THE EFFECT OF CARBOHYDRATE AND OF NITROGEN DEFICIENCY UPON GROWTH, FLOWERING, FRUIT SETTING AND DEVELOPMENT OF THE MALE AND FEMALE GAMETOPHYTES OF MUSKMELON, CUCUMIS MELO L.j PEPPER, CAPSICUM FRUTESCENS L. j AND LIMA BEAN, PHASEOLUS LUNATUS L.
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
JAMES CLINKSCALES HOFFMAN, B.S; M.Sc. The Ohio State University 1952
Approved by:
Adviser i
TABLE OF CONTENTS
Page
INTRODUCTION ...... 1
REVIEW OF LITERATURE...... 2
MATERIALS AND METHODS ...... 13
Cytologic&l procedure ...... 17
PRESENTATION OF D A T A ...... 19
Climatological factors ...... 19
Relation of cultural treatments to plant vigor ...... 22
Microsporogenesis and megasporogenesis and the male and female gametophyte development as affected by the different cultural treatments ...... 36
Abscission before anthesis ...... 38
Proportion of flowers reaching anthesis and anthers sheddind pollen or microspores ...... 35
Abscission of ovularies and fruit after anthesis as affecting fruit set ...... 56
Relation of fruit harvest to the flowers reaching anthesis ...... 59
Pollen germination, staining, and abortion ...... 61
Pollination and fertilization as affected by the various cultural treatments ...... 64
Cytological development of the embryo sac...... 83
Stage of development of lima bean ovularies abscising before anthesis...... 83
Stage of embryo sac development on the morning of anthesis ...... 84
Stage of embryo sac development after morning of anthesis until setting of fruit ...... 86
Stage of embryo sac development at period of abscission...... 93
SP2S758 ii
Page
Stage of embryo sac development of fruit that set and enlarge...... 98
Fruit set during period of sampling for cytological study . • ...... 102
Refractive indices and per cent soluble solids of peduncle and ovulary sap at time of anthesis...... 104
Fruit and seed development...... 106
Muskmelon and pepper seed germination counts .... 107
Counts of lima bean fruit, seed and ovules degenerating ...... Ill
DISCUSSION OF THE DATA...... 113
Plant vigor ...... 113
Male gametophyte responses ...... 115
Abscission before and after anthesis ...... 115
Relation of treatments to phenotypic viability of the pollen ...... 117
Pollination and fertilization ...... 118
Female gametophyte responses ...... 120
Abscission before anthesis ...... 120
Abscission after anthesis as related to fruit set and development ...... 121
Relation of fruit harvest to the flowers reaching anthesis...... 123
Embryo sac development ...... 124
Stage of embryo sac development of lima bean ovularies abscising before anthesis ...... 125
Stage of embryo sac development on the morning of anthesis...... 126
Stage of embryo sac development after morning of anthesis until setting of fruit ...... 127 iii
Page
Stage of embryo sac development at period of abscission ...... 129
Stage of embryo sac development of fruit that set and enlarge ...... 132
Relation of per cent soluble solids of peduncle and ovulary sap to the setting of fruit ...... 133
Relationship between fruit and seed development .... 134
Muskmelon and pepper seed germination counts ...... 134
Lima bean ovules degenerated at the time of fruit maturity ...... 136
SUMMARY...... 137
Male gametophyte responses ...... 141
Female gametophyte responses...... 142 LITERATURE CITED...... US
APPENDIX ...... 153
AUTOBIOGRAPHY...... 165 iv
ACKN0W1EDGMENTS
The writer wishes to gratefully acknowledge his appre ciation to Dr. Freeman S. Howlett, Head of the Department of
Horticulture, The Ohio State University, for his constant counsel and stimulating suggestions given during the course of the re search and preparation of this dissertation.
The writer is indebted to the Ohio Agricultural Experi ment Station for financial support given as a fellowship during part of the investigations and for facilities furnished during the experiments conducted at Wooster. - 1 -
THE EFFECT OF CARBOHYDRATE AND OF NITROGEN DEFICIENCY UPON GROWTH, FLOWERING, FRUIT SETTING AND DEVELOPMENT OF THE MALE AND FEMALE GAMETOPHYTES OF MUSKMELON, CUCUMIS MELO L.j PEPPER, CAPSICUM FRUTESCENS L.j AND LIMA BEAN, PHASEOLUS LUNATUS L.
INTRODUCTION
The abscission of ovularies before and after anthesis, also the developing fruits of muskmelon, pepper and lima bean often causes the grower great concern by reducing yields and final profits. This abscission of ovularies and young fruit generally ©coin’s each season but more severely some years, and with no degree of regularity between varieties. The factors affecting fruit set and development of these economic plants have not been definitely established. There has been much research carried out concerning the factors affecting fruit set and development of the tomato and apple. Cochran (10) has reported his findings on several factors which affect the fruit setting of the pepper. Very little work has been done in studying the factors which affect the fruit setting and development of the muskmelon and lima bean. The objective of the research herein described has been to de termine the effects of carbohydrate and nitrogen deficiencies upon microsporogenesis and megasporogenesis and the development of the gametophytes in the muskmelon, pepper and lima bean. Klebs* early re search and theory (26-31) suggest that carbohydrate and nitrogen metabolism is very important in the change from the vegetative to the reproductive phase of growth in plants. Since that time other con siderations have been shown to be involved, such as the interpretation given to the results of Sachs (47) of hormones being translocated through the plant to the flower primordial regions and that such - 2 -
materials necessary for fruit formation were produced in the leaves
in the presence of light.
REVIEW OF LITERATURE
Klebs (26-31) explained the behavior of plants by three
general conditions: inheritance, internal relations, and external
conditions. He has summarized his work by proposing the principle
that plant development is determined by internal relations, which, in
turn, may be changed by external conditions. For example, the quan
tity of available nutrients, especially those of a nitrogenous nature,
and light intensity, with their effects upon the synthesis of carbohy
drates, are the controlling factors in determining the type of plant
development. Nightingale (42) concluded in some of his later research
es on nitrogen nutrition of green plants that the organic nitrogenous
and carbohydrate constituents of the ,• nts can scarcely be considered
to be the immediate cause of flowering, but intelligent steps may be
employed in nitrogen nutrition to decrease or increase vegetative growth
and to hasten, delay or entirely eliminate flowering responses under many different conditions of environment. He (43) has used color
standards to correlate carbohydrate reserves in relation to nitrogen
nutrition in the pineapple, and indicates that this method has supplied
a sufficiently precise index of the carbohydrate content. Fischer (16)
studying the effects of varying amounts of carbon dioxide upon vege
tation and reproduction in plants, found that the supply of available
carbohydrates are relatively high in relation to the available nitro
gen, the plants were reproductive, and when the reverse was true the
plants were vegetative. Petri (45, 46) found that sterility in olives - 3 -
is largely the result of a lack of sufficient nitrogen. While a de
ficiency of nitrogen may cause a great number of flowers to form, the
ovularies fail to grow as fruit with fertilized egg cells. Branches that set fruit contained 2.12 to 2.37 per cent of nitrogen on a dry weight basis, but the unfruitful branches contained from 0.72 to 0.92 per cent nitrogen. He pointed out clearly that the conditions favor
able for fruitfulness do not always coincide with the best conditions
for flower development.
Lambeth (34) has shown that heavy side-dressing applications
of soluble nitrogenous fertilizers during the blossoming period in hibited pod set of the lima bean. The same author showed that the
average daily percentage set in snap beans during the first three weeks was as follows: 100 pound nitrogen level, 22.7 per cent, 200 pound nitrogen level, 26.9 per cent, and 19.4 per cent for the 400 pound nitrogen level. Where the 400 pound nitrogen application was used the detrimental effect was very serious for a period of approximately one week following the application.
Kraus and Kraybill (32) studied the vegetation and reproduc tion of the tomato and demonstrated Klebs’ theory by the control of fruiting. They showed that a plant may be changed by an environment that causes chemical changes within the plant. They recognized four
conditions that have a decided effect upon the behavior of the tomato plant with respect to fruit set. (1) An exceedingly low carbohydrate- nitrogen relationship accompanying a weekly vegetative condition. In this type, carbohydrates appear to be the limiting factor of growth, and the low relationship is due to the small amount of carbohydrates - u - present while the amount of nitrogen appears to be an indifferent fac tor. (2) A low carbohydrate-nitrogen relationship accompanying a vigorous vegetative condition. In this type there appears to be an available supply of both carbohydrates and nitrogen, but the balance between them is such as to produce the best vegetative conditions and
carbohydrates are not adequate for fruit set and development. (3) A high carbohydrate-nitrogen relationship accompanying abundant fruit set and production. In this type nitrogen compounds are available, but the high relationship is due to an excess of carbohydrates. (A) A very high carbohydrate-nitrogen relationship accompanying a weekly vegetative condition. Here nitrogen appears to be the limiting factor of growth and the high relationship is apparently due to the small amount of nitrogen present. Kraus and Kraybill have thus shown that the relation of the carbohydrate content of the tomato to the amount of nitrogen available strongly influences the vegetative and fruit
setting responses. Fruitfulness is associated neither with highest nitrates nor highest carbohydrates, but with a condition of balance between them.
Bushnell (7) stated that a plant normally bears many more flowers than it matures as fruits, and with the Hubbard squash a large percentage of the earlier flowers drop.
Dorsey (12) concluded that in the potato physiological in fluences operating independently of pollen or pistil development cause the ovularies to drop. He observed disintegration in some embryo sacs.
Results of Woo (59) with Amaranthus retroflexus and of Work
(60) with the tomato have shown that a large supply of nitrates in - 5 - the soil does not always force these plants out of the reproduction phase of growth. This would depend upon how large the supply was and the association of other climatic factors with the utilization of ni trogen.
Gurjar (17) concluded that the supply of nitrogen deter mined the relative proportion of carbohydrates and proteins in the tomato. Although the C/fa relationship was as low as 2 and as high as
19, fruiting took place only between the ratios of 4 and 6. These exact ratios should not be used; a relationship basis would be more desirable. Harvey and Murneek (19) studied the C/N relationship of apple spurs and concluded that spurs totally defoliated can set and mature a considerable number of fruits. The carbohydrates were ap parently still high in such spurs even after defoliation. In this respect the different varieties showed wide differences, but gener ally the defoliation decreased fruit set. A rather consistent in crease in the C/N relationship was effected after defoliation by de creasing the nitrogen. Young (61) found that varying environmental conditions may cause degeneration of the ovules and embryo sac in the potato at any stage of development.
Murneek (41) was unable to show any marked effect of repro duction on the rate of vegetative growth in the spider flower, Cleome sninosa. except in low nitrogen plants. The early developed fruits were directly responsible for the suppression of further flowering in this plant. The non-fruiting cycles were characterized by a periodic abortion of ovules. By destroying the first flowers, the spatial ar rangement of the alternating sterile and fertile cycles were interchanged.
Bradbury (5) indicated that degeneration of the ovules of sour cherry often began before fertilization. When cold killed a large proportion of the flower buds, the percentage of blossoms which set fruit was ordinarily larger than in other years. Mechanical thin ning of the flower buds increased the per cent set from 24 on unthin ned branches to 42 per cent on thinned branches. These results indi cate that the dropping of fruit of the early Richmond and Montmorency sour cherry varieties is closely correlated with the available food supply, and a stimulus acting upon the carpel or ovule, such as polli nation, fertilization, or embryo development. Kraybill (33) again working with the tomato found that the number of blossoms per cluster, the number of fruits which develop and grow per cluster, the number of fruits which remain undeveloped per cluster, and the number of blos soms which drop, can be influenced by modifying the amounts of avail able mineral nutrients under conditions of uniformity with reference to other external factors. The effectiveness of the treatments could be ascribed to changes in nitrogen level.
Cochran (8, 10) has studied the pepper and shown that plants which receive high-nitrogen treatments set a significantly higher per centage of blossoms them did plants receiving low-nitrogen, and that a combination of high nitrates and high soil moisture produced more flowers and a larger total number of fruits than did any other treat ment. The highest percentage of fruit set was obtained under long-day conditions. This increase in set of fruit, resulting from frequent applications of nitrate of soda, is in agreement with results of - 7 -
Woo (59) working with Amaranthus. This is in disagreement with Lloyd
(35) who worked in Illinois under field conditions and found that treatments of nitrate of soda in addition to irrigation were detrimen tal to fruit setting in the pepper. Apparently variations in light in tensity and temperature account for the differences between the results of Lloyd (35) and Cochran (10).
Binkley (4) has shown that the bean apparently produces many more blossoms than the plant could support as developed fruit if all the flowers were fertilized and pod development completed. The per cent of blossom and pod drop on the different varieties varied between
44*25 and 76.21 per cent. His data indicate that the blossoming and setting period appears to be a critical time when the bean plant is especially affected by wide variations in environmental conditions.
Howlett (23) shows that carbohydrate deficiency resulted in the suppression of the male organs, the production of microspore de generation and pollen sterility in the tomato, Lvcopersicum esculentum
Mill. In his experiments nitrogen deficiency affected microsporo- genesis and the development of the male gametophyte relatively little.
Nightingale and Farnham (44) grew sweet peas under varied nutrient concentrations and showed that plants which were relatively succulent had a high percentage of flower buds that abscissedj those plants that grew less succulent had a lower percentage of flower bud abscission.
Watts (54) in a detailed study of the tomato has shown that a condition of moderate vegetativeness is more closely associated with fruitfulness than extreme woodiness and a weak vegetative condition. - 8 -
Associated with moderately vegetative, but a very fruitful condition is a balance within the plant of carbohydrates and amino-nitrogen con tent. Extreme vegetativeness and non-fruitfulness is associated with relatively low carbohydrate content and high amino-nitrogen content.
Woody and weakly vegetative growth and non-fruitfulness is accompanied by low amino-nitrogen content, while low carbohydrate content, espec ially starch, is accompanied by moderate to high amino-nitrogen content.
Watts (55) has increased plant vigor of the tomato by partial deflora tion, and by removing the later forming clusters, the early yields, total yields and fruit size of the remaining clusters were increased.
Auchter and Harley (3) found that peppers were not particu larly sensitive to light intensity or length of daily illumination.
Shading with cheese cloth did not change the time of blossoming of the normal-day plants in the high nitrate series. The lack of nitrates de layed blossoming of peppers decidedly and length of day did not alter this condition. Shirley (50) found that the dry weight produced by tomato plants is almost directly proportional to light intensity up to about 20 per cent of full summer sunlight, and fruiting did not occur at all when the intensity fell below 8 per cent of full summer sunlight.
Watts (54) has shown that very vigorous and succulent tomato plants were unfruitful because the abortive blossoms and small "nubbins" that were produced during a period of limited light, grew and produced seedless "puffs" after the weather became less cloudy and the days longer. The results of these experiments also indicate that when the light conditions are unfavorable for the production of carbohydrates a limitation of the nitrogen supply may increase fruitfulness, - 9 -
©specially with respect to the percentage of the buds produced which set fruit. Cochran (8), working with pepper, found that vegetative growth was greater under the long day than under the normal length of day and fruit set was greater under normal day than the long days.
Howlett (23) has worked with the tomato in different seasons and found that under longer daylight periods during the fall more flowers reached anthesis and produced pollen of higher germinability than was the case with comparable clusters on the winter-grown plants.
Shaw and Sherwin (A9) and Hendry (22) observed that H m bean plants grew vigorously and produced many flowers but yield was low when the humidity was low. The low yield was associated with the abscission of the small pods.
Cordner (11) concluded that the blossom abscission of the
Henderson lima bean was associated with high air temperatures and dry atmosphere in Oklahoma, and that the detrimental effect of these con ditions depended upon the time of occurrence and the duration of such adverse conditions.
Andrews (1) found that under high temperature and low rela tive humidity lima bean plants failed to set and mature a normal crop of seed, but that under these conditions the Henderson variety pro duced a larger crop than did Fordhook. When these varieties were grown under relatively cool or moderate temperatures and high humidity,
Fordhook often yielded higher than Henderson. Andrews explained that the larger yields from Henderson as compared with Fordhook when grown under unfavorable conditions were associated with relatively greater root system and more efficient leaves of the Henderson variety as com- - 10 -
pared with Fordhook.
Lambeth (34) in controlled temperature experiments recorded
blossom drop and pod set of the lima bean, and the percentage pod set
was calculated for intervals of one week. In order to keep at a mini
mum the adverse effect of developing pods on subsequent set, the plants
were depodded weekly. Lambeth concluded that the minimum night tem
perature was as important as the maximum day temperature. The results
of Lambeth do not support the widespread belief that the failure to
set is indirectly affected by high temperatures. The set at 86° F.
under his conditions was not materially different from that at 72° F.
Buchholz and Blakeslee (6) using Datura stramonium, were
the first to correlate temperature with the rate of growth of the pol
len tube. The optimum temperature for pollen-tube growth in this
species was somewhere near 92° F. with a slight falling off in the
growth rate at 98.. 6° F. The growth rate at 92° F. was four and one-
half times that found at 52° F. Smith and Cochran (52) have shown
that temperature has a marked effect on the germination percentage of
tomato pollen, as well as on the rate of pollen-tube growth in the
style. The germination of pollen on the stigma reached a maximum of
66 per cent at 85° F. Germination was almost as high as 70°, was much lower at 50°, and exceedingly poor at 100°. The maximum germi
nation on the stigma at 50° was 21.5 per cent; at 100° the maximum wa3 6.3 per cent. The maximum rate of pollen-tube growth in the style
occurred at 70° F., with 85°, 50° and 100° ranging in decreasing
order.
Smith (51) found with the tomato that the temperature range - 11 -
approximately three days before anthesis appeared to have the great
est influence upon flowering. Blossom drop was greatly influenced by
hot dry winds and low humidity as well as by low soil moisture. On
10 plants under observation 47.4 per cent of the flowers aborted and
there was a lag of approximately three days between the time that the
temperature exerted an effect on blossom drop and the time that the
blossom drop was visible.
Cochran (10) concluded in his studies that unfavorable tem
perature and water supply were associated as basic factors in the drop
ping of buds, blossoms, and small fruits of the pepper. Low humidity
and high temperature resulted in excessive transpiration and a water
deficient plant, and under such conditions fruit failed to set. Low moisture supply in the soil was also an important factor associated with abscission. Forty plants held at 50° F. for about six months made very little growth producing only one flower that failed to set.
Plants that had produced blossoms at higher temperatures and were transferred to the 50 to 60° F. house at the time of anthesis set nearly 100 per cent parthenocarpic fruit.
In carefully controlled greenhouse experiments, Lambeth (34) found that the environmental factor which limited pod set of the lima bean to the greatest extent was available soil moisture. Pod set was lowered at soil moisture levels higher than field capacity or at levels approaching the wilting coefficient. Under field conditions
Lambeth has observed that frequent heavy rains at the beginning of the blossoming period seriously limited or completely prevented basal pod set. Lambeth did not show evidence but said that this condition was - 12 -
apparently caused by the rupture of the pollen grains prior to or
during germination, and that the difficulty was aggravated by close
plant spacing and by vigorous vegetative growth.
Considering the chain of events which take place before
the mature fruit is harvested, pollination and fertilization are cer
tainly vital factors to be studied in the ontogeny of the plant.
Muller-Thurgau (40) has shown that fruit set of the grape was evident
following the growth of the pollen tube into the style, and that fer
tilization was not necessary for a fruit to set, but carbohydrates
were necessary for the growth of the pollen tube into the pistil.
Evert (14, 15) working with the apple and pear in relation to fruit
set and food supply, indicated that fertilized ovularies have a great
nutritive advantage over the unfertilized, even though some ovularies
develop as partheno car pic fruit.
In the last decade considerable information has accumulated
on the effect of growth-regulating substances on fruit and seed de
velopment of several important horticultural plants. Since excessive
abscission of blossoms and small pods of certain varieties of lima
beans is usually directly associated with reduced yields, Hardenburg
(18), and Wester and Marth (56, 57) have tried growth-regulating sub
stances on limas with the object of reducing abscission and increasing
yield. Wester and Marth (57) reported increased yields t»y the use of
growth-regulating substances when the base of the lima bean flower was
scratched with a dissecting needle and indolebutyric and parachloro-
phenoxyacetic acids in lanolin paste was applied to this area. The
majority of treated flowers which later abscissed remained attached - 13 -
2 to 3 days longer than untreated flowers that abscissed. The number
of successful crosses was increased from 18.7 per cent to 28.8 per
cent. The treatment was also associated with a significant increase
in the average number of seed per fruit, from 1.95 to 2.43.
Whitaker and Pryor (58) reported that about one-third of
artificially pollinated flowers of the muskmelon set fruit. A sig -
nificant increase of about 22.0 per cent in the set of fruit was ob
tained by use of 4-chlorophenoxyacetic acid.
MATERIALS AND METHODS
These researches were conducted in 1940-41 in the green houses, gardens, and laboratories of the Department of Horticulture and Forestry at the Ohio State University at Columbus, and in 1946 in
similar locations of the Department of Horticulture, Ohio Agricultural
Experiment Station at Wooster. Two varieties of each vegetable have been studied, namely, Hearts of Gold and Honey Dew muskmelon, Hungarian
Wax and California Wonder pepper, and the Henderson and Fordhook U m bean. Uniform seed stocks of each variety were used in all of this work. They all proved to be highly homogeneous.
Brookston and Wooster silt loam soils were used for the
1940-41 and 1946 experiments respectively.
In Table 1, the date of planting and final harvest of all eaqperiments for the three kinds of vegetables are given. - 14 -
TABLE 1. - DATE OF PLANTING AND CONCLUSION OF EXPERIMENTS AS CON DUCTED, 1940-41 AND 1946
Location Kind Studied Date Planted Final Harvest Columbus Muskmelon June 20, 1940 October 15, 1940 Columbus Pepper June 28, 1940 October 16, 1940 Columbus Lima bean June 15, 1940 October 15, 1940 Columbus Muskmelon May 21, 1 9 a October 15, 1 9 a Columbus Pepper April 11, 1 9 a October 15, 1 9 a Columbus Lima bean May 21, 1 9 a October 15, 1 9 a
Wooster Muskmelon June 51 194-6 October 10, 1946 Wooster Pepper April 2, 1946 October 4, 1946 Wooster Lima bean June 5, 1946 October 5, 1946
The muskmelon and lima bean seed were planted directly in the field in all series of experiments. Plants for the pepper experi ments were started from seed sown in flats in rows 2 inches apart and placed in a greenhouse averaging approximately 80° F. Twenty-three days after planting the seedlings were transplanted to 4-inch pots, one plant to a pot and finally shifted to the gardens 30 days later.
For all kinds and varieties, 10 plants of each has been considered a sample in each year. Five extra plants were established in addition to the 10 for tissue tests and general histological work where active growing ovularies and fruits were removed. The pepper plants were se lected and graded for uniformity before being transplanted in blocks
2 by 3 feet apart. The muskmelons were thinned to three plants per hill in blocks 6 feet square. The lima beans were thinned to 4-inch spacings in the drill and the rows were 36 inches apart. The two va rieties of each kind were alternated in the rows to minimize any soil or water variations that might cause differences in plant response. - 15 -
There were three separate cultural treatments used in these experiments. Each treatment duplicated the conditions often occurring in commercial plantings that appear to he associated with fruit set and development in the muskmelon, pepper and lima bean.
1. All varieties and kinds were grown under field conditions with a fertilizer application of 150 pounds of phosphoric acid, and
100 pounds of potash to the acre. An attempt was made to maintain the nitrogen level below 10 p.p.m. by the incorporation of wheat straw in the soil. No nitrogen was added in the fertilizer.
2. All varieties and kinds were grown under a cheese cloth house with a fertilizer application of 200 pounds of nitrogen, 150 pounds of phosphoric acid, 100 pounds of potash, and 10 tons of manure to the acre. The nitrogen level was adjusted between 200 and 300 p.p.m.
3. All,varieties and kinds were grown under field condi tions with a fertilizer application of 200 pounds of nitrogen, 150 pounds of phosphoric acid, 100 pounds of potash, and 10 tons of manure to the acre. A nitrogen level between 200 and 300 p.p.m. was main tained as nearly as possible.
Cultural treatments 1 and 2 were used at Columbus and all three cultural treatments at Wooster.
The soil was tested at weekly intervals and nitrogen added if needed to maintain the desired concentrations. For these tests, diphenylamine solution, 0.12 of a gram of diphenylamine was dissolved in 100 milliliters of pure sulphuric acid. This solution, and color standards for comparison with nitrogen in concentrations ranging up - 16 - to 300 p.p.m. were freshly prepared for each test. All test samples were diluted to the same volume as the color standards. Tissue tests were compared with the soil tests; the only variation was the use of
75 per cent sulphuric acid.
The cloth houses used in these experiments were 6 feet in height, and covered with white cheese cloth 42 x 38 threads per inch warp and filler respectively. The light intensity in these houses as measured with a Weston light meter averaged 55.4 per cent of the normal, outside light throughout the growing seasons.
The soils at Columbus and Wooster varied from pH 6.0 to 6.5 respectively. Irrigation was not necessary at Wooster as adequate rain fell during the growing.season to maintain the soil moisture from
15 to 23 per cent. However, irrigation was used at Columbus during
1940 and 1941 to maintain adequate available water.
The striped cucumber beetle, Diabrotica vittata Fabricius, caused some minor damage to the muskmelons during the early stages of plant development but were controlled by dusting with 1 per cent rote- none mixed with talc and applied at the rate of 40 pounds per acre.
The red spider, Tetranvchus telarius Linnaeus, only appeared on the lima beans that were grown under cloth. Three daily applications of
1 per cent rotenone in mineral oil emulsified in water 1 to 400 gave satisfactory control. Apparently the eggs were not destroyed as there was often a re-occurrence of the adults.
Daily records were taken on all varieties grown with the dif ferent cultural treatments. For muskmelon, these records consisted of the number of male and perfect flowers reaching anthesis, male and - 1 7 -
perfect flowers not reaching anthesis, and total number of fruits
that set and developed. On pepper, counts were made of flowers reach
ing anthesis, flowers not reaching anthesis, and the total number of
fruits that set and developed. On lima beans, counts were made of
flowers reaching anthesis, flowers not reaching anthesis, number of
fruits that set and developed, number of seed developed, and total
number of aborted ovules.
Cvtological Procedure. From the experiments conducted at
Columbus pollen was germinated and stained daily, whereas weekly tests
were carried out at Wooster. Flowers that reached anthesis and lib
erated pollen from the microsporangia were examined at the time of
this dehiscence to determine the presence and viability of pollen on
the stigmatic surface and the growth of the pollen tubes through the
styles. Pollen of the muskmelon and pepper was germinated on culture
plates, whereas a greater percentage germination of lima bean pollen
took place in the microsporangia after placing the flowers in a moist
chamber at 80° F. for four hours. The pollen was later placed onto a
drop of saturated aceto-carmine and germination percentage calculated
by making section counts under the microscope. The culture medium
used for germination studies of muskmelon and pepper pollen consisted
of a mixture of 100 milliliters of distilled water, 1 gram of agar
agar and 5 grams of sucrose. This mixture was warmed on a water bath
until the agar agar dissolved, later stored in test tubes and placed
in a refrigerator at about 40° F. until needed. The culture medium was again warmed to liquid form as neededj and the right side of clean
slides was flooded with the culture solution, all surplus solution was - 18 - drained from the slide, leaving only a very thin homogeneous film. If the film was too thick, it tended to peel at a later stage or take up so much stain that the mount was rendered useless. After the material cooled and set, the pollen was sprinkled from the microsporangium onto the culture medium. The slides were then placed in petri dishes that were lined with moist filter paper after which time the pollen was in cubated at 80° F. until germinated. Pollen that did not stain with saturated aceto-carmine or analin-blue was considered non-viable as this type of pollen never germinated on the cultural plates or in the microsporangium of the lima bean. All stained grains did not germinate but all pollen that germinated stained.
