MORPHOLOGICAL STUDIES OP DIPLOID AUD AUTOTETRAPLOID
PLARTS OP PHYSALIS PRUIUOSA 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
ROBERT DAVID HEURY, B.S., M.S.
The Ohio State University
1958
Approved hy
Department of Botany and Plant Pathology ACKN OWLEDGMEUT S
The writer desires to take this opportunity to express his sincere thanks and appreciation to his adviser
Dr, G, W. Blaydes for his guidance, advice, criticisms, and encouragement during the course of this investigation and preparation of the dissertation. Thanks are also extended to Dr. K. K. Pandey for his helpful suggestions concerning the research and to Mr. A. S. Heilman for the photographic work.
ii TABLE OP COM'Ei'TTS
Page
IHTRODU CTI ON ...... 1
MATERIALS ARB M E T H O D S ...... 4
GENERAL MORPHOLOGY AND G R O W T H ...... 8
Observations and Results ...... 8
Discussion and Summary...... 21
PRE- ADD POST-PERTILIZATIOH MORPHOLOGY...... 27
Development of the Ovule and Emhryo Sac .... 27
Observations and Results ...... 27
Discussion and Summary ...... 33
Development of the Pollen ...... 37
Observations and Results ...... 37
Discussion and Summary ...... 42
Fertilization ...... 43
Observations and Results • ••••...• 43
Discussion and Summary ...... 44
Endosperm ...... 43
Observations and Results ...... 45
Discussion and Summary ...... 48
Embryogeny ...... 49
Observations, Results and Discussion . . . 49
Summary 57
Early Seed Coat Development •••••••••• 58
iii Table of Contents - Continued.
Page
AUTOTETRAPLOID STERILITY ...... 60
Observations and Results ...... 6l
Discussion and Summary ...... 70
PAETHENOCARPY ...... 74
SUMMARY ...... 79
LITERATURE CITED ...... 82
iv LIST OF TABLES
Table Page
1 Size of leaf tissues and size and frequency of
stomates in diploid and" tetraploid Ph.ysalis
pruinosa ...... 11
2 Flower and fruit characteristics of diploid and
tetraploid Physalis pruinosa- ...... 13
3 Relation of fruit size to number of seeds in
diploid and tetraploid Physalis pruinosa . . 18
4 Seed germination of diploid and tetraploid
Physalis pruinosa ...... 18
5 Rate of growth in height of diploid and tetra
ploid Physalis pruinosa ...... 20
6 Comparison between autotetraploid Physalis
pruinosa and autotetraploid Physalis
floridana ...... 22
7 Comparative data on the embryo sac of diploid
and tetraploid Physalis pruinosa...... 36
8 Fertilization and pre- and post-fertilization
abortion in diploid and tetraploid Physalis
pruinosa...... 68
9 Size of mature fruits and associated structures
in diploid and tetraploid Physalis pruinosa
formed as a result of self-pollination in
comparison to 0.25 per cent naphthaleneacetic
acid being applied to the stigma...... 75
v LIST OP ILLUSTRATIONS (All illustrations are of Physalis pruinosa L e)
Figure Page
1 Metaphase chromosomes in pollen mother cell of
d i p l o i d ...... 5
2 Metaphase chromosomes in pollen mother cell of
tetraploid ...... • ...... 5
3 Diploid and tetraploid plants • 10
4 Leaf cross-section of d i p l o i d ...... 12
5 Leaf cross-section of tetraploid ...... 12
6 Flowers of diploid and tetraploid ...... 14
7 Seeds of diploid and t e t r a p l o i d ...... 14
8 Pollen of diploid ...... 15
9 Pollen of tetraploid ...... * ...... 15
10 Megaspore mother cell, diploid ...... 31
11 Dyad (megaspore), diploid ...... 31
12 Tetrad of megaspores, d i p l o i d ...... 31
13 Functional megaspore, diploid ...... 31
14 2-nucleate embryo sac, diploid ...... • . • 32
15 4-nucleate embryo sac, diploid 32
16 8-nucleate embryo sac, diploid 32
17 Microspore mother cell, tetraploid ...... 39
18 Dyad (microspore), tetraploid ...... ••• 39
19 Tetrad of microspore nuclei, tetraploid . . . • . . 40
20 Tetrad of microspores, tetraploid ...... 40
21 2-nucleate pollen, tetraploid 41
22 Microcytes in microspore tetrad of tetraploid . . . 41
vi List of Illustrations - Continued
Figure Page
23 2-celled endosperm, diploid ...... 47
24 Embryo with. 8 cells in 6 tiers, d i p l o i d ...... 55
25 Embryo with 8 cells in 8 tiers, diploid ...... 55
26 Embryo with 8 cells in 7 tiers, d i p l o i d ...... 55
27 Embryo with 5 cells in 4 tiers, d i p l o i d ...... 55
28 Embryo with 6 cells in 6 tiers, 1 in metaphase,
-diploid ...... 55
29 Embryo with 8 cells in 6 tiers, a diploid variation . 56
30 Aberrant 6-celled embryo of diploid ...... 56
31 Embryo with 8 cells in 6 tiers, a tetraploid
variation ...... 56
32 Embryo with 8 cells in 7 tiers, a tetraploid
v a r i a t i o n ...... 56
33 Embryo with 8 cells in 6 tiers, a tetraploid
v a r i a t i o n ...... 56
34 Pollen tubes of tetraploid at base of style of
tetraploid ...... 67
35 Pollen tubes of diploid at base of style of diploid . 67
36 Fruits of diploid as a result of self-pollination and
of hormone placed on the stigma ...... 76
37 Fruits of tetraploid as a result of self-pollination
and of hormone placed on the stigma •••••.• 76
vii INTRODUCTION
Physalis is one of the genera of the Solanaceae, a family which contains some economically important crops including tomatoes, potatoes and eggplant# Although not generally considered of great economic importance, some species, including P. pruinosa# are culti vated for their fruits which are used in jams and relishes. The
Chinese lantern, P. alkekengi, is often cultivated for its orna mental calyces when in fruit. Physalis is often found as a common weed in temperate and tropical regions of the United States.
In this study reference will he made to some of the many morphological studies in the Solanaceae, hut apparently there has heen very little morphological work done in this genus. In 1896 it was monographed hy Rydherg and recently a cytological-genetic investigation was completed hy Menzel (l95l)» The megagametophyte of P . minima and P. peruviana as well as the emhryogeny of P. minima has heen studied hy Bhaduri (1935» 1936). Tognini (1 9 0) 0 described a few stages in the emhryogeny of P. edulis and Mascrd
(1921) conducted some studies of the anther in some genera of the
Solanaceae including Physalis.
Much has heen written on the utilization of tetraploids and other polyploids in plant breeding and agriculture hy Eigsti
(1957)» Eigsti and Dustin (l955)» Dermen (1940), Emsweller and
Ruttle (1941)t Stebbins (1956) and others. As these workers often mention, the advantages such as larger ecxmomically important plant organs and use in breeding for introduction of qualitative and quantitative changes in hybrids, are often offset by such disadvan- tages as slower growth, pollen sterility, and, particularly, low seed set.
A study of Physalis pruinosa was conducted to contribute to the morphological knowledge of the genus as well as of the family.
For example, there has heen some question on the development of the mega garnetophyte in this family (Schnarf 1931> Bhaduri 1935) • As a result of an accumulation of morphological information from the various investigations of many workers, a better understanding of the systematic and phylogenetic relationships may be obtained. A knowledge of the morphology may help in the analysis and understand ing of problems frequently encountered in commercial crop production in such an economically important family. The results of studies conducted on the development of the ovule, embryo sac, pollen, endosperm, young embryo and seed coat of Physalis pruinosa L. are reported in this dissertation.
In addition, due to the increased interest in polyploidy in agriculture, an investigation on the comparative morphology of diploid and colchicine-induced autotetraploid plants of Physalis pruinosa. including a study on the cause of low seed set in the latter, was thought to be a desirable addition to the morphology of polyploids that has accumulated and which in some cases has been inconsistent•
A preliminary study made on parthenooarpic fruit develop ment in this species is also contained in this report. In some economically important fruits, including grape®, oranges and grape fruit among others, parthenocarpy has been important. If there is 3
an increase in the utilization of Physalis as a fruit crop, parthe-
nocarpy may he desirable#
There is an abundance of literature on the subjects
studied in this investigation.' Obviously, it is impossible to cite
all of them. In the introduction to the major divisions of this
study (i.e., General Morphology, and Growth, Pre- and Post-fertiliza'
tion Morphology, Autotetraploid Sterility, and Parthenocarpy) re
ference is made to some relevant reviews and comprehensive works0
Along with the observations and discussion of the results of -this
study are included citations to some of the other works that are
relevant to the particular topic at hand. References cited are
often limited to the Solanaceae since this is the family concerned
in this study. MATERIALS AND METHODS
Diploid and autotetraploid plants of Physalis pruinosa L. were used in this study. Seed of "ground cherry" had heen obtained from the Pearce Seed Company of Moorestown, New Jersey. Through the courtesy of Dr. K. K. Pandey I obtained one diploid plant raised from this seed and one autotetraploid plant which had been produced from a colchicine—treated diploid plant from the same lot of seed.
