. E., P. C. Marth and L. P. Batjer. 1940.
Spraying with plant growth substances for
control of preharvest drop of apples. Proc.
Amer. Soc. Hort. Sci. 37:415-428.
19. Gustafson, Felix G. 1940. Parthenocarpic and normal
fruits compared as to percentage of setting
and size. Botan. Gaz. 102:280-286.
20. Hamner, C. L., J. E. Moulton and H. B. Tukey. 1946.
Effect of treating soil and seeds with 2,4-
Dichlorophenoxyacetic acid on germination
and development of seedlings. Botan. Gaz.
107(3):352-361.
21. Hamner, C. L. and Kiang Chi-Kien. 1948. Use of a
plastic material to increase the action of the
sodium salt of 2,4-D. Science 107(2786):572-
573.
22. Howlett, F. S. 1940. Effect of indole-butyric acid
upon tomato fruit set and development. Proc.
Amer. Soc. Hort. Sci. 39:217-227.


113
APPENDIX
TABLE 23
Analysis of Variance for Table 8
Source
D.F.
Sum of squares
Mean square
Total
24
46.56
Treatments
4
25.76
6.44 **
Replications
4
7.76
1.94
Error
16
13.04
0.815
L.S.D*
: 1.21
. F : 6.44/0.815
= 7.9
TABLE 24
Analysis of Variance for Table
9
Source
D.F. Sum
of squares
Mean square
Total
24
680.24
Treatments
4
329.84
82.46 **
Replications
4
201.84
50.46
Error
16
148.56
9.285
L.S.D. S 4.07 F : 82.46/9.285 = 8.88


38
of consistent size could be dispensed from this micrometer
pipette, which, although developed especially for this work,
may have a wider application in the field of microchemical
technique. It was found to be highly satisfactory and as
such may be described and illustrated here.
The pipette employs the micrometer principle and the
amount of fluid medium dispensed is in direct relation to
the displacement capacity of a calibrated screw which is
provided with a scale in order to make possible accurate
measurements of the amounts dispensed by each revolution or
fraction thereof. The micrometer pipette illustrated in
Pig, 3 was designed so that rotation of the arm (fixed to
the main screw) through one centimeter on the circular
scale would result in the delivery (from the end of the 27-
gauge hypodermic needle fragment) of a droplet of water at
4 C, weighing 0,97 mg., having the volumetric equivalent
to 0,0010 ml., with a constant correction factor of 3$ to
be added where extreme accuracy is required. Movement of
the arm through 1 mm. on the circular scale will result in
a droplet barely visible to the naked eye, having a volume
of 0.0001 ml. less 3$, for which no correction was made
for practical reasons in dealing with such small quantities.
The accessory screw activates a worm gear mechanism of fine
adjustment which makes possible a slow rotation speed with
more positive control. The accessory screw is easily re
moved by counterclockwise rotation when rapid turning of


55
by any other known technique of the same facility In use.
The aim was to find out whether small amounts of 2,4-D
solutions in high concentration were as effective as larger
amounts of 2,4-D solutions in lower concentration where
equivalent actual amounts of 2,4-D were concerned.
Pour different strengths of 2,4-D in the glycerine-
Karo mixture were used as follows:
1, 2,4-D, 3/4 saturation in a glycerine-Karo mixture (1:4)
Amounts used: 0,0002 ml,, 0,0003 ml,, 0,0004 ml.,
0,0006 ml,, and 0,0008 ml,
2, 2,4-D, l/2 saturation in glycerine-Karo mixture (1:4)
Amounts used: 0,0002 ml,, 0,0004 ml., 0.0006 ml.,
0,0008 ml., and 0,0010 ml,
3, 2,4-D, l/4 saturation in glycerine-Karo mixture (1:4)
Amounts used: 0,0002 ml., 0,0004 ml,, 0.0006 ml.,
0,0008 ml., and 0,0010 ml,
4, 2,4-D, 1/4 saturation in glycerine-Karo mixture (1:4)
Amounts used: 0,0004 ml., 0.0006 ml., 0.0008 ml.,
0,0010 ml,, and 0,0012 ml.
There were thus 4 different concentrations and 5
different amounts used as treatments. Each time, with
these treatments, a control was maintained which consisted
of treatment with the glycerine-Karo mixture alone. In
all, 10 flower buds were treated with each and with the
control and were allowed to be self-pollinated; the total


95
An increased number of parthenocarpic sets obtained with
hormone treatments may be thus explained
Many parthenocarpic pods were obtained with treatment
H.M with various species used as pollen parents during the
course of this study. Some of these were almost similar in
size to normal seed-containing pods but had no seeds at all.
Without hormone treatment and with the stimulation from the
pollen tube alone, both the number and lengths of partheno
carpic pods were comparatively much smaller. The reason
for the development of fewer and smaller parthenocarpic
pods in crosses between supposedly incompatible species
without use of hormones may be attributed to the lack of
the stimulation necessary to initiate the development of
ovarian and other tissues and also to the lack of stimulation
provided by developing ovules as in normal pods. In an
attempted cross, if the egg cell and the sperm cell are not
incompatible the hormone application should be able to over
come any lack of stimulation normally provided by the hormone
naturally occurring in the ovary which is activated by the
pollen tube.
A few sets of seed-containing pods have been obtained
from P. calcaratus. P. lathvroides and P. filiformis pollen
with the use of hormone treatment. This shows that the
sperm cells of these and the egg cell of P. vulgaris are
probably not incompatible and the reason for failure to
obtain sets from attempted crosses between these may possibly


118
23 Huang Tsung Chen. 1948. Chemical stimulation in pollen
germination and pollen tube growth. Botan.
Bull. Acad. Sinica. 2(4):282-290.
24. Kuehwein, Hans. 1948. Ueber Keimungsfordernde
substanzen in Pollen and Narben. Planta 35
(516):528-535.
25. LaRue, C. D. 1936. The effect of auxin on the abscission
of petioles. Proc. Nat. Acad. Sci. 22:254-259.
26. Lewis, D. 1946. Chemical control of fruit formation.
Jour. Pomo, and Hort. Sci. 22:175-183.
27. Linder, Paul J., James V. Brown and John V. Mitchell.
1949. Movement of externally applied phenoxy
compounds in bean plants in relation to
conditions favoring carbohydrate translocation.
Botan. Gaz. 110:628-632.
28. Linder, P. J., J. ¥. Mitehelland, J. ¥. ¥ood. 1950.
Effects of 2,4-Dichloro-5-iodo-phenoxyacetic
acid and its* derivatives as plant growth
regulators. Science 111:518-519.
29. Mitchell, A. E., ¥alter Toenjes and C. L. Hamner. 1948.
The use of dusts and concentrate sprays to
prevent preharvest drop of McIntosh apple.
Quart. Bull. Mich. Exp. Sta. 31(2):160-164.
30. Mitchell, John ¥. and Muriel R. ¥hitehead. 1942.
Effects of vaporous naphthoxyacetic acid on
development of tomato fruits with special
reference to their vitamin C contents. Botan.
Gaz. 104(2):362-365.
31. Mitchell, J. ¥. and J. ¥. Brown. 1946. Movement of
2,4-Bichlorophenoxyacetic acid stimulus and
its* relation to translocation of organic
food materials in plants. Botan. Gaz. 107:
393-407.
32. Muir, Robert M. 1942. Growth hormones as related to
the setting and development of fruit of
Nicotiana tabacum. Amer. Jour. Botan. 29:
716-720.
33. Mullison, ¥endell R. and Richard ¥. Hummer. 1949.
Some effects of the vapor of 2,4-D and
derivatives on various field erops and
vegetable seeds. Botan. Gaz. 111:77-85.


37
was showing response and was not too drastic at l/4 satu
ration* The precise quantities however were not controlled
in these trials and such control is undoubtedly an important
factor on which the effectiveness or inhibitory action de
pends. A need for a device for measuring a tiny drop with
accuracy and facility was apparent. Para-chlorophenoxyaeetie
acid saturated in Karo appeared to be too low in concen
tration. Even then, it did show a response in slight thick
ening of petiole and delay in abscission. It was evidently
safer to use while 2,4-D showed a narrow limit within which
the right effect could be expected.
1) Especially Designed Micrometer Pipette for Hpjrjpone
Application
For further studies with regard to the use of the
growth-regulators in hybridization of beans it became
necessary to develop a device for dispensing volumetrically
very minute quantities of these highly effective substances.
Standard micropipettes were found to be unsatisfactory for
handling viscous media and the necessity for frequent fill
ing because of their low capacities made their use cumber
some. They were also unable to dispense small enough
quantities with a sufficiently high degree of accuracy.
Accordingly a special pipette was devised to suit the
needs of the work. It was found that very minute droplets