For the study of pollen tubes in the styles and the stages of gametogenesis, ovularies or fruits with styles attached were col lected on the morning of anthesis before pollination, and at 1-, 2-, and 3-day intervals after pollination, for lima bean, pepper and musk melon respectively. Samples of ovularies or fruits were collected with styles attached, if possible, at first indication of abscission to de termine the stages of gametogenesis when abscission took place.
Location of ovularies and fruits with reference to their position on the plant was recorded at time of collection. The terminal flower was removed from the lima bean and in the case of collection of ovularies or fruits, one lateral flower was collected and one allowed to remain for development. Samples of muskmelon ovularies and fruits were collected from both the first and second axils of the fruiting branches. The pepper ovularies or fruits, on the main axis and from secondary branches, were collected for cytological study. - 19 -
Samples were taken of ovaries that were hand pollinated and also those that were normally pollinated as in nature.
Ten ovularies and fruit were fixed and killed in Navashin's fluid, dehydrated and infiltrated ty the tertiary buiyl alcohol method, and embedded in paraffin. Sections were cut 10 microns thick with a
Spencer rotary microtome, and stained with Heidenhain's iron-alum heimatoxylin as scheduled by Johansen (25).
Standard photomicrographic equipment at the Ohio Agricultu ral Experiment Station was used to take and develop all photomicro graphs.
Fruits from the three kinds were harvested at the stage of edible maturi-ty.
Any separate materials or methods employed in a single study will, be discussed in connection with the experiment involved.
• PRESENTATION OF THE DATA
T he experimental data are grouped according to year and lo cation (194-0 and 1941 at Columbus, 194-6 at Wooster) and crop. The data presented in detail are located in the appendix whereas only a summary form of the more elaborate tables are given in this section.
CLBIA.TOLOGICAL FACTORS
The climatological factors prevailing under the cloth house and in the normal outside environment are presented for comparison of results when analyzing the data for the three seasons. The average temperatures for each season as recorded by a thermograph during the critical growing period from planting until harvest are recorded in
Table 2. Even though the data in Table 2 show some differences be - 20 - tween the average temperature of the two locations and seasons, these
differences are considered very small and insignificant. The tempera
ture in the cloth houses was 2 to 4 degrees higher than the outside temperature during the hot part of the day. During moderate weather the temperature in the cloth houses averaged 1 to 2 degrees higher.
During the cool weather of September and October the cloth houses had a temperature slightly higher during daytime and lower at night by 1 or 2 degrees.
Average temperatures at Wooster as recorded by a thermograph during the period of fruit set and collection of samples for cytologi- cal study are listed in Table 3. The differences between the two lo cations were again very small, averaging approximately 1 degree for all periods of sample collection. The temperature factor as a variable of the two environments during fruit set and development is considered negligible and insignificant.
The soil temperature of the surface six inches at Wooster was cooler under shade with an average of 64..5 degrees F. compared to
69.5 degrees outside for the months of August and September.
TABLE 2. - AVERAGE TEMPERATURE AS RECORDED EE A THERMOGRAPH DURING THE CRITICAL GROWING PERIOD
Outside Cloth House Location Year Day Period Night Period Day Period Night Period (6 am -6 pm) (6 pm -6 am) (6 am - 6 pm) (6 pm -/6 am)
C olumbus 194-0 a .4. 68.3 79.2 68.8
Columbus 1941 73.8 60.1 70.5 61.1
Wooster 194-6 75.0 61.7 76.7 62.7 - 21 -
TABLE 3. - AVERAGE TEMPERATURE AS RECORDED BY A THERMOGRAPH DURING THE PERIOD OF FRUIT SET AND COLLECTION OF SAMPLES FOR CYTOLOGI- CAL STUDY, WOOSTER
Outside Cloth House Kind Day Period Night Period Day Period Night Period (6 am - 6 nm) (6 ot - 6 am) (6 am - 6 pm) (6 pm - 6 am)
Muskmelon. 75.8 63.9 76.5 65.6
Pepper 82.2 67.5 83.5 69.0
Lima Bean 78.0 65.9 78.6 66.8
TABLE A. - AVERAGE VAPOR PRESSURE, DEV/ POINT AND RELATIVE HUMIDITY AS RECORDED FROM PSYCHROMETRIC READINGS, WOOSTER
Environment Vapor Pressure Dew Point Relative Humidity ______(MM-Hg.)______(Fahrenheit)______(per cent)
Outside 10.07 52° AS.00
Cloth House 10.82 54° 51.00
The relationship of soil moisture to fruit setting and de velopment of the three kinds of vegetables studied is considered to be important. An endeavor was made to maintain the soil moisture between
65 and 85 per cent of the maximum field capacity at both locations.
The maximum field capacity was 23 and 25 per cent (dry weight basis) at
Wooster and Columbus respectively for the two soils used in the study.
Mulching with wheat straw was considered necessary at Columbus during both seasons to maintain the above soil moisture content. Soil mois ture conditions were near optimum at Wooster and mulching was unneces sary to maintain the soil moisture condition.
A series of light intensity readings made at two-hour inter vals in the cloth houses at both locations averaged 55.4- per cent - 22 - normal or a reduction of 44-*6 per cent light intensity throughout the daylight period. These readings were taken daily at the height of plant growth and averaged for three days during the period of anthesis.
The data presented in Table 4- are calculations of psychro- metric readings recorded at Wooster over a period of 25 days during the stage of anthesis. These data are considered representative in comparing the two locations for plant growth in all three years.
There was some slight variation in the actual vapor pressure of the two atmospheric environments which was considered a measure of the dif fusion pressure of water vapor from the plants. The temperature of the dew point and relative humidity vary with the air temperature.
Measurements of evaporation rates at Wooster were made with four calibrated atmoneters, two in each environment during the experi ments starting August 22, 194-6 and continuing for two months. The atmoneters were placed on stands with the porous clay spheres 16 inches from the soil. The porous clay spheres were examined daily and kept free of foreign matter. There was 51 per cent evaporation in the cloth house as compared to 100 per cent in the outside atmospheric experi mental area, or a reduction of 4-9 per cent evaporation in the cloth house as compared to the outside.
PETITION OF CULTURAL TREATMENTS TO PLANT VIGOR
It is necessary first to describe the plant characteristics of the three kinds of vegetables in order to compare the plants as grown under the three different cultural treatments. It will be noted later that extensive growth or formation of large numbers of leaf and branch axils is associated with the number of flower buds formed. - 23 -
The plant characteristics of both varieties of muskraelon are generally very similar. The plant is a hirsute, trailing annual, that may grow 10 to 1A feet in length under high nitrogen and reduced light conditions. The leaves are alternate, simple, palmately fine lobed, and somewhat angled when young, but become sub-cordate when fully grown. Tendrils are borne opposite the leaves. The plant consists of a primary stem that becomes procumbent upon attaining a length of
6 to 8 inches beyond the cotyledons. From the axils of the lower leaves arise A to 8 secondary stems, which, in their subsequent de velopment, may equal or surpass the main stem if the plant is extreme ly vegetative. From the first A to 6 leaf axils of these secondary stems may arise the same number of tertiary stems, which may, under favorable growing conditions, attain a length of A to 6 feet. On these tertiary stems, as well as on the outer portions of the second ary stems and on the primary stems, are borne short branches on which the bisporangiate flowers are formed, herein designated as flowering branches. In each leaf axil of the tertiary stems, as well as on the outer portion of the secondary stems and the main stem, is found a ten dril, a cluster of 3 to 5 staminate flower buds and a vegetative bud.
Staminate buds are found near the growing point of the branch, and one generally reaches anthesis before the growing tip is one inch beyond the node. The remaining staminate buds reach anthesis at intervals of
1 to 3 days thereafter, depending upon the rate of plant growth. At the time of anthesis of the first staminate flower, the vegetative bud in the same axil develops into a flowering branch. In the axil of the basal leaf of the flowering branch is borne a single flower bud that - 2U -
develops into a bisporangiate flower. This fruiting branch may cease
growing after the formation of the first flower, or it may continue
growth and form additional bisporangiate flowers as well as leaf axils.
Generally only two bisporangiate flowers reach anthesis on a single
branch, and if the flower on the first axil sets a fruit, the second
one usually abscises. Two bisporangiate flowers have rarely been noted
in a single leaf axil. The first flowering branches that form often
continue to grow and may reach a length of 6 feet. As the flowering
branches continue to grow, clusters of staminate flowers are produced
in each leaf axil together with a tendril. On vigorously vegetative
plants the flowering branches continue to develop, however, the last
ones that form generally fail to grow beyond a length of 6 or 8 inches.
The two varieties of muskmelon studied are andromonoecious, i.e., they bear staminate and bisporangiate flowers on the same plant.
The plant characteristics of both varieties of pepper are veiy similar in being erect, slightly compact and in developing forked branches repeatedly in a dichotomic manner. Both varieties are culti vated as annuals. The leaves are simple, flat and glabrous with entire margins. California Wonder has an ovate shaped leaf while those of
Hungarian Wax are slightly extended in length and narrow.
Generally a single flower bud is located in each axis fork but in both varieties two have been found to develop. The flower buds continue to develop as additional branches and axils are formed throughout the growing season. The flewers are bisporangiate and self pollination generally takes place if insects are prevented from visit ing the plants. - 25 -
Havis (20) has carefully described the plant habit of the
Henderson dwarf lima bean. He showed that in this variety the deter
minate type of growth could be changed to indeterminate by reducing
the light intensity.
The plant characteristics are generally similar in both va
rieties studied. The plants are annuals, with herbaceous stems that
become woody with age. Under normal conditions the plants are dwarf
with few branches but develop the runner habit as grown in a cheese
cloth house with the higher nitrogen level. Under this treatment the
plants of Henderson and Fordhook grew to 60 and 84. inches in height
respectively. Both varieties became prostrate and coiled from left
to right around supporting string. The stems branched from the third
to the fifth nodes but very seldom branched from later formed nodes.
The leaves are glabrous, and the leaflets of the trifoliate leaves
are cordate in shape. The inflorescence of the lima bean is a raceme,
the flowers having separate pedicels of approximately equal length.
The flower buds develop in the leaf axils in groups of three and in a
terminal inflorescence. The number of flower buds that form and reach
anthesis or abscise before anthesis depends upon the development and
extension of the primary, secondary and tertiary plant branches.
In comparing the plants as grown under the different cultu
ral treatments with reference to vigor and development of flower buds,
one must comprehend that there is a direct correlation between the
development of primary, secondary and tertiary plant branches with the total number of flower buds formed. Whether or not these flower
buds reach anthesis and develop mature fruit will be presented under - 26 -
the section on Gametophvte Responses.
In the experiments described, the general plant vigor of the
muskmelon, pepper and lima bean as grown under the three cultural
treatments are shown in figures 1 to 9 inclusive. The muskmelon, pep
per and lima bean plants were photographed 4 0 , 104 and 40 days res
pectively from the date of seeding. All plants grown with low nitrogen
of treatment 1 were weakly vegetative and grew slowly. Those plants
grown with high nitrogen and reduced light intensity of treatment 2 were extremely vegetative with a light green color, and extremely suc
culent growing points. Plants grown with high nitrogen of treatment 3 were vigorously vegetative with large, dark green stems and leaves.
Those plants grown under treatment 1 were nitrogen-deficient, appear
ing light green to yellow in color, grew slowly and produced the small est number of flowers and fruit. As evident by microchemical deter minations, the carbohydrate content of the stems and leaves was high, but nitrates were low. Plants of treatment 2 were carbohydrate-defi
cient, and high in nitrates, especially at the growing points. Starch, as indicated hy the iodine test, was extremely low in the active grow ing plant tips. Plants of treatment 3 were deficient in carbohydrates at the growing point but not 3 or 4 inches back from the meristematic area. The plants were moderately high in nitrates but not as high as those produced under treatment 2. -27-
Figure 1.- Muskmelon plants of treatment 1 growing in soil with a low nitrogen level, Wooster. — 28"
Figure 2.- Muskmelon plants of treatment 2 growing in .soil xrith a high nitrogen level and under reduced light inten sity, Wooster. ■29-
Figure 3»~ Muskmelon plants of treatment 3 growing in -soil with a high nitrogen level, Wooster. - 30-
Figure 4-- Pepper plants of treatment 1 growing in soil with a low nitrogen level, Wooster. ”31-
Figure 5.- Pepper plants of treatment 2 growing in soil with a high nitrogen level and under reduced light intensity, Wooster. -32-
Figure 6.- Pepper plants of treatment 3 growing in soil with a high nitrogen level, Woos ter. Figure 7.- Lima bean plants of treatment 1 growing in soil with a low nitrogen level, Wooster. “3 4'**
atigsaiafew PJS^I
Figure 3.- Lima bean plants of treatment 2 growing in soil with a high nitrogen level and under reduced light intensity, Wooster. -35-
Figure 9-- Lima bean plants of treatment 3 growing in soil with a high nitrogen level, Wooster. - 36 -
MICROSPQROGENESIS AND MEGASPOROGENESIS AND THE MALE AND FEMALE GAMETO- PHYTE DEVELOPMENT AS AFFECTED BY THE, DIFFERENT CULTURAL TREATMENTS
In the three species of plants studied, sporogenesis and the gametophyte development proceeded in the regular sequence. Four micro spores are formed try two divisions of the microsporocyte, each nucleus thus has the haploid number of chromosomes. The male gametoplyte de velopment begins when the microspore nucleus divides forming a genera tive cell and tube nucleus. The generative cell divides and forms two male gametes, this division generally takes place at about the time of anthesis, or just after pollination during the period of pollen tube growth in the style. This sequence of development appeared similar for the varieties of the muskmelon, pepper and lima bean studied.
The embryo sac development of muskmelon, pepper and lima bean is of the monosporic-normal type. This type of embryo sac has been critically studied by Schnarf (4-8) and Maheshwari (3 6 , 37 and
38). The lima bean ovary generally contains from 3 to 5 ovules, al though the muskmelon and pepper usually contain several hundred
(Table 22). In all three species a subepidermal cell enlarges and differentiates as a megasporocyte. This cell after two divisions forms four megaspores, each with the reduced chromosome number. Three of these disintegrate, leaving the one near the chalazal end, which enlarges and develops into the female gametophyte. This cell under goes three mitoses to form an eight-nucleate embryo sac. This re sults in four nuclei at each end after which one from each of the groups of four move to the center and are designated as polar nuclei.
The egg apparatus, consisting of an egg and the two synergids, is - 37 -
located at the micropylar end, the two polar nuclei lie in cytoplasm
near the center of the embryo sac, and the three antipodal cells, which
often degenerate before anthesis in these three plants, lie at the cha-
lazal end.
As previously noted, the usual development of the male
gametoplyte followed by pollination with final growth of the pollen
tube into the pistil are necessaxy before the megagamete unites with
the microgamete. In all cases observed in these three species the
polar nuclei first unite to form the secondary nucleus (figure 12 C,
F and G) while the male nucleus later fertilizes the secondary nu
cleus to form the primary endosperm nucleus. Figure 12 D and E show the stage of development just before the fusion of the polar nuclei.
Data are presented in Tables 5 to 13 inclusive (Appendix
Tables I to IX)that show the total number of flower buds formed, in
cluding both flowers reaching anthesis and those that drop before anthesis. The total plant growth is represented by these data, whether or not the flower buds developed fruit or abscissed before or after anthesis. There are no exceptions to this trend of greater flower bud formation with greater vegetative growth. This can be seen in the data presented as listed under total number of flower buds for all plants or average per plant. For example, as presented in Table
5, the Hearts of Gold muskmelon produced a total number of U ,896 and
8,506 staminate flower buds under treatments 1 and 2 respectively.
These tables present data showing those flower buds reaching anthesis and those that dropped before anthesis. The plants grown under these
treatments were classified as weakly vegetative and extremely vegeta- - 38 - tive respectively. All treatments for muskmelon, pepper and lima bean may be compared with reference to plant growth as has been pre sented in the case of the Hearts of Gold muskmelon. There was a defi nite positive correlation between plant growth and the number of flower buds formed on all varieties of the three kinds of plants studied.
Abscission Before Anthesis
Muskmelon is the only plant in this study that has stami nate flowers; these were studied in addition to the male parts of the bisporangiate flowers. The number of muskmelon flower buds listed as having dropped before anthesis (Tables 5> 6 and 7) are complete counts for the entire growing season. Microsporogenesis and the development of the male gametopbyte was consistently suppressed in the bisporan giate flowers of the muskmelon as compared to the staminate flowers.
The per cent of total drops of staminate buds as compared to bispo rangiate buds before anthesis was significantly lower under all treat ments. This can be observed in all the data as associated with flower abscission of the muskmelon, and is illustrated very definitely in
Table 7 with the Honey Dew muskmelon as grown with high nitrogen and reduced light intensity of cultural treatment 2. In this example
6.A per cent of the staminate buds dropped before anthesis, compared to 75.8 per cent bisporangiate buds. When the plants were growing rapidly, appearing succulent at the growing tips, a large percentage of the flower buds dropped.
Degenerated or undeveloped anthers were observed in the ma jority of flower buds that failed to reach anthesis. The outer wall, nutritive tapetum and central mass of sporogenous cells collapsed and - 39 - degenerated before the meiotic division. Mierosporocytes were pres ent in veiy few of the flower buds, and when present, appeared dis
organized and shrunken with irregular shapes. A few degenerated microspores were found in a single flower bud of Hearts of Gold that dropped before anthesis. It cannot be said that this flower bud failed to develop normally and was not injured by some biological means. It can be said with certainty that these small flower buds, figure 13 (2), generally failed to reach the meiotic division. This condition of development has also been observed in the tomato by
Hewlett (23).
Since abscission before anthesis of the bisporangiate flower buds has been presented as related to the development of the male gametophyte, this subject will be continued only as related to the female gametophyte.
The data in Tables 5, 6 and 7 indicate the total number of muskmelon flower buds abscising before anthesis for the entire grow ing season as produced under the three cultural treatments. As in dicated by these data muskmelon plants produced under the conditions of treatment 1 formed fewer bisporangiate flower buds when compared with those plants grown under treatments 2 and 3. With the single exception of Hearts of Gold as grown under treatment 1 at Wooster, the total number and per cent of bisporangiate flower buds abscis ing before anthesis were lower than the number and per cent abscis ing under treatments 2 and 3. The nitrogen deficiency was extreme under treatment 1 at Wooster, where A5.2 per cent of the bisporangiate TABLE 5. - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF MUSKMELON CROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1940
CULTURAL TREATMENT 4l CULTURAL TREATMENT #2 Honev Dew Hearts of Gold Honey Dew Hearts of Gold Period Flowers Dropped Flowers Dropped Flowers Dropped Flowers )rop])ed Reaching Before Reaching Before Reaching Before Reaching Jefd*e Anthesis Anthesis Anthesis Anthesis Anthesia Anthesis Anthesis mth(23 is Flower type** U PM P M PMPH PMPMP MP
Total 697 31 63 15 4802 278 94 89 8659 101 460 197 8107 245 *98 J75 Average Per Pleat 69.7 3.1 6.3 1.5 480.2 27.8 9.4 8.9 865.9 10.1 46.0 19.7 810.7 24.5 39.8 27.5 £ Per cent of Total 91.7 67.4 8.3 32.6 98.1 75.8 1.9 24.2 95.0 33.9 5.0 66.1 95.3 47.1 4.7 52.9 f Number Fruit Set 18 50 10 39 Per cent Set of Flowers Reaching Anthesis 58.1 18.0 9.9 15.9 Per cent Set of All Ovularies 39. 1 13. 6 3 .4 7. 5
* 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity.
** M - Staminate, and P - Bisporangiate. TABLE 6. - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF MUSKMELON CROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1941
^CULTURAL TREATMENT ^1 CULTURAL TREATMENT 42 Honey Dev ! Hearts of Gold j Honey Dev S Hearts of Gold Period Flowers Dropped Flowers Drop]sed i Flowers Dropped Flowers Dropped leaching Before Reaching Befra’6 Reaching Before Reaching Before Anthesis Anthesis Anthesis Anthc53 iS Anthesis Anthesis Anthesis Anthesis Flower True** M PMPPM PM PM PM PMP I J. Total L120 81 138 38 5608 531 30 21 1270 82 1220 286 6935 487 268 127 Average Per Plant L12.0 8.1 13.8 3.8 560.8 53.1 3.0 2.1 127.0 8.2 122.0 28.6 69315 48.7 26.8 12.7 Per cent of Total 39.0 68.1 11.0 31.9 99.5 96.2 0.5 3.8 51.0 22 3 49.0 77.7 96.3 79.3 3.7 JO.7 Number Fruit Set 6 60 4 40 Per cent Set of Flowers Reach ing Anthesis 7.4 11.3 4.9 8.2 Per cent Set of 1 j All Ovularies 5.0 10.9 1.1 6.f
* 1. Lov nitrogen level. 2. High nitrogen level and reduced light intensity.
** M - Staminate, and P - Bisporangiate. TABLE 7. - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF MUSKMELON CROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946
^CULTURAL T REAT M E N T #1 CULTURAL TREATMENT #2 Honev Dew Hearts of Gold Honey Dew Hearts of Gold Period Flowers Dropped Flowers Dropped Flowers Dropped Flowers Dropped Reaching Before Reaching Before Reaching Before Reaching Before Antheelis Anthesis Anthesis Antilesis Antheesis Anthesis Anthesis Anthesis
Flower Tyne** M PM P M PM P MPM P MP MP
Total 1402 74 3 14 3175 138 1 114 4394 239 300 747 5904 966 188 549 Average Per Plant 140.2 7.4 0.3 1.4 317.5 13.8 0.1 11.4 439.4 23.9 30.0 74.7 590.4 96.6 18.8 54.9 Per cent of Total 99.8 84.1 0.2 1-5.9 100.0 54.8 0.0 45.2 93.6 24.2 6.4 75.8 96.9 63.8 3.1 36.2 Number Fruit Set 10 14 26 38 Per cent Set of Flowers Reaching Anthesis 13.5 10.1 10.9 3.9 Per cent Set of All Ovularies 11.4 5.6 2.6 2. 5
* 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity.
** M - Staminate, and P - Bisporangiate. TABLE 7. (continued). - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF MUSKMELON ffiCOTN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946
^CULTURAL TREATMENT #3
Honav Dev Hearts of Gold Period Flowers Dropped Flowers Dropped Reaching Before Reaching Before Anthesis Anthesis Anthesis Anthesis Flower Tvoe** HPHP MPM P
Total 1820 108 189 189 3321 205 209 281 Average Per Plant 182.0 10.8 18.9 18.9 332.1 20.5 20.9 28.1 Per cent of Total 90.6 36.4 9.4 63.6 94.1 42.2 5.9 57.8 Humber Fruit Set 17 23 Per cent Set of Flowers Reaching Anthesis 15.7 11.2 Per cent Set of All Ovularies 5.7 4.7
* 3. High nitrogen level. ** M - Staminate, and P - Bisporangiate. -u- flower buds abscissed before anthesis. The data also show an appre ciably large percentage of the flower buds abscising before anthesis as grown under treatments 2 and 3 where the plants were extremely vege tative under treatment 2 and at the growing points under treatment 3.
With one exception (treatment 1 at Wooster) the amount of abscission before anthesis is greater in the case of Honey Dew than with Hearts of Gold. Generally, the development of the female gametophyte of the muskmelon was severely reduced by extreme nitrogen deficiency.
In the experiments conducted at Columbus in 19-41 there was an extreme difference between treatments 1 and 2 in the case of Honey
Dew muskmelon. During the period of July 29 to August 25, 194-1
(Appendix Table II) the plants under shade and with high nitrogen were so extremely carbohydrate deficient that a total of 785 staminate flowers failed to reach anthesis and for the entire growing season a total of 1,220 staminate flowers failed to reach anthesis. As a result of this weakened condition ascribed to carbohydrate deficiency, only
51.0 per cent of the staminate flowers reached anthesis and shed pol len as compared with 89.0 per cent in the case of treatment 1. As presented in Table 14., only 10.1 per cent of this pollen germinated when produced under treatment 2 as compared with 15.6 per cent for treatment 1.
Flower buds of the pepper and lima bean that dropped before anthesis usually varied more in per cent of total buds and stage of development than did the flower buds of muskmelon. With the exception of the Henderson variety of lima bean the growth of the pepper and lima bean flowering branches, and in turn, the formation of the - 4 5 -
flower buds were more adversely affected by temperature changes than
by light or nutrition. The proportion of total flower buds that drop
ped before anthesis (Tables 8 to 13 inclusive) show considerable varia
bility in any single variety of vegetable as grown tinder treatments 1
and 2. These variations in microsporogenesis and development of the
male gametopbytes of the bisporangiate flower buds of these two spe
cies appear very similar in that they respond very definitely and
quickly to minute changes in the environment, especially to tempera
ture.
The California Wonder pepper (Tables 8 to 10 inclusive)
under treatment 1, had percentages of flowers dropping before anthe
sis of 87.3, 19.2 and 14.1 in 1940> 1941 > and 1946 respectively. The
high percentage of dropped flowers at Columbus in 1940 was associated with cool nights aS well as an extended period of rain and high
humidity.
Fordhook lima bean (Tables 11 to 13 inclusive) under treat ment 2, had percentages of flowers dropping before anthesis of 26.6,
48.7 and 18.5 respectively during the same years, whereas Henderson
in the same comparison had flower bud abscissions of 27.7, 30.0 and
26.0 per cent respectively for these years.