This original autotetraploid plant was obtained by him by applying three drops of a 0.3 per cent colchicine solution on a piece of cotton placed on the unelongated epicotyl between the cotyledons of a young diploid plant for three consecutive days. Both the diploid and the autotetraploid plant were vegetatively propagated by stem cuttings in order to insure quantity and uniformity of material used.
For growth studies, however, plants raised from seeds of these selfed plants were used.
Rydberg’s (1 8 9) 6 monograph of the genus and his keys in
Small (1903, 1933) were used primarily to identify this species of
Physalis although the keys in Fernald (1950) and- Gleason (1952) were also used. Fernald’s key is based largely on Rydberg's monograph.
This plant was identified to be Physalis pruinosa L. Menzell's
(1951) cytotaxonomic investigation of the genus showed this species to have a 2n chromosome number of 24. As a part of this study, chromosome counts made of acetocarmine smears of dividing pollen mother cells from the diploid plant showed the ri number of chromo somes to be 12 (2n = 24) (Figure l). Twenty-four chromosomes (Figure
2) were found in dividing pollen mother cells of the tetraploid, thus in this plant 2n ** 4 8* • 4 5
o g Q £ < O^PnOO
& e * $ &
FI6. 2
Plate I. Figure 1. Chr ones ones ia dividiag micreepore mother cell (secead division) of diploid P. pruiaosa. Figure 2. Chronoecnes ia dividiag""aicreepere Bother cell (first division) of tetraploid P. pruiaoaa. 6
The plants grew in the botany department greenhouse. The material collected from them for microscopic study was fixed in FPA
(5 cc. formalin, 5 cc, propionic acid, 90 cc. 50i° alcohol) or
Havashin's Fixative (l g. chromic acid, 7 cc. glacial acetic acid,
92 cc. water mixed before using with equal parts of a mixture con taining 30 cc. formalin and 70 cc. water) and put under a partial vacuum for 24 hours to insure optimum penetration and fixation. The material was then dehydrated in a graded ethyl alcohol series and embedded in paraffin using toluene as the agent of infiltration. A
Spencer rotary microtome was used to cut sections 8 to 1 2 ^ thick depending on the material being studied. The ribbons were serially affixed to clean slides with Haunt's Adhesive, stained in Safranin 0
(in methyl-cellosolve) - Fast Green, cleared, mounted in Piccolyte and studied.
Pollen viability was determined by mounting fresh pollen in a drop of a combination of Aniline Blue in lactophenol (enough
Aniline Blue was added to give a dark blue solution) on a slide, covering with a cover glass and observing the degree of staining.
Bark-staining pollen grains were considered viable and non- or light-staining grains were considered to be non-viable since abortive grains were unstained or only lightly stained.
Studies of pollen tube grov/th in the style were made by fixing the styles for 24 hours in an acid-alcohol fixative (3 parts absolute alcohol, 1 part glacial acetic acid), placed in a 5 per cent pectinase solution (5 g. pectinase, 95 cc. distilled water, filtered, and adjusted to pH 4 with IN HCl) for 12 to 24 hours, stained with 7
acid fuchsin-light green ((Darlington and LaCour 1947) diluted 1:15 with 1 per cent aqueous light green) for 24 hours and mounted in
glycerine. The pollen tubes were easily visible as unstained
structures in the reddish-purple stylar tissue.
An ocular micrometer was used for microscopic measure
ments and a camera lucida was used for all drawings.
i GENERAL MORPHOLOGY AND GROWTH
Characteristics of polyploids have often heen investigated and reviewed (Blakeslee, Warmke and Avery 1939, Dermen 1940, Miint- zing 1936, Npggle 1946, Randolph 1941) and their economic value has been discussed by Eigsii (1957), Eigsti and Dustin (1955)> Stebbins
(1956) a m others. The most observable consequences of induced polyploids as Dermen (1940) points out are associated with increase in size of plant parts which are in turn economically desirable#
According to Muntzing (1936), in general, there tends to be a posi tive relationship between chromosome number and gigas characters.
Autotetraploids are usually characterized as being more robust in vegetative and floral parts. One usually finds larger leaves, flowers, fruits and seeds on the tetraploid plant than on the dip
loid plant. It has been shown that morphological changes in auto— tetraploids are due to alterations in cell size (Muntzing 1936)
¥*» although Dermen (1940) in his review states chromosome doubling may have no effect on the size of the cells thus being morphologically indistinguishable from the diploid. Some tetraploids may have larger cells but a decrease in the total number of cells, thereby resulting in a plant appearing similar to its diploid (Dermen 1940).
In this study leaf and pollen morphology, rate of plant growth and development, as well as seed, fruit and flower size of diploid and autotetraploid Physalis pruinosa plants are compared.
Observations and Results
The autotetraploid plant of Physalis pruinosa exhibited the usual larger morphological characters over the diploid (Figure
8 3). The leaves were about 30 per cent larger, 47 per cent thicker and also greener than the diploid (Table l). The increase in leaf thickness is due to larger cell size in the tetraploid and not an increase in the number of cell layers (Figures 4 and 5)» The darker green color is due to an increase in the number of chloroplasts, in this case the number being about twice that found in each diploid palisade cell. Kostoff (1938) found an increase in the number of chloroplasts per cell in tetraploid Petunia and tomato and with
Kendall (1934) observed likewise larger epidermal, palisade and spongy cells in tetraploid tomato. Larger guard cells have been observed in autotetraploid Datura (Blakeslee 1941) and tomato (Kostoff and Kendall 1934). In this study an increase in guard cell size as well as stomate length was found in tetraploid P. pruinosa. Hutton
(1954) also found larger stomates in tetraploid Physalis floridana.
If the larger size of an autotetraploid leaf is due to an increase in cell size rather than number of cells, the number of cells per unit leaf area of a diploid, in contrast to its autotetraploid, should be more. By comparing the number of stomates per square millimeter the frequency was found to be less in the tetraploid (Table l).
Similar observations have been made in maize (Randolph 1935)> trades- cantia and Secale (Sax and Sax 1937) and. tobacco (H. H. Smith 1939)•
Larger flowers, pollen and seed in tetraploid plants have been repoi'ted in many plants including Physalis floridana (Hutton
1954» Table 6), flax (Ross and Boyce 1946), and tomato (Kostoff 1938,
Lindstrom and Humphrey 1933)* Similar results were found in this study for Physalis pruinosa (Table 2, Figures 6, 7* 8, 9)* 10
Figure 3« Diploid (left) and tetraploid (right) plants of Physalis pruinosa» Table 1. Size of leaf tissues and size and frequency of stomates in diploid and tetraploid Physalis pruinosa. Each figure represents an average of 10 measurements. aa Upper Palisade Number Number Spongy Number Lower Leaf Midrib Leaf epidermis layer of of layer of epidermis blade thick- blade thickness thickness palisade chloroplasts thickness spongy thickness thickness ness width (microns) (microns) layers per pal.cell (microns) layers (microns) (microns) (microns) (cm)
Diploid 16.6 67.6 1 20.4 64.9 4.1 10.4 159.7 685.1 5.3
Tetra ploid 26.7 123.5 1 44.6 134.9 3.8 15.6 300.8 872.9 7.5 H H
Cell diameter Guard cell length Storaate length Number of stomates (microns) (microns) (microns) per square millimeter Upper Lower Upper Lower Upper Lower Upper Lower ______epidermis epidermis epidermis epidermis epidermis epidermis epidermis epidermis
Diploid 61.8 55.7 26.4 25.4 14.5 ' 11.4 136 431
Tetra ploid 86.9 84.0 43.9 38.3 30.0 26.5 86 231 12
Figure 4« Cross-section of leaf of diploid P. -pruinosa (175X) o
Figure 5» Cross-section of leaf of tetraploid P. pruinosa (l75X)» Table 2. Flower and fruit characteristics in diploid and tetraploid Physalis pruinosa*
Average Range Number observed Diploid Tetraploid Diploid Tetraploid Diploid Tetraploid
Diameter of open flower (millimeters) 9.6 1 1 .6 9.0-1 0 .0 10.0-13.0 120 60
Diameter of mature fruit (millimeters) 11,7 1 1 .0 8.0-14.5 6,5-1 5 .0 312 295
Length of seed (millimeters) 1.75 2.1 1.5-2.0 2.0-2.25 100 100
Diameter of pollen grains (microns) 21.8 27.6 17.2-23.4 23.4-32.1 200 350
Pollen tube width (microns) 5.58 7.3 4.9-6.1 4.9-9.8 160 100 1
Style width (microns) 404.5 550.0 330-440 517-572 90 90
Frequency of 4-pored pollen grain (fo) - - 0 6.2 527 450
Humber of ovules at the megaspore 10 10 mother cell stage 77.4 49.2 71-86 37-60 ovularies ovularies
Number of ovules when flower opens 102.3 78 70-149 41-138 18 18 ovularies ovularies
211 193 Number of seeds per fruit 76.9 20.7 27-147 0-46 fruits fruits Figure 6. Flowers of diploid (left) and tetraploid (right) P. pguinosa. Scale is in millimeters.