TABLE 13
Results of Attempted Crosses Between P, vulgaris (A) and Various Other Species.
Number of Pods set and Average Length of Pods (in cm.) Obtained with Various
Treatments,
1,1
pcPK
rMmzm
ts-c
UfP
... Control
No, of
No, of
Av,
No.
of
Av,
No.
,of
Av,
No,
Of
Av,
Kind of cross
flowers
pods
1,
pods
1*
pods
1,
pods
1,
trea ted
Set £
of
set
of
set
of
set
Of
nod
P
s
pod
, Em
S
pod
F
s
PQd
p. xaUagU (a) X
P. lathyroides (6)
20
6 1
5,6
5
-
6.5
6
2
7,9
4
1
6,4
£ mUftKlg U)
P. aureus 10)
X
10
6 -
5.0
3
a
6.1
2
-
5,0
3

4,8
£* IHifiSEiS. £ Aaaa.tus (h)
X
15
4 -
4,4
8
-
6,5
7
mm
5,7
4
-
3,9
p. xaiMEia (a)
P. punco (N)
X
4
1 -
5.2
2
1
7.6
mm
-
-
1
mm
5,7
£. mUaE.§ (a)
£* aasate*8
X
10
1 -
4.1
1
-
4.0
1
mm
4,2
*
-
p* xaU&gl8 (a)
£ coc^ne^s (C)
X
5
1 -
8.5
2
Ml
3.5
N
1
12,6
1
-
4,6
g* matear (a)
P. angularis (M)
X
4
* Ml
-
1
-
7.1
-
-

1
5,2
Total
68
19 1
22
1
16
3
14
1
* P Parthenocarpic
* S Seed-containing


97
acid, Howiett (22) obtained increased fruit-set of
tomatoes from weakly viable pollen with the help of indole-
butyric acid. Wester and Marth (53) have also obtained an
increased pod-set of hand-pollinated lima beans with a mix
ture of para-chlorophenoxyacetic acid and indole-butyric
acid. These hormones have thus repeatedly shown favorable
responses in obtaining higher pod-set. It would certainly
have been interesting to know the effect of indole-butyric
acid separately as well, since it has shown such favorable
response in conjunction with PCPA.
Effect on Seed-set
Hormone treatment which gave a significant increase
in the number of pods and seeds set from crosses between
P. vulgaris and P. coccineus (Tables 8 and 9) did not,
however, increase the number of seeds per pod appreciably.
A small increase over the control was however obtained with
PCPA&IBA (3.3 as compared to 3.1). In case of self-polli
nated P. vulgaris flowers, those treated with PCPA gave an
increased pod-set as well as a higher number of seeds per
pod. PCPA&IBA also gave an appreciable increase in pod-set
(though not statistically significant) and also a small
increase in number of seeds per pod (Table 15). The effect
of hormones on the number of seeds per pod has not been
statistically studied, and so no conclusive statement can be


2
causes such as improper germination of the pollen, too slow
growth and penetration of the pollen tube, lack of stimu
lation to development of ovary and surrounding tissues,
abscission of style before fertilization, or the abscission
of the whole flower before pod and seed set. For success
and ease in hybridization a plant breeder is desirous of
obtaining a high percentage of success in crossing resulting
in healthy and well-developed seed of good germination
capacity. In bean breeding both types of difficulties are
met. Many species have not been successfully crossed with
the common improved types or the percentage of set obtained
has been very low, necessitating pollination of numerous
flowers. Furthermore, unfavorable environmental conditions
such as extremes of light and heat also result in high
abscission rate. The exact causes and nature of incompati
bility between certain species are not definitely known and
may be many and various.
Hormones have assumed great importance in horticultural
research during the recent past and have been used in many
ways to stimulate growth and other physiological processes
in plants. Checking the abseisslon of fruits and supplement
ing normal fruit-set have been the two most important uses
of hormones with apples and tomatoes. Although hormones
have been generally reported as inhibiting fertilization,
by regulating the amount and method of application they may
encourage development of seed and fruit, reduce abscission


11
parthenocarpy of cherries, plums and Oenothera. He says
that incompatibility is a genetically controlled character.
It may be because of the failure of the pollen tube to reach
the ovary before abscission of the flower or style. Fertili
zation thus cannot take place and ovules do not develop.
Using alpha-naphthaleneacetamide he studied its effect on
pollen tubes, the mechanism of abscission of the flowers
and style, and the development of fruits and seeds with and
without pollen. In case of Prunus avium the application of
an aqueous solution of alpha-naphthaleneacetamide to flowers
resulted in delayed abscission of the style. This species
is self-incompatible and untreated flowers, both unpolli
nated and self-pollinated, lost their styles two days earlier
than the treated ones. The growth of pollen tubes of com
patible pollen was, however, not affected by the hormone
treatment. With Oenothera the aqueous solution showed no
effect, but application of 1% lanolin emulsion to the lower
part of the ovary delayed abscission. Time of treatment,
from four days before up to the time of flower opening, show
ed no significant difference in effectiveness. Incompatible
tubes showed many abnormalities; with 1% emulsion causing
swelling or even bursting. The compatible tubes showed a
more remarkable effect in stopping growth at 4 to 5 mm* and
their ends bursting instead of reaching the 20 ram. length
of untreated ones.
Lewis thus found that alpha-naphthaleneacetamide treat-


25
pollination, it is quite prolific, and it is a representative
type of the commercially grown varieties with many good
horticultural characteristics.
The two most serious greenhouse pests were mildew and
red spider-mite. The former was controlled by dusting with
sulphur and the latter through the use of the newer miticides
especially E.P.N. miticide, generously provided by the Station
Department of Entomology. The E.P.N. miticide was applied
as a spray at the rate of 1 lb./lOO gals, of water. The soil
at first was sterilized with formalin or D-D, and later on
was better sterilized with steam to eliminate nematodes.
When the plants were established, a tablespoonful of 4-7-5
fertilizer mixture was applied to each plant at monthly
intervals.
Inasmuch as the success in any hybridization work depends
mainly on the proper technique of emasculation and pollination,
the technique found to be very satisfactory during the course
of this work needs to be described in some detail.
B. The Technique of Emasculation and Pollination
The largest flower buds obtainable about one day previ
ous to anthesis are selected for emasculation (Pig. 1 A).
The suture which closes the banner around the other flower
parts is opened with extreme care in order not to tear the


10
Vitamins, hormones, pyramidines and purines shoved stimu
lating effects. He tried 33 different growth-promoting
substances, of which 16 showed significant increases in
germination or tube growth. But the growth of pollen with
these substances was not comparable to that in stigma
exudate. In the latter case the tubes had an average
length of 16 diameters as compared with from 3 to 6 diame
ters in various other media,
Chen (23), however, could not find any favorable
effect on germination and tube growth with auxins on Prunus
armeniaca which showed favorable responses to additions of
micro-nutrient elements to the culture medium.
The work of Kuehlwein (24) throws further light on the
activity and stimulating power of growth-promoting substances
on pollen and stigma. His results are of especial interest
because they suggest the possibility that the pollen tube
growth of certain species might be favorably stimulated with
certain hormone treatments. By adding the pollen and stigma
extracts of various species to the culture media and observ
ing the growth of pollen tubes of various species in differ
ent media, he found that the growth-promoting substances of
pollen and stigma are "species or organ specific"; some pro
ducing a more favorable increase in growth or germination
with the pollen of certain species than with the pollen of
others.
Lewis (26) studied the problem of incompatibility and


79
TABLE 17
Germination of Seeds Resulting from Crosses between
E* iriUSgJjjjL **d E- (B) with
Various Hormone Treatments,
Ho, of germinated seeds and growing
seedlings out of 20 sown on 6/9/50,
Bate when
observed
POPA
PCPA&IBA
H.M.
KNA
Control
6/13/50
2
1
1
1
1
6/14/50
3
2
1
3
1
6/15/50
5
2
1
3
1
6/16/50
9
4
3
4
1
6/17/50
10
4
3
7
6
6/19/50
13
6
6
10
6
6/20/50
13
7
8
11
6
Germination
percentage
65
35
40
55
30
6/29/50
8
6
5
7
2


24
EXPERIMENTAL MATERIAL AND METHODS
A* General
As pointed out before, the aim of this study was three
fold. The primary object was to find out if hormone treat
ment could be used effectively in controlled cross-polli
nation of beans. Since the most effective method of hormone
application for treatment of individual flowers so far used
and recommended is the lanolin paste method, a secondary
objective was to work out some other equally effective method
which would permit a greater control of the quantity of
hormone used with each flower. This precision is desirable
so that the treatment may be repeated with consistent results.
The third objective was to cross as many species of Phaseolus
as possible within the limitations of time, etc., with a
standard variety of snap bean, Cherokee Wax (P. vulgaris),
so as to obtain hybrids which could be tested later.
Except for some of the preliminary trials, all the work
was done in the greenhouse at the Florida Agricultural Ex
periment Station, Gainesville, from August, 1949 to August,
1950. In all preliminary trials lima bean was also used as
one of the parents, but later on it was found desirable to
confine efforts to one variety of P. vulgaris, which was the
Cherokee Wax. The reason for selecting and using this
variety was that its flowers are very convenient for hand-