These data show the large variations of California Wonder and Hungarian Wax peppers and Fordhook lima bean to environmental
change, whereas Henderson lima bean is very unresponsive to small
changes in the environment. It will be noted that the experiments in
1940 at Columbus were conducted under much higher temperature than those conducted at Columbus in 1942 and at Wooster. TABLE 8. - SUMMARY CF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1940
*CUttURAt TREATMENT#! CULTURAL TREATMENT #2 Hungarian Wax California Wonder Hungarian Wax California Wonder Period Flowers Dropped Flowers Dropped Flowers Dropped Flowers Dropped Reaching Before Reaching Before Reaching Before Reaching Before Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis
Total 83 89 10 69 188 157 86 148 Average Per Plant 8.3 8.9 1.0 6.9 18.8 15.7 8.6 14.8 Per cent of Total 48.3 51.7 12.7 87.3 54.5 45.5 36.7 63.3 Number Fruit Set 21 5 a 8 Per cent Set of Flowers Reaching Anthesis 25.3 50.0 21.8 9.3 Per cent Set of All Ovularies 12.2 6. 3 11.9 3.4
* 1. Lew nitrogen level. 2. High nitrogen level and reduced light intensity. TABUS 9. - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER CROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1941
^CULTURAL TREATMENT#1 CULTURAL TREATMENT #2 Hungarian Wax California Wonder Hungarian Wax California Wonder Period Flowers Dropped Flowers Dropped flowers Dropped Flowers Jropped Reaching Before Reaching Before leaching Before Reaching 3efore Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Inthesis
Total** 828 141 2473 588 1096 365 1885 1236 Average Per Plant 92.0 15.7 274.8 65.3 121.8 40.6 209.4 137.3 Percent of Total 85.5 14.5 80.8 19.2 75.0 25.0 60.4 39.6 Number Fruit Set 149 334 47 74 Per cent Set of Flowers Reaching Anthesis 18.0 13.5 4.3 3.9 Per cent Set of All Ovularies 15.4 10.9 3.2 2 .5
*1. Lot nitrogen level. 2. High nitrogen level and reduced light intensity. **Based on 9 plants. TABLE 10. - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER 310HN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946
^CULTURAL TREATMENT &L CULTURAL T :lEATMENI^ Hungarian Wax California Wonder Hungarian Wax California Wonder Period Flowers Dropped • Flowers Dropped Flowers Dropped Flowers Dropped Reaching Before Reaching Before Reaching Before Reaching Before Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis
Total 920 116 391 64 2098 72 1251 52 Average Per Plant 92.0 11.6 39.1 6.4 209.8 7.2 125.1 5.2 Per cent of Total 88.8 11.2 85.9 14.1 96.7 3.3 96.0 4.0 Number Fruit Set 411 90 728 34 8 Per cent Set of Flowers Reaching Anthesis 44.7 23.0 34.7 27.8 Per cent Set of All Ovularies 39.7 19 .8 33.6 26 .7
* 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABLE 10 (continued). - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 194-6
"CULTURAL TREATMENT #3 Hungarian Wax California Wonder Period Plowers Dropped Flowers Dropped leaching Before Reaching Before Anthesis Anthesis Anthesis Anthesis
Total 1079 70 606 81 Average Per Plant 107.9 7.0 60.6 8.1 Per cent of Total 93.9 6.1 88.2 11.8 Number Fruit Set 432 171 Per cent Set of Flowers Reaching Anthesis 40.0 28.2 Per cent Set of All Ovularies 37 .6 24 .9
* 3 . High nitrogen level. TABLE U . - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1940
SB' U L m n TREAT TETT"#! FTT L 'fU T lT T I \ E A T M E NT#2 Fordbook Henderson Fordb pok Hende:rson Period Flowers Dropped Flowers Dropped Flowers Dropped Flowers Dropped Reaching Before Reaching Before Reaching Before Reaching Before Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis
Total 694 157 696 129 729 252 769 295 Average Per Plant 69.4 15.7 69.6 12.9 72.9 25.2 76.9 29.5 Per cent of Total 81.6 18.4 84.4 15.6 73.4 26.6 72.3 27.7 Number Fruit Set 88 117 126 93 Per cent Set of Flowers Reaching Anthesis 12.7 16.8 17.4 12.1 Per cent Set of All Ovularies 10. 3 14.2 12. ‘3 8. 7
* 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABLE 12. - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROTN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1941
*C » L T OT'L' (f R FT' t TT e n t 'TT fttJtlftlRAL TREATMENT #2 Fordl100k Henderson Fordllook Henderson Period Flowers Dropped Flowers Dropped Flowers Dropped Flowers )ropped Reaching Before Reaching Before Reaching Before Reaching before Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Inthesis
Total 672 130 707 67 495 470 285 122 Average Per Plant 67.2 13.0 70.7 6.7 49.5 47.0 28.5 12.2 Per cent of Total 83.8 16.2 91.3 8.7 51.3 48.7 70.0 30.0 Number Fruit Set 98 157 87 97 Per cent Set of Flowers Reaching Anthesis 14.6 22.2 17.6 34.0 Per cent Set of All Ovularies 12. 2 20.,3 9.,0 23. 8
* 1. Low nitrogen level.
2. High nitrogen level and reduced light intensity. TABLE 13. - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946
*C U 'L' T UR A L T REA*ttoENT#l c u t, i u ft'xr t R-m u e r r e Fordhook Henderson Fordhook Henderson Period Flowers Dropped Flowers Dropped Flowers Dropped Flowers Dropped Reaching Before Reaching Before Reaching Before Reaching Before Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis
Total 718 321 998 230 2033 462 1967 691 Average Per Plant 71.8 32.1 99.8 23.0 203.3 46.2 196.7 69.1 Per cent of Total 69.1 30.9 81.3 18.7 81.5 18.5 74.0 26.0 Number Fruit Set 87 170 220 286 Per cent Set of Flowers Reaching Anthesis 12.1 17.0 10.8 14.5 Per cent Set of All Ovularies 8.4 13.8 8.8 10. 8
* 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABLE 13 (continued). - SUMMARY OF REPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946
^CULTURAL TREATMENT #3 Fordhook Henderson Period Flowers Dropped Flowers Dropped Reaching Before Reaching Before Anthesis Anthesis Anthesis Anthesis
Total 631 706 1060 439 Average Per Plant 63.1 70.6 106.0 43.9 Per cent of Total 47.2 52.8 70.7 29.3 Number Fruit Set 94 229 Per cent Set of Flowers Reaching Anthesis 14.9 21.6 Per cent Set of All Ovularies 7.0 15.3
* 3 . High nitrogen level - 54 -
Degenerated microsporocytes and raicrospores of pepper and
lima bean were usually present in the anthers of flower buds that
dropped before anthesis. In a few of the more mature flower buds,
degenerated pollen grains were present. Pollen collected from the
anthers of flower buds that dropped before anthesis always failed to
germinate.
The data in Tables 8, 9 and 10 show the total number of
pepper flower buds abscising before anthesis for the entire growing
season as produced under the three cultural treatments. The per
centage abscission of flower buds from the weakly vegetative pepper
as grown under treatment 1 at Columbus in 194-0 and at Wooster in
194-6 was greater than those grown under treatments 2 and 3. The ab
scission of flower buds (Table 9) from plants grown under treatment 1
at Columbus in 1941 was less before' anthesis than from those of treat
ment 2. The total number of flower buds abscising increased as the
total number of buds increased, which was, of course, associated with
axil formation and an extension in growth of the branches.
Data in Tables 11, 12 and 13 indicate that with one excep
tion (Fordhook at Wooster) a larger percentage of the flower buds of
lima bean abscissed before anthesis under treatment 2 than under
treatment 1. At Wooster the abscission before anthesis of the flower
buds of Fordhook under treatment 3 was 52.8 per cent, compared to the
30.9 and 18.5 per cent produced under treatments 1 and 2 respectively. - 55 -
Proportion of Flowers Reaching Anthesis and Anthers Shewing Pnl 1 pm or Microspores
In all treatments daily counts of flowers reaching anthesis were made on the varieties of the three vegetables studied (Appendix
Tables I to IX inclusive). These data are presented in weekly totals.
Anthers in flowers reaching anthesis usually dehisced, shedding pollen and microspores. Whether the microspores were degenerated or the pollen was sterile or not will be discussed later. There was no predominat ing evidence of dichogany as associated with the male gametophytes of these plants. With very few exceptions, the anthers dehisced on the morning of anthesis. These exceptions were observed on mornings during late September and early October after rain or heavy dew, with tempera tures below 65° F. Later, in the afternoon period, when the humidily was lower and the temperature higher, the anthers shed both pollen grains and microspores.
There was no predominating trend of dichogany as associated with the female gametoplytes of these plants. With very few exceptions the bisporafigiat*: flower buds reached anthesis before 9:00 a.m. The exceptions were observed on mornings during late September and late
October after a very cool night with a minimum temperature of below
50° Fj later in the day the flower buds would reach anthesis.
Under the section, Abscission Before Anthesis. the total and percentage of flower buds that dropped have been presented. The percent age of flower buds that dropped before anthesis and the percentage that reached anthesis include all flower buds on a basis of 100. These data - 56 - have been calculated and listed as per cent of total flower buds
(Tables 5 to 13 inclusive).
Generally the Honey Dew variety of muskmelon has produced fewer bisporangiate; flowers than the Hearts of Gold variety. These results were consistent in all years and treatments. The plants under the high nitrogen and reduced light treatment produced the largest number of flowers, but on a proportional basis these flowers did not set fruit.
With the exception of the results secured at Wooster, the
Hungarian Wax pepper had a greater number of flowers that reached anthesis than had California Wonder. However, the percentage set of all flowers reaching anthesis was consistently greater for Hungarian
Wax.
There was very little difference between the number of flowers reaching anthesis for the two varieties of lima beans. The slight variation between seasons is associated with climatic factors as reviewed ty Andrews (l).
Abscission of Ovularies and Fruit After Anthesis as Affecting Fruit Set
The per cent of flower buds reaching anthesis is not neces sarily associated with the number or per cent that finally set and develop as mature fruit. The abscission of ovularies or young fruit of lima bean, pepper and muskmelon occurs regularly on the 2nd, Uth and 8th day respectively after anthesis. This date only varied slightly in the muskmelon since the ovularies or young fruits do not definitely abscise in some instances but remain on the plant and degenerate after double - 57- fertilization. The abscission layer was no more sharply defined when
16.7 per cent of the eggs were fertilized than when 100 per cent of the eggs were fertilized (Table 17). These data indicate that abscis- sed fruits did not necessarily have fewer eggs fertilized and that in the case of the muskmelon the formation of the abscission layer was not directly associated with a lack of fertilization of the eggs. Mann and Robinson (39) also working with the muskmelon indicated that abscis- sed fruits which showed early growth frequently lengthened at the same rate as fruits which matured, but that these abscising fruits ceased e- longation quite suddenly and always remained green, turgid, and attach ed for several days after growth stopped. Mann and Robinson also in dicated that although more extensive data are needed, there was no evidence that abscissed fruits had fewer eggs fertilized.
The data presented in Tables 5 to 13 inclusive give the total number of fruit that set and developed for the entire growing season for each year and for all treatments. The per cent of flowers that reached anthesis and later developed to mature fruit is comparable to the per cent of all ovularies setting, which include in the calcula tions both flower buds that abscissed before anthesis and those flow ers reaching anthesis. These data show the final effect of the three cultural treatments upon the fruiting of the muskmelon, pepper and lima bean.
The data in Tables 5, 6 and 7 at Columbus show that muskmelon plants as grown under treatment 1 set a greater percentage and number of ovularies than those grown under treatment 2 . Under conditions of treatment 3 at Wooster, both Honey Dev; and Hearts of Gold set the largest - 58 -
percentage of fruit of any treatment. The total number of fruit
setting does not always appear to be associated with the per cent
set of all ovularies, as the total number of bisporangiate flower buds formed are considered in the calculations. It will be noted that under conditions of treatment 1 at Wooster, significantly fewer fruit of Honey Dew or Hearts of Gold set as compared to treatments
2 and 3 (Table 7), but does not vary accordingly in percentage. These plants as grown under treatment 1 were extremely nitrogen deficient and were very weakly vegetative. Generally, the percentage of ovularies abscising after anthesis as grown under treatment 2 (Table 7) is un usually high for Hearts of Gold. Only 3.9 per cent of the flowers reaching anthesis set fruit, as compared with 10.9 per cent for Honey
Dew. When the per cent set of all ovularies is considered, which in cludes those that dropped before anthesis, both Honey Dew and Hearts of
Gold compare very closely in that 2.6 and 2.5 percent dropped respect ively.
The data as presented in Tables 8, 9 and 10 for the years
194-0, 1941 and 194-6 respectively, indicate that pepper plants as grown under treatment 1, with one exception (California Wonder, Wooster, 1946, treatment 2), set a greater percentage of ovularies, but during 1940 at
Columbus and 1946 at Wooster, treatment 2, the largest number of fruit set on both Hungarian Wax and California Wonder. The largest pepper plants were produced during the growing season of 1946 at Wooster under treatment 2, which is also indicated in Table 10 by the total number of flower buds formed. These plants were extremely vegetative, with -59-
Hungarian Wax setting a significantly lower percentage of its fruit than were set under treatments 1 and 3. With California Y/onder the per cent differences were not so great between treatments 2 and 3 > but somewhat lower under treatment 1. With one exception (Columbus
194-0> treatment l) there was a trend which indicates that the abscis sion in percentage after anthesis of California Wonder v/as larger as compared to Hungarian Wax.
The lima bean data as presented in Tables 11, 12 and 13 concerning total number of fruit setting and percentage set of those ovularies reaching anthesis were not consistent during all years.
The general trend was for a larger number and percentage of fruit to set that reached anthesis as grown under treatments 2 and 3. The one exception on a percentage basis is Henderson as grown under treatment
1 at Columbus in 194-0, where 16.8 per cent of the ovularies that reached anthesis set fruit, as compared to 12.1 per cent for treat ment 2. However, there were several exceptions to this condition in total number setting at Columbus (Fordhook, treatment 1 in 1941;
Henderson, treatment 1 both years at Columbus.)
With one exception (at Columbus in 1940, treatment 2) the abscission of ovularies and young fruits after anthesis was larger in both number and percentage in Fordhook than in Henderson.
Relation of Fruit Harvest to the Flowers Reaching Anthesis
The harvesting of mature fruit as related to the stimula tion of flowers reaching anthesis was studied at Columbus in 1941 and at Wooster in 1946. There was no increase in number of flowers reaching anthesis as a result of harvesting the mature fruit of - 60 - either muskmelon or lima bean.
In the case of pepper, however, as shown by the data pre sented (Appendix Tables V and Vl), a definite increase was observed in the number of flowers reaching anthesis after each fruit harvest.
Mature fruit were harvested (Appendix Table V, Columbus 194.1)
August 12, September 16, and at the end of the experiment. There was a very significant increase in number of flower buds reaching anthesis especially after the first harvest date. This condition was more prevalent with California Wonder than with Hungarian Wax and was more pronounced under treatment 2 than treatment 1. For ex ample, it will be noted (Appendix Table V, treatment 2) that 47
California Wonder flower buds reached anthesis from August 5 to
August 11. After the mature fruit were harvested on August 12, a gradual increase in number of flower buds reaching anthesis was ob served. This stimulation of flower buds reaching anthesis as. a re sult of the harvest of mature fruit was also found for California
Wonder as grown under treatment 1. The number of flower buds reaching anthesis from August 5 to August 11 were 16, and a gradual increase occurred after the fruit harvest on August 12. The second fruit harvest was not quite so stimulating to flower bud formation as the number of flower buds reaching anthesis increased only slightly. Mature pepper fruits were harvested (Appendix Table VI at Wooster) July 10, August 14» September 17, and at the end of the experiment. Especially after the first two harvest dates, there was an unusually significant increase in the number of flower buds reaching anthesis. This condition was again more prevalent with - 61 -
Calif ornis Wonder than with Hungarian Wax and was more pronounced under treatment 2 than under treatments 1 or 3. As indicated in
Appendix Table VI, treatment 2, California Wonder had 64 flower buds to reach anthesis from August 8 to August 14, but after harvesting
69 fruits August 14 > the number of flower buds reaching anthesis had increased to 201 for the period of August 22 to August 28. Hungarian
Wax, under the same treatment and dates, showed a slight increase of from 175 to 217 flower buds reaching anthesis.
Pollen Germination. Staining, and Abortion
The term, "grains," includes both pollen and microspores.
Because of the magnitude of the work involved in collecting these data it was most difficult to separate microspores or partially de veloped pollen grains from mature pollen. The data include both grains that appeared to be normal when stained with aceto-carmine and those that had germinated. As earlier described for the tomato by
Hewlett (23), there are in all three of these vegetables visibly aborted pollen grains. The percentage of visibly aborted grains and grains which stained but did not germinate may be calculated from the data presented in Table 1 4 . By subtracting the per cent stained from 100, the per cent not stained (visibly aborted grains) can be calculated. The per cent stained minus the per cent germinated gives proportion of grains stained but not germinating.
The majority of visibly aborted grains were raic r os pores.
These microspores appear colorless, shrunken and extremely irregular in shape, with nucleus and cytoplasm degenerated. A single degen erated grain is shown in figure 10,B, with a somewhat shrunken con - 62 - dition of the cytoplasm. In some of the experiments, this type of grain represents a very large number as shown by percentages in
Table 14, treatment 2 at Wooster. California Wonder pepper had 46.0 per cent of the grains stained and 54.0 per cent appearing visibly aborted. In this experiment California Wonder plants were extremely vegetative, and appeared with all the symptoms of carbohydrate de ficiency, figure 5 . As opposed to this in the same location where plants were grown under treatment 3 (figure 6 ) were vigorously vegetative with only a mild carbohydrate deficiency at the very grow ing tip, 56.0 per cent of the grains stained normally and 44.0 per cent were visibly aborted. California Wonder pepper, grown as nitrogen deficient plants, treatment 1 , figure 4 , produced 66.0 per cent grains that stained well and 34.0 per cent that were visibly aborted. Under treatments 1, 2 and 3 at Wooster, 48.0, 32.0 and
39.0 per cent respectively of the grains germinated.
It is of considerable interest to note in the case of Honey
Dew and Hearts of Gold muskmelon that even though the per cent of visibly aborted grains was small and the per cent stained grains was large, this did not necessarily mean that a large per cent of the grains germinated. Germination of muskmelon pollen was very low in all experiments but always averaged slightly lower under treatment 2 .
Plants grown under treatment 1 , nitrogen deficient and weakly vege tative, produced pollen that germinated usually as well or even higher than the pollen produced under treatments 2 and 3. The Ford hook lima bean was exceptional in consistently producing slightly TABLE 14..- PER CENT MUSKMELON, PEPPER, AND LIMA BEAN POLLEN GERMINATION TO GRAINS STAINED WITH ACETO- CARMINE
.... ! Honev Dew Hearts of Gold nusjsme-Lou j stained Germinated Stained Germinated *Cultural Treatment } 1 | 2 3 1 | 2 | 3 112 J 3 1 I 2 3 Columbus 1940 j 78.6} 86.0 19.3 } 12.3 ! 78.4- ! 82.0 16.61 12.6 Golumbus 19A1 1 67.3 75.0 1 5 .6 } 1 0 .1 } 67.4 175.0 15.11 13.5 Wooster 1946...... 1 72.0. . 78.2., 82-2 1 7 .0.1 70.1 1 78.0 80.8 16.0! K . 2_ M,3
Pepper !------■a. ------} Stained Germinated 1 Stained Germinated 1 T i n 1 0 1 } 1 ! 2 1.3 j I 2 1 3 1 1 .1 2 1 3 1 j 2 1 3 i Columbus 1940 1 76.0 1 70.0 \ 55.1 50.2 I 60.1 ! 57.2 j 51.3 i 45.1i Columbus 1941 i 86.0 { 76.0 1 66.2 60.1! 1 65.2 ! 63.1 i 56.4 1 47.2 ! Wooster 19£6 1 78.0 ! 72.0 1 74.2 56.0 1 59.1 1 66.0 1 £6.0 ! 56.0 48.0.1 32.0..1 39,0-
— Fordhook 1 Henderson Lima Bean Stained 1 Germinated 1 Stained Germinated 1 2 1 3 !l 12 i 3 il ! 2 3 1 2 .3 _ Columbus 1940 74.6 82.9 . 53.3 1 54.2 1 86.8 i 85.6 69.0 62.7 Columbus 1941 70.1 79.2 53.1 i 55.1 i 86.6 1 80.4 70.1 60.1 Eopster 1 9 4 6 ...... 72,0 8J,3 79,4 l 50.2J_52,1j 84,1 ! .79.2- 83,0 66.3 58.3 64.?
1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. - 64 - more germinable pollen when grown under treatment 2 .
Degenerated pollen grains were often found on the stigmatic surface; figure 10A, shows a single grain that illus trates this degeneration. The percentage of grains that stained but had failed to germinate is considered generally high; especially is this consistently true in the muskmelon. The percentage of stained pollen grains was generally higher for both varieties of muskmelon and for the Fordhook lima bean under conditions of reduced light- high nitrogen, and of high nitrogen; however, Henderson lima bean and the varieties of pepper under the first treatment consistently produced a higher percentage of stained grains. This large per centage of stained grains in proportion to the per cent germinated is variable between varieties and species of the plants studied.
All grains were usually degenerated in the anthers of a flower on the day of anthesis, figure 12A. This condition was very often observed in all anthers of the lima bean flower that reached anthe sis, shed grains but dropped two days after anthesis.
Pollination and Fertilization As Affected by the Various Cultural
Treatments
The muskmelon is chiefly insect-pollinated, usually by- honeybees . Pollen from both the bisporangiate and staminate flowers of the muskmelon are equally viable, therefore autogamous or geitonogamous pollination may occur.
The pepper and lima bean are both usually self-pollinated; however, the lima bean is frequently visited and often pollinated by honeybees. Erwin (13) conducted experiments on pepper which showed - 6 § - either self or cross-pollination may occur, and that the major por tion of the blossoms reached anthesis within three hours after sun rise. Cochran (10) has shown that pollination and fertilization is not essential for fruit setting in the pepper and that degeneration or abortion of the embryo sac in abscissed ovularies was associated with, but not the cause of, abscission. In all the experiments of the different treatments herein reported, pollination appeared complete and adequate. Where flowers had reached anthesis, grains were usually on the stigmatic surface in the late afternoon.
Honeybees visited the muskmelons growing in the cloth house and ap parently effected daily pollination. As a check on insect pollina tion, 100: muskmelon flowers were hand pollinated and isolated from bees. These hand pollinated flowers set 10 per cent, whereas 11 per cent set under' insect pollination on the same day as grown with high nitrogen and reduced light intensity of treatment 2 , indicat ing that hand pollination was not necessary.
For all species the germination of the pollen grains on the stigmatic surface and growth of the pollen tubes into the pistil did not always occur with equal regularity or rapidity. As has been mentioned, the visibly aborted grains and those stained but not germinating were apparent on the stigmatic surface in ap proximately the same proportion - 66-
* * - V -
-END
EMB EMB END
F - 4 - - E M B ■T^’-END DSN -67-
FIGURE 10. -PHOTOiVlICROGRAPHIC REPRESENTATION OF MUSKMELON GAME-
TOPHYTE DEVELOPMENT AND DEGENERATION UNDER VARIED ENVIRONMENTS:
A - Pollen grain on stigma with developing pollen tube growing in the pistil on morning of anthesis. B - Undeveloped
grains and degenerating pollen tubes on the stigma one day after
anthesis. C - Degenerated egg and synergids, two polar nuclei and
degenerated antipodals on morning of anthesis. D -Embryo and endo sperm six days after anthesis. E - Embryo and three endosperm cells nine days after anthesis showing slow development and in the initial stage of degeneration. F - Embryo and endosperm showing a slow rate
of development nine days after anthesis with an expanded embryo sac, the young fruit was undeveloped and undoubtedly would have dropped.
G - Embryo and endosperm eight days after anthesis with expanded embryo sac which indicates fertilization, fruit set and development.
H - Embryo and endosperm with expanded embryo sac eight days after anthesisj the fruit had developed to three-fourths of an inch in diamater before degeneration was observed, which indicates develop ment as in G. but degeneration is apparent by the affinity of the endosperm and embryo tissue for the stain. I - Embryo and endosperm nine days after anthesis showing signs of degeneration with a re tarded development and little expansion of the embryo sac or en largement of the fruit. J - Embryo and endosperm of a fruit that had not made normal development, nine days after anthesis, showing signs of degeneration after embryo sac expansion and development;
degeneration is apparent by the affinity of the endosperm and embryo tissue for the stain. K - Endosperm nine days after anthesis, - 68 -
FIGURE 10. - Con't. showing signs of degeneration after advanced development. L - Embryo sac seven days after anthesis with egg apparatus showing signs of degeneration and no division of the secondary nucleus. M - Secondary nucleus showing signs of degeneration seven days after anthesis with egg apparatus already degenerated. N - Embryo sac seven days after anthesis showing signs of slow development resulting in apparent abscission of the fruit. 0 - Embryo sac seven days after anthesis showing signs of slow development and expansion with degenerating endosperm resulting in apparent abscission of the undeveloped yellow fruit. P - Embryo sac eight days after anthesis showing normal de velopment of the endosperm v/ith apparent degeneration of the embryo; however, the fruit had developed normally but apparently this ovule would not have continued to develop and possibly abscission would have taken place later. Q - Embryo sac nine days after anthesis showing complete degeneration of contents.
X380 -69-
& — DPT
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-DPT
mm A i ^ % V ■ *rii
Z'tvZ. — EMB >^1T ;’»i* .vr *a,.--M-EMB EMB I7, 4 — Hfh— END r-END
m x s m M i- >*># -EMB >r- t» -EMB m
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F< *.■!(■ -70-
FIGURE 11. - PH0T0MICR0GRAPHIC REPRESENTATION OF PEPPER GAIvIIPTO—
PHXTE. DEVELOPMENT AND DEGENERATION UNDER VARIED ENVIRONMENTS:
A - Pollen tubes of Hungarian Wax growing in the style, showing signs of degeneration one day after anthesis. B - Pollen tubes of Hungarian Wax growing in the style as produced under treat ment 2 , showing signs of an advanced stage of degeneration two days after anthesis. C - Pollen tubes of Hungarian Wax 1.5 millimeters in length growing in the style as produced under treatment 2 , show ing signs of degeneration two days after anthesis. D - Pollen tube of Hungarian Wax 3.5 millimeters in length growing in the style as produced under treatment 2, showing signs of normal development two days after anthesis. E - Pollen tube of Hungarian Wax A.5 milli meters in length growing into the ovule one day after anthesis.