juL uij u iij d Figure 7* Seeds of diploid (left) and tetraploid (right) pruinosa. Scale is in millimeters. Figure 8. Pollen of diploid P. pruinosa (370X)
Figure 9« Pollen of tetraploid P. pruinosa (370X). Note greater variation in size and 4-pores in some. 16
A high degree of pollen abortion is often found in auto- tetraploids and this will be discussed later in more detail. About one—half of the pollen was found to be aborted in this study (Table
8). Associated with this is the more frequent variation in the size of pollen grains. The viable tetraploid pollen grains are generally larger than the diploid. This variability in tetraploid pollen grains as observed here and in other plants (H. H. Smith
1939» Kostoff and Kendall 1934» Lindstrom and Koos 193l) is appar ently due to unequal chromosome distribution as a result of meiotic abnormalities. Randolph (1935) in maize, however, observed abundant pollen of uniform size. Warmke and Blakeslee (1939) reported the presence of 4-pored pollen in tetraploid tobacco and although none were observed in diploid P. pruinosa» 6 per cent of the pollen grains in the tetraploid were 4-pored, the rest being 3—pored (Figure 9)«
The tetraploid pollen tubes were also larger (Table 2), this having been likewise observed in Petunia and tomato (Kostoff 1938). Al though the tetraploid seeds are larger than the diploid, the charac teristic low seed set of autotetraploids was present, the reduction being about 70 per cent (Table 2). A cause of this will be dis cussed later. In P. floridana Hutton (1954) found the autotetra ploid to have 84 per cent fewer seeds than the diploid.
As with other organs of the autotetraploid plant, an in crease in size of the fruit would be eapected but smaller fruits have been reported in Datura (Blakeslee 1941) and Solanum (Jorgensen
1928). In tomato, Jorgensen likewise found that the tetraploid fruits usually were not larger than the diploid, often being smaller. 17
He suggested this may he due to a fewer number of seeds. As shown in Table 2 after measuring about 300 fruits, each from diploid and tetraploid plants, the size averaged about the same. In general, the fruits from the two plants were similar and could not be ex ternally distinguished. Ths range of size variation^ however, was larger in the tetraploid fruits (Tables 2, 9). A study of fruit size in relation to the included number of seeds showed a definite relationship to be present (Table 3). As the number of seeds in both diploid and tetraploid fruits increase, the size of the fruit usually increases. As a result of the low seed set in the tetra ploids, the size of the fruits formed is smaller than might normally be expected, resulting in size of fruits that is generally similar to that of the diploid (Tables 2, 9)«
Hesse (1938) reports that the rate of germination of tetraploid Petunia seeds is equal to or higher than the diploid. In tomato slightly earlier germination of tetraploid seeds has been re corded (Gustafsen 1944) as well as being 3 to 5 days slower (Jorgen sen 1928). In tetraploid flax Pandey (1956a) found slower germina tion of the tetraploid seed. Tetraploid seeds of P. pruinosa were found to germinate slower than diploid seeds (Table 4)» Germination of the seeds of the diploid started 6 days after planting, of the tetraploid, 8 days. Half of the diploid seed had germinated in 8 to
9 days after planting whereas in the tetraploid it was 13 to 14 days.
The rate of growth is generally slower in autotetraploids than in their diploids (Stebbins 1950)• Data based on a study of 10 diploid and 15 tetraploid P_. pruinosa plants from seed germination 18
Table 3. Relation of fruit size to number of seeds in diploid and tetraploid Physalis pruinosa. These data are "based on 76 diploid and 76 tetraploid fruits. Num'ber of Diameter of fruit seeds per . , -in millimeters ______fruit______Diploid Tetraploid
0 — 6.8 0-9 — 10.2 10-19 — 9.95 20-29 10.0 12.0 30-39 10.1 12.7 40-49 1 0 .4 14.5 50-59 1 0 .5 — 6O-6 9 9 .8 — 70-79 11.0 — 8O-8 9 11.1 - 90-99 11.8 - 100-109 12.5 - 110-119 13.5 - 120-129 13.9 - 130-139 14.3 — 140-149 14.0 —
Table 4* Seed germination of diploid and tetraploid Physalis pruinosa. Results on basis of 100 seeds each placed on
Days Number of seeds Per cent seed after germinated_____ germination "planted11 Diploid Tetraploid Diploid Tetraploid
5 0 0 0 0 6 18 0 18 0 7 30 0 30 0 8 45 7 45 7 9 58 12 58 12 10 64 19 64 19 11 70 27 70 27 12 76 48 76 48 13 78 49 78 49 14 78 52 78 52 15 80 55 80 55 19 until they were 7 weeks old are given in Table 5* The average rate of growth in height was less in the tetraploids indicating the usual slower rate of growth. It is interesting to note that in all aged plants, although the minimum size range as well as the average and median is larger in the diploid than in the tetraploid, the maxi mum range starting approximately 3 weeks after germination is larger in the tetraploid. The height of the tallest diploid and tetraploid plants can thus he compared. Out of the 15 tetraploid plants there were two which began to grow more rapidly than the other diploid and tetraploid plants and thus became taller. This is assumed to be due in part to genetically different tetraploid seeds.
Associated with slower growth in height of tetraploids is a longer time before flower formation. Blakeslee (l94l) noted this in Petunia as did Kostoff (1938) in both Petunia and tomato. Levan
(1942) however observed approximately the same flowering date in both diploid and tetraploid flax and Kostoff and Kendall (1931) noticed a tetraploid Petunia plant which flowered 8 days before the diploid. In this study the diploid flower buds were externally visible 30 to 36 days after germination, the median being 30 days; the tetraploid range was 32 to 49 days, the median being 38 days.
The first diploid flower opened 43 days after germination, .all 10 plants being in flower 55 days after germination. The first tetra ploid flower opened 50 days after germination and the last plant in flower 70 days after germination. The median, i.e., half of the plants being in flower, was 45 and 59 days respectively for the dip—
; loid and tetraploid. As can be seen, flower bud formation and open- 20
Table 5* Rate of growth in height of diploid and tetraploid Physalis pruinosa. Data on basis of 10 diploid and 15
Weeks after seed _____ Diploid______Tetraploid germination Ave . Median Range Ave. Median Range
2 4.0 3.7 3.0- 5.0 3.2 3.2 2.8- 3.9 3 8.2 8.2 7.0- 9.2 6.4 8.5 4.1-10.0 4 13.5 13.4 11.0-15.5 9.3 9.0 6.1-17.5 5 18.2 18.7 15.2-21.0 12.6 12.0 8.5-22.5 6 22.5 2 3 .0 18.2-26.2 15.9 14.6 11.8-29.3 7 25.9 2 6 .0 22.0-29.2 19.5 17.3 13.5-35.5
ing was not only earlier in the diploid but extended over a shorter length of time. This lag in time also points to slower growth and development characteristic of tetraploids. This is observed also in that the flower buds and anthers of the tetraploid open slower than do those of the diploid.
The two tetraploid plants mentioned above as having an increased rate of vegetative growth over the diploids were later in flower bud development and opening as compared to the diploids.
Although these plants were the first of the tetraploid plants to flower, the first flower bud formation was observed to be 2 days later than that of the diploid and this was 2 days longer than the diploid median. The first flower opened 7 days later than that of the diploid which was 5 days more than the median. This suggests that an increased chromosome number may affect vegetative and re productive growth differently. Further study on this point should be initiated. This later blooming characteristic of autotetraploids may be desirable in some plants of floricultural importance. (Ems— weller and Ruttle 1941 lO 21
As can be seen in Figure 3, the diploid appears more branched than the tetraploid. This has been observed to be a general
character observed during the course of this study. This also has been noticed in Solanum (Jorgensen 1928), flax (Levan 1942), Petunia
(Hesse 1938), and iJicotiana (H. H. Smith 1939)* In autotetraploid
rye less tillering is considered a disadvantage since this results
in less heads per plant (Stebbins 1956).