49
TABLE 6
Results of Attempted Crosses on P*. vulgaris with
Hormone Treatments
Kind of cross
Treatment
No. of
Buds
treated
No. of sets
obtained
Partheno-
Seed con-
carpic
taining
p*. ratorJjg
PCPA
2
,
_
X
POPA & IBA
2
1
-
P. coecineus
H.H.
2
-
1
KNA
2
-
-
Control
2
-
-
ZM.lfiaris
PCPA
12
5
2
X
PCPA & IBA
12
4
-
P. atroournureus
H.M.
7
5
1
KNA
11
2
2
Control
12
1
-
P. vulgaris
PCPA
2
2
-
X
PCPA & IBA
-
-
-
Et ealcaratus
H.M.
-
-
-
KNA
2
1
-
Control
2
-
m


TABLE 11
Total Number of Pods Set from Attempted Crosses between vulgaris and
P, flliformis (E) with Various Treatments, and the Average Length of
Pods Obtained,
Treatment
No, of flowers
treated
No, of pods
with seeds set
No, of parth-
enocarpic
pods-set
Average
length of
pods in
cm.
PCPA
50
1
15
5.7
PCPA&IBA
50
-
22
6.1
H.M.
50
-
21
6.7
KNA
50
3
6
6.1
Control
50
5
5,7


Ill
pose.
Treatment of flowers with para-chlorophenoxyacetic
acid and indole-butyric acid mixture in glycerine-Karo
significantly increased the percentage of sets from attempt
ed crosses between £. vulgaris and P. coccineus by 26# over
the control. A significant increase of 14# was obtained
with par-chlorophenoxyacetic acid. The mixture of the two
also resulted in an increase in seed-set from 14.2 with
control to 21.2 with the hormone treatment per 10 flowers
treated.
Pods containing seeds were obtained from attempted
crosses between P. vulgaris as pistillate parent and P.
calcaratus. P. filiformis. P. atropurnureus. P. acutifolius
and P. lathyroldes as staminate parents. It has not yet
been established by planting and observation whether these
are valid crosses. With some species 0.5# potassium salt
of naphthaleneacetic acid in glycerine-Karo mixture gave
increased set of seed-containing pods. With incompatible
species hormone treatment resulted in increased set of
parthenocarpic pods.
Ihese hormones, in the concentrations and amounts used,
did not inhibit fertilization and ovule development nor did
they affect adversely the germination capacity of resulting
seeds.
With 2,4-D it was found that the concentration of the
hormone in the medium was more important than the amount of


16
frility.:
The earliest reeord of the effective use of hormone
treatment to overcome Incompatibility and obtain fruit- and
seed-set with successful fertilization is found in the work
of Eyster (15), A strain of Golden-rose petunia was found
to be self-sterile, a genetic character behaving as a simple
recessive. The incompatibility was caused by a very slow
rate of growth of the pollen tube and formation of the
abscission layer much earlier than usual. On the basis of
Yasuda*s findings (5fr) it was stated that the placenta of
Petunia violcea secretes a special substance which diffuses
into the style causing inhibition of growth of pollen. Spray
ing the flowers with 10 ppm. aqueous solution of alphanaphtha-
leneacetamide immediately after or before pollination resulted
in development of capsules and viable seeds. The explanation
of this is found in the work of Lewis (26) reported earlier.
This instance gives reason to believe that the hormone treat
ment may prove to be useful in overcoming self-or cross
sterility of other plants as well.
A highly significant increase in fruit-set of hand-
pollinated flowers of Cucumis melo obtained by Whitaker and
Prior (54) with hormone treatment deserves special mention
here. Although self- or cross-sterility is not a problem
with melons, about one third of the hand-pollinated flowers
do not set fruits, which is a great handicap to breeding


TABLE 22
Analysis of Covariance Considering PCPA and Control
Source
B.F.
Sx2
Sxv
Sy2
Errors of Estimate
D* F. S.S. M.S.
Total
13
69,7143
84.0786
102.5857
Treatment
1
0.0
0,0
0.0492
Average in
Treatment
12
69.7143
84.0786
102.5365
11
1.1339
Treatment 2
6
34.8572
40.23
46.7006
5
0.2697
Treatment 3
6
34.8572
43.8486
55.8359
5
0.6766
10
1
0.9463 0.0946)
0.1876 0.1876)
F=lf2f: 1.98
F.05 4*96


28
A proprietary name for such a latex suspension is Goodrite
VL 100, manufactured by B. F. Goodrich Chemical Company,
Cleveland, Ohio. In most cases the use of latex is un
necessary if the flover parts are only slightly disarranged
during the emasculation process and the bud is returned to
its original closed condition after pollination. Where
closure is difficult or conditions of low atmospheric
humidity prevail, the use of latex is more essential in
order to prevent the withering of the style before the
pollen tubes have had a chance to grow through it. The
successive steps in the process of emasculation and polli
nation of bean flowers are illustrated in Fig. 1.
C. Preliminary Studies
Instead of arbitrarily selecting hormones and concen
trations for treatments or using the chemicals reported to
be effective, it was thought better to find out through
several trials which chemicals and concentrations showed
some kind of stimulation without causing any undesirable
effect. It was therefore necessary to make numerous pre
liminary trials which consisted of treatment of several
hundred flowers both in the field and in the greenhouse.
As the method of sealing the pollinated flowers with
latex suspension is very effective and convenient, the first


51
applied at the point of union of the peduncle and the
receptacle without making any scratch. In all 1272 snap
bean flower buds were emasculated and treated with hormones
after pollinating them with pollen from different species.
410 buds were allowed to be self-pollinated and then were
treated with hormone mixtures or used as controls. As re
ported by workers in the past (53, 54), for any reliable
inferences, a large number of buds should be treated. The
percentage of sets being so low, and the variability being
so high, this evidently is imperative. The time required
for emasculating and treating individual flowers and the
limitation set by availability of uniform sized buds re
stricted the planning of the entire experiment on a
statistical basis. The variability in such work is large
and less under the control of the experimenter. Size, age,
position of the buds on the plant, location, age and stage
of growth of the plant, time of emasculation and pollination,
all may be expected to affect the sets. A proper design for
statistical analysis was, however, used with some inter
specific crosses.
Experiment 1. Interspecific Crosses
In the experiment designed for statistical analysis
three species were used as pollen parents: P*. coccineus.
E*. fillformis and atropurpureus. The number of flowers


NO. OF POD
77
Effect of Different Concentrations and Amounts of 2,4-D in
Glycerine-Karo Mixture on Pod-set of P. vulgaris (A).
(Data from Table 16)


120
46. Swartley, J. E. and L. C. Chadwick. 1942. Effects of
synthetic growth substances on cuttings, seeds
and transplants. Ohio Agr. Exp. Sta. Bi
monthly Bull. 27(217):125-144.
47. Tukey, H. B., C. L. Hamner and Barbara Imhofe. 1945.
/ Histological changes in bindweed and sowthistle
following applications of 2,4-D in herbicidal
concentrations. Botan. Caz. 107(1):62-73.
48. Tullis, Edgar C. and Wm. C. Davis. 1950. Persistence
of 2,4-D in plant-tissues. Science 3:90.
49. Weintraub, R. L., J. B. Brown and J. N. Yeatman. 1950.
Recovery of growth regulator from plants treat
ed with 2,4-D. Science 3:493-494.
50. Went, F. W. 1928. Wuehstoff and Wachstum. Rec. trav.
botan, neerland. 25:1-116.
51. Went, F. W. 1939. Further analysis of pea test for
auxin. Bull. Torrey Botan. Club. 66:391-410.
52. Wester, R. E. and Paul C. Marth. 1947. Effect of some
growth regulators on yield of bush lima beans.
Proc. Amer. Soc. Hort. Sci. 49:315-319.
53. Wester, R. E. and Paul C. Marth. 1949. Some effects
of a growth regulator mixture in controlled
cross-pollination of lima bean. Proc. Amer.
Soc. Hort. Sci. 53:315-318.
54. Whitaker, T, W. and Dean E. Pryor. 1946. Effect of
plant growth regulators on set of fruits from
hand pollinated flowers in Cucumis melo.
Proc. Amer. Soc. Hort. Sci. 48:417-422.
55. Wittwer, S. H. and A. E. Murneek. 1947. Hormone sprays
and dusts for snap beans. Market Growers Jour.
76:8-36.
56. Yasuda, Sadawo. 1932. Physiological researches on the
fertility of Petunia violcea. Botan. Mag.
(Tokyo)46(548):519-517.
57. Zimmerman, P. W. and F. Wilcoxon. 1935. Several
growth substances which cause initiation of
roots and other processes in plants. Contrib.
Boyce Thompson Inst. 7:209-229.