F - Pollen tube of Hungarian Wax as produced under treatment 3 , show ing signs of degeneration two days after anthesis. G - Pollen tube of Hungarian Wax as produced under treatment 2 , showing signs of degeneration as collected at first indication of abscission three days after anthesis. H - Egg and polar nuclei of California Wonder as produced under treatment 1, showing no signs of fertilization four days after anthesis. I - Embryo sac of California Wonder as produced under treatment 1, showing signs of complete degeneration of the egg apparatus on the morning of anthesis. J - Embryo sac of California
Wonder as produced under treatment 2, showing the normal development
of the secondary nucleus by the morning of anthesis; the egg had also developed normally. K - Developing embryo sac of California Wonder six days after anthesis showing delayed development of the embryo as -71-
FIGURE 11. - Con't. produced under treatment 1 , with the endosperm cells formed along the embryo sac wall. L - Developing embryo sac of California Wonder six days after anthesis, showing delayed development as produced under treatment 1, with the izygote not yet divided. M, N, 0 - Embryo sacs of California Wonder six days after anthesis as grown under treatment
1 , showing normal expansion and development, with the endosperm cells forming along the embryo sac wall. P, Q - Two sections of the same embryo sac of California Wonder taken from a fruit that had set and enlarged to 2 centimeters in diameter, as grown under treatment 1 , seven days after anthesis, showing division and enlargement of the em bryo and endosperm respectively. K - Embryo sac showing extreme en largement with several normal nuclei of the embryo seven days after anthesis and apparent fruit set. S - Embryo sac showing extreme en largement seven days after anthesis and apparent fruit set, with the developing embryo in the anaphase of mitosis. X3S0 -72-
AW . ^ -A I'*. H ' < — -E --DPG "‘l --PN • * v .— p t SN . #•< ■PG - * = £ • • • X'
D • i*V> i — - s —os .? * '•," • ■’ ’ * ■ — E ' \ **- ' -- e — W - s * ~ X . 4?..V ft 7-~ E S N •V.*. O — - P N — PN -EMB .1 A ------EMB ^ EMB ‘ * END V# . -■EMB ■ n t f ' — END J } i EMB -END —-END ' >' I s*- -Ar -ENO ■\* I e J M -END ••iViKij* ;’•* - I e n d m - y i i ■:;•»' i -EMB ------END N Q *■- ‘ .a< : f :■■;. • - • ? . i '*/ ■v&V.. ' O' ” : ■\.*.£ V-|^--EMB :V#r--EMB r ^ T T - - ™ -EMB .*•£.£ ■'— -S.---END f * .« * ??■ % - - A v END ’•■<* ■ A. A Li. END / ^ V------/ r ^ . — END • * . . • T -73- FIGURE 12. - PKOTOIyilCROGRAFHIG REPRESENTATION OF LIMA BEAK GAME- TOPHYTE DEVELOPMENT AND DEGENERATION UNDER VARIED ENVIRONMENTS: A - Pollen showing no germination two days after anthesis, ovulaiy abscissed with mature embryo sacs. B - Pollen germinating one day after anthesis as produced under treatment 2. C - Mature embryo sac of Henderson with egg and secondary nucleus formed on morning of anthesis. D - Mature embryo sac of Fordhook with egg showing signs of degeneration, and two polar nuclei before fusion on the morning of anthesis. E - Mature embryo sac with complete egg apparatus, and two polar nuclei fusing to form the secondary nucleus on morning of anthesis. F - Mature embryo sac of Henderson as pro duced under treatment 2, appearing normal on morning of anthesis with degeneration of synergids, egg present, secondary nucleus formed and antipodals no'longer present. G - Mature embryo sac of Henderson as produced under treatment 3, showing signs of degeneration of the synergids and of egg on morning of anthesis, with a secondary nucleus formed and the antipodals no longer present. H - Embryo sac on morn ing of anthesis showing signs of expansion, with enlarged egg and polar nuclei before fusion. I - Embryo and endosperm of Henderson one day after anthesis as produced under treatment 3, showing a rapid rate of development. J - Embryo and endosperm of Henderson two days after anthesis as produced under treatment 2, with developing embryo and endosperm. K - Embryo and endosperm of Henderson two days after anthesis as produced under treatment 3 > with rapid developing embryo and endosperm. L - Embryo and endosperm of Henderson two days after anthesis as produced under treatment 3, with an extremely rapid -74- FIGURE 12. - Con't. developing embryo and endosperm. M - Development of Henderson embryo sac three days after anthesis as produced under treatment 1. N, 0 - Same embryo sac of Henderson as produced under treatment 3> showing after three days, a rapid development of the embryo and endosperm respectively. P - Embryo of Fordhook six days after anthesis as pro duced under treatment 2, with embryo sac well developed and fruit apparently set and enlarging in size. Q - Stem end embryo sac of Henderson as produced under treatment 2, ovulary collected at first indication of abscission two days after anthesis, egg showing signs of degeneration and polar nuclei not fused. R - Stem end embryo sac of Fordhook as produced under treatment 3, ovulary collected at first indication of abscission two days after anthesis, egg and polar nuclei appear normal and enlarged but not fertilized. S - Embryo sac of Henderson showing slow development as produced under treatment 1, abscissed fruit collected two days after anthesis. T - Embryo sac of Henderson showing degeneration as produced under treatment 2, ab scissed fruit collected two days after anthesis. U - Embryo sac of Fordhook showing degeneration and disorganization of embryo and no endosperm development as produced under treatment 1, abscissed fruit collected two days after anthesis. A and B, X188; others X380. “ 75“ Figure 13,- Muskmelon fruiting branches. (1) normal development (a) degeneration and abscission apparent, Wooster. -76- as the data show in Table 14. However, it is very important to observe the degeneration of the pollen tube contents, illustrated in figure 11, A, B, C, F and G, as was so often apparent with pepper when grown under treatment 2 and to a limited degree under treatment 3. These degenerated pollen tubes usually appear with enlarged penetrating ends that contain large amounts of cytoplasm that accumulated in the end during the period of degeneration and disorganization. After this degeneration, such pollen tubes no longer grow in length in the pistil. In the sectioned style, figure 11A, approximately 120 degenerated pollen tubes were found in one 10 micron section. The pistils that con tained large numbers of such degenerated pollen tubes were produced under treatment 2 on extremely vegetative plants. The degeneration of muskmelon pollen tubes in abscissed pistils was very prevalent under conditions of treatment 2 (Tables 15 and 17), while this degeneration was observed to a lesser extent tinder treatments 1 and 3. The data presented in Tables 16 and 19, treatment 2, show that the lima bean ovularies, and fruits which abscissed were associated with the degeneration of the pollen grain and tube contents, and that this condition commonly occurred in the case of Fordhook but not to such a great extent with Henderson. • During the 3 years of the investigation in treatment 2 there were 5.1, 8.0 and 0 per cent of the eggs of Fordhook lima bean that were fertilized in the ovularies or fruits that definitely abscissed, whereas 41.0, 35.0 and 74.2 per cent of the eggs of Henderson were -77- fertilized. Lambeth (34) recently indicated that the frequent failure to obtain a satisfactory pod set with the large-seeded lima varieties such as Fordhook was attributed directly to inadequate fertilization of the eggs, and that in order to prevent abscission of the flower within the forty-eight hour period following pollination, it was necessary that at least one egg in the ovary be fertilized. Poor fertilization most frequently resulted from a failure of the micro gamete to reach the egg while the egg was receptive. The results of Lambeth are in agreement with those data presented for treatment 2 at Wooster, but not entirely in agreement with the results presented in all treatments at Columbus. Lambeth did not mention in his conclu sion the effect of different cultural treatments upon fertilization, which possibly accounts for the variations between his data and those presnted here. In the case of Henderson, which is a small seeded lima bean, data are presented in Tables 16 and 19 for all years, which show that even though fertilization occurs this does not neces sarily mean that the pods will set and develop. In treatment 1 at Wooster as presented in Table 19, nitrogen was extremely deficient, and the ovularies or fruits of the Fordhook variety that abscissed 2 days after anthesis had 20 per cent of the eggs fertilized. This would indicate that nitrogen deficiency was associated with the de velopment of the embryo and final fruit set of the Fordhook lima bean. Therefore Lambeth's suggestion that fruit set is exclusively the result of fertilization is not supported by these data. TABLE 15. DEVELOPMENT OF THE EMBRYO SAC IN TOO VARIETIES OF MUSKMELON AND PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS Honey Dew Hearts of Gold -Cultural **Embryo Sac Nuclei Embryo Sac Nuclei Collection Period Treatment Time Collected Development Development 8 Fertilized 8 Fertilized August 20-30, 1940 1 Abscission-8 Days After Anthesis 8.0 92.0 40.0 60.0 August 20-3C, 1940 2 Abscission-8 Days After Anthesis 86.0 14.0 61.0 39.0 July 15-25, 1941 1 Abscission-8 Days After Anthesis 15.0 85.0 36.0 64.0 July 15-25, 1941 2 Abscission-8 Days After Anthesis 77.0 23.0 67.0 33.0 Hungarian Wax California Wonder Sept.3-0ct.16, 1940 1 Abscission-4 Days After Anthesis 100.0 79.0 21.0 Sept.3-Oct.l6, 1940 2 Abscission-4 Days After Anthesis 100.0 100.0 July l-Oct.15, 1941 1 Abscission-4 Days After Anthesis 82.0 18.0 91.0 9.0 July l-Oct.15, 1941 2 Abscission-4 Days After Anthesis 100.0 100.0 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. ** Sample of 100 ovularies of each variety for each year and from each treatment. TABLE 16. DEVELOPMENT OF THE EMBRYO SAC B! TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS * Cul ...... ' 1... Fordhook Henderson tural i **Embrvo Sac Nuclei Development Embrvo Sac Nuclei Development Collection Period treat Time Collected j iFerti- i 1 i iFerti- ment ! 0 2 4 8 jlized 0 j 2 j 4 j 8 jlized A u g .5-Sept.22,1940 1 Abscission beforej50.0 21.4. 2 8 .6 i 40.Oi i 30.Oi 30.Oi I t i l 1 Anthesis [ 1 I I I 1 Aug.5-Sept. 22,1940 2 " J40.0 20.0 39.0 l.oi 35.Oi20.Oi35.oi 10.Oi 1 July 1-Sept.3,1941 1 " J45.0 10.0 45.0 1 30.Oi i 36.0} 3 4 . 0 i July 1-Sept.3,1941 2 . J30.0 -501.0 . 2 0 . 0 1 . 2S.Dll.0-,0j.4O.0i 22.Oi Aug.10-30, 1940 1 Morning of j 100.01 1 1 i100.0i i t i l 1 Anthesis j 1 I I I 1 Aug.10-30, 1940 2 11 |1 1.2 97.61 1.2 i i i100.0i v II 1 July 10-30, 1941 1 | 2.0 9 8 .oi i j iioo.oj Julv 10-30. 1941 . 2.. - It | 1 0 0 . 0 1 100.0 Aug.5-Sept.22,1940 1 Abscission-2 Daysj n 98.cTi 2.0 I 1 1 81.4! 18.6 After Anthesis | 1 i l l 1 Aug.5-Sept.22,1940 2 11 1 94-9* 5.1 j j j 59.oj 41.0 July 1-Sept.3,1941 1 11 1 96.0* 4-0 ! j ! 75.01 25.0 July., 1-Sept, 3,194I, _ 2 __ _ ii i . ... ' 92.0! 8.0 . . 1 .. 1 ' 65.0' 35.0 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. ** Sample of 100 ovularies or fruit of each variety for each year and from each treatment. TABLE 17. DEVELOPMENT OF THE EMBRYO SAC IN TWO VARIETIES OF MUSKMELON (21 OTTO UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946. Honey Dew Hearts of Gold Date Collected ^Cultural Time Collected **Erabryo Sac Nuclei Embryo Sac Nuclei Treatment Development Development 4 8 Fertilized 4 8 Tertilized August 2 1 Morning of Anthesis 5.0 95.0 100.0 August 2 2 Morning of Anthesis 8.3 91.7 100.0 August 2 3 Morning of Anthesis 16.7 14.3 83,7 August 6 1 3 Days After Anthesis 78.8 21.2 46.7 53.3 August 6 2 3 Days After Anthesis 100.0 81.3 18.7 August 6 .. . 3 . 3 Days After Anthesis 50.0 50.0 50.0 50.0 August 10 1 o Days After Anthesis 20.0 80.0 18.2 81.8 August 10 2 6 Days After Anthesis 71.4 28.6 100.0 August 10 3 6 Days After Anthesis 100.0 6.2 93.8 August 14 1 9 Days After Anthesis 100.0 5.0 95.0 August 14 2 9 Days After Anthesis 60.0 40.0 16.7 83.3 August 14 3 9 Days After Anthesis 16.7 83.3 13.8 84.2 August 18 1 Fruit Set-9 Days After Anthesis 100.0 100.0 August 18 2 Fruit Set-9 Days After Anthesis 83.3 16.7 100.0 August 18 ... . 3 Fruit Set-9 Days After Anthesis 100.0 100.0 August 19 1 Abscission-8 Days After Anthesis 44.4 55.6 100.0 August 19 2 Abscission-8 Days After Anthesis 63.7 36.3 83.3 16.7 August 19 - ...3 Abscission-8 Days After Anthesis 20.0 80.0 100.0 *1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. **Sample of 10 ovularies or fruit of each variety from all treatments. TABLE 18. DEVELOPMENT OF THE EMBRYO SAC IN TWO VARIETIES OF PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946. Hungarian Wax California Wonder Date Collected •*Gultural Time Collected t*Embryo Sac Nuclei Embryo Sac Nuclei Treatment Development Development 8 Fertilized 8 Fertilized July 8 1 Morning of Anthesis 100.0 100.0 July 8 2 Morning of Anthesis 100.0 100.0 Julv 8 .. 3 Morning of Anthesis. 100.0 100.0 July 11 1 2 Days After Anthesis 100.0 92.9 7.1 July 11 2 2 Days After Anthesis 94.3 5.7 60.0 40.0 July 11 3 2 Days After Anthesis 100.0 89.5 10.5 July 14 1 4 Days After Anthesis 66.7 33.3 50.0 50.0 July 14 2 4 Days After Anthesis 77.8 22.2 21.4 78.6 July 14 _____ 3 4 Days After Anthesis 61.9 38.1 4 ! . 2 58.8 July 14 1 Abscission-4 Days After Anthesis 100.0 63.6^ 36.4 July 14 2 Abscission-4 Days After Anthesis 91.2 8.8 78.3 21.7 July 14 3 Abscission-4 Days After Anthesis 87.5 12.5 93.8 _ 6.2 July 18 1 Fruit Set-6 Days After Anthesis 18.2 81.8 6.1 93.9 July 18 2 Fruit Set-6 Days After Anthesis 30 j8 69.2 40.0 60.0 July 18 3 Fruit Set-6 Days After Anthesis 23.1 76.9 76.9 23.1 July 20 1 6 Days After Anthesis 61.9 38.1 8.9 91.1 July 20 2 6 Days After Anthesis 92.9 7.1 61.1 38.9 July 20 _____ 2 . 6 Days After Anthesis 43.5 56.5 73.6 26.4 * 1. Low nitrogel level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. ** Sample of 10 ovularies or fruit of each variety from all treatments TABLE 19. DEVELOPMENT OF THE EMBRYO SAC IN TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946. Fordhook Henderson Date Collected ^Cultural Time Collected **Embryo Sac Nuclei Development Embryo Sac Nuclei Development Treatment Ferti Ferti 1 2 4 8 lized 1 2 4 8 lized July 16 1 Morning of 2.5 97.5 4.1 95.9 Anthesis July 16 2 » 100.0 2.2 97.8 July 16 3 9 100.0 98.0 2.0 July 18 1 1 Day After 3.1 71.9 25.0 5.6 94.4 Anthesis July 18 2 9 t.2 4.2 62.5 29.1 76.9 23.1 July 18 3 9 6 . 9 75.9 17.2 17.4 82.6 July 19 1 2 Days After 34.2 6 5 .£ 3.4 96.6 Anthesis July 19 2 » 75.0 25.0 18.2 81.8 July 19 3 » 3.2 19.4 77.4 24,1 75.9 . July 19 1 Abscission-2 10.0 10.0 60.0 20.0 83.3 16.7 Days After July 19 2 Anthesis 100.0 25.8 74.2 July 19 3 9 88.9 11.1 72.2 27.8 July 20 1 3 Days After 3.4 96.6 4.2 95.8 Anthesis July 20 2 " 82.4 17.6 100.0 Julv 20 3 » 6.1 93.9 4.8 95.2 . July 23 1 Fruit Set-6 Days 9.1 90.9 100.0 After Anthesis July 23 2 9 100.0 7.7 92.3 Julv 23 3 " 93.8 6.2 6.2 - 2 M _ * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. ** Sample of 10 ovularies or fruit of each variety from all treatments. -83^ CYTOLOGICAL DEVELOPMENT OF THE EMBRYO SAC Stage of Development of Lima Bean Ovularies Abscising Before Anthesis Ovularies of lima bean that abscissed before anthesis were studied cytologieally to determine the various stages of embryo sac nuclei development. This study was made only with this species grown at Columbus. The data are presented in Table 16 from a total sample of 100 ovularies for each treatment and variety. All ovules of each ovulary were examined and the stage of embryo sac nuclei development is recorded in percentage of total ovules. The percentage listed as zero, under embryo sac nuclei development, are examples where the reduction division had not taken place. Actually, the female garneto- phyte had not yet developed from the megaspore. Considering the embryo sac nuclei development of all ovu laries that abscissed before anthesis, the variety Henderson appeared to be in the more advanced stages of development. As indicated by these data for Henderson, eight nuclei, or mature embryo sacs had formed in 34- .0 per cent of the cases where the plants were grown with a low nitrogen level under treatment 1 at Columbus in 1941. With the same conditions the previous year Fordhook formed 28.6 per cent of mature embryo sacs. The stage of development, with the ex ception of Fordhook in 1941» was slightly more advanced as produced under full sunlight and low nitrogen or low nitrogen as compared with high nitrogen level and reduced light intensity. In the majority of cases the nuclei of these ovularies that abscissed before anthesis appeared to be in the early stages of degeneration. Such nuclei had -84- a great affinity for the stain and generally appeared shrunken, with their contents disorganized. Stage of Embryo Sac Development on the Morning of Anthesis The muskmelon flowers reaching anthesis were collected be tween 9:00 and 10:00 a. m. each morning to determine the stage of embryo sac nuclei development. Data from samples of 100 sectioned ovularies are presented in Table 16. Results from 10 ovularies from all other cultural treatments handled in a similar manner are pre sented in Tables 17, 18 and 19. Ovularies were cut serially and only those with a complete series of sections were studied, since if a portion were lost there would be a possibility of error in the final nuclear count. Mature embryo sacs with 8 nuclei were visually formed by the morning of anthesis for all species. There was some slight de lay in the development of the muskmelon embryo sac for both varieties (Table 17, treatment 3) > and of the Honey Dev; variety as produced under treatments 1 and 2. Degeneration and disorganization of the embryo sac was often observed in the case of all three species. In the case of muskmelon, figure 10 C shows the egg and antipodals in a state of degeneration. This embryo sac in an ovulary of Honey Dew was pro duced under treatment 3 with a high nitrogen level. The same state of development on the morning of anthesis was observed more fre quently when the plants were grown under a low nitrogen, and a high nitrogen and reduced light intensity. -85- The data as presented in Table 18 indicate that both Hungarian Wax and California Wonder, as produced under all cultural treatments, have mature embryo sacs with 8 nuclei on the morning of anthesis. However, mary of these mature embryo sacs were showing signs of degeneration on the morning of anthesis. Figure 11J shows an embryo sac of California Wonder (treatment l) degenerating at this time. The egg apparatus and antipodals have completely degen erated. The polar nuclei have not fused normally to form the secondary nucleus as illustrated in figure 11 J. As indicated in Tables 16 and 19, the embryo sacs of both Fordhook and Henderson lima beans are usually mature with 8 nuclei on the morning of anthesis. Figure 12 C and D show the embryo sacs of Henderson and Fordhook on the morning of anthesis as produced under treatment 1 or low nitrogen. The polar nuclei have fused as shown in figure 12 C, but not in 12 D where the egg is also showing signs of degeneration. Figure 12 E is a photograph of the embryo sac of Henderson as produced under high nitrogen and reduced light intensity. The polar nuclei are in the process of fusing, and the synergids have not yet degenerated. The embryo sac of Henderson as produced under this treatment has degenerated synergids and the secondary nucleus formed (figure 12 F). Figure 12 G and H are of embryo sacs of Henderson on the morning of anthesis as produced under treatment 3 (high nitrogen). The synergids have degenerated in both embryo sacs (G and H). Polar nuclei have fused to form the secondary nucleus in figure 12 G, but not in figure 12 H. An unusual condition is observed in figure 12 H where there are two polar nuclei in posi - 86- tion before apparent fusion to form the secondary nucleus. One of the polar nuclei has two nucleoli which is very uncommon in the case of the lima bean. Stage of Embryo Sac Development After Morning of Anthesis Until Setting of Fruit These experiments were conducted at Wooster during the sum mer of 194.6. Each kind of plant was studied and collections of ovu laries or young fruit were made at varying intervals depending upon the rapidity of embryo sac development. Muskmelon ovularies or young fruits were collected on the third, sixth and ninth day after anthesis to determine the stage of embryo sac nuclei development. The embryo sac nuclei development as calculated in percentage for each date is given in Table 17. The data were collected from 100 embryo sac counts from each of 10 ovu laries or fruit samples of each variety and as grown under all treatments. The data show that the variety Honey Dew was more affected by the cultural treatments than was Hearts of Gold, and in the like manner, Honey Dew as grown under high nitrogen and reduced light in tensity was more affected than under other treatments. This treat ment greatly delayed and reduced fertilization and development of the embryo of Honey Dew; as 0, 28.6 and 40.0 per cent fertilization (Table 17) was apparent after the third, sixth and ninth day res pectively; as compared with 18.7, 100.0 and 83.3 per cent for Hearts of Gold. In comparing the embryo sac development as produced under -87- low and under high nitrogen, no unusual differences were found in the case of Hearts of Gold, but in contrast to this, by the third day low nitrogen significantly delayed the fertilization to 21.2 per cent for Honey Dew as compared with 53.3 per cent for Hearts of Gold. However under the high nitrogen development was practically the same, up to and including the ninth day after anthesis for both varieties. As can be seen from the data in Table 17, fertilization of both Honey Dev; and Hearts of Gold muskmelon often took place by the third day after anthesis but the proportion of eggs fertilized usually increased up to and including the sixth day after anthesis. This delay was mainly associated with high nitrogen and reduced light intensity. Even though fertilization took place, this did not always mean that normal fruit development followed. As already mentioned, the per cent of flower buds achieving anthesis that set fruit was low for the muskmelon when compared to the per cent fer tilization indicated in Table 17. Some representative variations in development and de generation after anthesis are illustrated in figure 10. The embryo sac shown in figure 10 D was collected six days after anthesis and illustrates normal development of the embryo and endosperm. An embryo sac showinig signs of complete degeneration nine days after anthesis can be seen in figure 10 Q. This Hearts of Gold fruit with the embryo sacs in a state of complete degeneration was produced under low nitrogen, and appeared to develop normally for six days; however, by the ninth day the growth rate had reduced and the young - 88 - fruit was definitely yellow in color. The embryo and endosperm to be found in figure 10 E, collected nine days after anthesis, showed a slow rate of development and was in the initial stage of degenera tion. This embryo sac is from Hearts of Gold as grown under high nitrogen and reduced light intensity. Figure 10 F represents an ex panded embryo sac the embryo and endosperm of which had a slow rate of development nine days after anthesis. This was from young and undeveloped Honey Dew fruit produced under low nitrogen and reduced light. An embryo sac of Honey Dev/ produced under high nitrogen and normal light is shown in figure 10 I, with embryo and endosperm be ginning to degenerate and with an extreme slow rate of development nine days after anthesis. The embryo sac failed to expand as usual, and the fruit did not develop to normal size. Figure 10 J is of an embryo sac of Honey Dew as produced under high nitrogen and normal light. The embryo and endosperm is from a fruit that had not made normal development nine days after anthesis, but the embryo sac had expanded and developed. Degeneration is apparent by the affinity of the endosperm and embryo tissue for the stain. Figure 10 K shows the endosperm of Honey Dew nine days after anthesis as produced under the same treatment. The upper portion of the endosperm tissue shows signs of degeneration after this advanced stage of develop ment . The embryo was not definitely located in this embryo sac, which may indicate that it did not form or that it had degenerated. Ovularies and young fruits of the pepper were collected on the second, fourth and sixth day after anthesis to determine the - 89 - stage of nuclei development in the embryo sac. The nuclei develop ment as calculated in percentage for each stage is given in Table 18. The data were collected from counts of 100 embryo sacs from each of 10 ovularies or fruit samples of each variety as grown under all treatments. Mature embryo sacs of the pepper were developed in all ovularies of both varieties ty the morning of anthesis. As indicat ed ty the data (Table 18) fertilization of both Hungarian Wax and California Wonder pepper took place by the second day after anthesis but generally increased up to and including the fourth or sixth day after anthesis, varying with the variety and cultural treatment. These data also indicate that the variety Hungarian Wax, with the one exception (treatment 3, six days after anthesis) had not ad vanced as rapidly in per cent fertilization as had California Wonder by the second, fourth or sixth day after anthesis. The regularity of fertilization of both varieties was affected by the conditions of the cultural treatments. A delay in fertilization was noted in the embryo sac two days after anthesis in both California Wonder and Hungarian Wax under conditions of low and high nitrogen. Six days after anthesis 38.1, 7.1 and 56.5 per cent of the Hungarian Wax egg cells were fertilized as produced under treatments 1, 2 and 3 res pectively, and in the like manner 91.1, 38.9 and 26.4- per cent of the California Wonder egg cells were fertilized. This low percent age for the extremely vegetative Hungarian Wax plants is not un usually low when compared with 22.2 per cent fertilized under treat- -90- raent 2, four days after anthesis. The high per cent of fertili zation of California Wonder egg cells is not entirely in proportion to the per cent set of flowers reaching anthesis as presented in Table 10. These data indicate that only 23.0 per cent of the flow ers of California Wonder reaching anthesis set fruit as produced under treatment 1, whereas 27.8 and 28.2 per cent set fruit duced under treatments 2 and 3 respectively. There is evidence that less variation exists between treatments when comparing per cent set of flowers reaching anthesis for both California Wonder and Hungarian Wax than when comparing per cent fertilization six days after anthesis. These data again indicate that in the pepper even though a large percentage of the eggs of a fruit are fertilized, a fruit will not necessarily set and develop to maturity. Some representative variations in development after an thesis are indicated in figure 11. The embryo sac as shown in figure 11 H was collected four days after anthesis. The egg and polar nuclei of this embryo sac taken from a California Wonder ovu lary produced under treatment 1 showed no signs of fertilization. This is very typical of many embryo sacs at the time when the pollen tubes and contents degenerate in the style (Figure 11 F). Since fertilization did not mean normal development of the embryo sac or fruit, there was a very decided difference between those embryo sacs of pepper fruit that developed for four to six days after which the embryo sac degenerated and those that set after six days. The for mer embryo sac had a smaller internal area. Figure 11 K and L shows -91- embryo sacs of California Wonder six days after anthesis as produc ed under treatment 1. Delayed development of the zygote and endo sperm with little expansion of the embryo sac is typical of the con dition that is usually found when a fruit abscissed approximately six days after anthesis. Figure 11 M, N and 0 are of embryo sacs of California Wonder six days after anthesis as produced under treatment 1, displaying normal expansion of the embryo sac, and enlargement and division of the embryo. The total number of endosperm cells formed in these embryo sacs was usually greater than the total number formed under treatment 1, illustrated in figure 11 K and L. Lima bean ovularies or young fruits were collected on the first, second and third day after