Discussion and Summary
Size increase of plant parts such as leaves, stomates,
flowers, pollen and seeds usually associated with polyploids (Mint—
zing 1936, Noggle 1946) was found to be true in autotetraploid
Physalis pruinosa. The increase in leaf size was due to an increase
in cell size rather than cell number and presumably this is-true for
the other parts. According to Dermen (1940) most polyploid changes
are due to an increase of cell size and not the total number of
cells. This change in cell size is probably due to changes in the
amount of various substances associated with cell growth and elonga
tion brought about by a doubling of the chromosomes (Stebbins 1950).
A comparison of diploid and autotetraploid plants of
Physalis pruinosa as a result of this study and of P. floridana
(Hutton 1954) is given in Table 6. In both plants the diploid
chromosome number is 24* The size increase of the P» pruinosa
tetraploid over its diploid is, in general, larger than that of P.
floridana. Pollen size and degree of seed set are, however, smaller
in P. pruinosa. In tetraploid Niootiana rustica, tabaoum and glauca 22
Table 6. Comparison between autotetraploid Physalis pruinosa and autotetraploid Physalis floridana» Figures are per cent increase over their respective diploids. Data on P_. floridana based on Hutton (l954)» P. pruinosa P. floridana Tetraploid Diploid Tetraploid Diploid _ Leaf thickness 47?° - 1 7 ? Leaf size 30?° 13.1 ? ° — Guard cell size W ° - — Stomate size 34? 405& — Flower diameter 1 8.2# 1896 — Pollen size 22»2>?° 4 0 ? — Pollen sterility 49.4# 4 0 ? - Seed reduction 73*1?° 841° - Start of flowering tetraploids coincident about 1 week later than diploids Period over which first flowers open 20 days 12 days 12 days 7 days
H. H. Smith (1939) found smaller leaves whereas YTarmke and Blakeslee
(1939) in H. sanderae found broader leaves than in the diploid. In
all cases the leaves were thicker. In comparison with the diploid
Smith found smaller flowers in IT. rustioa and larger flowers in IT. tabacum and glauca tetraploids. Warmke and Blakeslee also observed larger flowers in tetraploid H. sanderae. Effect of chromosome
doubling on the growth physiology of different genotypes (species)
as well as environmental differences could account for these dif ferences.
A majority of the diploid and tetraploid fruits of tomato were found to be indistinguishable in size and shape by Kostoff and
Kendall (1934)• Similar observations were made by Lindstrom and
Humphrey (1933) and they mentioned this may be due to 30 to 40 per
cent less seed set in the tetraploid which may cause the tetraploid 23 fruits not to "be as large as would be expected. During the course of this investigation fruit size in relation to content of seeds was studied and larger fruits were found to contain more seeds.
Thus the tetraploid fruit appears to "be smaller due to low seed set. This relationship can "be explained "by the fact that growth hormones will cause parthenocarpy whereas without them (and no pollination) the fruit does not develop (Table 9)» Apparently during seed maturation hormones are produced which cause fruit de velopment. Y/ith fewer seeds, then less hormone and smaller fruits would be expected.
According to Boggle (1946) there is general agreement among botanists that polyploidy brings about a slower rate of growth but that an increase of plant size may or may not be a re sult. Stebbins (1956) in his general review on polyploid crops mentions slow growth is one of their disadvantages, low seed set being another. Plants which are the result of chromosome doubling are on the whole as often smaller and less vigorous as they are larger and more vigorous than their diploids (Demerec 1947)• Dip—
-loid and tetraploid Tradescantias are indistinguishable from each other externally (Swanson 1957). Tetraploid fiber flax grows slower than its diploid all the time whereas tetraploid oil—flax, although in early growth was slower than its diploid, was growing faster by the end of the season (Levan 1942). Pandey (1956a) found also that the tetraploid linseed variety has a greater growth rate in height than the diploid but the tetraploid fiber flax grows slower than the diploid. In the same species then, the rate of growth of the 2 4 tetraploid in relation to its diploid is not the same. H. E. Smith
(1939) found that tetraploid Nicotiana rustica, tahacum, and glauca
were smaller than the diploids whereas Warmke and Blakeslee (1939)
found tetraploid N. sanderae to he taller and larger than the dip-
; loid. Hutton (1954) starting with 6-inch high diploid -and tetra
ploid plants of Physalis floridana found at time of flower opening
which was coincident, the tetraploids were ahout 14«8 per cent
taller.
In Physalis pruinosa all of the tetraploid plants were
initially slower in growth in height than the diploids hut after
several weeks two of the 15 tetraploid plants became larger than
any of the diploids, the others remaining smaller. As has been re
viewed here, there is variation in growth between the diploids and
their autotetraploids as well as among the autotetraploids them
selves and the results are not consistent. This emphasizes the need
for more critical work on this problem. The exact reason for these variable results is unknown as far as I am aware. It is obvious
that these variations do occur. Several partial explanations have been offered. H. H. Smith (1939) suggests the tetraploid fficotiana plants may be smaller due to nuclettB.'.-cytoplasm ratio disturbance,
less frequent cell division or to "other causes." Gustafson (1944)
found 5^.8 per cent as much growth hormone in tetraploid tomatoes
as in diploid, although he says the general appearance of the tetra
ploid plant gives no hint as to its less hormone content. Ingaome
tetraploids slower rate of growth and development is thought to be •
due to a decreased rate of cell division (Noggle 1946). Hoggle men— 25 tions a slower rate of respiration and thus a lower rate of metabo lism as found in tetraploid barley may be involved in slower growth.
Kostoff (1938) observed in Petunia and tomato that the period from seed germination to flov/ering increases with ploidy.
Tetraploid tomatoes flowered 10 to 60 days later than diploid
(Kostoff and Kendall 1934). The same workers (1931) found an ex ception in tetraploid Petunia in that one grew faster and flowered
8 days before the diploid and explained this may be due to a varietal difference (1934). Polyploid plants in general though, flower later than the diploids (Demerec 1947). In this study tetraploid Physalis pruinosa flowered about 1 week later than the diploids. In P. floridana Hutton (1954)» starting with 20 uniform 6-inch high dip—
; loid and tetraploid plants, found the starting of flowering was coincident. As in P. pruinosa he found the first flowers appeared over a longer period of time than did the diploids. However, the length of this period was shorter than in P. pruinosa (Table 6).
Since growth and size of plants is due to the number and size of cells, and an increase in size of plants as well as size and number of cells does not always accompany chromosome doubling, the effect of the latter is variable in various plants as well as in the same plant. The physiological brocesses which are responsible for the phenomena of growth are controlled by the heredity and en vironment of the plant as a whole as well as of each cell of it. Any genetic unbalance or irregularity as a result of chromosome doubling could cause variations in the processes through their control on production and utilization of substances involved in growth. The 26 effect of the double chromosome complement per se on the growth processes through its effect on the growth regulating substances is also involved. External or internal environmental variations down to the molecular level can likewise affect the basic physio logical growth processes of each plant resulting in growth varia tions between individual plants. Although the precise cause of variation in growth and development in diploid, tetraploid or any plant cannot always be determined, it must be based on the complex interrelationships and effects of the heredity and environment on the physiological processes within the plant. PEE- ABE POST-FERTILIZATION MORPHOLOGY
v
The development of the pollen, archesporial cell, integu
ment, embryo sac, fertilization, endosperm and embryo in the Sola-
naceae has been adequately reviewed by Schnarf (1929, 1931) • Since
1931 many other morphological investigations have been made in the
family among which the more comprehensive are Barnard (1949) on
Capsicum, Bhaduri (1932, 1935> 1936) on Solanum, Physalis, Withania,
Oestrum, Nicotiana, Salpiglossis, Lycopersicum, Datura, Petunia,
Brunfelsia, Clarke (1940) on Solanum, Cochran (1938) on Capsicum,
Cooper (1931) on Lycopersicum, Dorasami and Gopinath (1944) on
Capsicum, Goodspeed (1947) Nicotiana, Magtang (1936) eggplant,
Rees-Leonard (1935) Solanum and 0. Smith (1935) on Lycopersicum,
In general the results of these and other investigations
are in accord with what has been found in earlier studies on the family although there have been a few variations reported. The results of this study and discussion of these results in relation
to relative investigations will be reported under the following headings: Development of the Ovule and Embryo Sac, Development of
the Pollen, Fertilization, Endosperm, Embryogeny, and Early Seed
Coat Development.