33
further consideration in the matter of establishing definite
dosages which would be effective in preventing abscission
without producing an over-stimulation which would be detri
mental to seed development. It is not soluble in the common
vegetable fats which are fluid enough for ease in appli
cation, but it is soluble in Karo, a commercial preparation
of corn-syrup which appeared to be very convenient for
application. Instead of weighing out the chemical and using
different measured concentrations it was easier to make a
saturated solution with excess of 2,4-D in Karo which could
be separated out by centrifuging or filtration with the aid
of heat* This was used as a basis for various dilutions in
the viscous media of the Karo type.
The effective range of 2,4-D was found to be very
narrow during the course of further trials. Para-ehloro-
phenoxyacetic acid, which has been reported to be effective
with lima beans by Wester and Marth (53), was also included
in the trials hereafter* It was also saturated in Karo and
the excess separated out by centrifuging. The treatments
then tested were:
1. Saturated 2,4-D in Karo,
2. Vfo 2,4-D in lanolin,
3. Saturated para-chlorophenoxyacetic acid in Karo and
4. Control, (Karo only).
These were used with both snap bean and lima bean flowers


34
after emasculating and pollinating them with pollen from
various species. Saturated 2,4-D being toxic, the strengths
were gradually reduced to l/2 saturation, and then to l/4
saturation. The l/4 saturation appeared to be the safer
concentration to use. 2,4-D in lanolin was applied in a
scratch by a needle on the calyx at the base of the ovary.
2,4-D and para-chlorophenoxyacetic acid in Karo were applied
at the same place with and without a scratch. Ordinary
glass tubing with one end drawn out to a thin capillary tube
and with a piece of a rubber tubing attached to the other
end was used at this stage as applicator for the Karo mix
ture. It was helpful in releasing a minute drop of the
chemical in Karo with uniformity within certain limits.
The number and kind of crosses made and the number of
sets obtained are given in Table 2.
None of the numerous crosses between lima bean (a Ford-
hook selection) and a wild type of lima bean and also be
tween snap bean and P. lathvroides set any pods when 2,4-D
was used, either saturated in Karo or 1% in lanolin.
Several crosses between the two types of lima bean (P.
lunatus) with reduced strength of 2,4-D were also made.
These have been summarized in Table 3.
Although these preliminary trials did not show results
suggestive of a very favorable effect of the hormone appli
cation, they at least gave sufficient indication that 2,4-D


5
is translocated and brings about certain growth responses
within the plant body (8, 9). This provided an explanation
to Darwins earlier observation that shoots of grass failed
to bend towards light if their tips were cut off (4). In
1928 Went demonstrated the bending of decapitated oat
coleoptiles to one side when agar jelly containing some un
known hormones was applied. This method is still the most
widely used and probably the best method for measuring
concentrations of unknown hormones (50). By 1934 auxintriolic
acid (auxin-a), auxinolonic acid (auxln-b) and indole-acetic
acid (heteroauxin) were isolated from plants. Indole-acetic
acid is known to occur in higher plants and has been obtained
from leaves, endosperm of seeds, etc. (4). Zimmerman and
Wilcoxon (57, 58, 59) and later others, found that many other
chemical compounds such as indole-butyric, indole-propionic,
naphthaleneacetic, phenylacetic, and indolepyruvic acids
acted as stimulants in various growth processes and so may
be called synthetic hormones. Many other compounds have
since then been added to the list but the chlorophenoxy
acids and their derivatives have been most widely used.
The plant breeder, desirous of crop improvement by
hybridization, is primarily interested in a higher percent
age of fruit-set with his crossing, and in production of
viable seeds. The use of hormones to accomplish this end
is thus of Interest and should be considered from two points
of view: l) their use as directly helping the actual pro-


82
TABLE 18
Germination of Seeds from Flowers of P. vulgaris (A)
Self-pollinated and Given Various Hormone Treatments.
Date of
observation
No.
of seeds germinated out of 20
6/9/50
sown on
PCPA PCPA&IBA
H.M.
KNA
Control
6/13/50
12
10
15
14
15
6/15/50
18
17
18
17
19
6/15/50
19
17
18
18
19
6/16/50
19
18
18
17
19
Germination %
95
90
90
85
95
TABLE 19
Average Height (in inches) of Seedlings from Seeds of
P. vulcaris Selfed and Treated with Various Hormones.
Time when
observed
PCPA PCPA&IBA
H.M.
KNA Control
7 days
after sowing
3.98 3.58
2.56
2.55 2.1
8 days
after sowing
6.2 5.8
4.9
4.2 3.6


22
corn, spring wheat and sugarbeet, Swartley and Chadwick
(46), using naphthaleneacetamide and indole-butyric acid
with 41 garden perennials, obtained significant increase in
percentage of germination with 11,
The property of retarding the germination and growth
of seedlings exhibited by some chemicals suggested the
possibility of using them effectively for weed control,
2,4-D had shown such effect with various species,
Hamner et al, (20) used 2,4-D, applying it both to the
soil and seeds, to study its effect on germination of
various kinds of seeds, With 1 gm, of the chemical per pot
of the soil, they found complete retardation of germination
of clover and cabbage seeds. The germination of wheat seed
obtained, however, was 85$. No effect on germination was
observed with 0,001 gm, of the acid per pot. Application
of the chemical to the seeds (soaked for 4 hours) at various
concentrations from 1 to 100 ppm, also showed effect on
germination and growth of the seedlings. Bean seeds treated
with 1 ppm, of 2,4-D in aqueous solution showed "the
characteristic formative effects and virus' like symptoms0
in the seedlings. At 10 ppm, the seedlings were "severely
checked* and exhibited swelling in the hypocotyl, while 100
Ppm, of 2,4-D completely checked growth. The authors con
cluded that soil treatment with 2,4-D at one ppm, adversely
affects seed germination and growth of seedlings in many


110
SUMMARY
Incompatibility between certain species and a low per
centage of pod-set from attempted crosses are great handicaps
in bean hybridization. Some workers have reported successful
use of hormones in overcoming incompatibilities and in in
creasing fruit-set of beans, melons and lily.
An attempt was made to study the effectiveness of
hormones in reducing abscission of hand-pollinated bean
\
flowers and in increasing the set of pods with viable seeds
from attempted crosses between P. vulgaris (Cherokee Wax)
and various other species of Phaseolus.
Preliminary trials with several hundred flowers showed
that some of the chemicals were effective in producing
favorable responses such as checking abscission, increasing
pod-set, and production of parthenocarpic pods.
A mixture of glycerine and Karo in the proportion of 1:4
was found to be a very satisfactory medium for hormone
application. Many chemicals which are not appreciably
soluble in water, when saturated in glycerine and then
diluted with 4 times of its volume of Karo, produced desirable
effects without being toxic. These could be applied either
on the calyx or on the peduncle without making any scratch.
A 0.0004 to 0.0006 ml. size droplet of the mixture was the
amount used in each application. It could be easily dispensed
with the help of the micrometer pipette designed for the pur-


4
REVIEW OP LITERATURE
General
Although hormone research is comparatively a recent
venture in the field of horticulture, the literature on the
subject is voluminous. It is neither possible nor desirable
to attempt a complete review of this literature here. As is
well known, hormones have been demonstrated to be useful in
favorably affecting the physiological activities of plants
in various ways. The most common uses of economic import
ance are: the stimulation of the rooting of cuttings, the
control of fruit-drop by reducing abscission, the thinning
of blossoms and buds, the production of parthenocarpic
fruits, the inhibition of sprouting, and the killing of
weeds. While considerable work has been done on the above
mentioned aspects, the literature on the use of hormones
for stimulating fruit-set and viable seed production is very
meager. In fact there is evidence to the effect that hormones
actually inhibit fertilization (37), An attempt is made here
to review the pertinent literature on hormone research re
garding their use as an aid to fruit-set, fertilization,
overcoming incompatibility and any other physiological
activity that directly or indirectly helps in self or cross
fertilized fruit development,
Boysen Jensen in 1910 showed that some chemical substance


107
vith this pipette has previously been described. It is not
only useful for work of this nature but may prove to be
suitable for use in micro-chemistry where minute measure
ments such as this are also necessary.
With regard to the growth-regulating substances used
in this study and found satisfactory in the glycerine-Karo
medium, the effective concentrations have not been designated
in parts per million because it was determined early that,
except for 2,4-D, maximum concentrations attainable were
desirable since the whole plant was not subjected to treat
ment and only extremely small localized areas of application
were involved. It was found desirable rather to regulate
the dosage by regulating the actual volumetrlcally measured
amounts than to determine relative concentrations in terms
of parts per million. The concentration of chemicals
expressed as percentage saturation in glycerine should be
adequate inasmuch as the mixtures of this strength can be
prepared with consistent accuracy.


121
58, Zimmerman, P. W. and A. E, Hitchcock. 1941. Formative
effects induced with h-naphthoxyacetic acid.
Contrih. Boyce Thompson Inst. 12:1-14.
59. Zimmerman, P. V. and A. E. Hitchcock. 1942.
Substituted phenoxy and benzoic acid growth
substances and the relation of structure to
physiological activity. Contrib. Boyce
Thompson Inst. 12:321-343.