anthesis to determine the stage of embryo sac nuclei development. The embryo sac nuclei development as calculated in percentage for each stage is given in Table 19. The data were collected from the total counts of all embryo sacs from 10 ovularies or fruit samples. Mature embryo sacs of the lima bean were usually formed ty the morning of anthesis. There was some slight delay, but this was of no significance. As indicated ty the data (Table 19), fertilization of both Fordhook and Henderson lima bean took place by the first day after anthesis but generally in creased up to and including the third day after anthesis, and varied v/ith the variety and cultural treatment. In contrast to this, the variety Fordhook was more extremely affected ty the condi tions of the cultural treatments than was Henderson. The variety Henderson showed a delay in fertilization one day after anthesis as produced under high nitrogen and reduced light intensity, with 23.1 -92- per cent of the eggs fertilized as compared with 94.4 and 82.6 per cent when produced under low and high nitrogen treatments. The Henderson ovules which were fertilized under treatment 2 increased to 81.8 per cent ty the second day after anthesis. A lag in ferti lization of Fordhook eggs was found on the second and third day after anthesis as produced under treatment 2. For example, 65.8, 25.0 and 77.4 per cent of the eggs were fertilized ty the second day after anthesis under treatments 1, 2 and 3 respectively. Similarly, 96.6, 17.6 and 93.9 per cent were fertilized by the third djy. A comparison between the percentages of fertilization and of fruit set on the third day is desirable (Table 13). These data show that only 12.1, 10.8 and 14.9 per cent respectively set fruit of the total number of flowers reaching anthesis in spite of the high percentage of eggs fertilized as listed above. These data again indicate that, in the lima bean even though a large percent age of the eggs were fertilized, the fruit did not necessarily set and develop to maturity. Some representative variations in development after an thesis are illustrated in figure 12. The lima bean embryo sac if developing normally, when produced under treatment 1, usually ap peared three days after anthesis as shown in figure 12 M. Figure 12 J shows an embryo and endosperm of Henderson two days after an thesis as produced under treatment 2. The embryo sac shown in figure 12 I demonstrates normal development of the embryo and en dosperm of Henderson one day after anthesis as produced under -93- treatment 3. The embryo and endosperm of Henderson in figure 12 K and L made a rapid development by two days after anthesis as produced under treatment 3* Figures 12 N and 0 are of the same embryo sac of Henderson as produced under treatment 3, showing the rapid develop ment that had been made by three days after anthesis. Stage of Embrvo Sac Development at Period of Abscission The stages of muskmelon embryo sac development at the period of abscission in all experiments are presented in Tables 15 and 17. Definite abscission or cessation in growth of the ovulary or young fruits was usually apparent by the eight day after anthesis, and often could be detected by the seventh day after anthesis. The ovularies or young fruits that abscissed eight days after anthesis produced under high nitrogen and reduced light intensity showed very much less fert ilization as compared to those ovularies produced under low and high nitrogen. The data in Table 14 generally indicate that the per cent germination of pollen was lower under this treatment (2) than under treatments 1 and 3. Even though this trend of pollen germination was in the same direction as the per cent fertilization when comparing treatments, the differences between per cent fertilization was much greater than the differences between pollen germination when comparing these treatments. The abscissed fruit of Honey Dew produced under treatment 3 (Table 17) had 80.0 per cent of the eggs fertilized as compared to 55.6 and 36.3 per cent fertilization when produced under treatments 1 and 2 respectively. It should be noted that of those fruits abscising eight days after anthesis extreme variations do exist in the per cent fertilization when considering cultural treat- -94- inents for both Honey Dew and Hearts of Gold. Hearts of Gold at Wooster, Table 17, had 16,7 per cent fertilization under treatment 2 , while there was 100 per cent fertilization under treatments 1 and 3. Honey Dew (Table 15) had 92.0 per cent fertilization under treat ment 1 as compared with 14.0 per cent under treatment 2 , and for 1941 > 85.O and 23.0 per cent tinder treatments 1 and 2 respectively. Even though these percentages are very wide and fertilization often reached 100 per cent, the fact still remains that all fruits ab scissed after cessation of growth. Some representative variations in embryo sac development of the muskmelon at the period of abscission are indicated in figure 10 . The expanded embryo sac (figure 10 H) shows an embryo and endosperm eight days after anthesis. The fruit developed normally for about six days and had reached three-fourths of an inch in diameter before degeneration began. Degeneration is apparent ty the affinity of the endosperm tissue for the stain. An embryo sac of an ovulary abscis- sing seven days after anthesis, that had an egg apparatus showing signs of degeneration and no division of the secondary nucleus, is shown in figure 10 L. The degeneration of the secondary nucleus of an embryo sac that was taken from an abscissed fruit seven days after anthesis is shown in figure 10 M. The egg aoparatus had also degen erated, but is not present in this figure. Figure 10 N and 0 repre sent embryo sacs of abscissed fruits seven days after anthesis show ing signs of slow development and expansion with degenerating endo sperm resulting in abscission of the undeveloped and yellow ap- pearing fruit. The stages of embryo sac development in the pepper at the period of abscission as studied in all experiments, are presented in Tables 15 and 18. Usually, definite abscission of the ovulary or young fruit occurred by the fourth day after anthesis. In a very few cases fruit containing several hundred fertilized ovules would abscise on the sixth day after anthesis. These data, as presented in Tables 15 and 18, indicate that abscission of pepper four days after anthesis was very closely associated with the proportion of eggs that had not been fertilized. It is not the intention here to indicate that all flower buds that abscissed four days after an thesis contained a small percentage of fertilized eggs. There were some cases where 95.0 per cent of the eggs were fertilized and the fruit still abscissed four or six days after anthesis. Since the majority of eggs of pepper fruits that abscissed after anthesis were unfertilized, it is important to know the condi tion of the pollen and pollen tubes, and whether or not pollination was complete. Pollination, with very few exceptions, was complete in both varieties of pepper as grown under all treatments. Pollen tubes, as shown in figure 11 A, B, C, F and G, are very typical of those present in the style of pepper fruit buds that abscissed four days after anthesis. The majority of such pollen tubes never grew more than 2 millimeters in length before degenerating. The styles of California Wonder and Hungarian Wax averaged U and 8 millimeters in length respectively as produced in these experiments. Considering all treatments, the variety California Wonder -96- had a slightly greater percentage of fertilized eggs than did Hun garian Wax at the time of abscission, four days after anthesis. Dur ing the two seasons at Columbus, California Wonder had 21.0 and 9.0 per cent of its eggs fertilized respectively, as compared to no fer tilization at the time of anthesis under conditions of treatment 2 or high nitrogen and reduced light intensity. Hungarian Wax had no fertilized eggs at the time of abscission under treatments 1 and 2 at Columbus in 194-0. As indicated for Wooster, Table 18, Calif ornia Wonder had 36.A per cent of its eggs fertilized at the time of abscission when produced under treatment 1, as compared with no fer tilization of Hungarian Wax eggs. When grown under conditions of high nitrogen, Hungarian Wax had 12.5 per cent fertilized eggs as compared to 6.2 per cent for California Wonder. Two extreme variations in embryo sac development in pepper at the period of abscission are illustrated in figure 11 H, K and L. Figure 11 H shows an embryo sac of California Wonder produced under low nitrogen with the egg and polar nuclei before fusion, displaying no signs of fertilization four days after anthesis. This embryo sac is very typical of those found in ovularies with unfertilized eggs at the time of abscission four days after anthesis. The other type of embryo sac development is the one that has been fertilized, shown in figure 11 K and L. Here the embryo sac of California Wonder has de layed development as produced under treatment 1, with the zygote not yet divided, but with some endosperm division. The cell enlargement of the embryo sac seldom increases normally in width, but usually does expand in length. -97- The stages of lima bean embryo sac development at the period of abscission as studied in all experiments are presented in Tables 16 and 19. Definite abscission of the ovularies or young fruits usually occurred on the second day after anthesis. With one exception (treatment 1 of Table 19) Henderson had a larger percent age of fertilized eggs than Fordhook at the time of abscission, two days after anthesis. Fordhook had 20.0 as compared with 3.6.7 per cent fertilized eggs for Henderson under this low nitrogen treatment. Under conditions precluding fertilization, pollen usually failed to germinate on the stigmatic surface. Such non-germinating pollen grains on stigmas where the ovulary abscissed tv/o days after anthesis are shown in figure 12 A. Even though a fruit may have all its eggs fertilized, this does not necessarily mean such a fruit will set. All variations in' the proportion of eggs fertilized were observed in those fruit of both Fordhook and Henderson that abscissed two days after anthesis. With one exception (Fordhook, Table 19) a greater percentage of fertilized eggs developed under treatment 2, where abscission occurred two days after anthesis. Here abscission continued until the sixth day after anthesis. In the abscissed fruits of Henderson, 74.2 per cent of the eggs were fertilized at Wooster (Table 19) under treatment 2; however, this per cent of fer tilization of the eggs of abscissed fruits is not as high as the 92.3 per cent fertilization of the eggs of those fruits that had definitely set under the same treatment six days after anthesis. Some representative variations in embryo sac development of the lima bean at the period of abscission, two days after - 98 - anthesis, are indicated in figure 12. The embryo sacs shown in figure 12 Q and T are from fruits of Henderson as produced under treatment 2 collected at the first indication of abscission, two days after anthesis. The egg in figure 12 Q is in a state of de generation and the polar nuclei have not fused to form the secondary nucleus. This is the stem end embryo sac of the abscissed fruit. Figure 12 T illustrates an embryo that developed very rapidly during two days following anthesis but is showing very definite signs of deterioration as indicated by the affinity of the embryo for the stain. Figure 12 R shows the stem end embryo sac of Fordhook as produced under treatment 3. In this case the egg and polar nuclei appear normal after enlargement but had not been fertilized. The embryo sac of a Henderson fruit that abscissed (figure 12 S) showed signs of disorganization and lack of development as produced under treatment 1. The embryo sac of an abscissed Fordhook fruit as pro duced under treatment 1 and collected two days after anthesis is shown in figure 12 U with a degenerated and disorganized embryo. This embryo sac shows a lack of expansion with practically no endo sperm development. As with the other vegetables these photomicro graphs of the lima bean show that even though a fruit may have fer tilized eggs, this does not necessarily mean such a fruit will set. Stage of Embryo Sac Development of Fruit That Set and Enlarge The stage of muskmelon embryo sac development observed in fruit that definitely set and enlarged by the ninth day after anthe sis was studied at Wooster. Fruit of Honey Dew or Hearts of Gold that measured one inch in diameter at this time and had no yellow -99- appearance was considered to have set and developed normally. Very seldom would such a fruit abscise subsequently. Twenty of these fruits were selected from each cultural treatment and variety on the ninth day after anthesis. Ten were collected for cytological study and 10 tagged to observe the future development. In every case, the 10 tagged fruit developed to maturity, even including Honey Dev/. This was true even under treatment 2 where only 16.7 per cent of the eggs were observed to be fertilized. The data presented in Table 17 for fruit set nine days after anthesis indicate 100 per cent ferti lization of the eggs, with the one exception (Honey Dew, treatment 2). As presented in Table 17 (August 14-th collection) the per cent fertilization on the ninth day after anthesis is high, with the exception of Honey Dev/ when produced under treatment 2. This excep tion agrees very well with those data for Honey Dev/ fruit that defi nitely set but had only 16.7 per cent of the eggs fertilized. As discussed later, the germination of the Honey Dev/ seed as produced under treatment 2 was very low as compared to the germination of Honey Dew seed produced under treatments 1 and 3. Bnbryo sacs of a fruit that have definitely set usually appear as in figure 10 G v/here the embryo is circular in cross sec tion. The endosperm formed along the embryo sac wall was usually expanded to an oval shape. Those embryo sacs of Honey Dev/ fruit that set and developed, as grown under high nitrogen and reduced light intensity, often appeared as in figure 10 P. While the endo sperm developed normally, the embryo had a decidedly greater af finity for the stain. But, as can be seen from the data, fertiliza - 100 - tion visually failed to take place. The pollen and pollen tubes under such conditions appeared as in figure 10 B. In this instance, very few of the pollen tubes grew more than 1 millimeter into the pistil before ceasing to develop. The stage of pepper embryo sac development observed in fruit that definitely sex, and enlarged by six days after anthesis was studied at Wooster. Fruit of Hungarian Wax or California Wonder that measured 1 and 2 centimeters in diameter respectively by this time and did not drop after being touched lightly were considered to have set and developed normally. Very seldom would such a fruit abscise subsequently. Twenty of these fruits were selected from each cultural treatment and variety on the sixth day after anthesis. Ten were collected for cytological study and 10 tagged to observe the future development. In every case, the 10 tagged fruit developed to maturity. Even this included California Wonder from treatment 3? where only 23.1 per cent of the eggs were observed to be fertilized. The data presented in Table 18 indicate a greater percent age of fertilized eggs for both Hungarian Wax and California Wonder as produced under low, nitrogen for those fruit that appeared to have definitely set six days after anthesis. For Hungarian Wax 69.2 per cent fertilized eggs were observed as produced under treatment 2, as compared with 60.0 per cent for California Wonder. In the like man ner, Hungarian Wax and California Wonder had 76.9 and 23.1 per cent fertilized eggs as produced under treatment 3. As has been presented (Table 18), the per cent fertiliza tion on the sixth day after anthesis, when collected at two day in - 101 - tervals, shows a low trend of fertilization with 7.1 and 38.9 per cent fertilized eggs for Hungarian Wax and California Wonder respec tively under treatment 2, and with 58.5 and 26.4 per cent when pro duced under treatment 3. The same embryo sac of California Wonder (figure 11 P and Q) is typical of a normal development and was taken from a fruit that had set and enlarged to approximately 2 centimeters in diameter, as grown under treatment 1 seven days after anthesis. Figure 11 R and S show embryo sacs as developed seven days after anthesis, with a still greater width and apparently the fruit had developed normally. The stage of lima bean embryo sac development observed in fruit that definitely set and enlarged by the sixth day after anthe sis was studied at Wooster. Fruit of Fordhook or Henderson that measured 20 and 15 millimeters in length respectively by this time and did not drop after being touched lightly, were considered to have set and developed normally. Very seldom would such fruit ab scise subsequently. Twenty of these fruits were selected from each cultural treatment and variety on the sixth day after anthesis. Ten were collected for cytological stucfy- and 10 tagged for the purpose of observing the future development as before. One fruit of Fordhook (treatment l) abscissed on the eighth day after anthesis, all other tagged fruit developed to maturity. There appear to be no unusual differences between per cent fertilization as presented in Table 19, when comparing varieties and treatments. As presented later (Table 24), the per cent of total ovules that degenerated by the time of fruit maturity was consistently higher than the per cent of eggs that were not fertilized. This is supported by the data (Table 19) where only a low percentage of mature eight-cell embryo sacs lacked ferti lization six days after anthesis. When comparing the per cent ferti lization of Fordhook under treatment 2, 17.6 per cent of the eggs were fertilized by the third day after anthesis, whereas 100 per cent of the eggs were fertilized in those fruit that had set by the sixth day after anthesis. It must be mentioned here that Fordhook as pro duced under treatment 2 continued to abscise ovularies or young fruits up to and including the sixth day after anthesis. The embryo of a fruit that had definitely set usually ap pears as in figure 12 P. This embryo of Fordhook had developed six days after anthesis and is very typical of normal development. The embryo sacs in figure 12 I, K, N and 0 illustrate normal development on the first, second and third day after anthesis respectively. Fruit Set During Period of Sampling for Oytological Study The results of an experiment concerning the per cent fruit set during the period of sampling for cytological study of the two varieties of pepper and lima bean as grown under all treatments at Wooster can be found in Table 20. These data are presented as per cent set of flowers reaching anthesis and are similar to the data concerning total flowers reaching anthesis as presented in Tables 10 and 13. Ten flowers of each variety of pepper as produced under each treatment were tagged July 15 > and the per cent set represent those that grew to maturity. These samples are small and are not as repre sentative as the complete data to be found in Table 10 and 13. A total of 100 flowers of lima bean were tagged during the -103- period of July 17 to 26. Ten flowers of each variety as produced under each treatment were tagged daily and the per cent set as pre sented are those that grew to maturity. It is evident from these data (Table 20) and also those presented in Table 13, that Henderson consistently set a larger percentage and total number of fruit than did Fordhook. As produced under treatment 1, both varieties set the largest percentage of fruit during the 10-day sampling period. Ford hook when appearing extremely vegetative under treatments, set 8.8 per cent of the flowers reaching anthesis as compared to 70.0 and 54.0 per cent when produced under treatments 1 and 3 respectively. For Henderson there are no unusual differences between treatments 2 and 3, however, under treatment 1 this variety set a significantly higher percentage of its fruit as compared to treatments 2 and 3. There is a slight proportional relationship between fruit set of pepper (Table 20) with the cytological development of the em bryo sac six days after anthesis (Table 18). Hungarian Wax had 70.0, 60.0 and 80.0 per cent fruit set as grown under treatments 1, 2 and 3 respectively when compared with 38.1, 7.1 and 56.5 per cent ferti lized eggs. The trend is the same for California Wonder which in the like manner had 90.0, 50.0 and 50.0 per cent fruit set as com pared with 91.1, 38.9 and 26.4 per cent fertilized eggs. For both varieties the proportional relationship between fruit set and ferti lized eggs show a similar trend. The proportional relationship between fruit set of lima bean (Table 20) and the cytological development of the embryo sac three days after anthesis (Table 19) shows a relationship in the case - 104 - of Fordhook but not for Henderson. Fordhook had 70.0, 8.8 and 54-0 per cent fruit set as produced under treatments 1, 2 and 3 respec tively when compared with 96.6, 17.7 and 93.9 per cent fertilized eggs. In the like manner Henderson had 94.0, 70.6 and 68.0 per cent fruit set as compared with 95.8, 100.0 and 95.2 per cent fertilized eggs. REFRACTIVE INDICES AND PER CENT SOLUBLE SOLIDS OF PEDUNCLE AND OVULARY SAP AT TIME OF ANTHESIS The refractive index and per cent soluble solids of the o peduncle and ovulary sap was determined at 20 Centigrade by the use of the Abbe Refractometer. During the experiments at Wooster samples of 100 ovularies with peduncles attached were collected from each va riety as produced under each cultural treatment. Ten ovularies of each variety and treatment were sampled each day for 10 days during the period of collection for cytological study. The sap samples were extracted with a hydraulic press and the index of refraction read within one hour after the samples were harvested. All samples were harvested between 9:00 and 10: a.m. The average of 10 readings for the total period for all varieties, as produced under the different cultural treatments, are presented in Table 21. The differences in per cent soluble solids are very small between the varieties Honey Dew and Hearts of Gold. Both varieties of muskmelon consistently had a lower percentage of soluble solids under conditions of treatment 2, The differences between treatments 1 and 3 were very narrowj however, the trend was of a higher percent age soluble solids for both varieties as produced under treatment 1, -105- TABLE 20. PER CENT FRUIT SET DURING THE PERIOD OF SAMPLING FOR CYTO- LOGICAL STUDY OF THE EMBRYO SAC DEVELOPMENT OF TWO VARIETIES OF PEPPER AND LIMA BEAN GROW UNDER VARIED LIGHT AND NITRO GEN SUPPLY, WOOSTER | Pepper Lima Bean ! (July 3 to 21) (July 17 to 26) "‘Cultural j Hungarian California Treatment I Wax Wonder Fordhook Henderson i 1 j 70.0 90.0 70.0 94.0 2 j 60.0 50.0 9.0 71.0 3 j 80.0 50.0 54.0 68.0 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. TABLE 21. REFRACTIVE INDEX AND PF.R CENT SOLUBLE SOLIDS OF SAP EX- TRACTED FROM THE PEDUNCLE AND OVULARIES OF TWO VARIETIES OF MUSKMELON, PEPPER AND LIMA BEAN GROW UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER Honey Dew Hearts of Gold ^Cultural I Refractive Per cent 1 Refractive Per cent Treatment i Index Soluble Solids i Index _ . Soluble Solids 1 i 1.3374 3.2 j 1.3378 3.4 2 j 1.3364 2.3 j 1.3364 2.3 .3 . 1..1.3371___ 2,9_____ 1 1,3373 _ 3.1 Hungarian Wax California Wonder 1 I 1.3468 9.2 j 1.3468 9.2 2 1.3485 10.8 j 1.3490 10.9 3 ... 1 .1.3471.. 9.7 i 1.3460 9,0 Fordhook Henderson 1 | 1.3487 10.7 1.3490 10.8 2 1.3452 8.4 | 1.3469 9.5 -3 . 1 It3502 .. . 1 U 5 ..... 1...._l_-.33-0.4_.. 11.6 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. - 106 - when compared with treatment 3. There were no outstanding differences between Hungarian V/ax and California Wonder peppers as produced under the different cultu ral treatments. The cultural treatment differences indicate a higher percentage soluble soxids for both varieties when produced under treatment 2. These results concerning treatment 2 are not in agree ment with those presented for muskmelon. The differences between treatments 1 and 3 are again very narrow and the trends for the two varieties are in opposite directions. The differences between.the lima bean varieties, Fordhook and Henderson, in per cent soluble solids are insignificant except when produced under treatment 2. Fordhook had 8 . A per cent soluble solids as compared to 9.5 per cent for Henderson. In all indexes of refraction, this trend was the same. In comparing treatments for both varieties the lower percentage of soluble solids was formed un der treatment 2. For both varieties, treatment 3 consistently pro duced the highest percentage of soluble solids. There appear to be a few similar trends when comparing the per cent soluble solids of muskmelon, pepper and lima bean; as for example the low percentage for muskmelon and lima bean when produced under treatment 2. Treatment is important for all three kinds of plants in the production of the highest percentage soluble solids. Muskmelon produced the highest percentage of soluble solids under treatment 1, pepper under treatment 2, and lima bean under treat ment 3. FRUIT AND SEFD DEVELOPMENT -107- Muskmelon and Pepper Seed Germination Counts At the time of edible maturity 10 muskmelons were harvested from each variety and from each cultural treatment as produced during the experiments at Wooster. The total numbers of seed taken from each muskmelon are presented in Table 22. As determined by analysis of variance, Snedecor (53), the number of seed produced by Honey Dew was significantly higher than the number produced by Hearts of Gold. Honey Dew muskmelons grown under high nitrogen and reduced light in tensity produced a significantly larger number of seed than those pro duced under treatments of low and high nitrogen. There were no sig nificant differences between cultural treatments in number of seed produced by Hearts of Gold. A random sample of 100 seed were taken from each fruit and germinated after one month. The percentages that germinated are given in Table 22. It takes a difference of 16.8 per cent between averages to be significant at the 5 per cent point. The germination of Hearts of Gold seed, as indicated, is significantly higher than the germination of Honey Dew seed. As produced under high nitrogen and reduced light intensity, seed of both Honey Dev/ and Hearts of Gold germinated significantly lower than the seed of fruits produced under both high and low nitrogen. As shown in figure 14-, Honey Dew when well supplied with nitrogen but with reduced light, produced seed of low germination. The actual germination as shown in Table 22 is 13.8 per cent. This germination percentage is significantly lower than any of the other germination data presented. These data also correlate with the low percentage fertilization of Honey Dew -108- H O N E Y DEW h o n e y D E W HIGH NITROGEN h ig h n i t r o g e n REDUCED L IG H T . NORMAL LIGHT Figure 1^..- Muskmelon seedlings on left produced from seed of harvested fruit from treatment 2 and those on right from treatment 3, Wooster. TABLE 22.-SEED DEVELOPMENT OF INDIVIDUAL FRUIT OF TWO VARIETIES OF MOSKMELON MID PEPPER GROWN Ul'IDER VARIED LIGHT AMID NITROGEN SUPPLY, WOOSTER Jlf 1 - -.r UOJjl U iullJ iiu: j2ij.rj£iuix irJ. ""J-*":"'.*. .... UU --- i Honev Dew Hearts of Gold Honev Dew Hearts of Gold Konev Dew Hearts of Gold Fruit J No. Per cent No. Per cent No. Per cent No. Per cent Ho. Per cent No. Per cent No. ; Seed Germi Seed Germi Seed Germi Seed Germi Seed Germi Seed Germi i nation nation nation nation nation nation 1 J 465 19.0 332 95.0 948 - 00.0 434 86.0 637 80.0 370 96.0 2 \ 404 70.0 228 93.0 652 21.0 446 97.0 452 55.0 392 97.0 3 1 437 79.0 328 78.0 642 3.0 322 85.0 638 95.0 332 85.0 4 | 619 54.0 386 98.0 744 12.0 325 70.0 400 68.0 374 96.0 5 i 487 96.0 338 88.0 633 31.0 379 21.0 452 77.0 400 88.0 6 ! 453 31.0 253 71.0 330 31.0 434 51.0 402 85.0 252 93.0 7 ! 285 75.0 314 93.0 716 18.0 404 43.0 634 28.0 372 94.0 8 i 390 44.0 394 91.0 967 00.0 381 78.0 436 57.0 295 95.0 9 i 356 68.0 269 99.0 716 8.0 4-58 90.0 454 73.0 321 84.0 H> | 204 89.0 383 86.0 608 14.0 __ 3_75_ 40.0 429 74.0 333. 96.0 Total{ 4100 625.0 3225 892.0 6956 138.0 3958 661.0 4934 692.0 3441 924.0 Av.#*l 410.0 62-iLJ .322.5 89.2 695.6 ... 13.8 1.395.8 66.1 493.4 69.2 344.1 92.4 ! Hungarian Wax Calif. ..Wonder . Hungarian Wax Calif. Wonder Hungarian Wax Calif . Wonder 1 j 285 98.0 433 96.0 254 95.0 33 100.0 184 72.0 307 93.0 2 i 141 100.0 84 96.0 269 96.0 164 100.0 213 85.0 254 94-0 3 ! 