Development of the Ovule and Embryo Sac
Observations and Results
Since the manner and sequences of the stages of develop
ment recorded here were similar in both the diploid and tetraploid
material, no distinction between the two will be made in the follow-
27 28
ing description; the observations applying to both of them unless
noted.
In the ovularies of very young flowers the axile.placenta
is composed of homogeneous appearing cells, the placenta as a whole
appearing smooth on the surface. Due to cell division in the epi
dermal and sub-epidermal layers at intervals over the placenta, a
series of bumps being the young ovules are initiated, resulting in
an uneven appearance of the placenta. As the ovule becomes larger
there is unequal lateral growth which results in the erect ovule becoming curved and resulting by the time the embryo sac is mature
in the ovules being in all stages from campylotropous to anatropous, most, however, being nearly anatropous.
When the flower bud is approximately 2.0 mm. wide many
ovules are visible and at this time a hypodermal cell at the tip of
each ovule becomes evident from the other uniform ovule cells by
enlarging and becoming quite conspicuous (Figure 10). It contains
a large nucleus and dense cytoplasm. This is the archesporial cell.
After the archesporial cell has differentiated the single
integument, characteristic of the Solanaceae (Schnarf 1931)> starts
to differentiate (Figure 10). Around the periphery of the ovule,
due to divisions of the epidermal and subepidermal cell layers, a
ridge of tissue becomes apparent which is the young integument.
This continues to enlarge and elongate until it surrounds and there
by embeds the archesporial cell within it• The ends of the integu
ment do not fuse thus leaving a pore, the micropyle, between. The
rest of the ovular tissue is termed the nucellus and thus at this 29 stage the ovule consists of a single integument (with a micropyle), a nucellus and an archesporial cell* Since the archesporial cell is an apical hypodermal cell the nucellus is a uniseriate layer of cells over the top and sides of the archesporial cell and more massive helow it, thus being tenuinucellate. The flower hud gener ally is about 2*5 to 2.8 mm. wide when the ovules are in this stage but variations have been found.
The archesporial cell does not divide into a primary parietal cell and a primary sporogeneous cell but morphologically is the megaspore mother cell and by the time the flower bud is about
3.0 mm. wide is a comparatively large cell. At this time the mega- spore mother cell divides transversely twice in rapid succession.
This results in first a dyad (Figure 11) and finally a linear tetrad of four megaspores (Figure 12). This division of the megaspore mother cell is a meiotic division; each resulting megaspore has the n_ number of chromosomes. The four megaspores are similar in appear ance but soon the chalazal megaspore becomes larger while the other three begin to disintegrate and appear as a red-staining amorphous mass on top of the chalazal megaspore.
The functional chalazal megaspore (Figure 13) enlarges and its nucleus divides into two which become separated toward the ends of the enlarging cell. This structure is the 2-nuoleate embryo sac (Figure 14)* Each of these two nuclei divide again resulting in a 4-nucleate embryo sac (Figure 15) followed by a division of each of these four nuclei resulting in the formation of an 8-nucleate embryo sac (Figure 16). Thus Just before or when the flower opens 30
each ovule contains an 8-nucleate embryo sac. This type of embryo
sac development corresponds to the monosporic 8-nucleate Polygonum
or normal type. This type is commonly found in the Solanaceae
(Schnarf 1931).
As the embryo sac matures, one nucleus from each end moves
toward the center of the embryo sac. Each of these is a polar
nucleus. When the flower opens the polar nuclei may or may not have
fused. At this time there is a higher amount of fusion of the polar
nuclei in the diploid (4&f°) than in the tetraploid (18^). Of the unfused polar nuclei 9&»4 per cent in the diploid and 89.8 per cent
in the tetraploid were next to each other. This shows a slower rate
of migration and fusion of the polar nuclei in the tetraploid as
compared to the diploid. Two days after the flower opens 9 6 , 4 per
cent of the polar nuclei were fused in the diploid and 95*5 per
cent in the tetraploid. At this time all of the unfused polar
nuclei of the diploid and 91 P®*“ cent of those of the tetraploid were next to each other.
The group of three nuclei at the micropylar end of the
embryo sac is known as the egg apparatus consisting of an egg and
two synergids. The three nuclei at the chalazal end are the anti- podals. In the young 8-nucleate embryo sac the antipodals are
evident but they are ephemeral and thus degenerate after formation
and are not generally found in a mature embryo sac. As the embryo
sac matures the synergids elongate and become pear or triangular
shape in appearance. The egg becomes elongated and its nucleus often
projects above and between the synergids. The fusion nucleus or the 31
Plate II• All figures are diploid P_. pruinosa. Figure 10• Ovule with megaspore mother cell and young integument. Figure 11. Dyad. Figure 12. Tetrad of megaspores. Figure 13. Functional megaspore with three disintegrating ones. 32
FIG. 16
Plate III. All figures are diploid P. pruinosa. Figure 14« 2-nucleate embryo sac. Figure 15. 4-nucleate embryo sac. Figure 1 6. 8-nucleate embryo sac. 33 two polar nuclei are usually found "beside the egg or less frequently at the edge of the embryo sac lateral to the egg.
During megasporogenesis and the development of the embryo sac the nucellus degenerates and when the embryo sac is mature the nucellus has completely degenerated and the embryo sac lies ad jacent to a layer of integument cells which during the disintegration of the nucellus become enlarged and contain prominent nuclei. This layer of cells is known as the integumentary tapetum or endothelium and has been observed in many Solanaceous plants (Bhaduri 1935)*
Discussion and Summary
The development of the ovules, nucellus, integument, archesporial cell, megaspores and embryo sac as here described for
Physalis pruinosa is similar to that which has been found character istic in other plants of the Solanaceae.
Bhaduri (1935) has studied stages in ovule development from its origin through the embryo sac stage in Physalis minima and P_. peruviana as well as species of the following Solanaceous genera: Solanum, Withania, Oestrum, Nicotiana, Salpiglossis, Lyco persicum, Datura, Petunia, and Brunfelsia and has found them all to be similar in development which in turn is similar to what has been found in this investigation on Physalis pruinosa. Barnard (1949) in Duboisia, Cochran (1938) in Capsicum, Cooper (1931) and 0. Smith
(1935) in tomato, Rees-Leonard (1935) in potato, Goodspeed (1947) in Nicotiana and others have found essentially similar results in their investigations. Since the development as described here for
Physalis pruinosa is basically the same as found in other investi- 34 gated Solanaceous plants, only variations in development will "be noted here,
Bhaduri (1935) found in Oestrum campylotropous ovules and in Nicotiana and Petunia anatropous ones, Goodspeed (1947) found anatropous ovules in Nicotiana. Cochran (1938) and Dorasami and
Gopinath (1944) "both found anatropous ovules in Capsicum. In tomato, Bhaduri found half-anatropous ovules whereas 0, Smith
(1935) and Cooper (1931) found anatropous ovules. In Solanum,
Bhaduri found half-anatropous, Rees-Leonard (1935) found amphitropous ovules and Young (1923) ovules transitional from anatropous to campylotropous. In Physalis and the other genera studied by Bhaduri
(except Oestrum, Nicotiana and Petunia) half-anatropous ovules were found, these being apparently transitional stages between campylo- tropy and anatropy. In Physalis pruinosa most of the ovules would correspond with Bhaduri’s half-anatropous designation although both campylotropous and anatropous ovules were observed.
There have been very few descriptions of embryo sac de velopment in the Solanaceae that have not been described as normal, i.e., monosporic 8-nucleate type of development as has been found here in P. pruinosa. Solanum tuberosum was described as having a
"lily—type" by Young (1923) but upon reinvestigation Rees-Leonard
(1935) found it to be of the normal type. Snarf (1931) suggests that the "lily-type" reported for £^, muricatum should also be re investigated. Lengel (1954) found a bisporic embryo sac in Capsicum frutesoens var. Japanese Variegated Ornamental and it appeared to be well substantiated. Normal types have been found in C. frutescens 35
(Cochran 1938, Dorasami and Gopinath 1944) and in C_. annum
(Banerji 1931).
Sc&mrf (1931) says the antipodals are variable in the
family. He mentions that in Hyoscyamus niger and Datura laevis
they are small and disappear early whereas in Datura metel they
remain long after fertilization. Dorasami and Gopinath (1944)
find them cellular and synergid-like in Capsicum frutescens.
Bhaduri (1935) found in general that in the species he has in vestigated they degenerate by the time of fertilization as have many other workers. They are short-lived in Physalis pruinosa.