78
mixture, which apparently does not seem to he toxic in effect
in any of the different amounts used in this experiment, gave
no increased pod-set over the control. This may possibly be
due to the fact that the conditions during the course of this
experiment were particularly suitable for pod-set and ab
scission was at a minimum, which is suggested by the high
percentage of pod-set, as much as 70$, without the use of
hormone treatment.
Experiment 4, Germination Tests
The results of germination tests and observations of
growing seedlings have indicated that the hormone treatment
of flowers after pollination does not adversely affect the
germination percentage of seeds obtained from some crosses.
The results of tests with the seed obtained as a result of
crosses between P. vulgaris (A) and P. coccineus (b), which
are summarized in Table 17, show that, in this particular
case, the germination percentage is actually increased.
From Table 17 it is seen that the maximum number of
seedlings was secured in a period of 11 days after sowing.
Seeds obtained from flower buds treated with PCPA gave the
highest germination percentage which was 65, Hormones also
increased the germination percentage of seeds as compared
to the control, with which it was the lowest. These hybrid
seeds were found to be low in germination capacity as com-


TABLE 2
Results of Attempted Crosses between P,. vulgaris and coccineus
Kind of cross
Treatment
No, of flowers
treated
No, of
pods set
P, vulgaris
1.
2,4-D l/2 saturation in
Karo with puncture
6
X
P. coccineus
2.
2,4-D l/2 saturation in
Karo without puncture
19
6
3,
2,4-D 1/4 saturation in
Karo without puncture
10
6
4*
2,4-D 0,5% in lanolin in
a scratch
35
16
5.
Para-chlorophenoxyacetic
acid saturated in Karo
19
9
6.
Control, Karo alone
35
12


57
regulator mixtures on treatment of buds causes any effect on
the rate of development of pods, a subsidiary experiment was
run using vulgaris material. A number of buds of uniform
sise were allowed to be self-pollinated and treated with PCPA
and 2,4-D (l/4 saturation) and also with control. At each
application 0.0006 ml. of PCPA and 0.0004 ml. of 2,4-D mix
ture were used. Measurements of 4 pods from each treatment
were taken daily for a period of 8 days, and the experiment
was replicated 3 times. Usually the pods reach their maximum
size in about 8 to 10 days after they have started develop
ing. The aim was to compare the rate of development of pods
with and without hormone treatment to determine if there was
any initial or continued effect of hormones on the develop
ing pods. The data regarding this study are recorded in
Table 20.


71
PCPA&IBA and that with control, although not more than the
L.S.D., is very nearly equal to it, Sinoe this treatment
has PCPA as its active agent and has also given an increas
ed set of pods and seeds with the interspecific erosses be
tween P. vulgaris and P. coccineus. it should not be regard
ed as merely accidental,
Table 15 shows that the treatment PCPA also gave a
significantly higher number of seeds as compared with
control. The average number of seeds obtained per 10
flowers treated in the experiment was 29.75 with PCPA and
17.50 with the control. The difference is much higher than
the 7.16 needed for significance at the 5$ level. PCPA&IBA
also seems to have a favorable effect on seed- and pod-set
as compared to the control, although the difference is not
quite significant when compared with the L.S.D. This
experiment thus definitely showed that the hormone treat
ment, in the amounts used, has no inhibitory effect on pod
or seed development. On the contrary PCPA treatment of
flower buds very significantly increased set of both pods
and seeds. From Table 15 it is also noticed that hormone
treatment did not decrease the number of seeds per pod.
There was, in fact, a small increase in the number of seeds
per pod with PCPA and also with PCPA&IBA and KNA. The
difference, however, is not so significant as in the numbers
of pod and seed set. Unless shown to be statistically
significant it cannot be considered to be a definite ad-


59
and 6.4 with PCPA and PCPA & IBA respectively as against
3.8 with control. The least significant difference was
calculated as 1.21,
increase in set.
so that
both
treatments
gave
significant
TABUS
8
Results of Attempted Crosses of P
(B) Using Various
. vulaaris
Treatments
X

P. coccineus
Replication
Number
of pods containing seeds obtained from
treating 10 flower buds.
PCPA
PC PM IBA
H.M.
KNA
Control
I
6
7
5
4
2
II
5
6
6
4
6
III
4
6
3
3
3
IV
5
7
5
4
5
V
6
6
5
3
3
Total
26
32
24
18
19
Average
5.2
6.4
4.8
3.6
3.8
% Pod-set
52
64
48
36
38
L.S.D.


1.21
(Analysis of variance given in Appendix Table 23)


12
ment without compatible pollen did not stimulate development
of fruits in cherries, plums or pear. In Oenothera, however,
smear treatment of the ovary resulted in parthenocarpic fruit
development. The ovules grew to normal size but had no
embryos. He suggests that for fruit formation the necessary
stimulation is provided by the pollen tubes as they enter the
ovary as well as by the developing ovules. The pollen tubes
probably give a greater stimulus than the ovules in many-
seeded fruits. Therefore parthenocarpic fruit development
with chemical treatment is more successful in many-seeded
fruits like cucumbers than in fruits like plums. Lewis also
shoved that hormone treatment checks that abscission which
is due to the change in the walls of the existing cells and
not that which is due to rapid cell division. The abscission
of the style and of mature fruits is of the former type while
the premature flower and fruit drop is abscission of the
latter type.
Besides the evidences quoted above, there are numerous
instances where hormones have been demonstrated to check
abscission of fruits. LaRue (25) found that the fall of
Coleus leaves can be considerably reduced by hormone appli
cations. Date flowers also show the same reaction, (35).
As early as 1939-40 the use of indole-acetic acid,
indole-butyric acid, indole-propionic acid and naphtha-
leneacetic acid was demonstrated to be effective in check-


65
be necessary to establish the validity of the crosses. The
effect of hormone treatments is very evident in giving an
increased number of parthenocarpic sets with PCPA, PCPA&IBA
and H.M. Hie slightly greater average length of pods follow
ing treatment with H.M. suggests that this mixture has a
greater stimulation to growth than any other treatment.
Other Interspecific Crosses
The results of the attempted crosses between P*. vulgaris
(Cherokee Wax) as pistillate parent and different species of
Phaseolus and their varieties as staminate parents are re
ported in Tables 12 and 13* The number of pods containing
seeds and parthenocarpic pods (more than one inch in length)
have been separately recorded and also the average lengths
of pods set with different treatments have been given.
Table 12 clearly shows that hormone treatment aids in
obtaining increased set of pods containing seeds, at least
with calcara tus (F) With this species KNA and PCPA&IBA
again gave increased pod-set of 20$ as against 5$ with the
control. H.M. and PCPA also gave an increased number of pods
containing seeds, the percentage of set being 15$ and 10$
respectively. In order to get more precise information,
several hundred flowers should be treated. The hormone
treatments, except KNA, also gave a higher number of


73
TABLE 15
Number of Seed Obtained from 10 Plovers of P. vulgaris (A)
Allowed to Be Self-pollinated and
Given Various Hormone Treatments.
Number of seeds obtained and
Replication the treatments used.
PCPA
PCPA&IBA
H.M.
KNA
Control
Total
I
19
21
14
25
16
95
II
30
15
10
10
11
76
III
37
36
21
26
28
148
IV
33
19
12
21
15
100
Total
119
91
57
82
70
419
Average
29.75
22.75
14.25
20.5
17.5
No. of seeds
per pod
4.1
3.64
3.16
3.73
3.33
L.S.B. :
7.16
(Analysis of variance given in Appendix Table 27)


19
used several chemicals. They demonstrated that bud, blossom
and pod drops of string beans are reduced by hormone treat
ment. These bud, blossom and fruit drops were said to be due
to hot, dry weather and insects such as the tarnished plant-
bugs and the potato leaf-hopper. They induced abscission by
higher temperature, by use of illuminating gas and by caging
insects over the plants. Alpha-naphthaleneacetle acid and
2,4-D were found to be most effective, giving best results
as a dust (40 to 80 ppm.). An increase of 40$ in yield of
wax beans was obtained, resulting from a "greater number of
small, high grade beans rather than larger beans".
Murneek et al. (34), working with snap beans, also
obtained similar increases in yield with hormone treatment
but found that the increase in yield was obtained only when
the weather was hot. In the fall crop, hormones actually
decreased the yield. The authors believed that the yield
increase, when obtained, was due to increase in the
chlorophyll content of leaves and a stimulation of carpel-
growth brought about by the hormone treatment. They report
that, at least under certain environmental conditions, the
number of pods per plant and their rate of development can
be increased by hormone treatment. The same workers further
continued the trials using various other chemicals, e.g.,
the substituted phenoxy-benzoic acids and chlorine-substi
tuted phenoxy acids, and in 1947 (55) reported most con-


80
pared to those obtained as a result of selfing P. vulgaris
and also took longer time to emerge. Observations of the
growing seedlings showed that the seeds obtained from the
attempted crosses between P. vulgaris (A) and P. coccineus
(B) resulted from valid crosses, the elevation of the
cotyledons of the hybrid seedlings was, in general, inter
mediate between the two parental extremes but in no case
was it as high as in the P. vulgaris pistillate parent.
Hiere were many abnormalities noticed in the seedlings such
as the curvature of the stem, difficulty in the tip breaking
through the soil and the cotyledons, and upside down germi
nation of seeds with roots coming up. The seedlings were
weak and delicate and, as is evident from the observations
made on 6/29/50 in Table 17, many of them died early. The
growth of seedlings from seeds with hormone treatment of
flower buds before fertilisation was more accelerated in
the beginning than that of the control but this effect
gradually became less noticeable later on. Ibis test sug
gested that the seed obtained as a result of this cross
should be germinated in a soft medium such as vermiculite
which does not offer much resistance to the emerging tip.
The germination tests of the seed obtained as a result
of selfing P. vulgaris (A) and treating the flowers with
hormone mixtures, reported in Table 18, conclusively show
that hormone treatment of flowers does not have a dele-