229 95.0 340 100.0 244 96.0 279 98.0 189 85.0 364 88.0 4 ! 222 99.0 423 99.0 273 95.0 145 96.0 237 91.0 298 99.0 5 i 206 98.0 377 97.0 152 96.0 53 100.0 I65 96.0 253 96.0 6 i 193 94.0 329 93.0 217 99.0 291 100.0 217 95.0 108 98.0 7 ! 125 94.0 416 59.0 113 100.0 496 98.0 209 95.0 480 92.0 8 ! 179 95.0 420 95.0 171 100.0 I64 96.0 99 90.0 105 94.0 9 I 221 97.0 296 98.0 193 84.0 213 99.0 213 68.0 377 96.0 10 ! 178 91.0 _l 3.19 91.0 63 98.0 3.85 96.0 202 1.0 ... 497 96.0 Total i 1979 961.0 3437 924.0 r 1949 959.0 2223 983.0 1928 778.0 3043 946.0 Av.«# ! 197-9 96.1 jl343>.7 . 3 2 * 1L j -324*2. .95.9 . 2 ^ L __ 2.8, 3_... J22.-8 .. 77,8 . 3.04,3. 94.6 *1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. tt*Minimum significant difference, 19:1. Muskmelon and Pepper, No. Seed 89.2, 88./+; Per cent Germination 16.8 and 11.6,respectively. ‘ABIE 23.- FRUIT, SEED, MID OVULE DEGENERATION COUNTS OH TWO VARIETIES OF LIMA BEAK GROIJ1I TOILER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS *Cul- F 0 R D H 0 0 K H E 11 D E R SON tural Fruit Seed Ovules Per cent I Fruit | Seed Ovules Per cent Year treat Per Per Degen Ovules ! Per j Per Degen Ovules ment Plant Plant erating Degen ! Plant j Plant erating Degen 1 i erating ..... I erating I 1940 1 8.3 20.8 1.3 5.9 U . 7 | 28.8 1.2 4.0 1940 2 12.6 26.0 3.4 11.6 9.3 25.3 0.9 3.4 1941 1 9.8 19.3 3.7 16.1 15.7 S 35.3 5.4 13.3 1941 2 8.7 21.7 3.5 13.9 ! 9.7 j 22.6 4.8 17.5 ...... _.J i_ _ — i Note: - Data are listed as average from 10 plants. * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABLE 24.- FRUIT, SEED, MID OVULE DEGENERATION COUNTS ON THO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SuPPIY, WOOSTER 1 F 0 R~ D HOOK j HENDERSON !r— rrt-r-- ;• rr.--- n— rrr----- rr—r--- —rr------r r1----- n — r-rrz---r -;— t _----- — r- '^Cultural j Fruit Seed Stem End Per cent}Total Per cent] Fruit Seed jStem End]Per cent Total Per cent Treatment J Per Per Ovules Stem EndjOvules Total | Per Per ‘Ovules 'Stem End Ovules Total jPlant Plant Degen Ovules jDegen- Ovules i Plant Plant!Degen- 'Ovules Degen Ovules i i erating Degen- j erating Degen- | i erating !Degen- erating Degen i eratinv i eratinv j j j eratinv erating 1 j 8.7 27.3 3.5 40.2 1 4.7 14.7 il7.0 44.I 4.6 | 27.1 7.5 14.5 2 122.0 67.5 10.4 47.3 ! 14.9 18.1 '28.6 71.7 j 12.8 j 44-3 17.6 19.7 .. J_._.. J_9.,4 j[_2D._6_j 4.0 .... iiU6_._ _L._5.. 4 L5.9,iU .7..1. 1J1,_Q... 10.7 15,3 Note:- Data are listed as average from 10 plants. * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. 3. High nitrogen level. -111- jis indicated under treatment 2, presented in Table 17, where only 16.7 per cent of the eggs were fertilized. Pepper were harvested at the red stage of maturity. Ten fruits were taken from each variety and from each cultural treatment as produced during the experiments at ?/ooster. Counts of the total number of seed taken from each pepper fruit are presented in Table 22. The number of seed produced by California Wonder, with the ex ception of when produced under treatment of high nitrogen and re duced light intensity, was significantly higher than the number pro duced by Hungarian Wax. California Wonder pepper as grown under low nitrogen significantly produced a larger number of seed than those formed under high nitrogen, but not under the conditions of high ni trogen and reduced light intensity of treatment 2. There were no significant differences between cultural treatments, in number of seed produced, by Hungarian Wax. A random sample of 100 seed were taken from each fruit where possible; if 100 seed were not available in any single pepper fruit, the total of 100 was completed from the bulk quantity from each variety as produced under the cultural treatment concerned. These seed samples were stored for one month before planting in a germination test. The percentages of seed that germinated are pre sented in Table 22. It takes a difference of 11.6 per cent between averages to be significant at the 5 per cent point. Hungarian Wax produced under high nitrogen formed a significantly lower percentage of seed that germinated than any variety under any treatment. Counts of Lima Bean Fruit. Seed and Ovules Degenerating -112- Total counts of lima bean fruit, seed and ovules degenerat ing are presented in Table 23 for the Columbus experiments. These data were not in all cases consistent, for example the number of fruit per plant for Fordhook show no single trend. The difference in number of seed per plant are insignificant. The ovules degenerating are those that do not develop into mature seed and for Fordhook or Henderson are not consistent. Henderson under both series has pro duced more fruit and seed per plant as grown under low nitrogen. Those data presented in Table 24 are consistent and show a trend in the same direction for both varieties. Henderson signifi cantly produced more fruit and seed per plant than did Fordhook. These data agree with those presented in Table 23, with the exception of the experiment at Columbus in 1940, as grown with high nitrogen and reduced light intensity of treatment 2. Fordhook had a larger percentage of stem end ovules which degenerated as compared with Hen derson when produced with low nitrogen of treatment 1 and high nitro gen of treatment 3 , and a slightly higher percentage as produced with high nitrogen and reduced light intensity of treatment 2. There are no significant differences between varieties in per cent of total ovules degenerating. The per cent stem end ovules degenerating (Table 24) is very high for both varieties, when compared to per cent total ovules degenerating, and slightly higher for Fordhook. Counts of fruit, seed and ovules degenerating are significantly higher as produced with high nitrogen and reduced light intensity of treatment 2 ; and as produced with high nitrogen of treatment 3 are consistently higher -113- than those produced with low nitrogen of treatment 1 . The lima bean fruits from all series and cultural treat ments, had formed at the dry stage of maturity, at least one fully developed seed. DISCUSSION OF THE DATA PLANT VIGOR The number of flower buds that visibly formed, whether they reached anthesis or abscissed before anthesis, depended upon the de velopment and extension of the primary, secondary and tertiary plant branches. This development and extension did not necessarily corre late with plant vigor as the plants produced with high nitrogen and reduced light intensity were extremely vegetative but spindly and would not be classified as vigorously vegetative. The plants grown with low nitrogen were weakly vegetative, grew slowly and generally showed signs of nitrogen deficiency. Plants of treatment 2 were ex tremely vegetative when the first flower buds reached anthesis, the plants being light green in color with extremely succulent growing points. The main axis and branches were small in diameter relative to length and extension in growth. Only plants as grown with high nitrogen of treatment 3 at Wooster were vigorously vegetative with large, dark green stems and leaves. The stems were large in diameter relative to length. Kraus and Kraybill (32) have classified tomato plant growth and vigor as associated with their carbohydrate-nitrogen relation ship. Under their classification, the plants grown in these experi ments with low nitrogen of treatment 1 during all years had a very -114- high carbohydrate content associated with a weakly vegetative condi tion and corresponded to their class XV type.. Nitrogen was here the limiting factor of growth and the high carbohydrate content was asso ciated with the limited amount of nitrogen present. The plants grown with high nitrogen and reduced light in tensity of treatment 2 are here classified as similar to class I de scribed by Kraus andKraybill. All the plants of the three species were extremely vegetative, but were not considered weakly or vigo rously vegetative. The plants had a very low carbohydrate content accompanying an extremely vegetative condition. Here carbohydrates appeared to be the limiting factor and the low relationship was due to carbohydrate deficiency while the nitrogen supply appeared to be adequate. The plants grown with high nitrogen of treatment 3 are considered as class II with only localized carbohydrate deficiency in the meristematic areas. At these growing points, the plants had a low carbohydrate content accompanying a vigorous vegetative condi tion. Here carbohydrates were deficient at the time the first flower buds reached anthesis. For vegetative growth there was an available supply of both carbohydrates and nitrogen but the relation ship was such as to produce a vegetative condition at the time the first flower buds reached anthesis. Under the conditions of this treatment, localized carbohydrate deficiency existed for approximate ly 15 days after the first flower buds reached anthesis. This lo calized deficiency of carbohydrates continued to extend with the newly formed branches upon which flower buds were initiated. - us- as indicated by microchemical analysis, the carbohydrate content of the stems and leaves of plants grown with low nitrogen of treatment 1 was considered high, but nitrates were generally low and they appeared to be the limiting factor in plant growth and vigor. Plants as grown with high nitrogen and reduced light inten sity of treatment 2 were consistently low in carbohydrates and high in nitrates, especially at the growing points. This condition ex isted for approximately 25 days after the first flower buds reached anthesis. Again tinder conditions of high nitrogen of treatment 3 y this localized deficiency of carbohydrates continued to extend with the newly formed branches upon which flower buds were initiated. With development of age, the plants became less succulent but some what vigorously vegetative. As indicated, plants grown with high nitrogen of treatment 3 were deficient in carbohydrates at the ex treme growing point. After this period of localized carbohydrate deficiency, the plants of all three species as produced under treat ment 3 appeared to be very typical of the class III tomato plants as described by Kraus and Kraybill (32). MALE GAMETOPHYTE RESPONSES Abscission Before and After Anthesis Microsporogenesis and the male gametopbyte development pro ceeded in the regular sequence where the mature male gametophyte formed, disregarding degeneration caused by physiological deficien cies. The mature male gametophyte, as has been indicated, often failed to function normally and in many cases the flower buds ab scissed before anthesis. Under these conditions, carbohydrates or -116- nitrogen were deficient. Where the staminate and bisporangiate flower buds of musk melon were studied separately, there was an indication that micro- sporogenesis and the development of the male gametophyte, as grown under all treatments, was consistently suppressed by abscission before anthesis of the bisporangiate flower buds as compared to the stami nate flower buds. As has been indicated (Table 7) with the Honey Dew muskmelon when grown with high nitrogen and reduced light intensity (treatment 2 ), 6 .4- per cent of the staminate buds dropped before an thesis, as compared to 75.8 per cent of bisporangiate buds. This abscission of the staminate and bisporangiate flower buds of musk melon was associated with, if not caused by, carbohydrate deficiency, and to a much less extent, nitrogen deficiency, as correlated with the vegetative growth of the plants. When the plants of all three species were growing rapidly, appearing succulent, and carbohydrate- deficient at the growing tips, a larger percentage of the flower buds abscissed than when the plants approached maturity. Since micro- sporocytes were seldom present in muskmelon flower buds that abscis sed before anthesis it is plausible that the first meiotic division failed to take place. Where severe nitrogen deficiency existed as with treatment 1 at Columbus in 194-0, Hungarian Wax and California Wonder pepper had a per cent of total drops before anthesis of 51.7 and 87.3 respective ly. The same varieties under carbohydrate-deficient conditions of treatment 2 had a total drop before anthesis of 45-5 end 63.2 per cent. California Wonder was affected by the cultural treatments as -117- grown in all series much quicker and to a greater extent than was Hungarian Wax. The large size fruit of California Wonder as compared to Hungarian Wax was associated with the per cent of buds that drop ped before and after anthesis. The Fordhook lima bean, a large plant type, showed more variations in undeveloped microsporocytes than did the variety Hen derson which is a small plant type. Carbohydrate deficiency as ex isted under treatments 2 and 3 vitiated the male gametophyte develop ment to a greater extent than did nitrogen deficiency. Relation of Treatments to Phenotypic Viability of the Pollen It is of utmost interest to compare the pollen that germi nated with the per cent of stained grains. All pollen grains that germinated stained with aceto-carmine, but not all stained grains germinated. The visibly aborted grains were usually associated with carbohydrate-deficient plants or at least localized deficiency. Generally the per cent of visibly aborted pepper grains as formed with low nitrogen of treatment 1 was lower than the percentage formed under the other cultural treatments. Where the percentage is high, it is associated with severe nitrogen deficiency. As previously pointed out in the case of muskmelon, it has been shown that there was a high percentage of stained grains and inversely a very low germination percentage. The muskmelon pollen germinated very poorly as produced under all cultural treatments but always averaged slightly lower when produced by carbohydrate-defi cient plants. With the exception of the Fordhook lima bean, nitrogen- deficient, weakly vegetative plants produced pollen that germinated as -118- well or even better than the pollen of the carbohydrate-deficient plants. Fordhook lima bean consistently produced slightly more stained and germinable pollen as grown under carbohydrate-deficient conditions of treatment 2. Even though this difference is not so great, the trend of this large number of counts for all years is unusual and indicates that varietal differences are important when studying male gametophyte responses. Nitrogen deficiency had a more pronounced effect upon the development of the male gametophyte of Fordhook than that of Henderson. Carbohydrate deficiency at the point of the terminal inflorescence, but not in the leaf axils, of both varieties of lima bean when grown under treatment 2 was a known fact. As shown in figure 12 A, all grains were degenerated in the anthers of many of these flowers on the day of anthesis. Mature de generated pollen grains were consistently present on the stigmatic surface and the number was generally high for both varieties of musk melon and for California Wonder pepper when grown under carbohydrate- deficient conditions. Pollination and Fertilization Pollination was usually complete on the day of anthesis for all of the varieties studied. However, germination of the pollen grains on the stigmatic surface and growth of the pollen tubes into the pistil did not always occur with equal regularity. The visibly aborted grains and those that stained but failed to germinate were always apparent on the stigmatic surface. However, the important factor was the degeneration of the pollen tube and contents in the pistil after germination. This condition was very much apparent with -ii9- both varieties of pepper but to a greater extent with Hungarian Wax when carbohydrate-deficient plants were grown with high nitrogen and reduced light intensity and to a limited degree with high nitrogen. Degenerated pollen tubes were seldom found in the pistil of pepper when collected from a nitrogen-deficient plant, even though fertili zation in the case of Hungarian Wax failed to take place where the ovularies abscissed four days after anthesis (Table 18). That carbo hydrate deficiency is associated v/ith if not the cause of these pepper pollen tubes degenerating is an apparent certainty. The researches of Muller-Thurgau (4.0) supports this work by indicating that fertilization is not necessary for fruit set of the grape, but for growth of the pollen tube into the pistil, carbohydrates are necessary. The degeneration of muskmelon pollen tubes in abscissed pistils was very prevalent when collected from carbohydrate-defi cient plants as grown under treatment 2, Tables 15 and 17, while to a lesser extent as grown under treatments 1 and 3. Here again, as with pepper, carbohydrate deficiency is associated v/ith, if not the cause of, the degeneration of these muskmelon pollen tubes. Degeneration of the pollen grain and tube contents was ap parent in the case of Fordhook lima bean but not to such a great ex tent with Henderson. This degeneration of the pollen and tube con tents of Fordhook was very severe for the flowers that abscissed two days after anthesis from carbohydrate-deficient plants as grown under treatment 2 . Under the same conditions as the muskmelon and pepper -120- were grown, carbohydrate deficiency apparently caused the degeneration of the Fordhook lima bean pollen and tube contents, but not to such a great extent with Henderson, It can be said v/ith certainty, that if the germinated pollen of these plants as produced under all cultural treatments had grown into the pistil and each had actively fertilized an egg, the male gametophyte response would have been adequate for a satisfactory fruit set, if fruit set is dependent upon fertilization. A plausible explanation of the data presented indicates that carbohydrate defi ciency is detrimental to the development of the male gametophytes of muskmelon, pepper and the Fordhook lima bean, but apparently is more limited in its effects upon the variety Henderson. FEMALE GAMETOPHYTE RESPONSES Abscission Before Anthesis Where bisporangiate flower buds of the three kinds of plants studied abscise before anthesis, such a condition necessarily affects both the male and female gametophyte development. In the case of the muskmelon, nitrogen-deficient plants usually had fewer bisporangiate flower buds that abscissed before anthesis and therefore had a larger number of flowers to reach an thesis than did the carbohydrate-deficient plants. The trend of the data indicate that nitrogen-deficient muskmelon plants may abscise as large a percentage of bisporangiate flower buds before anthesis as do carbohydrate-deficient plants. This trend is usually greater in the case of Honey Dew than with Hearts of Gold. There is evidence that both carbohydrate and nitrogen deficiency have a similar effect -121- in reducing the development cf the muskmelon flower buds before anthe- °-is Weakly vegetative, nitrogen-deficient pepper plants usually had a larger percentage of abscission of the total flower buds formed than did carbohydrate-deficient plants. However, the total number of flower buds abscising increased as the total number of leaf axils and buds formed increased. This was always greater in the case of carbohydrate-deficient plants. Carbohydrate-deficient lima beans of both varieties ab- scissed a larger number and percentage of flower buds before an- thesis than did the nitrogen-deficient plants. The data indicate that nitrogen dificiency severely af fected the development of the female gametophyte of muskmelon and pepper, but of lima'bean to a lesser extent. Carbohydrate defi ciency has been more pronounced in affecting the development cf lima bean flower buds before anthesis and to a lesser extent upon the development of the bisporangiate flower buds of muskmelon and pepper. Abscission After Anthesis as Related to Fruit Set and Development It is highly significant that ovularies or young fruit of lima bean, pepper and muskmelon have a regular period of abscission on the 2nd, 4-th and 8th day respectively after anthesis. This date often varied slightly with the muskmelon as the abscission layer was not so sharply defined as with pepper or lima bean. The abscis sion layer that was formed on a regular schedule for the three vege tables may be associated v/ith pollination and fertilization but is certainly not entirely caused by these factors. There is substantial -122- evidence that nitrogen and carbohydrate deficiency are associated with, if not the cause of, ovulary and young fruit abscission after anthesis in the case of aid. three. Carbohydrate deficiency has apparently affected muskmelon ovulary and fruit abscission after anthesis to a slightly greater extent than nitrogen deficiency. However, it must be understood that nitrogen deficiency has had a detrimental effect as associated with abscission and development of the muskmelon fruit. As has been in dicated, under conditions at Wooster in 1946 > treatment 1, fewer fruit of both muskmelon varieties set and developed as compared to the same varieties when grown under the other treatments. These muskmelon plants as grown at Wooster showed all the signs of extreme nitrogen deficiency and a very weakly vegetative condition. There is a possibility that when nitrogen deficiency is very pronounced as at Columbus in 1940 and Wooster in 1946, treatment 1, both Hungarian Wax and California Wonder set and developed fewer fruit than under conditions of carbohydrate deficiency. Nitrogen de ficiency of pepper was not so pronounced as carbohydrate deficiency during the experiments at Columbus in 1941. Under these conditions, carbohydrate deficiency is associated with a reduction in fruit set and development. All indications point to the fact that both nitro gen and carbohydrate deficiency affect the fruit set and development of pepper, but the severity of the deficiency must not be overlooked, especially the localized deficiency at the point where the ovularies or young fruits are developing. As with pepper, both Fordhoolc and Henderson lima bean are -123- affected by carbohydrate and nitrogen deficiency. The severity of this deficiency with reference to localization is again apparent.. The group of flowers forming the raceme always failed to set fruit even though the flower buds in the leaf axils were setting fruit at the same time. These racemes produced upon the tall climbing plants as grown with high nitrogen and reduced light intensity of treatment 2, were extremely succulent and deficient in carbohydrates. Because the flowers in the leaf axils were least deficient in carbohydrates, they tended to set a greater percentage of fruit than did the flowers of the same location on nitrogen-deficient plants. Fruit set and development on the variety Fordhook was more severely affected than on Henderson by both carbohydrate and nitrogen deficiency. Relation of Fruit Harvest to the Flowers Reaching Anthesis Bradbury (5) found that thinning sour cherries increased the per cent set from 24 on unthinned branches to 42 on thinned branches. Mann and Robinson (39) found that on unthinned vines of muskmelon, about 10 per cent of the bisporangiate flowers set fruit, and on thinned vines, 60-70 per cent of the flowers set. Cordner (ll) found that lima bean fruits on the base of racemes were associated with reduced fruit set toward the apical end, and that apical set was in creased by basal defloration or removal of the small fruit. The results of these three workers seem to suggest that the available food or nutrient supply appears to be associated with, if not the cause of, the increased fruit set introduced by thinning. As the data collected at Wooster show, there was no in crease in number of flowers reaching anthesis as a result of harvest -124- ing the mature fruit of muskmelon or lima bean. However, this in crease of flowers reaching anthesis after fruit harvest is very pro nounced in the case of both varieties of pepper and especially with California Wonder. Pepper is a long season crop as compared with either muskmelon or lima bean. In frost-free areas, as for example some parts of southern Florida, peppers usually grow for nine months before they are considered unprofitable from a production stand point, whereas muskmelon and lima bean are completely harvested after approximately four months. Being a long season crop and having an active meristem that continues growth after the fruit are harvest ed, it is conceivable that pepper plants should form additional flower buds in new leaf axils. These data also indicate that carbo hydrates, and to a greater extent nitrogen, are limiting factors for the formation of new flower buds for approximately three weeks before the mature fruits are harvested. Apparently the fruits that first set and develop utilize the available carbohydrates and nitrogen. This later limited the supply for new vegetative growth and flower bud formation. EMBRYO SAC DEVELOPMENT The embryo sac development of muskmelon, pepper and lima bean as influenced by carbohydrate or nitrogen deficiency had not been fully investigated. Cochran (10) concluded that it seems prob able that unless degeneration of the embryo sac of pepper is due to some hereditary factor, it is brought about by a lack of proper nutrition. However, Cochran did not present any quantitative data for embryo sac development as influenced by a lack of proper nutri -125- tion, but showed that pepper plants receiving a high-nitrogen supply- set a significantly higher percentage of ovularies than did plants receiving low nitrogen. This set of fruit was not associated with embryo sac development as related to nitrogen deficiency. In fact, as has already been mentioned, he showed that pollination and ferti lization were not essential for fruit setting in the pepper and that degeneration or abortion of the embryo sac in abscissed. ovularies was associated with, but was not the cause of, abscission. These fruits of pepper that set without pollination or fertilization were subjected to low-temperature treatments, so his experiments are not related to any of the cultural treatments of this study. Stage of Etnbryo Sac Development of Lima Bean Ovularies Abscising Before Anthesis Of the three vegetables, only the embryo sac of the lima bean (Table 16) was studied before anthesis. The stage of embryo sac development of lima bean ovularies abscising before anthesis ap peared to be influenced by the carbohydrate content. The variety Henderson was less affected by both carbohydrate and nitrogen de ficiency than Fordhook. Carbohydrate deficiency, with one excep tion (Fordhook under treatment 2 at Columbus in 1941-) affected the advanced stage of embryo sac development in lima bean flowers that abscissed before anthesis to a slightly greater extent than did nitrogen deficiency. These data on lima bean indicated v/ith the ex ception noted, that carbohydrate deficiency retarded early mitosis in the embryo sac to a slightly greater extent than did nitrogen deficiency. -126- Stage of Embryo Sac Development on the Morning of Anthesis With the exception of muskmelon, as indicated by the data (Table 17) mature embryo sacs were usually formed by the morning of anthesis. If one examines the per cent of fertilized eggs of musk melon fruit that set, it can be said that this slight delay of de velopment by the morning of anthesis of a small percentage of the embryo sacs would not influence fruit set. The data (Table 17) in dicated by later collections (3, 6 and 9 days) after anthesis that all the embryo sacs of muskmelon were mature. Degeneration of the egg as observed in figure 10 G was produced more frequently by nitrogen-deficient plants and to a slightly less extent by carbo hydrate-deficient plants. As with muskmelon, ?