Fusion of the polar nuclei before fertilization has been
often observed (Cooper 1931, Clarke 1940, Rees-Leonard 1935>
0. Smith 1935) and. although it generally takes place before opening of the flower it may be delayed until the pollen tube enters the
embryo sac (Bhaduri 1935). When the flower opened, in diploid P. pruinosa about half and in the tetraploid less than one—fourth of the polar nuclei had fused; 2 days later when fertilization has usually occurred about all had fused in both the diploid and tetra ploid (Table "j). The slower rate of migration and fusion of the polar nuclei was also observed in autotetraploid Trifolium (Pandey
1955) and in both cases appears to be another manifestation of
slower growth in the tetraploid as compared to the diploid.
The development of the megasporangium, megasporogenesis
and megagametogenesis were found to be the same in diploid and auto—
tetraploid plants of Physalis pruinosa. In diploid and autotetra ploid Trifolium megasporogenesis and megagametogenesis were also found to be similar (Pandey 1935). 36
Table 7. Comparative data on the embryo sac of diploid and tetra ploid Physalis pruinosa. Size is given in microns. Average Range Rumber observed Diploid Tetraploid Diploid Tetraploid Diploid Tetraploid
Embryo sac length 68.2 82.4 61.7-79 74.1-86.4 45 50 Polar nucleus diameter 5.4 7.0 4.9-7.4 4.9-9.8 25 26 Fusion nucleus diameter 7.4 9.5 -7.4 8.6—9.8 40 25 Triple fusion nucleus diameter 11.3 12.3 9.8-12.3 ±12.3 24 10 Egg nucleus diameter 4.4 4.9 3.7-4.9 ± 4.9 38 35 Synergid nucleus diameter 3.9 4.3 2.4-5.0 3.7-4.9 30 25 i<> polar nuclei fhsed when flower opens 46 18 283 216 io polar nuclei fused 2 days after flower opens 96.4 95.5 405 274
In Table 7 are given comparative measurements of the
embryo sac and the included nuclei. Since in each tetraploid
nucleus the chromatin material is larger in amount over that present
in the diploid* the expected increase in the tetraploid nuclei size 37
over that of the diploid is here confirmed. As Bhaduri (1935)
found for Physalis minima and peruviana, the egg nucleus ia smaller
than the polar nucleus and ahout the same as the synergid nucleus.
The 2n fusion nucleus and the 3n triple fusion (endosperm) nucleus
in each case is larger than the n egg and synergid nuclei. In
turn, the diploid nuclei are smaller than the corresponding tetra ploid nuclei. The tetraploid embryo sac also is longer than the diploid. In comparison with what Bhaduri (1935) found in Physalis minima and peruviana, the eggj synergid, and polar nuclei in Physalis pruinosa turned out to be within the same general magnitude but of a slightly smaller average size. Bhaduri records average dimensions
of the egg nucleus, synergid nucleus, and polar nucleus as 4.6l{ *
4*5 and 6.0 JL{ , respectively for P. minima and 4«9p( > 4*5 K and 6.4 U for P. peruviana. I found for diploid P. pruinosa 4»4J»{ »
3.9 K > and 5*4/X > respectively. The overlap of the measurements in the "range" column as well as the slight differences in size may in some cases be due in part to the plane and location of the sec tion, unequal or incomplete e:xpansion or growth of the embryo sac or nuclei, variation in the amount of chromatin material in the nuclei, or variations in shape and form.
Development of the Pollen
Observations and Results
Sporogenous tissue in the anthers can be observed in flower buds in which the ovules have not started to differentiate
in the ovulary. By the time the ovules are forming, microspore mother cells are evident. At the time when stages previous to and 38
during the differentiation of the megaspore mother cells are pre
sent in the young ovule, stages from the microspore mother cell
through tetrad formation are present in the anthers. By the time the megaspore mother cell is fully differentiated and the integu ments are forming, microspores can he seen. Thus during mega
sporogenesis and emhryo sac development, microspores are present in the anthers. Some 2-nucleate pollen grains are observed in the anthers when megaspores are present in the ovules. When the flower opens, 2-nucleate pollen grains are present. The microspore mother cell nucleus (Figure 17) divides twice in rapid succession. The first division results in a dyad (Figure 18) and the second division results in a tetrad of microspores within the mother cell wall
(Figure 19)• The nuclear division is meiotic since the number of chromosomes in each microspore is one-half that of the mother cell.
There is no cell wall formed between the nuclei of the dyad. Wall formation is simultaneous between the four microspore nuclei. The microspores then enlarge, and separate and a thick wall is then formed around each one. The microspore nucleus then divides into a generative and a tube nucleus; a 2-nucleate pollen grain results
(Figure 21). All the diploid pollen grains had three germ pores;
6.2 per cent of the tetraploid pollen grains had four germ pores and the rest had three.
The pollen grains of the diploid were generally uniform
in size while those of the tetraploid were more variable in size.
Although the cytological details of microspore formation were not
studied, this variation in pollen size and the frequent presence of (17QX)
Figure 18. Dyads in anther of tetraploid, (170X). • 40
Figure 19. Tetrad, of microspore nuclei (tetra^ ploid), (170X).
Figure 20. Tetrad of microspores (tetraploid), (170X). Figure 21. 2-nucleate uollen (tetraploid), (3150X).
Figure 22. Microcytes in microspore tetrad of tetraploid, (170X). 42 microcytes in a tetrad (Figure 22) could "be explained "by the apparently unequal distribution of nuclear material as a result of irregularities in the meiotic division. In the course of studying microsporogenesis, occasional lagging chromosomes were observed which, at least in part, would indicate this to be true.
Discussion and Summary
The earlier development of the pollen formation stages in the anther in contrast to the megagametophyte formation in the ovularies in the same plant has been observed before. Barnard
(1949) in Duboisia found tetrads or pollen in the anther when the megaspore mother cell divided and 0. Smith (1935) found in tomato
2-nucleate pollen present at the 4“Celled embryo sac stage.
Simultaneous wall formation as found in Physalis pruinosa agrees with that characteristic of the Solanaceae (Schnarf 1931) and has been found to be tsue in later investigations of Solanaceous plants (Cochran 1938, Goodspeed 1947» Magtang 1936, 0. Smith 1935»
Young 1923).
Two-nucleate pollen grains in mature anthers as found in this study have been observed in Nicotiana tabacum and Datura laevis
(Schnarf 193l) and in Capsicum frutescens (Cochran 1938). Lengel
(1954) found 3-nucleate pollen in Capsicum frutescens var. Japanese
Variegated Ornamental,
Pollen grains were 3-pored in diploid Petunia and diploid tomato but in polyploid Petunia (Ferguson and Coolidge 1932, Stout and Chandler 1941) and tetraploid tomato (Jorgensen 1928) 4—pored pollen grains were present as they were in this study. Variability 43
in size and shape of pollen grains of the tetraploid was character
istic of tomato (Lindstrom and Koos 1931, Kostoff and Kendall 1934)
and Petunia (Ferguson and Coolidge 193^)•
The development of pollen in Physalis pruinosa appears to
"be similar to that described for other Solanaceous plants. The
pollen of the tetraploid, as has been found.true in other species,
is generally characterized by more variability in size and shape,
due in part to irregular chromosome distribution, and by the in
creased number of germ pores.
Fertilization
Observations and Results
Pollen tubes can be observed in the ovulary and embryo
sac of both diploid and tetraploid plants 24 hours after self polli nation indicating fertilization may then first occur at this time.
Rarely any pollen tubes can be seen in the ovulary of any plant
less than 24 hours after pollination. Studies of pollen tube growth
in the style show that although 2 hours after pollination a few
grains start to bulge at the pores but rarely germinate, it was not until 16 hours after pollination in styles of both the diploid and
the tetraploid that pollen tubes were readily visible and then were
only in the upper one-fourth of the style. Twenty-four hours after
self-pollination the majority of the pollen tubes were from three-
fourths of the length to the bottom of the style. The growth rate
of the diploid and tetraploid pollen tubes thus appears to be simi
lar. The styles were found to be of the closed type as are found
in Datura (Maheshwari 1950)* The styles are firmly attached to the 44 ovularies 24 hours after the flower opens hut after 48 hours the styles usually have abscised or are very weakly attached. There fore, most fertilization in both the diploid and tetraploid occurs between 24 and 48 hours after pollination. At the time of fertiliza tion the antipodals and nucellus have disintegrated, the embryo sac being surrounded by the prominent integumentary tapetum. The synergids are probably destroyed by the pollen tube since they are not commonly apparent after fertilization. In ovules containing unfertilized eggs they are generally very prominent. Porogamy was observed in all cases.