119
34. Murneek, A. E., S. H. Wittwer and B. D. Hemphill. 1944.
Hormone sprays on snap beans. Market Growers
Jour. 73:197, 215, 218, 221.
35. Nixon, R. W. and P. E. Gardner, 1939. Effects of certain
growth substances in inflorescences of dates.
Botan. Gaz. 100:868-871.
36. Overbeek, J. Van, M. E. Conklin and A. E. Blakeslee.
1941. Hormone controls growth of ovules.
Amer. Jour. Botan. 28:647-656.
37. Overbeek, J. Van. 1944. Growth regulating substances
in plants. Annual Review of Biochem. 13:
631-666.
38. Pomery, C. S. and V. V. Aldrich* 1943. Set of citrus
fruit in relation to application of certain
growth substances. Proc. Amer. Soc. Iiort.
Sci. 42:146-148.
39. Randhawa, G. S. and H. C. Thompson. 1949. Effect of
application of hormone on yield of tomatoes
grown in the green house. Proc. Amer. Soc.
Hort. Sci. 53:337-344.
40. Rice, E. L. 1948. Absorption and translocation of
ammonium 2,4-Dichlorophenoxyacetic acid by
bean plants. Botan. Gaz. 109:301-314.
41. Skoog, F., C. L. Schneider and P. Malan. 1942. Inter
actions of auxins in growth and inhibition.
Amer. Jour. Botan. 29:568-576.
42. Smith, Paul F. 1942. Studies of the growth of pollen
with respect to temperature, auxins, colchicine
and vitamin-B. Amer. Jour. Botan. 29:56-65.
43. Snedicor, George V. 1948. Statistical Methods. Fourth
edition, The Iowa State College Press, Ames,
Iowa, pages 318-331.
44. Stewart, V. M. S, and Ira J. Condit. 1949. The effect
of 2,4-D and other plant growth regulators on
the Calirayrna fig. Amer. Jour. Botan. 36(4):
332-335.
45. Swarbrick, T. 1943. Progress report on the use of
naphthoxyacetic acid to increase the fruit set
of strawberry variety Tardive de Leopold. Ann.
Report Agr. Hort. Res. Sta. Long Ashton
(Bristol) 1-32.


ACKNOWLEDGEMENTS
The author wishes to acknowledge with deep gratitude
the kindly advice of Dr, A, P. Lorz, under whose guidance
and constant help this study was conducted.
He wishes to express his thanks and sincere appreci
ation to the members of his committee as well as to the
members of the staff of the University of Florida whose
assistance and encouragement were a constant source of
inspiration.
He particularly acknowledges the kindly advice and
criticisms of Dr* H. S, Wolfe, Head Professor of Horti
culture and Mr, I, W, Ziegler, Assistant Professor of
Horticulture who went through the manuscript and Mr,
W. D, Hanson, Assistant Professor of Agronomy who gave
valuable suggestions regarding the statistical analysis
of the data.
ii


44
to those already reported were observed and abscission was
delayed In both cases. This showed that applications at
both places were equally effective.
Having observed the effectiveness to be the same
whether the hormone was applied on the abscission zone or
on the calyx, with Pg. coccineus flowers, the method had to
be further tested with snap bean flowers using the usual
pollination technique. A small preliminary trial using 66
flowers with snap bean as pistillate parent and P*. coccineus
as staminate was made. Two places of application as mention
ed before were tried. The treatments and number of polli
nations made and sets obtained are given in Table 5. The
hormones were applied without making any puncture.
These trials showed that application of a hormone drop
on the abscission zone was as good on the peduncle as on
the ovary and probably was safer as less inhibitory effect
on the ovule development could be expected. The best point
of application was decided to be the point where the
receptacle joins the peduncle. Besides giving information
regarding the place of application this trial also suggested
the possibility that the hormone treatment may prove useful
in obtaining a greater number of sets when the abscission
rate is high. During the course of these trials the
environmental conditions seemed ideal for pod setting and a
very low percentage of abscission was manifest with untreat
ed flowers.


17
work. These authors used various hormones and also ether
extract of cantaloupe pollen, Hie hormones were applied In
lanolin paste, where and in most cases 10 to 15 mg. per
gram of lanolin was the optimum concentration range. 2,4-D
and 4-ehlorophenoxyacetic acid were found to he most effect
ive and gave very highly significant results. An increase
in set of 27$ has been reported with the use of hormone
treatment.
Emsweller and Stuart (14) successfully overcame the
self-incompatibility of Creole, Craft and Ace varieties of
Easter lily (Lilium longiflorum) with hormone treatment of
flowers. They also tried these treatments with inter
specific crossing. Various chemicals were used after dis
solving them in lanolin at concentrations from 0.1 to 1$.
The paste was applied in a wound produced by removing one
petal and also at the base of the style. Effectiveness was
found to be the same in both cases. While untreated, hand-
pollinated flowers did not set a single fruit, 1$ naphtha-
leneacetamide treatment resulted in production of viable
seeds although they were weak in germination. Treatment
with the hormone caused a swelling of pedicel and delay in
abscission by several weeks. Although hormone treatment
was not very effective in species and varietal crossing,
the authors say that it can help in making some difficult
crosses possible. Chemical analysis of young developing
fruits after treatment showed a higher sugar content which


18
is suggestive of greater mobilization of food material caus
ed by the hormones. The authors believe that this may be
responsible for the delay in abscission and may also check
rapid degeneration of embryo sacs, keeping the egg cell
viable for a longer period.
There appears to be only one instance in the literature
where hormones have been used with beans with favorable
responses in controlled cross-pollination. Wester and Marth
(53) used 1$ concentration of a mixture of indole-butyric
acid and p-chlorophenoxyaeetic acid in proportion of 4 : 1
and applied it in three ways to cross-pollinated flowers of
lima beans. As an aqueous solution, the mixture was sprayed
on the stigma and as an emulsion in lanolin, it was applied
on the pedicel, with and without scratching the tissue.
When applied in a scratch, the treatment resulted in 28.8$
of successful sets as against 18.7$ for the control. The
degree of success depended also on parental combinations.
The most interesting feature of the trials was an increase
in the average number of seeds per pod from 1.95 to 2.43.
Pse of Hormone in Increasing Yield of Beans:
Hormones have been definitely shown to be effective in
increasing yield of beans. It would be desirable here to
review briefly the literature concerning this aspect.
Fisher et al. (2, 16) conducted extensive studies and


41
the main screw is desired. All parts except the hypodermic
needle and the screws are fabricated from polysterene
plastic resin. In the growth regulator studies it was not
necessary to use quantities smaller than 0.0002 ml., which
were large enough for the practical purposes of this work.
2) Trials on Phaseolus coccineus (Scarlet-runner bean)
A few plants of Scarlet-runner bean (P*. coccineus)
were growing vigorously in the greenhouse and flowered pro
fusely and continuously during fall and spring. All flowers,
however, abscissed without producing any pods. Evidently
there was some incompatibility resulting from some peculiarity
of the environmental condition within the greenhouse because
the same material growing outside in a frost-protected
location was capable of pod development. It was an excell
ent plant material for trials and tests to study the nature
of the effect of 2,4-D, para-chlorophenoxyacetic acid and
also other hormone mixtures. The pollen was definitely
viable because sets were obtained easily on P*. vulgaris with
P. coccineus pollen.
In order to study the effect of l/4 saturation of 2,4-D
in Karo and saturated para-chlorophenoxyacetic acid in Kero,
and simultaneously to see if the self-incompatibility of P.
coccineus could be overcome with hormone treatment, several
flower buds were treated with the hormone and sealed. Also


Fig. 1. The Successive Stages of Emasculation
and Pollination of Bean Flowers.
(Explanation given in the text)


30
4, and 5 gave responses in the majority of flowers treated.
The flowers treated with these lasted for more than four
days while the others as well as the controls (treated with
latex hut without hormone) dropped within that time. Al
though no definite growth stimulation or swelling was noticed,
the pedunele did appear to he straight and stiff. When the
trials were repeated, however, the responses were not con
sistent. Inasmuch as none of the hormones excepting Nos. 3
and 4 are soluble in water or the latex suspension, they
could hardly he expected to he effective consistently, be
cause the quantity applied in each ease was not definite and
uniform. Alcohol coagulated the latex; therefore, it could
not he used to dissolve the hormones before mixing with the
latex suspension.
Of all the hormones used only 2,4-D and potassium salt
of naphthaleneacetic acid are appreciably soluble in water.
To study the nature of the effect of these two, saturated
solutions of each were made in water. A number of emascu
lated flower buds of snap bean were sealed with latex and a
little of the water solution of each was applied by dipping
a needle in the solution and making a small scratch at the
base of the ovary with it. Controls were maintained. The
effect was spectacular. All treated flowers stayed long
after the controls dropped. Many parthenocarpic pods de
veloped, the 2,4-D showing greater effect. Abscission was