/hen nitrogen was deficient, (figure 11, l) pepper embryo sac degeneration on the morning of anthesis was very pronounced. When Hungarian Wax and California Wonder were pro duced under carbohydrate-deficient conditions there was no apparent degeneration of the embryo sac contents on the morning of anthesis, although this does not preclude the possibility that later degenera tion took place. Embryo sac development of both Fordhook and Henderson lima bean appeared to have reached the mature stage of development by the morning of anthesis. Of those flowers reaching anthesis, carbohy drate or nitrogen deficiency did not have an apparent influence upon the embryo sacs of lima bean on the day of anthesis. Generally, where the development of the 8-nucleate embryo sac showed signs of delay, or degeneration of the embryo sac contents was apparent, ovu- -127- laries of flower buds containing such embryo sacs abscissed before anthesis. Stage of Embryo Sac Development After Morning of Anthesis Until Setting of Fruit The embryo sacs of muskmelon as presented in Table 17 showed mature development on the third day after anthesis under all cultural treatments. However, by the third day after anthesis carbo hydrate deficiency as produced with cultural treatment 2 had greatly retarded egg fertilization. The data indicated that Honey Dev/ had a lower percentage fertilization as produced by carbohydrate-deficient plants than Hearts of Gold. The majority of the pollen tubes in the pistils of carbohydrate-deficient plants had aborted. Ity’ the third day after anthesis, carbohydrate deficiency apparently had a more pronounced influence on the male than upon the female garnetophyte development. Under the conditions of these experiments, Honey Dew, a more extremely vigorous growing variety than Hearts of Gold, con tinued to be very severely affected by carbohydrate deficiency as- compared with Hearts of Gold. As illustrated in figure 10 Q, degen eration of the embryo sac and contents often took place when pro duced by nitrogen-deficient plants but to a lesser extent by carbo hydrate-deficient plants. There is additional evidence (Table 18) to indicate that the process of fertilization of the pepper was severely retarded by carbohydrate deficiency. Again, the aborted pollen tubes were lo cated in the pistil, which indicated a more pronounced influence of carbohydrate deficiency upon the male than upon the female gameto- -128- pbyte. There is a possibility that some hormonial activity causing retardation was associated with this lack of pollen tube growth and in turn fertilization, but if this were true, the lack of or re tardation of such hormonal activity was directly associated with car- hydrate deficiency. Nitrogen deficiency had only a very slight effect in retarding the rapidity of fertilization of California Won der fruit six days after anthesis, but it retarded to a greater ex tent the fertilization of Hungarian Wax. Even though all the eggs of a fruit may be fertilized, this does not make it certain that such fruit would set and develop to maturity. The expansion of the embryo sac, as shown in figure 11 M, N and 0, is very significant when compared to those of figure 11 K and L, where a slow rate of expansion is associated with embryo sacs of fruit that abscissed approximately six days after anthesis. Ni trogen deficiency was usually associated with, if not the cause of, this delayed development and lack of expansion. This does not ne cessarily mean that all the embryo sacs of pepper expanded normally when produced upon carbohydrate-deficient plants, but it is pointed out that nitrogen deficiency caused slightly more delay in embryo sac expansion than did carbohydrate deficiency. As related to embryo sac development after anthesis, lima bean showed much variation in per cent fertilization up to and in cluding the third day after anthesis (Table 19). Approximately 75 per cent of the drops on the second and third day after anthesis contained fertilized eggs. In contrast to this, Fordhook showed a trend up to the third day after anthesis of a low fertilization per- -129- centage as influenced by carbohydrate deficiency of treatment 2. For example, the fruit with only 1 or 2 eggs fertilized usually abscissed before the sixth day after anthesis, but approximately 5 per cent of such fruit as produced on carbohydrate-deficient plants developed to maturity. Henderson was slightly affected by carbohy drate deficiency up to and including the first day after anthesis. Ety- the second day after anthesis, the ovularies containing unfertiliz ed eggs and the majority of the fruit with 1 or 2 eggs fertilized had abscissed. This early abscission is what caused the apparent in crease in per cent fertilization at later dates, for example 6 days after anthesis for both Henderson and Fordhook. The low fertiliza tion percentage of lima bean eggs as produced on carbohydrate-defi cient plants was associated with, if not caused by, the relationship of carbohydrate deficiency to the degeneration of the male gameto- phyte and therefore in turn the final union of the microgamete with the megagamete. Nitrogen deficiency had apparently some detrimental influence upon the female gametophyte development of lima bean where the ovularies and fruit were collected at one day intervals after anthesis. As presented in (Tables 11, 12 and 13) for each year a high percentage of the ovularies and fruit abscissed two days after anthesis when the plants were produced with low nitrogen of treat ment 1. Also there was a delayed development of the embryo sac in about 20 per cent of the ovules of Fordhook but not with Henderson where abscission took place two days after anthesis (Table 19). Stage of Embryo Sac Development at Period of Abscission Abscission or at least cessation in growth of the ovulary -130- or young fruit of muskmelon on approximately the eighth day after an thesis was a very usual condition, and was associated with both car bohydrate and nitrogen deficiency. As indicated by the data of all three years, carbohydrate deficiency was very detrimental in reducing the fertilization of those eggs of fruits that abscissed eight days after anthesis. This reduction was again associated with carbohy drate deficiency as it affects male gametopbyte development. It is evident that nitrogen-deficient Hearts of Gold (Table 17) produced fruit which abscissed eight days after anthesis that had 100 per cent of the eggs fertilized. The same complete fertilization took place in the case of Hearts of Gold when produced at V7ooster with high nitrogen of treatment 3 where carbohydrate deficiency was localized at the growing points. The fact remains that even though the eggs of a muskmelon fruit may be 100 per cent fertilized, such a fruit may abscise approximately eight days after anthesis. Apparently fruit development of the muskmelon was not entirely associated with fertilization, but was more closely associated with the carbohydrate- nitrogen relationship of the plant. Mann and Robinson (39) also working with the muskmelon indicated that there was no evidence that fruit drop was brought about by the disfunction of embryo sac deve lopment or pollen tube growth, and that the mechanism by which to control fruit set was not determined. Abscission of the ovularies or young fruit of pepper usually occurred on the fourth day after anthesis. Abscission of pep per ovularies and young fruit at this time was usually closely asso ciated with the lack of fertilization, while in turn resulted from -131- degeneration of the pollen tubes within the pistil after germina tion. Incomplete fertilization was very pronounced under conditions of both carbohydrate and nitrogen deficiency and to a slightly greater extent with Hungarian Wax than with California Wonder. These data do not entirely agree with those of Cochran (10) but it must be remembered that Cochran subjected his pepper plants to low tempera tures and his results demonstrated that pollination and fertilization were not essential for fruit setting. He further showed that degen eration or abortion of the embryo sac in abscissed fruit was asso ciated with, but was not the cause of, abscission. Abscission of the ovularies or young fruits of lima bean occurred regularly on the second day after anthesis. There was never above 20.0 per cent of the eggs of Fordhook fertilized at this time. Where the eggs were completely unfertilized, (Fordhook, treat ment 2 at Wooster) the pollen had failed to germinate and was largely associated with a carbohydrate-deficient condition. Often, these ovularies continued to abscise for six days after anthesis. It is evident that fertilization was consistently lower in those fruits where abscission occurred two days after anthesis as compared to those fruits that definitely set six days after anthesis (Table 19). This abscission of the Fordhook lima bean, two days after anthesis, was very closely associated with unfertilized eggs, which in turn was closely related to carbohydrate deficiency and to a slightly less extent nitrogen deficiency. The abscissed fruits of Henderson as produced with high nitrogen and reduced light intensity of treat ment 2 had 4.1.0) 35.0 and 74..2 per cent fertilized eggs for the three -132- years (Tables 16 and 19). Here carbohydrate deficiency was asso ciated with abscission in spite of the fact that fertilization took place. Also nitrogen deficiency was even more detrimental to fer tilization and development of the embryo of Henderson as indicated by the dropping of fruit approximately two days after anthesis. The embryo sacs of lima bean ovularies and fruits that abscissed two days after anthesis, and were produced by nitrogen-deficient plants, generally expanded slowly as indicated by figure 12 U. Stage of Embryo Sac Development of Fruit That Set and Enlarge The fruit of muskmelon that set and enlarged by the ninth day after anthesis (Table 17), with one exception (Honey Dew, treat ment 2) had an apparent 100 per cent fertilization. The carbohydrate- deficient Honey Dew plants produced fruit with an average of 16.7 per cent fertilized eggs. This low percentage egg fertilization as pro duced by carbohydrate-deficient plants is associated with degenera tion of the pollen tubes within the pistil. Such degenerated grains and pollen tubes that were so closely associated with carbohydrate deficiency are shown in figure 10 B. Pepper fruits that abscissed usually showed such conditions by the sixth day after anthesis when the formation of an abscission layer was definitely apparent. Embryo sac development of pepper fruit that had definitely set six days after anthesis showed a larger percentage fertilization as produced with low nitrogen than when pro duced with high nitrogen, or high nitrogen and reduced light inten sity. The low percentage egg fertilization of pepper fruit that had definitely set, produced with high nitrogen and reduced light inten- -133- sity of treatment 2, was due to the degeneration of the male gameto- phyte as associated with carbohydrate deficiency. There appeared to be no unusual differences between per cent egg fertilization of the lima bean fruit of either variety that had definitely set six days after anthesis. Where the carbobydrate- nitrogen relationship influenced the per cent egg fertilization of lima bean up to the third day after anthesis, no such indications ex isted six days after anthesis. It is definite that lima bean fruit with a low percentage of egg fertilization, caused by carbohydrate deficiency, usually abscissed by the sixth day after anthesis. As produced under the cultural treatments of these experiments, the union of the megagamete with the microgamete appeared to be necessary for final fruit development of the lima bean. However, this union and complete egg fertilization of an ovulary does not necessarily indi cate that a fruit will develop to maturity. RELATION 01' PER CHIT SOLUBLE SOLIDS OF PEDUNCLE AND OVULARY SAP TO THE SETTING OF FRUIT The peduncle and ovulary sap of both varieties of muskmelon and lima bean had a significantly low percentage of soluble solids when the plants were grown with high nitrogen and reduced light in tensity. This would be expected as the reduced light intensity under the conditions of this experiment was undoubtedly associated with a reduction in the rate of photosynthesis and in turn the production of soluble solids per unit of leaf area. The large plants and leaves produced under this treatment shaded the fruiting branches or flow ers, thereby reducing the light intensity to approximately 50 per -134- cent normal as recorded in the cloth house. That light intensity is the factor involved appears certain if one compares the soluble solid percentages of plants grown with high nitrogen and reduced light in tensity with those grown only with high nitrogen. The higher percentage soluble solids of both varieties of pepper as produced with high nitrogen and reduced light intensity of treatment 2 is at variance with the data for both muskmelon and lima bean. It appears likely that the photosynthetic-respiratory ratio may have been increased in the pepper by the reduced light intensity of treatment 2. This species produces a normal crop in southern Florida during mid-winter when the light intensity is much reduced, whereas the muskmelon and lima bean often do not. Muskmelon produced the highest percentage of soluble solids with low nitrogen of treatment 1, pepper with high nitrogen and re duced light intensity of treatment 2, and lima bean with high nitrogen of treatment 3. Because of these variations in per cent soluble solids, there appeared to be an association between the physiologi cal activity of the different species of plants as related to the photosynthetic-respiratory ratio. RELATIONSHIP BETWEEN FRUIT AND SEED DEVELOPMENT Muskmelon and Pepper Seed Germination Counts Seed germination in the muskmelon increased as the per cent fertilization increased, although a slight amount of degeneration was observed between the time of definite fruit set and fruit maturity. This apparent degeneration after fertilization and fruit set was asso ciated with both carbolydrate and nitrogen deficiency but to a slight -135- ly greater extent with the latter. The Honey Dew fruit having seed that completely failed to germinate were as large in size as those fruit containing seed that germinated 31 per cent produced under high nitrogen and reduced light intensity of treatment 2. These results were not entirely in disagreement with those of Heinicke (21) who in dicated that the weight of the apple is in proportion to the number of seed in the fruit. It is of interest that the two largest fruits, as produced with high nitrogen and reduced light intensity of treat ment 2 , had 967 and 94-8 as the highest count of seed; however, none of these germinated. The seeds contained no embryos, but appeared empty and flat. The low percentage germination of Hungarian Wax pepper seed as produced with high nitrogen of treatment 3 was due to three fruits that had internal morphological abnormalities as described by Cochran (9). All fruit were selected at random for the experiment at Wooster-, so these abnormal fruit were not discarded. Carbohydrate-deficient plants of California Wonder produced fruit containing seed with a sig nificantly lower germination percentage than the nitrogen-deficient plants. There were pronounced indications that carbohydrate defici ency affected the development of the male gametopbyte as associated with fertilization and final seed development of the California Wonder to a greater extent than that of Hungarian Wax. These results with pepper are in agreement with those of Howlett (23) who studied the effects of carbohydrate and nitrogen deficiency upon microsporo- genesis and the development of the male gametopbyte in the tomato. That variations do exist between varieties of pepper as associated -136- with male gametopbyte development is substantiated by Hewlett (24.), who found modifications of flower structure and variations between varieties of tomatoes as influenced by the environment. Lima Bean Ovules Degenerated at the Time of Fruit Maturity Henderson tended to produce more fruit and seed per plant compared to Fordhook in all years of the experiments. This trend was in agreement with those data presented by Andrews (2 ) who concluded that Fordhook is less consistent in yield than Henderson, a relatively consistent yielding variety. Andrews presented data indicating that when Henderson plants were grown under hot, dry conditions they had a greater root-top ratio, more numerous and extensive roots, obtained more water from the soil, had more open stomates, a higher rate of transpiration, earlier pollen germination, higher chlorophyll concen trations, and a more consistent pod-set and yield than Fordhook. Under conditions of less rapid water loss from the plant tissues, when grown with high nitrogen and reduced light intensity of treatment 2 of these experiments, Fordhook approached Henderson in stomatal opening, root development, pod-set, and yields. In addition to the factors studied by Andrews, these experiments indicated that Fordhook was consistently lower in carbohydrates than Henderson, especially as produced with high nitrogen and reduced light intensity of treat ment 2 . The proportion of ovules that degenerated by the time of fruit maturity was consistently higher than the proportion of eggs that were not fertilized six days after anthesis. This indicated that fertilized eggs of both lima bean varieties degenerated between -137- the sixth day after anthesis and fruit maturity. This degeneration of fertilized eggs of lima bean XS ociatec with both carbohydrate and nitrogen deficiency but to a slightly greater extent with carbo hydrate deficiency. When comparing the three treatments, those plants which were extremely carbohydrate-deficient had a decided and consis tent increase in per cent ovule degeneration as associated with incom plete fertilization by the microgamete. This condition appeared to be not so severe when the plants were nitrogen-deficient or showed a lo calized carbohydrate-deficient condition at the growing points. The large percentage of lima bean stem end ovule degenerat ed, as compared to total ovules per fruit, was definitely because of the lack of fertilization. That carbohydrate deficiency was asso ciated with, if not the cause of, this low fertilization percentage appears certain. At the time of maturity all lima bean pods contained at least one or more fully developed seed. This seed development in dicates that under the conditions of the experiments, fertilization with the formation of at least one seed hy either of the two varieties of lima bean v/as necessary for complete pod development. SUMMARY This was a study of the effects of carbohydrate and nitrogen defi ciency on growth, flowering, fruit setting and development cf the male and female gametophytes of muskmelon, p e p p e r and lima bean. The main objective has been to determine the effects of carbohydrate and nitrogen deficiencies upon microsporogenesis and megasporogenesis and the development of the gametophytes of these plants. Special em- - 128 - phasis was placed upon the relationship of carbohydrate and nitrogen deficiency to ovulary and fruit abscission as associated with the male and female gametopbyte development. The researches were conducted during three separate seasons, 1940 and 1941 in the greenhouses, gardens and laboratories of the De partment of Horticulture r.nd Forestry at The Ohio State University at Columbus, and in 1946 in similar locations of the Department of Horti culture, Ohio Agricultural Experiment Station at Wooster. Studies were made of the developing ovularies and. fruits of two varieties of each vegetable, namely, Hearts of Gold and Honey Dew muskmelons, Hungarian Wax and California Wonder peppers, and the Henderson and Fordhook lima beans. Ten plants of each variety were studied during each of the three years and for each treatment. Five plants were grown, in ad dition to these 10 plants, for tissue tests and general histological work utilizing the growing ovularies and fruits. Three separate cultural treatments were employed in these experiments. All varieties and species were grown under treatment 1 with a fertilizer application of 150 pounds of phosphoric acid, and 100 pounds of potash to the acre. The nitrogen level was maintained below 10 parts per million by the addition of wheat straw when neces sary. This treatment was designed to give nitrogen deficiency. Treatment 2 consisted of a cheese cloth house with the light intensity reduced to an average of 55.4 Per cent normal, and a fertilizer application of 200 pounds of nitrogen, 150 pounds of phos phoric acid, 100 pounds of potash and 10 tons of manure per acre. The -139- nitrogen level was maintained between 200 and 300 parts per million. This treatment was designed to give carbohydrate deficiency. Treatment 3 consisted of the same fertilizer applications as treatment 2 but with no shade. Under this treatment the nitrogen supply was adequate and in the like manner the carbohydrate supply with the exception of the growing tips of vigorously vegetative plants. In such active meristematic areas the carbohydrate supply was often deficient. Diphenylamine was used to determine the nitrate level, and iodine reagent to determine the starch level. By the use of these reagents it was possible to determine the relative amounts of nitrates and starch present in different areas of the plants. The tests were made at the growing points and in the leaf axil areas where the flower buds were reaching anthesis. The following daily records were taken on all varieties as- grown under the three cultural treatments. For muskmelon, these re cords consisted of the number of male and bisporangiate flowers reaching anthesis, male and bisporangiate flowers not reaching anthe sis, and total number of fruit that set and developed. On pepper, counts were made of flowers reaching anthesis, flowers not reaching anthesis, and total number of fruit that set and developed. On lima bean, counts were made of flowers reaching anthesis, flowers not reaching anthesis, number of fruit that set and developed, number of seed developed and total number of aborted ovules. Pollen was germinated and stained daily during the 194-0 and 194-1experiments and at weekly intervals during the 194-6 experiments . -Ho- Pollen cf the muskmelon and pepper was germinated on culture media, whereas lima bean pollen germinated best in the microsporangium after placing the flowers in a moist chamber at 80° F. for four hours. Ovu laries or fruits with styles attached were collected for histological study the morning of anthesis before pollination, and at 1 , 2 and 3 days intervals after pollination from lima bean, pepper and muskmelon respectively. Samples of ovularies or fruits were collected with styles attached at the first indication of abscission to determine the stages of gametogenesis when abscission took place. Plants grown under conditions of low nitrogen were weakly vegetative, grew slowly and showed the expected signs of nitrogen de ficiency. Those plants grown under lor/ light intensity were carbo hydrate deficient up to and including the period of approximately 25 days after the first flower buds reached anthesis. Localized defi ciency of carbohydrates continued to extend with the newly formed branches upon which the flower buds were initiated. High nitrogen and normal sunlight produced vigorously vegetative plants with large, dark green stems and leaves, the stems being large in diameter rela tive to length. These plants had a localized carbohydrate defi ciency in the meristematic areas up to and including approximately 15 days after the first fruits set. Muskmelon ovularies and peduncles of nitrogen-deficient plants contained the highest percentage of soluble solids. The pep per on the other hand had the highest percentage of soluble solids when grown as carbohydrate-deficient plants, and lima bean when grown with a localized carbohydrate deficiency at the growing points. -Hl- Under all conditions abscission after anthesis of ovularies or young fruits of lima bean, pepper and muskmelon usually occurred regularly on the 2nd, 4th and 8th day respectively after anthesis. Male Gametophvte Responses The abscission before and after anthesis of the staminate and bisporangiate flower buds of muskmelon was associated with, if not caused by, carbohydrate deficiency. Nitrogen deficiency had only a slight effect. Those muskmelon flower buds that abscissed before anthesis as a result of carbohydrate deficiency seldom contained microsporocytes, apparently because reduction division failed to take place. Carbohydrate deficiency vitiated the male gametopbyte de velopment of lima bean to a greater extent than did nitrogen defi ciency, and Fordhook lima bean was more severely affected in this respect than Henderson or either variety of pepper. All pollen grains that germinated took the aceto-carmine stain; however, all of the grains that stained did not germinate. The visibly aborted grains were usually associated with carbohy drate-deficient plants or at least localized deficiency. The per centage of visibly aborted grains in pepper was often high when the plants were severely nitrogen deficient. Muskmelon pollen produced on carbohydrate-deficient plants germinated somewhat less than similar pollen produced on nitrogen deficient plants. Nitrogen deficient plants of both pepper varieties and the Henderson variety of lima bean produced pollen that germinated as well or even better than the pollen of similar carbohydrate-deficient plants. -H2- Although nitrogen deficiency affected male gametopbyte development to a relatively small extent, carbohydrate deficiency re sulted in a marked suppression of the male gametophyte development of muskmelon, pepper and the Fordhook variety of lima bean. Pollination took place on the day of anthesis for the species and varieties studied. Degenerated pollen tubes were some times found in the pistil. For example, carbohydrate deficiency re sulted in the degeneration of large numbers of muskmelon and pepper pollen tubes in the pistil, and to a considerable extent of Fordhook lima bean two days after anthesis. Degeneration was seldom observed when pollen was produced on nitrogen-deficient muskmelon or pepper plants. Female Gametophyte Responses Carbohydrate and nitrogen deficiency had a similar effect in reducing the size of the muskmelon bisporangiate flower buds be fore anthesis. Nitrogen-deficient pepper plants had a greater per centage of total flower buds which abscissed before anthesis than did carbohydrate-deficient plants, but the total number of flower buds abscising increased as the total number of leaf axils and in cluding flower buds formed increased. This was always greater when the plants were deficient in carbohydrates. Carbohydrate-deficient lima bean plants had a larger num ber and percentage of flower buds that abscissed before anthesis than did the nitrogen-deficient plants. Nitrogen deficiency adversely affected the development of the female gametophytes of muskmelon and pepper. On the other hand, -143- carbohydrate deficiency interfered more with the development of lima bean flower buds before anthesis, than with the bisporangiate flower buds of muskmelon and pepper before anthesis. When extreme nitrogen deficiency existed practically no muskmelon fruits developed. Pepper and lima bean plants of both va rieties that showed a very pronounced nitrogen deficiency set and de veloped fewer fruits than plants deficient in carbohydrates. Ap parently both nitrogen and carbohydrate deficiency affected the fruit set and development of pepper, especially the localized deficiency at the point where the ovularies or young fruits were formed. After removing the mature fruit of pepper, new growth was initiated and a considerable number of new flower buds reached anthe sis . The effect was more pronounced on California Wonder than on Hungarian Wax. Fruit harvest had little affect on initiation of new growth of the other species studied. With the exception of Fordhook lima bean in 194.1, an ex amination of the embryo sac development of those lima bean ovularies abscising before anthesis showed that carbohydrate as well as nitrogen deficiency retarded early megaspore division. Results with Fordhook indicated the importance of varietal differences in the effect of the degree of carbohydrate or nitrogen deficiency on embryo sac develop ment. When nitrogen was deficient, pepper embryo sac degeneration on the morning of anthesis was very severe. Carbohydrate deficiency on the other hand, caused no apparent degeneration of the embryo sac contents of pepper on the morning of anthesis. -i u - Carbohydrate or nitrogen deficiency had no apparent in fluence upon the embryo sac development of lima bean ovularies by the day of anthesis. When the development of the 8-nucleate embryo sac showed signs of delay, or v/hen degeneration of the embryo sac contents was evident, ovularies of flower buds containing such embryo sacs did not reach anthesis but abscissed at an earlier stage. Honey Dev/ as compared, to Hearts of Gold muskmelon had a lower percentage of fertilization when produced by carbohydrate-defi cient plants. This lack of fertilization was the result of the pro nounced degeneration of the male gametophyte from carbohydrate-defi cient plants. Degenerated pollen tubes of pepper were observed in the