Discussion and Summary
Fertilization occurs in Physalis pruinosa for the most part between 24 and 48 hours after pollination. Fertilization has been reported to occur in potato in 36 hours (Clarke 1940) and generally 2 to 4 days after pollination in the Irish Cobbler variety
(Arnason 1943)* In Capsicum fertilization is 42 hours after pol lination at 70°F (Cochran 1938), in Petunia 24 to 32 hours (Cooper
1946) and in tomato 0. Smith (1935) never observed fertilization less than 50 hours after pollination. He observed an inactivity of - the pollen grains for several hours after they were placed on the stigma. A similar observation was made in this study although the apparent time of inactivity was longer. In general, it appears that the time of fertilization is similar in these plants. Maheshwari
(1950) in his discussion of pollen tube growth mentions that tempera ture is an important factor. If the temperature were the same for all of these plants, a more accurate comparison could be madeo 45
Fertiliza'tion in the Solanaceae is porogamous as reviewed
"by Schnarf (1931) and this has been found to he true in later in vestigations (Barnard 1949» Cooper 1931, Dorasami and Gopinath
1944, Magtang 1936, 0. Smith 1935) of plants in this family in cluding the present study on Physalis pruinosa. At the time of fertilisation, the synergids have likewise been found to degen erate in Duboisia (Barnard 1949) and eggplant (Magtang 1936) as well as in other plants.
Endosperm
Observations and Results
The endosperm arises as a result of the division of the triple fusion nucleus. There was never any endosperm observed in ovules of non-pollinated flowers, in ovules of flowers to which a hormone had been applied to the stigma, or in ovules of flowers that had been pollinated for 6 hours, the style being removed after that period of time.
The first division of the endosperm nucleus in both the diploid and tetraploid was always transverse with a cell wall form ing between the daughter nuclei in the center of the embryo sac
(Figure 23). Since the subsequent divisions of the endosperm nuclei were always followed by cell wall formation, a cellular endosperm was formed. Helobial or free nuclear endosperm was not observed. The plane of division of each of the nuclei of the 2- celled endosperm was not always the same in each seed of diploid and tetraploid fruits. In both it was observed that the two nuclei may 4 6 divide transversely, resulting in a linear row of four endosperm cells, or one nucleus may divide transversely and one vertically, or both nuclei may divide vertically. The divisions of the 2-celled endosperm nuclei may be simultaneous but frequently they are not; thus 3-celled endosperm is observed. Although successive divisions of the endosperm were not followed in detail, it was apparent that there appeared to be no uniform pattern of spindle orientation since the distribution of endosperm cells in one seed does not necessarily correspond to that of another seed.
The triple fusion nucleus seems to divide shortly after formation. Rarely is endosperm observed in seeds one day after pollination in either the diploid or tetraploid since the pollen tube usually does not enter the embryo sac until -at least 24 hours after pollination. Three days after pollination 2— to 8-celled endosperm was frequent in diploid seeds whereas in tetraploid seeds 2- to 3-oelled endosperm was frequent. This supports the fact that cell division appears to be slower- in tetraploids than in diploids as was first indicated by slower growth and less height of the tetraploid plants. Predominately 2- to 12—celled endosperm in the diploid and 2- to 4-celled endosperm in the tetraploid was found 4 days after pollination. Although endosperm counts were not made in older seeds because of the difficulty in accurately counting cells in serial sections, it was observed the endosperm in both the diploid and tetraploid was highly and densely cellular and appeared in general, similar in both. It is possible that as is true of vege tative growth, after an extended length of time the amount of endo sperm formation may be similar in diploid and tetraploid plants. o © 47
Figure 23. Longitudinal section through the embryo sac of diploid P. pruinosa showing 2—celled endosperm. 48
Discussion and Summary
Cellular endosperm, the type, here found in Physalis pruinosa, appears to he the most common type of endosperm found in the Solanaceae (Schnarf 1929» 1931). Bhaduri (1936) has since ob served cellular endosperm in Physalis minima, Withania and Hicotiana plumbaginifolia. Exceptions are nuclear endosperm in Schizanthus pinnatus and Salpiglossis picta and helobial endosperm in Hyoscyamus niger. Recently Barnard (1949) has found endosperm development of a different type in Duboisia in which both free nuclei and cell wall formation occur, this apparently being of a helobial type.
Transverse wall formation following the first division of the endosperm nucleus has been found in Batura metel and laevis,
Petunia (Schnarf 1931)> eggplant (Magtang 1936) and other plants.
As was found in this study of Physalis, the plane of the next di visions may vary even within genera as is reviewed by Schnarf (l93l), so apparently there is no uniform method of further endosperm forma tion! in the family.
The endosperm nucleus divides in Physalis pruinosa before the zygote. This has been reported for other plants including
Puboisia (Barnard 1949)» potato (Clarke 1940), Capsicum (Cochran
1938), Physalis minima, Y/ithania, Ficotiana, Petunia (Bhaduri 1936)
and tomato (0. Smith 1935). endosperm nucleus has been found
to divide within a short time after fertilization in tomato (0.
Smith 1935) and potato (Clarke 1940) as well as in this present
study on Physalis. Ferguson (1927) reported in Petunia that the
endosperm nucleus divided before discharge of the sperms from the 49
pollen tube "but Cooper (1946) upon reinvestigation found double
fertilization as usual.
Cooper and Brink (1945) found that in tetraploid tomatoes
the endosperm starts growth more slowly hut by 8 days is on a par
with the endosperm of diploid tomatoes. This slower rate of tetra
ploid endosperm cell division is similar to that found in this
study and again emphasizes the slower cell division and growth
commonly associated with tetraploids.
Bmbryogeny
Observations, Results and Discussion
In all cases, the zygote divides first transversely form
ing an embryo composed of two cells arranged in two tiers. Each
of these cells then divides transversely resulting in a 4-celled
embryo with the cells linearly arranged in four tiers. This 4-
celled embryo is typical of the Solanaceae and is referred to as
the Solanad type of embryo development (Johansen 1950). Depending
on the plane of division in the cells of the 4-celled embryo, the
appearance of the 8-celled embryo varies and, accordingly, varia
tions in the embryo development are, in part, classified on the
basis of the arrangement and destination of the cells of the third
cell generation. In Physalis minima. Bhaduri (1936) has observed
four types of variation in the development of the 8-celled embryo
from the 4-celled embryo. Because of this fact, it was decided to
investigate here the development of the embryo in Physalis pruinosa up to the 8-celled embryo. The Soueges system of nomenclature has
been followed (Soueges 1922). The lower cell of the 2-celled embryo, 50 i.e., the one nearest the micropyle, is designated as cb, the one at the distal or chalazal end as ca. The derivatives of ca are 1 and 1* and of cb are ra and ci. Therefore, at the end of the second cell generation there is a 4-celled embryo, the cells of which are designated as 1, 1*, m and ci, reading from the cell nearest the chalazal end to the cell nearest the micropylar end of the embryo sac.
The zygote after formation elongates "before the first transverse division. In the diploid, this first division was ob served 5 days after pollination in a few cases and 6 to 7 days after pollination in most of the embryos. In the tetraploid, the first division was observed 6 days after pollination in a few cases and 7 to 8 days in the majority of cases. This slower development of the embryo of the tetraploid was further substantiated by the observation that 7 days after pollination the diploid had many 2- and 4-celled embryos but the tetraploid had no 4-celled embryos, few 2-celled embryos and many elongated zygotes. Nine days after pollination the seeds of the diploid contained 4- to 20-celled embryos, the tetraploid contained 2- to 10—celled embryos. This apparent slower rate of embryo cell division in the tetraploid as compared to the diploid may be due in part to the fewer number of endosperm cells and hence less food supply in the former, but pro bably this reflects the same inherent relatively slower growth of the tetraploid plant as was observed previously for the growth in length of the plant and for endosperm development. 51
In the diploid, 18 8-celled embryos were observed in the
material sectioned. In the 120 seeds sectioned, embryos with more
and with fewer number of cells were found but only 8-celled embryos
were studied. Thirteen of these 8-celled embryos had the eight
cells arranged in six tiers (Figure 24). This is the Mcotiana
type of embryo of Soueges (1922) and as is summarized in Johansen
(1950) is found in Ilicotiana, Petunia, Withania, Datura, Schizanthus,
Atropha, and Solanum. Bhaduri (1936) found this type of develop
ment in some embryos of P. minima. This is what Bhaduri calls his
first type of development.
Four of the 8-celled embryos were characterized by having
eight cells in eight tiers, i.e., a linear row of eight cells
(Figure 25). Bhaduri observed this type of embryo as common in
P_. minima. Johansen (1950) refers to this as the Physalis I varia
tion and it is also found in Solanum sisymbrifolium. Bhaduri calls
this his second type of development.
One 8-celled embryo was composed of eight cells in seven
tiers (Figure 26). This type of cell disposition is found in both
the Physalis II and Physalis III variation according to Johansen.