23
cases. Grass seeds are more resistant than the seeds of
many of the vegetables,
Tukey et al, (47) studied the histological changes in
bindweed and sowthistle on treatment with 2,4-D at 1000 ppm,
as spray. Among the effects produced were plasraolysis of
pollen grains, checking of chlorophyll development,
plasmolysis of cells of leaves, increase in cell division
in all cambial zones, enlargement and rupture of cortical
cells and disappearence of starch from all parts of the
flower* The chemical also cheeked starch hydrolysis,
Mullison et al, (33) treated the seeds of various field
and vegetable crops using the vapors of 2,4-D and its
derivatives. Bean seeds subjected to vapor treatment with
2,4-D derivatives did not show much reduction in germination,
although the methyl ester caused a reduction in germination
by 12$ as compared to the control. Isopropyl 2,4-Dichloro-
phenoxyacetate vapor treatment of lima bean seeds resulted
in germination of 44$ as against 68$ with untreated controls.
Without going further into the voluminous literature
on weed control by hormones, it will be evident that any use
of hormones for effecting greater pod and seed-set may affect
the viability of seed and the nature of development of
seedlings. This fact, therefore, needs to be borne in mind
while studying the effects of hormone treatment of flower
buds with a view to obtaining a greater set of fruit.


76
TABLE 16
Effect of Different Concentrations and Amounts of 2,4-B Mixture on
Pod and Seed-set of P. vulearis Self-iJollinated.
-
Concentrations of 2.4-D in glyserine-Karo mixture
Amount
of the
Mixture
3/4 Saturation
1/2 Saturation
1/4 Satura
tion
l/8 Saturation
No 2,4-D Control
used
. . (
Flowers
Sets
Flowers
Sets
Flowers
S,.
Flowers
Set*
i .....
Flowers
Sets
treated
Pods Seeds
treated
Pods Seeds
treated
Pods
Seeds
treated
iPods
Seeds
treated
Pods Seeds
0.0000 ml*
-
-
wm
mm
-
-
-
-
-
-
-
-
10
7 30
0*0002 ml*
10
0
0
10
4
\
12
10
3
11
-
-
mm
-
-
0.0003 ml.
10
3
5
-
Wf '
-
-

-
mm
m
-
0.0004 ml*
10
2
7
10
2
8
10
2
10
10
5
22
0.0006 ml*
10
2
8
10
0
0
10
3
15
10
7
22
0.0008 ml*
10
1
2
10
2
10
10
2
11
10
7
26
0.0010 ml*
mm
am-
mm
10
2
6
10
1
2
10
6
18
0.0012 ml.
m,
mm
pa

*
10
8
24


58
RESULTS
Experiment 1. Interspecific Crosses
It has been pointed out that the planning of the experi
ment with crosses between vulgaris (Cherokee Wax) as
pistillate parent and various other species of Phaseolus as
staminate parents was done on a statistical basis only with
three species, viz., P^ coccineus. P. atropurpureus and P.
filiformis. The number of sets obtained with P^ atropurpureus
as staminate parent was exceedingly low, making any statisti
cal interpretation impractical, and therefore the results
with this species are grouped with those of others. The
results of crosses with P*. coccineus and P^ filiformis have,
however, been statistically analysed.
The results of attempted crosses with P^ coccineus (B)
as staminate parent on P*. vulgaris (A) as pistillate are
given in Tables 8 and 9. PCPA and PCPA & IBA treatments
gave highly significant increases in pod-set and PCPA & IBA
gave a similar increase in the number of seeds obtained as
compared with control. Without the hormone treatment 38$
of the pollinated flowers set pods containing seeds, while
treatments PCPA and PCPA & IBA gave pod-sets of 52$ and 64$
respectively. As is clear from the table 8, the average
number of pods obtained per 10 flower buds treated was 5.2


14
setting of tomatoes In the greenhouse helped in overcoming
the reduced fruit-set resulting from cold weather. Using
indole-butyric acid, he found that an emulsion at 0.3$
concentration was effective as a paste. The cross-polli
nated flowers with viable pollen, on treatment, developed
fruits superior to the untreated. In case of self-polli
nated flowers, only 60$ of the untreated flowers set fruits
while the percentage was 100$ with treated flowers. Treated
flowers had a poorer seed development than untreated flowers.
The vapors of naphthoxyacetic acid have also been shown to
produce a high percentage of seedless fruits (50 to 98$) in
greenhouse tomatoes (30). Randhawa and Thompson (39) used
various other hormones, viz., beta-naphthoxyacetie acid
(50 ppm.), alpha-O-chlorophenoxypropionic acid (50 ppm.),
p-chlorophenoxyacetic acid (25 ppm.), 2,4-5-trichloro-
phenoxyacetic acid (10 ppm.) and 2,5-dichlorobenzoie acid
(100 ppm.). Spraying the flowers, either when they were
half open or when all were open, gave a higher set of fruits
than untreated controls. That the effectiveness of differ
ent hormones varies according to species is evident from the
work of Crane (13) who found that the aqueous sprays of
indole-acetic acid (1500 to 2670 ppm.) gave parthenocarpic
fruit-set of figs and the fruits were not inferior to those
caprified. Naphthoxyacetic acid and 2,4-D were not effect
ive. Stewart (44), however, has obtained seedless figs with


60
Since the total number of seeds obtained as a result
of a cross is important rather than the number of pods set,
the data regarding the number of seeds obtained with various
treatments are given in Table 9.
TABLE 9
Number of Seeds Obtained as a Result of Attempted Crosses
between P. vulgaris and Pt eoceineus (B)
Using Various Treatments.
Replications
Seeds
obtained per 10
flowers
treated
PCPA
PCPA&IBA
H.M.
KNA
Control
I
16
17
15
15
7
II
16
24
16
13
20
III
9
20
10
5
9
IV
15
28
17
13
15
V
16
17
13
8
8
Total
72
106
71
54
59
Average
14.4
21.2
14.2
10.8
11.6
Average number
of seeds per
pod
2.77
3.31
2.96
3.0
3.1
L.S.D.


4.07
(Analysis of variance given in Appendix Table 24)
The data presented in Table 9 show that the treatment


81
terious effect on germination capacity of the resulting seeds
hut may possibly be responsible for weakness in the stem of
the seedlings* The germination percentage of seeds from
PCPA-treated flowers was found to be 95, the same as for the
control* The germination percentages of seed from flowers
treated with PCPA&IBA, H.M., and KNA were found to be 90, 90,
and 85 respectively* A very marked difference in the rate
of growth of seedlings from hormone treatments was observed
in the early stages of growth. Seeds obtained from flowers
treated with hormones gave seedlings which grew faster in
the beginning than did those from the control. This is
evident from Table 19 in which the average heights of
seedlings measured on the seventh and eighth day after sowing
are recorded. Figure 5, which is a photograph of the
seedlings growing in a flat taken on the ninth day after sow
ing, also clearly shows that the hormone treatment resulted
in Increased growth rate of seedlings in the initial stages,
which was more pronounced with PCPA and PCPA&IBA.
Experiment 5. Effect of Hormone Treatment on the Rate of
Development of Pods
As has been described earlier, daily measurements of
pod length were taken after treating the flowers with 2,4-D,
PCPA and control. Each day 4 pods were measured from each of


93
3 Stimulation provided by the hormone supplementing any
that may have resulted from the pollen tube in causing
greater ovule development.
During the course of the preliminary trials as well as
in the final experiments, the effective part the chemicals
play in checking abscission has been definitely observed.
Reduced yield of beans due to high temperature conditions
resulting in a high abscission rate has been reported to be
a problem by Fisher et al.(16) and Vittwer and Murneek (55)
The reason for obtaining increased yields of beans with
hormone treatment has been explained to be its property of
checking abscission. The findings of this study are in
agreement with the results of these workers. In hand poll!
nation with beans this tendency of the flowers to absciss
is increased even more, probably, due to handling of buds
and the unavoidable injury caused to the flower parts dur
ing emasculation. In Florida the weather conditions are
often favorable for high abscission rate due to high
temperatures and bright sunlight. The principal causes
of a low percentage of sets obtained from hand-pollinated
flowers thus can be said to be the high rate of abscission
of flowers before pod-set or following pod-set if adverse
conditions prevail later. Whether the abscission of the
style also is a cause of incompatibility and failure of
pod-set can not be said with certainty, but this appears
unlikely.


72
vantage* Among the various treatments used only H*M, gave
a slightly smaller number of seeds per pod than the control.
Since all other treatments have given at least some increase
in the number of seeds per pod, it can very definitely be
inferred that at least the hormones other than H.M. in the
concentrations used in this study do not inhibit fertili
zation and seed development.
TABLE 14
Number of Seed-containing Pods Set from 10 Flowers Allowed
to be Self-pollinated and Subjected to Various Hormone
Treatments.
Replication
No.
of pods set out
of 10
flowers treated
PCPA
PCPA&IBA
H.M.
SNA
Control
Total
I
5
6
4
5
4
24
11
7
5
5
4
5
26
III
9
8
6
7
7
37
IV
8
6
4
6
5
29
Total
29
25
19
22
21
116
Average
7.25
6.25
4.75
5.5
5.25
L.S.D.
: 1.
,12
(Analysis of variance given in Appendix Table 26)


83
Fig. 5
Photograph of the Growing Seedlings Obtained from P. vulgaris
Flowers Self-pollinated and Treated with Various Hormones and
Control. (The photograph was taken on the ninth day after
sowing.)