pistil. There was evidence that the final degeneration of the embryo sac and contents was directly associated with a lack of fertiliza tion, because of the effects of carbohydrate deficiency upon the de generation of the male gametophyte. Embryo sacs of pepper produced by nitrogen-deficient plants showed more delay in expansion than em bryo sacs of carbohydrate-deficient plants. The low percentage of fertilization of lima bean eggs pro duced upon carbohydrate-deficient plants was associated with, if not caused by, the inhibition of the development of the male gametophyte, and in turn with a failure of the union of the microgamete with the megagamete. Those embryo sacs of lima bean from nitrogen-deficient plants examined during thr three day period after anthesis showed no visible signs of degeneration. Fruits from nitrogen-deficient and localized carbohydrate- -US- deficient Hearts of Gold muskmelon plants showed signs of abscission eight days after anthesis when 100 per cent of the eggs were ferti lized. This indicated that development of the muskmelon ovulary was not primarily associated with fertilization, but was more closely as sociated with a direct carbohydrate-nitrogen relationship within the plant. Abscission of pepper ovularies and young fruits four days after anthesis was usually associated with a lack of fertilization. This lack of fertilization resulted from degeneration of the pollen tubes within the pistil. Incomplete fertilization six days after anthesis was very pronounced in both carbohydrate and nitrogen-defi cient pepper plants, and to a slightly greater extent with Hungarian Wax than with California Wonder, In like manner, fertilization was greater in carbohydrate-deficient plants than in nitrogen-deficient plants. The abscirsion of the lima bean two days after anthesis was usually associated with unfertilized eggs, which in turn was closely related to carbohydrate deficiency and to a lesser extent nitrogen deficiency. Embryo sacs of ovularies and fruits of lima bean from ni trogen-deficient plants thau abscissed two days after anthesis usually expanded more slowly than those embryo sacs on carbohydrate-deficient plants. Carbohydrate-deficient Honey Dew muskmelon plants produced fruit with an average of 16.7 per cent fertilized eggs. This low fertility was associated with degeneration of the pollen tubes within the pistil. The fertilization percentage was also in agreement with - 1 4 6 - the low rate of seed germination. Embryo sac development of pepper fruits that had definitely- set on nitrogen-deficient plants six days after anthesis had a higher percentage of fertilized eggs than similar fruits on carbohydrate- deficient plants. This smaller percentage of fertilized eggs was as sociated with the degeneration of the male gametophyte of carbohydrate- deficient pepper plants. Lima bean fruits usually abscissed two days after anthesis but generally no later than the sixth day after anthesis. These ab scissed fruits, especially Fordhook, had a low egg fertilization percentage caused by carbohydrate deficiency as related to male gametophyte degeneration, and female gametophyte degeneration as- related to nitrogen deficiency. With both varieties of lima beans the union of the megagamete and the microgamete nuclei appeared to be necessary for final fruit development; however, fertilization did not necessarily mean that a fruit would develop to maturity. At the time of maturity all lima bean pods contained at least one or more ful ly developed seeds. The apparent degeneration after fertilization of the musk- melon and pepper ovules was associated with both carbohydrate and nitrogen deficiency but to a slightly greater extent with nitrogen deficiency. The size of Honey Dew muskmelon fruit from carbohydrate-de ficient plants was not associated with the percentage fertilization but with the total number of ovules present which later developed as viable or non-viable seed. -14.7- Fertilized eggs of both varieties of lima bean often de generated between the sixth day after anthesis and the date of final fruit maturity. This degeneration of the fertilized eggs was asso ciated with both carbohydrate and nitrogen deficiency. A larger per centage of stem end ovules of lima bean degenerated as compared to the percentage of total ovule degeneration per fruit. This degener ation resulted from a lack of fertilization, which was caused by carbohydrate deficiency and its effects upon the development of the male gametophyte. -148- LITERATURE CITED 1 . Andrews, F. S. Physiological factors associated with the fruit ing of the bush lima bean. Proc. Amer. Soc. Hort. Sci. 34s 498-501. 1936. 2. Andrews, F. S. Root-top ratio of the bush lima bean as an index of adaptability to ecological conditions. Proc. Amer. Soc. Hort. Sci. 37: 752-738. 1939. 3 . Auchter, E . C ., and Harley, C . P . Effect of various lengths of day on development and chemical composition of some horticul tural plants. Proc. Amer. Soc. Hort. Sci. 21: 199-214. 1924. 4 . Binkley, A. M. The amount of blossom and pod drop on six varieties of garden beans. Proc. Amer. Soc. Hort. Sci. 29 : 489-492. 1933. 5. Bradbury, D. Notes on the dropping of immature sour cherry fruits. Proc. Amer. Soc. Hort. Sci. 22: 105-110. 1925. 6 . Buchholz, J. T., and Blakeslee, A. F. Pollen-tube growth at various temperatures. Amer. Join-. Bot. 14s 358-369. 1927. 7. Bushnell, John W. The fertility and fruiting habit in Cucur bits. Proc.,Amer. Soc. Hort. Sci. 17: 47-51. 1920. 8 . Cochran, H. L. Factors affecting flowering and fruit-setting in the pepper. Proc. Amer. Soc. Hort. Sci. 29: 434-437. 1932. 9. Cochran, H. L. Morphology of an internal type of abnormality in the fruit of the pepper. Bot. Gaz. 97: 408-415. 1935. 10. Cochran, H. L. Some factors influencing growth and fruit- setting in the pepper (Capsicum frutescens L.). Cornell University, Agr. Exp. Memoir 190. July 1936. 11. Cordner, H. B. External and internal factors affecting blossom drop and set of pods in lima beans. Proc, Amer. Soc. Hort. Sci. 30: 571-576. 1933. 12. Dorsey, M. J. A note on the dropping of flowers in the potato. Jour. Heredity 10: 226-228. 1919. 13. Erwin, A. T. The peppers. Iowa Agr. Exp. Sta. Bui. 293. 1932. 14. Ewert, R. DieParthenokarpic Oder Jungfernfruchtigkeit der Obstbaume. -Berlin; 1-57. 1907. -149- 15. Ewert, R. Neuere Untersuchungen uber Parthenokarpie bei Obstbaumen und einigen andern fruchttragenden Gev/achsen. Landw. Jahrb. 38: 767-839. 1909. 16. Fischer, Hugo. Zur Frage der Kohlensauer-einahrung der Pflanzen. Gartenflora. 65: 232-237. 1916. (Cited in Bot. Gaz. 67: 446 . 1919) 17. Gurjar, A. M. Carbou-nitrogen ratio in relation to plant metabolism. Science. 51: 351-352. 1920. 18. Hardenburg, E. V. Effect of hormone dust on pod set and yield in beans. Proc. Amer. Soc. Hort. Sci. 45: 367-370. 1944. 19. Harvey, E. M., and Murneek, A. E. The relation of carbohydrates and nitrogen to the behavior of apple spurs. Ore. Agr. Exp. Sta. Bui. 176. 1920. 20. Havis, Leon. Some factors which influence the fruiting habit of Henderson's bush lima bean (Phaseolus lunatus). Ohio Agr. Exp. Sta. Bui. 535. 1934- 21. Heinicke, Arthur J. Factors influencing the abscission of flowers and partially developed fruits of the apple (lyrus malus L.). Cornell Univ. Agr. Exp. Sta. Bui. 393: 41-114. 1917. 22. Hendry, G. W. Bean culture in California. Calif. Agr. Exp. Sta. Bui. 294. 1918. 23. Howlett, F. S. The effect of carbohydrate and of nitrogen deficiency upon microsporogenesis and the development of the male gametophyte in the tomato, Lycopersicum esculentum Mill. Ann. Bot. 50: 767-803. 1936. 24. Howlett, F. S. The modification of flower structure by environ ment in varieties of Lycopersicum esculentum. Jour. Agr. Res. 58: 79-117. 1939. 25. Johansen, Donald A. Plant Microtechnique. McGraw-Hill Book Company, Inc. New York. 1940. 26. Klebs, George. Uber die Organisation der Gallerte bie einigen Algen unt Flagellaten. Untersuch Botan Inst. 2: 333-417. 1888. ft 27. Klebs, George. Uber die vermehrung von Jfydrodictyon retreculatum. Flora. 73: 351-410. 1890. 28. Klebs, George. Willkurliche Entwickelungsanderungen bei Pflanzen. Jena. 1903. -150- 29. Klebs, George. Uber Probleme der Entwicklung. Biol. Centrbl. 24: 257-267, 289-305, 449-465, 545-559, 601-614. 1904. 30. Klebs, George. Alterations in the development and forms of plants as a result of environment. Proc. Roy. Soc. London, 82: 547-558. 1910. tf 31. Klebs, George. Uber Waehstum und Ruhe tropische Baumarten. Jahrb. Wiss. Bot. 56: 734-792. 1915. 32. Kraus, E. J., and Kraybill, H. R. Vegetation and reproduction with special reference to the tomato. Ore. Agr. Exp. Sta. Bui. 149. 1918. 33. Kraybill, H.„R. Effect of nutrition on the number of blossoms per cluster and the dropping of blossoms in the tomato. Proc. Amer. Soc. Hort. Sci. 22: 371-374. 1926. 34. Lambeth, Victor N. Some factors influencing pod set and yield of the lima bean. Univ. Mo. Agr. Exp. Sta. Res. Bui. 466. 1950. 35. Lloyd, J. W. Some tests in the culture of peppers. 111. Agr. Exp. Sta. Bui. 274: 329-336. 1926. 36. Maheshwari, P. A critical review of the types of embryo sacs in angiosperms. New Phytologist. 36: 359-417. 1937. 37. Maheshwari, P. Recent work on the types of embryo sacs in angiosperms-A critical review. Jour. Indian Bot. Soc. 20: 229-261. 1941. 38. Maheshwari, P. The angiosperra embryo sac. Bot. Rev. 14: 1-56. 1948. 39. Mann, Louis K., and Robinson, Jeanette. Fertilization, seed development, and fruit growth as related to fruit set in the cantaloupe (Cucumis melo L.). Amer. Jour. Bot. 37: 685-697. 1950. 40. Muller-Thurgau, H. Abhangigkeit der Ausbildung der Thauben- beeren und einiger anderer Fruchte von der Entwicklung der Samen. Landw. Jahrb. Schweiz. 12: 135-205. 1897. 41. Murneek, A. E. Physiology of reproduction in Horticultural plants. II. The physiological basis of intermittent sterility with special reference to Spider Flower. Mo. Agr. Exp. Sta. Res. Bui. 106: 1-30. 1927. 42. Nightingale, G. T. The nitrogen nutrition of green plants. Bot. Rev. 3: 85-174. 1937. -151- 43. Nightingale, G. T. Nitrate and carbolnydrate reserves in rela tion to nitrogen nutrition of pineapple. Bot. Gaz. 103: 4.09-456. 1942. 44. Nightingale, G. T., and Farnham, R. B. Effects of nutrient concentration on anatomy, metabolism and bud abscission of sweet pea. Bot. Gaz. 97: 477-517. 1936. 4$. Petri, L. Studi sulla malattei dell1 olive-V Richerche sulla biologia e pathologia firoale dell* olive raern, Della R. stazione Di patologia vegetale. Roma. 1914. 46. Petri, L. Investigation on the nitrogen nutrition of the olive, Atti. R. Acad. Econ. Egr. Georg. Firenze. 5. Ser. 13. 1916. 47. Sachs, Julius. Stoff und Form der Pflanzenorgane. Bot. Inst. Wurzburg Arb. 2: 452-488, 689-718. 1882. 48. Schnarf, K. Contemporary understanding of embryo sac develop ment among angiosperms. Bot. Rev. 2: 565-585. 1936. 49. Shaw, G. W., and Sherwin, M. E. Production of the lima bean. Univ. Calif. Agr. Exp. Sta. Bui. 224. 1911. 50. Shirley, H. L. The influence of light intensity and light qual ity upon the growth of plants. Amer. Jour. Bot. 16: 354-390. 1929. 51. Smith, 0. . Relation, of-temperature to anthesis and blossom drop of the tomato, together with a histological study of the pistils. Jour. Agr. Res. 44s 183-190. 1932. 52. Smith, 0., and Cochran, H. L. Effect of temperature on pollen germination and tube growth in the tomato. Cornell Univ. Agr. Exp. Sta. Memoir 175. 1935. 53. Snedecor, G. W. Statistical methods. The Iowa State College Press. Ames, 1946. 54. Watts, Victor M. Some factors which influence growth and fruit ing of the tomato. Ark. Agr. Exp. Sta. Bui. 267. 1931. 55. Watts, Victor M. Growth and fruiting responses to pruning and defloration of tomato plants. Ark. Agr. Exp. Sta. Bui. 347. 1937. 56. Wester, R. E., and Marth, P. C. Effect of somegrowth regu lators on yield of bush lima beans. Proc. Amer. Soc. Hort. Sci. 49: 315-319. 1947. -152- 57. Wester, R. E., and Marth, P. C. Some effects of a growth regu lator mixture in controlled cross-pollination of lima bean. Proc. Amer. Soc. Hort. Sci. 53: 315-318. 194-9. 58. Whitaker, T. W., and Pryor, D. E. Effect of plant-growth regulators on the set of fruit from hand-pollinated flowers in (Cucumis melo L.). Proc. Amer. Soc. Hort. Sci. 4.8 : 4-17-4.22. 1946. 59. Woo, M. L. Chemical constituents of Amaranthus retroflexus. Bot. Gaz. 68: 313-344-. 1919. 60. Work, Paul. Nitrate of soda in the nutrition of the tomato. Cornell Univ. Agr. Exp. Sta. Memoir 75. 1924. 61. Young, W. J. The formation and degeneration of germ cells in the potato. Amer. Jour. Bot. 10: 325-335. 1923. APPENDIX TAB IE I . - REPRODUCTIVE RESPONSES OF TWO VARIETIES 01* MUSKMELON GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLI, COLUMBUS, 1 9 4 0 *CUL1URAL TREATMENT#! CULTURAL T R E A T M E N T #2 Honey Dew 1 Hearts of Gold Honey Dew T Hearts of Gold Period Flowers Dropped t Flowers Dropped Flowers Dropped JFlowers Dropped Reaching Before Reaching Before Reaching Before 'Reaching Before iUrthegjg Anthesis j Anthesis Anthesis ABtifaasto .. Anthesis Anthesis Flower type** M M - } P M JL JL MP 1 August 14-20 70 0 1 1 1 1 2 °! 465 j 26 685 9 8 | 680} 4 28 August 21-27 128 1 1 1 816 J120 3 7 1181 7 17 45 J1065} 15 10 33 August 28-September 3 162 3 9 5} 1184j 90 2 34 1865 3 34 10 }1690} 23 34 58 September 4-10 181 2 0 1707} 15 10 38 2614 35 94 63 }2655| 59 58 43 4! i September 11-17 142 14 8 3} 493 21 15 9 2181 39 93 53 }1449| 38 119 65 September 18-24 14 11 43 2} 137 j 6 63 0 106 8 206 18 351} 61 151 44 September 25-Oct. 1 0 0 0 0} 0| 0 0 0 27 0 15 0 I 199} 42 25 4 October 2-4 0 0 0 o| 0| 0 0 0 0 0 0 18* 0 0 i 1 u I Total 697 31 63 » i 4802 }278 } 94 89 8659 1101 460 197 }8107{245 398 *275 Average Per Plant 69.7 3.1 6.3 1.5} 480 278} 9.4 8.9 865.9:10.1 46.0 19.7} 810.7} 24.5 398} 27.5 Per cent of Total 91.7 67.4 8.3} 32.6} 98.1175.8} 1.9 24.2 95.0}33.9 5.0 66.1} 95.3147.1,'4.7}52.9 1 Number Fruit Set 18 1 50 } i 10 I 39 1 Per cent Set of 1 Flowers Reaching 1 I ! Anthesis 582 1 18.0} 9.9 1 15.91 Per cent Set of All 1 i 1 Ovularies 39.3 1 13.61 1 3.4 7.5i ____ L J__L * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. *# M - Staminate, and P - Bisporangiate. TABLE II.- REPRODUCTIVE RESPONSES OF TWO VARIETIES OF MUSKMELON GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1 9 4 1 "CULTURAL TREATMENT #1} CULTURAL TREATMENT #2’ Honey Dew J Hearts of Gold Honey Dew Hearts of Gold Period Flowers 'Dropped •Flowers Dropped { Flowers {Dropped •Flowers Dropped Reaching {Before {Reaching Before |Reaching {Before {Reaching Before Anthesis {Anthesis Anthesis Anthesis {Anthesis •Anthesis Anthesis Flower Type** M__ LP M__LP MM P | M • M i-M IF— M LP 1 I July 1-7 6 0 10 0 177 5 0 1 0 i 84 0 i 208,' 1* 20 0 July 8-14 158 3 8 2 451 19 1 1. 18 0 I 37 1 i 410j 28 0 1 July 15-21 341 34 8 16 879 158 6 5 I 109 3 I 51 3 i 980 I 36 20 28 July 22-28 344 30 42 9 1110 112 0 8 i 145 12 i 150 42 1102j 60 23 27 July 29 - Aug. 4 143 2 0 0 773 77 0 0 j 29 0 i 274 87 1173 I 32 53 i 24 August 5-11 86 6 23 6 942 136 4 2 I 225 20 | 193 45 955 il53 29 i 11 August 12-18 40 6 30 4 810 18 6 2 362 24 1 174 63 750 Jl38 31 i 4 August 19-25 2 0 15 l 357 6 8 2 250 12 ! 144 33 617 i 36 40 I 4 August 26 - Sept. 0 0 2 0 109 0 5 131 ill 113 12 661 i 3 44 j 23 September 2-4 0 0 0 0 0 0 0 0 1 0 ■ 0 0 79 0 8 i 5 Total 1120 81 138 38 5608 531 30 i 21 1270 ! 82 1220 286 6935 487 268 127 Average Per Plant 112.0 8.1 13.8 3.8 560 & 53.1 3.0! 2.1 127.Oi 8.2 122 .d 28.6 693.5 48.7 26.8 12.7 Per cent of Total 89.0 68.1 11.0 31.9 99.5 96.2 0.5!3.8 51.0 i 22.3 49.0 77.7 96.3{ 79.3 3.7 20.7 Number Fruit Set 6 60 ! * ! 40 Per cenySr Flowers! 1 Reaching Anthesis! 7.4 11.3 | 4.9 8.2 Per---- centx Set of n 2 1 1 Ovularies 1 5.0! • 10.9 ll.l 6.5 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. ** M - Staminate, and P - Bisporangiate. TABLE III.REPRODUCTIVE RESPONSES OF TWO VARIETIES OF MUSIO-ELON GROWN UNDER VARIED LIGHT AND NITROGEN SUPPIZ, WOOSTER, 194.6 ^CULTURAL TREATMENT #1 CULTURAL TREATMENT #2 Honey Dew Hearts of Gold Honev Dew Hearts of Gold Period Flowers Dropped Flowers Dropped |Flowers Dropped Flowers { Dropped Reaching Before Reaching Before {Reaching Before Reaching { Before Anthesis Anthesis Anthesis Anthesis!Anthesis Anthesis Anthesis ! Anthesis Flower Tyne** MP M P M . P M p ! M P M P M P 1 MP f t July 9-15 2 0 0 0 25 0 0 0} 9 0 0 0 114 0{ 0 0 July 16-22 78 0 0 0 173 0 0 0{ 188 0 0 0 233 0{ 0 0 July 23-29 158 0 0 0 322 0 0 0! 435 2 10 33 241 35' , 5 0 July 30-Aug. 5 270 4 0 0 365 15 0 o{ 534 20 0 31 384 74 I 0 0 August 6-12 269 16 3 .10 445 30 1 4{ 497 33 70 72 569 108 ! 2 8 August 13-19 235 17 0 4 447 26 0 o{ 513 41 53 128 796 146 ! 29 54 SSI- August 20-26 247 21 0 0 589 48 0 o{ 655 37 53 175 976 186 ! 24 50 Aug. 27 - Sept. 2 125 14 0 0 471 14 0 28! 771 49 34 150 975 160 { 26 147 Sept. 3-9 18 2 0 0 270 5 0 8 2 ; 579 33 54 108 740 149 ! 40 67 Sept. 10-16 0 0 0 0 64 0 0 o{ 140 22 24 29 455 50 { 42 97 Sept. 17-23 0 0 0 0 4 0 0 o{ 73 2 2 21 375 52 i 19 111 Sent. 24 0 0 Q 0 ___ a 0 0 0! 0 0 Q 0 46 6 { 1 15 Total 1402 74 3 14 3175 138 1 11414394 2391 300 747 5904 966 {188 549 Average Per Plant 140.2 7.4 0.3 1.4 317.5 13.8,0.1 11.41439.4 23.9 30.0 74.7 590.4 96.6 18.8 54-9 Per cent of Total 99.8 84.1 0.2 15.9 100.0 54.810.0 45.2J 93.6 24.2 6.4 75.8 96.9 63.8 3.1 36.2 Number Fruit Set 10 14 ! { 26 38 Per cent Set of Flowers Reaching Anthesis 13.5 lO.i; 10.9 3.9 Per cent Set of All 1 Ovularies ______11.41 -■l.vftj. 2.61 2.51 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. ** M - Staminate, and P - Bisporangiate•. TABLE IIl(continued). -REPRODUCTIVE RESPONSES OF TWO VARIETIES OF MUSKMELON GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, WOOSTER, 1946 Hone1)Dew _j Hearts of Gold Period Flowers Dropped j Flowers | Dropped Reaching Before j Reaching | Before Anthesis _ Anthesis { Anthesis } Anthesis is i r* M P M P ! M P i M P July 9-15 15 0 0 0 83 0 | 0 0 July 16-22 132 0 0 0 291 0 0 0 July 23-29 313 3 0 0 360 5 0 1 July 30 - August 5 406 21 0 1 462 25 0 0 August 6-12 341 29 20 10 613 49 0 0 August 13-19 234 20 72 97 460 38 23 30 August 20-26 192 17 38 47 527 34 90 140 -9St- August 27 - September 2 111 U 59 28 350 30 57 83 September 3 - 9 51 7 0 6 155 21 36 27 September 10-16 6 0 0 0 17 2 3 0 September 17-23 19 0 0 0 3 1 0 0 Sentember 24 0 0 0 0 0 0 0 0 Total 1820 108 189 189 3321 205 209 281 Average Per Plant 182.0 10.8 18.9 18.9 332.1 20.5 20.9 28.1 Per cent of Total 90.6 36.4 9.4 63.6 94.1 42.2 5.9 57.8 Number Fruit Set 17 23 Per cent Set of Flowers i Reaching Anthesis 15.7 11.2 i Per cent Set of All Ovularies 5.7 4.7 i .1 i i I * 3. High, nitrogen level. #* M - Staxninate, and P - Bisporangiate.. TABLE IV.- REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLY, COLUMBUS, 1940 "CULTURAL TREATMENT#!* CULTURAL TREATMENT I 2 Hungarian Wax ( California Wonder Hungarian Wax California Wonder Period Flowers | Dropped j Flowers j Dropped Flowers j Dropped Flowers j Dropped Reaching J Before j Reaching j Before Reaching Before Reaching! Before Anthesis | Anthesis j Anthesis } Anthesis Anthesi3 Anthesis Anthaaisj Anthesis September 3 - 9 6 0 0 99 1 12 22 September 10 - 16 12 3 1 24 6 25 5 September 1 7 - 2 3 21 13 19 29 25 28 40 1 H September 2 4 - 3 0 16 29 29 6 59 1 36 vjt Total 83 69 10 69 188 157 86 146 I Average Per Plant 8.3 8.9 1.0 | 6.9 18.8 15.7 8.6 14.8 Per cent of Total 46.3 51.7 ! 12.7 ! 87.3 54.5 45.5 36.7 63.3 Number Fruit Set 21 u 8 I Per cent Set of Flowers 1 Reaching Anthesis 25.3 50.0 21.8 9.3 ! Per cent Set of All I Ovularies 12.2 6.3 11.9 3.4 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABLE V.- REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLI, COLUMBUS, 1941 *0 U L T P R A L TREATMENT#!} CULTURAL IRE AIMEN T~#2 Hungarian Wax California Wonder Hungarian Wax Californiar Wonder Period Flowers t Dropped Flowers Dropped Flowers Dropped Flowers Dropped Reaching j Before Reaching Before Reaching Before Reaching i Before Anthesis \ Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis! Aj&M aia July 1 - 7 192 40 64 66 235 124 48 I 153 July 8 - 1 4 140 17 40 11 203 16 13 34 July 1 5 - 2 1 108 16 109 15 176 8 42 17 July 2 2 - 2 8 61 15 99 61 102 36 132 157 68 116 July 29 - August 4 31 21 38 116 34 184 i i-1 August 5 - H 27 10 16 9 48 7 47 32 Vn 81 o> August 1 2 - 1 8 45 15 67 33 20 29 83 i August 19 - 25 52 5 124 23 43 23 177 61 August 26 - September 1 64 2 196 17 63 6 261 30 September 2 - 8 59 0 580 81 53 17 434 91 September 9 - 1 5 9 0 423 58 17 10 136 89 September 1 6 - 2 2 5 0 384 26 25 18 145 68 September 23 - 29 10 0 164 40 30 15 108 78 September 30 - October 6 17 0 94 24 10 17 78 89 October 7 - 1 3 8 0 64 8 3 5 52 63 October 1 4 - 1 5 ______£L _Q_ _JJL __ Q_ __ 0 __ Q_ _12_ __ 9 Total*# 828 141 2473 588 1096 365 1885 1236 Average Per Plant 92.0 15.7 274.8 65.3 121.8 ! 40.6 209.4 137.3 Percent of Total 85.5 14.5 80.8 19.2 75.0 I 25.0 60.4 39.6 Number Fruit Set 149 334 47 1 74 Per cent Set of Flowers Reaching Anthesis 18.0 13.5 4.3 I . 3.9 Per Cent Set of All I Ovularies 1 JLQjJL Jk/L J__ *1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. ** Based on 9 plants. TABLE VI.-REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLI, WOOSTER, 194.6 "CULTURAL TREAIMEMI 111 CULTURAL TREATMENT #21 ——---- — r "r! , .. , 1 Hungarian Wax 1 California Wonder Hungarian Wax CalifomjLa Wonder j Period Flowers Dropped Flowers Dropped Flowers Dropped Flowers Dropped j Reaching Before Reaching Before Reaching Before Reaching Before j Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis Anthesis} June 20 - 26 6 1 1 0 26 0 2 0 ! June 27 - July 3 26 3 5 0 61 2 42 0 ! July 4- - 10 71 9 13 0 163 1 76 4 ! July 1 1 - 1 7 92 5 35 0 245 8 146 15 i July 1 8 - 2 4 120 18 60 6 246 20 131 ! 17 i, July 25 - 31 147 9 91 2 212 18 105 10 fc August 1 - 7 137 24 96 21 11 120 241 1 F August 8 - 1 4 68 11 i 43 25 175 4 64 1 August 1 5 - 2 1 70 27 1 25 10 173 7 78 | 4 1 August 2 2 - 2 8 48 0 17 0 217 1 201 \ 0 i August 29 - Sept. 4 50 0 3 0 161 0 127 1 0 i September 5 - 1 1 34 0 1 0 82 0 111 ! 0 1 September 1 2 - 1 8 40 3 1 0 66 0 48 1 0 i September 1 9 - 2 1 11 6 0 0 30 0 0 I 0 i L ______...-.. 1 _ 1 Total 920 116 391 64 2098 72 1251 52 { Average Per Plant 92.0 11.6 39.1 6.4- 209.8 7.2 125.1 5.2 J Per cent of Total 88.8 11.2 85.9 14.1 96.7 3.3 96.0 4*0 j Number Fruit Set All 90 728 348 1 Per cent Set of Flowers j Reaching Anthesis 44-7 23.0 34.7 27.8 1 Per cent Set of All Ovularies 39.7 19.8 33.6 26.7 j ------L * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABLE VI (continued).- REPRODUCTIVE RESPONSES OF TWO VARIETIES OF PEPPER GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLI, WOOSTER, 1946 ^CULTURAL TREATMENT#! ^ ^ A I.Fa m J a m Hungarian Wax T California Wonder Period Flowers j Dropped j Flowers J Dropped Reaching j Before 1 Reachingj Before Anthesis^ _ Anthesis, j&j&egj-JL; Anthesis June 20- 26 June 27 - July 3 July 4 - 1 0 July 1 1 - 1 7 July 1 8 - 2 4 -160- July 25 - 31 August 1 - 7 August 8 - 14 August 15 - 21 August 22-28 August 29 - Sept. 4 September 5 - 1 1 September 1 2 - 1 8 September 1 9 - 2 1 Total 606 Average Per Plant 107.9 60.6 Per cent of Total 93.9 Number Fruit Set Per cent Set of Flowers Reaching Anthesis Per cent Set of All Ovularies * 3. High nitrogen level. TABLEVII.-REPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN ’ SUPPLI, COLUMBUS, 1940 "CULTURAL TREATMENT#! | CULTURAL TREATMENT #2 Fordbook T Henderson [ For^hook____ Henderson_____ Period Flowers Dropped Flowers f Dropped j Flowers j Dropped Flowers {Dropped Reaching Before j Reaching Before Reaching j Before Reaching | Before Anthesis Anthesis} Anthesis Anthesis j Anthesis Anthesis } Anthesis. i August 5 - 1 1 69 25 53 35 j 82 27 25 28 i August 1 2 - 1 S 137 30 378 41 I 151 6 333 26 i i August 19 - 25 237 8 204 20 i 173 17 279 53 i August 26 - September 1 123 40 61 33 ti 166 71 121 140 i i September 2 - 8 109 45 0 0 i 98 41 11 48 i i -191' September 9 - 1 5 19 0 0 t 46 18 0 0 i i September 1 6 - 2 2 0 0 0 i 13 72 0 0 Total 694 157 696 129 729 252 769 295 Average Per Plant 69.4 15.7 69.6 12.9 72.9 25.2 76.9 29.5 Per cent of Total 81.6 18.4 84.4 15.6 73.4 26.6 72.3 27.7 Number Fruit Set 88 117 126 93 Per cent Set of Flowers Reaching Anthesis 12.7 16.8 17.4 12.1 Per cent Set of All Ovularies 10.3 14.2 12.9 8.7 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABIfiVjIJ-RBPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLI, COLUMBUS, 1941 "CULTURAL TREATMENT#! CULTURAL TREATMENT #2 Fordhook Henderson Fordhook Henderson Period Flowers 1, Dropped j Flowers Dropped Flowers Dropped Flowers Dropped Reaching} Before j Reaching Before Reaching Before Reaching Before Anthesis i Anthesis Anthesis Anthesis . A a & e s i s . Anthesis. Antheais Anthesis July 1 - 7 72 10 27 3 25 1 4 4 July 8-14 142 a 337 19 79 14 45 21 July 15-21 166 22 195 21 118 30 65 15 July 22-28 134 22 139 14 108 52 98 12 July 29 - August 4 33 12 3 6 16 81 23 17 August 5-11 70 7 6 0 50 78 15 11 August 12-18 33 11 0 2 9 76 12 12 H August 19 - 25 20 5 0 2 53 81 4 8 August 26 - September 1 2 0 0 0 33 54 15 18 September 2 - 3 0 0 0 0 4 3 4 4 Total 672 130 707 67 495 470 285 122 Average Per Plant 67.2 j 13.0 j 70.7 6.7 49.5 47.0 28.5 12.2 Per cent of Total 83.8 | 16.2 j 91.3 8.7 51.3 48.7 70.0 30.0 Number Fruit Set 98 j ! 157 87 97 Per cent Set of Flowers Reaching Anthesis 14.6 22.2 17.6 34.0 Per cent Set of All Ovularies 12.2 20.3 9-0 23.8 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TAB IE IX. -REPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN * SUPPLI, WOOSTER, 194-6 T * c T R E A T M E N T # 1 1 C U L ~T~~U R A L TREATMENT #2i 1 Fordhook 1 Henderson Fordhook Henderson j Period ! Flowers Dropped j Flowers Dropped Flowers Dropped Flowers { Dropped j | Reaching Before j Reaching Before Reaching Before Reaching { Before j . .... _ ! Anthesis. Anthesis Anthesis Anthesis Anthesis Anthesis 1 Anthesis} July 13-19 0 3 0 0 0 July 20 - 26 160 1 87 0 467 1 682 0 July 27 - August 2 280 6 248 2 476 0 660 73 August 3 - 9 175 71 364 47 503 84 334 304 August 10-16 69 92 226 99 287 156 84 228 August 17 - 23 15 126 70 60 132 199 11 86 i August 24-30 3 25 0 22 37 22 0 0 August 31 - Sept. J 0 0 0 0 3 0 0 0 Total 718 321 998 230 2033 462 1967 691 Average Per Plant 71.8 32.1 99.8 23.0 203.3 46.2 196.7 69.1 Per cent of Total 69.1 30.9 81.3 18.7 81.5 18.5 74.0 26.0 Number Fruit Set 87 170 220 286 Per cent Set of Flowers Reaching Anthesis 12.1 17.0 10.8 14.5 Per cent Set of All I Ovularies 8.4 13.8 8.8 10.8 * 1. Low nitrogen level. 2. High nitrogen level and reduced light intensity. TABLE XX (continued).-REPRODUCTIVE RESPONSES OF TWO VARIETIES OF LIMA BEAN GROWN UNDER VARIED LIGHT AND NITROGEN SUPPLI, WOOSTER, 1946 *C II L T P R A L T R E A T M E N T #3 | - t — — ------• — - --- JefidepPIL. I I ^ Period j Flowers ! Dropped | Flowers i Dropped i Reaching { Before I Reaching Before ! Anthesis Anthesis i AnthesisAnthesis July 13 - 19 19 July 20 - 26 197 July 27 - August 2 188 August 3 - 9 166 August 10 - 16 51 August 17 - 23 10 August 24-30 0 August 31 - Sept. 2 0 Total 631 Average Per Plant 63.1 106.0 Per cent of Total 47.2 Number Fruit Set 94 Per cent Set of Flowers Reaching Anthesis 14-9 Per cent Set of All Ovularies * 3. High nitrogen level. -165- AUTOBIQGRAPHY I, James Clinkscales Hoffman, was born in Blythewood, South Carolina, June 29, 1915. I received ny secondary school education in the public schools of the village of Blythewood, South Carolina. %■ undergraduate training was obtained at Clemson, from which I received the degree Bachelor of Science in 1938. In June 1938, I was av/arded an Ohio Agricultural Experiment Station fellowship in the Department of Horticulture and Forestry of The Ohio State University, which I held until November 1941. From The Ohio State University, I received the degree Master of Science in 1939. In November 1 941, I was appointed Assistant Horticulturist in The Florida Agricultural Experiment Station, in which capacity I served until February 1, 1942. I entered the United States Army as a Second Lieutenant of Infantry February 4, 194.2, and after serving in the European and Asiatic theaters was separated from the service as a Major of Infantry in 194.6. I returned to The Ohio State University in the winter quar ter and was in residence as a veteran student until December 20, 194-6. I returned to The Florida Agricultural Experiment Station in the capacity of Associate Horticulturist and held this position until April 1, 194.9. At that time I was appointed Horticulturist at the Southeastern Vegetable Breeding Laboratory, Bureau of Plant Industry, United States Department of Agriculture, at Charleston, South Carolina. I held this position while completing the require ments for the degree Doctor of Philosophy.