101
flowers were treated in each replication with various hormone
mixtures and the control. Vith species which cross easily
this number may be considered to be the minimum needed, but
in case of species with which the percentage of pod-set is
very low many times this many flowers should be treated in
each replication.
Medium and Method of Hormone Application
The medium and method of hormone application is an
important factor on which the successful use of these
chemicals depends. The medium and method should not only
be effective in producing desirable results but should also
be convenient* Unlike the use of hormones on a large
number of plants in a wide area, treatment of individual
flowers in hybridization work calls for a very precise
control of amount and concentration of the chemical used.
Very minute amounts of l/4 saturation of 2,4-D have been
found to be enough to produce an effect which tends to be
come toxic* Lanolin has been the most common medium used
by most workers so far, and has proved to be effective*
However only a limited control of the amount used in each
application can be achieved with this medium.
Vaster and Marth (53), Whittaker and Prior (54) and
Emsweller and Stuart (14) have used lanolin paste with
various chemicals for treatment of individual flowers, but


40
Pig. 3. Photograph of the Micrometer Pipette with
a few Refinements.


15
2,4-D at the concentration of 250 ppm.
Hormone treatment has not always shown increased fruit
set. Evidences of a negative effect are also met witliin the
literature, eg., with greenhouse potatoes (11), Washington
navel oranges and Marsh grapefruit (38). There are many more
references in the literature which report effectiveness of
hormones in causing increased fruit-set with various other
crops. These, however, in general, also report that hormone-
induced fruits, though normally pollinated, have a lower seed
content. For example, drench spraying with aqueous solution
of naphthoxyacetic acid (20 ppm.) applied to strawberries
when in full flower, has been reported by Swarbrick (45) to
result in a 17$ increase in yield. He reports, however,
that the fruits did not have the normal number of seeds and
that the increase in yield was due to increase in size rather
than the number. Burrel and Whitaker (10) used 1$ indole-
acetic acid in a lanolin paste with success in obtaining an
increased fruit-set with muskmelons. The low fruit-set in
muskmelons, due to heavy flower drop, is checked by the
application of hormone to one lobe of the stigma. They
suggest that this method may be useful to the plant-breeders
who are interested in a larger number of fruits by hand-
pollination.
Hormones as an Aid in Hybridization by Checking Incompatj-


TABLE 3
Result of Attempted Crosses between lunatus and a Wild Lima Bean
Kind of oross
Treatment No.
of crosses
made
No. of sets
obtained
Lima bean (P.
lunatus)
1.
2,4-D l/2 saturation in
Karo with puncture
10
-
X
2.
2,4-D l/2 saturation in
Karo without puncture
23
2
Wild lima
3.
2,4-D 1/4 saturation in
Karo without puncture
4
2
4.
2,4-D 0.5$ in lanolin in
a scratch
32
4
5.
Para-chlorophenoxyacetic acid
saturated in Karo
4
1
6.
Control, Karo alone
37
6


116
LITERATURE CITED
1. Addicott, Fredrick T. 1943. Pollen germination and
pollen tube growth as Influenced by pure
growth substances. Plant Physio. 18 (2): 270-
79.
2. Allen, T. C. and E. H. Fisher. 1943. Plant hormones
increase yield of wax beans. Wis. Agri. Exp.
Sta. Bull. 460:54-55.
3. Amlong, H. U. and G. Naundorf. 1939. Wuchsstoff und
Pflanzenertrag. Forschungsdienst 7:465-482.
4. Avery, George S. and Elizabeth Bindloss Johnson. 1947.
Hormones and horticulture. McGraw Hill Book
Company Inc. pg 8-9.
5. Ibid pg. 186-205.
6. Batjer, L. P. and A. H. Thompson. 1948. A comparison
of naphthaleneacetic acid and 2,4-D sprays for
controlling preharvest drop of Bartletts'
pears. Proc. Amer. Soc. Hort. Sci. 51:71-74.
7. Batjer, L. P. and A. H. Thompson. 1949. The trans
mission effect of naphthaleneacetic acid on
apple drop as determined by localized appli
cation. Proc. Amer. Soc. Hort. Sci. 51:
77-80.
8. Boysen Jenson, P. 1910. Ueber die Leitung des
phototropisehen Reizes in AvenaKeimpflanzen.
Ber. Deut. Botan. Ges. 28:118-120.
9. Boysen Jenson, P. 1911. La transmission de l*irritation
phototropique dans l'Avena. K. Danske Videnskab.
Selskab. Forh. No. 3:1-24.
10. Burrel, P. C. and T. W. Whitaker. 1940. The effect of
indole-acetic acid on fruit setting in musk-
melons. Proc. Amer. Soc. Hort. Sci. 37:829-830.
11. Clark, H. E., W. C. Edmundson and P. M. Lombard. 1941.
Seed setting in potatoes as affected by spraying
alpha-naphthaleneacetamide and by light. Amer.
Potato Jour. 18:273-279.


50
ready been given. The different species of Phaseolus and
their sources, used in this study were as follows:
A. Phaseolus vulgaris (Cherokee Wax).
B. Phaseolus coccineus (Scarlet-runner) P. I. 165421.
Collected by Ware and Manning in Mexico.
C. Phaseolus coccineus var albus from 0. H. Parson
Hs
(Bartel "bush lima").
D. Phaseolus atropurnureus M557 from 0. W. Horwell,
E. Phaseolus filiformis 51-2 op from 0. W. Morvell.
F. Phaseolus calcaratus (Korean rice-bean, long day
form), from E. M. Meader, New Hampshire.
G. Phaseolus lathvroides M 248-1 op from 0. W. Morvell.
II. Phaseolus lunatus A Fordhook lima selection.
I. Phaseolus acutifolius 81 op 1 op from 0. W. Morvell.
J. Phaseolus acutifolius (white seeded) from Rex Thomas
Beltsville, U. S. Department of Agriculture.
K. Phaseolus acutifolius 257 from 0. W. Morvell.
Ii. Phaseolus angularis P. I. 174907.
M. Phaseolus angularis (Adzuki) from Rex Thomas, Belts
ville.
N. Phaseolus mungo from E. C. Stair.
O. Phaseolus aureus from E. C. Stair.
In all crosses attempted the amount of hormone prepa
ration used was within a range of 0.0004 0.0006 ml.,
applied with the special pipette. The chemicals were


3
of the flowers before fertilization and thus overcome the
above mentioned difficulties without inhibiting fertili
zation. Very little work has been done with regard to the
use of hormones in this respect but there is evidence in
the literature which suggests that hormones may be used with
advantage in hand-pollination to obtain a greater number of
fruits set and healthier seeds.
The results of investigations and trials with hormones
on beans with a view to obtaining various interspecific
crosses are reported in the following pages. The aim of
the study has been threefold. In the first place, as many
species as possible have been used as the staminate parent
in attempted crosses with the standard varieties of snapbean
in an effort to obtain hybrids which may show desirable com
binations of characters or may at least prove helpful in
bridging the gulf between them so that they may be later
used for still further crossing as one of the parents.
Secondly, an attempt has been made to study the effects of
different hormone treatments, their concentration and method
of application on hand-pollinated snapbean flowers with a
view to working out an effective treatment which would over
come some of the incompatibilities and difficulties involved
in obtaining good pod-set. Thirdly, the aim of this study
has also been to evolve a technique for applying the hormone
treatment which would permit measurement and regulation of
the quantity applied in order that the procedure might be re
peated.


Use of Growth Regulating Substances as an Aid
In Hybridization of Phaseolus
By
RAMESHWER PRASAD JYOTISHI
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
September, 1950


54
Experiment 2. Effeet on Fertilisation and Ovule Development
There is evidence in the literature that hormone treat
ment inhibits growth of ovules and their fertilisation.
Whether this tendency can be overcon by regulating the
amount and using proper chemicals was important to investi
gate. An experiment with snap bean, the parent used for
all crosses attempted in this study, was conducted in which
the buds were allowed to be self-fertilised. Ten buds of
uniform sise were subjected to each of the treatments, and
each treatment was replicated 4 times for statistical
analysis. The amount and method of application were the
same as with the species crosses. Hie results of this
experiment are reported in Tables 14 and 15. Hie entire
experiment consisted of treatment of 200 buds.
Experiment 3. Effective Range of 2.4-P Concentration
Although the preliminary studies showed that 2,4-D,
which caused quick responses, was likely to be too drastic
in effect, it was decided to work out the effective ranges
with respect to both amount applied in each application
and strength of the concentration used. With the help of
the special pipette it was possible to control the sise of
the drop with a higher degree of accuracy than is obtained