Bhaduri found this type of embryo in P. minima. He separated them
on the basis of the ontogeny of the embryo into his Type 3 (Physalis
II) and Type 4 (Physalis III) and found the former occasionally in
P. minima and the latter rare. Therefore, to ascertain which type
of embryo was present, pre-8-celled embryo stages were studied on
the basis of Bhaduri*s classification. The basis of separation is
that in Bhaduri's Type 3» 1 divides longitudinally generally after 52
1', m, and oi divide transversely and in his Type 4» 1 divides longitudinally "before l 1, m, and ci divide transversely, i.e., 1 divides longitudinally in the 4-celled stage.
One embryo which consisted of five cells in four tiers
(Figure 27) was observed. Here was a configuration in which 1 divided longitudinally in the 4-celled stage and before 1*, m, and ci had divided transversely. Thus with the mentioned trans verse divisions occurring, the Bhaduri Type 4 embryo could be formed.
One embryo with six cells in five tiers and one with seven cells in six tiers were observed which would be the expected series to be found in this sequence to the embryo with eight cells in seven tiers.
Bhaduri's Type III requires that 1 generally divides after
1', m, and ci divide longitudinally. One embryo was observed in which there were six cells in six tiers and 1 was in metaphase
(in Bhaduri Type 1 1* generally divides earlier than l), the plane of which was orientated longitudinally (Figure 28). In this case
1 is definitely dividing longitudinally after transverse division of two of the three cells l1, m, and ci. Although one cell has not divided transversely, this apparently occurs since he mentions Ino divides generally before the other three cells. In any case, 1 divided at a later stage than in the 4-celled stage which is characteristic of his Type IT. It appears then that all four types of 8-celled embryos, that Bhaduri found in P. minima are present in
P. pruinosa.
Figure 43 on page 288 of Bhaduri (1936) shows an 8-oelled embryo (with the lower cell in metaphase) in six tiers and is 53 identified as a later stage in proembryo development. In this embryo 1 has apparently not divided. L f has divided transversely into l£, and 1^, each of which in turn has divided longitudinally.
By referring to Figure 42, one may infer that he may mean to imply that 1 has actually divided and the resulting cell is under the one drawn in Figure 43* Nevertheless, Figure 43 does show only an 8— celled embryo. Two such 8—celled embryos of this configuration were found (Figure 29) and of course they are a different type from the others described,.
One young embryo was observed which could not be a fore runner of any of the four types of the 8—celled embryos described.
It was a 6—celled embryo with the fourth cell from the distal end divided longitudinally thus appearing as six cells in five tiers
(Figure 30). As might be expected, deviations from the previously observed cell.division sequence could occur and further observations on the development of such deviations should be conducted.
In the tetraploid, 21 8-celled embryos were observed out of 97 seeds sectioned. Sixteen of these were the Nicotiana Type or
Bhaduri Type I. Four were Bhaduri Type 2 and one with eight cells in seven tiers could have been either Bhaduri Type 3 or Type 4* By finding an embryo with five cells in four tiers (the apical cell having divided longitudinally) one could verify the presence of a
Bhaduri Type IV embryo. One such embryo was observed. Observation was not made of any 7-celled embryo with 1 in longitudinal division so definite confirmation on the presence of Bhaduri Type III was not obtained although at least four embryos with seven cells in six tiers, the apical cell having divided longitudinally, were observed0, o o these apparently being either Type III or Type IV.
Three of the four types of 8—celled embryos found in the diploid were found in the tetraploid and possibly also the fourth type. The frequency of each type is the same in diploid and in tetraploid plants in that the Bhaduri Type I (Hicotiana Type) was the most frequent, Type II less frequent with Types III and IV much less frequent.
Figure 17 on page 286 of Bhaduri (1936) shows an aberrant type of development of the proembryo. This shows a 7-celled. embryo
(with a probable eighth cell) in which 1 has divided longitudinally and one of these daughter cells has apparently subsequently divided transversely. An 8-celled embryo in six tiers, with the one daughter cell of the initial longitudinal division of 1 in a transverse orientated metaphase, was observed in this study (Figure 31)• Since subsequent divisions of the daughter cell resulting from the initial division of 1 are longitudinal, this transverse division would be irregular.
An embryo with eight cells in seven tiers was observed in two instances in which the third cell from the distal end had divided longitudinally, thus forming a different configuration of an 8-oelled embryo (Figure 32). Another type of 8—celled embryo had eight cells in six tiers, but in this case both the apical cell and the second from the apical cell had divided longitudinally
(Figure 33)* In the tetraploid, as in the diploid, deviations in the sequence of cell division of developing embryos could be expected o
55
0®
a
FIG. 2 4 FIG. 2 5 FIG. 26
FIG. 2 7 FIG. 28
Plate IV* All figures are of diploid P. pruinosa* Figure 24* Embryo ■with 8 cells in 6 tiers* Figure 25* Embryo with 8 cells in 8 tiers. Figure 26* Bribryo with 8 cells in 7 tiers* Figure 27* Embryo with 5 oells in 4 tiers* Figure 28. Embryo with 6 oells in 6 tiers, 1 being in metaphase* 56
0
FIG. 3 0
FIG. 29 2 *H FIG. 31
Q
'9* FIG.3 2 FIG. 33
Plate V. Figure 29. Diploid embryo variation with 8 cells in 6 tiers. Figure 30. Aberrant diploid embryo. Figure 31• Tetraploid embryo variation with 8 cells in 6 tiers. Figure 32. Tetraploid embryo variation with 8 cells in 7 tiers. Figure 33• Tetraploid embryo variation with 8 cells in 6 tiers. 57 and the effect of this on later embryological differentiation and development should he investigated.
Summary
The zygote does not divide immediately after formation hut there is a delay before a 2-celled embryo is formed in Physalis pruinosa. The first 2-celled embryo v/as observed 5 days after pollination (about 3 days after fertilization) in the diploid and
6 days after pollination (about 4 days after fertilization) in the tetraploid. Cooper and Brink (1945) noticed in tomato a lag of development in the tetraploid over the diploid also. Although no time was given, Bhaduri (1936) observed a delay in the division of the zygote in Physalis minima, Withania, Nicotiana, and Petunia as did Magtang (1936) in eggplant. In potato, Clarke (1940) did not find a 2-celled embryo until 4 days after pollination (about 2.5 days after fertilization), Cochran (1938) in Capsicum noticed the first division of the zygote 24 to 36 hours after fertilization
(approximately 3 days after pollination) and 0. Smith (1935) in tomato noticed it 44 hours after fertilization (approximately 4 days after pollination). In all cases the first tv/o divisions are trans verse resulting in a 4-celled linear embryo characteristic of the
Solanaceae (Schnarf 1931» Soueges 1922).
As Bhaduri (1936) found in Physalis minima, as a result of this study four types of development of 8-celled embryos were found in diploid Physalis pruinosa and definitely three types and possibly the fourth in the tetraploid. The comparison of the fre— 58 quency of these types of embryo development in P. pruinosa and in
P. minima can be made only by the use of Bhaduri's relative terms as "some" embryos in P. minima develop according to his Type I
(Excotiana Variation), Type IX is "common," Type III occurs "occa
sionally," and Type IV is "rare." In this? investigation it appears that the "common" type of development (in terms of the most fre quent) in diploid and tetraploid P. pruinosa is the Eicotiana Type
(Bhaduri Type i) with his Type II occurring in "some," Types III and IV being "occasional" to "rare."
Several embryos that do not follow the described embryo
structure were observed and variations might be expected on the basis of Johansen's (1950) statement that "the Solanaceae are an
exceedingly difficult group to diagnose embryonomically, mainly because of the excessive variation in the location and planes of the divisions marking the transition from the second to the third
cell generation."
2arly Seed Goat Development
Simultaneous with the growth of the embryo and endosperm
in the seed is the enlargement of the seed coat (integument). In
seeds of both the diploid and tetraploid the cells composing the
integumental tissue appeared to be in nearly parallel rows thus
indicating an abundance of periolinal divisions. This organization
of cells was especially evident from just back of the tapetum to
about the fourth subepidermal layer of cells. In older seeds sev
eral cell layers adjacent to the tapetum were crushed and in some
a slight crushing of the tapetum itself was noticed. 0 Smith (1935) 59 found in enlarging tomato seeds that the integument cells start to collapse at the tapetum. In this study a definite meristematic
call layer could not he distinguished in the integument hut a pre dominance of periclinal divisions were observed in the four or five t cell layers behind the tapetum. Although the enlargement of the integument appeared to he due to many periclinal divisions in this area, these divisions were not limited to this area since peri— clinal cell divisions were observed throughout the integument be tween the epidermis and the tapetum. As a result of the division and enlargement of these cells, the seed coat became thicker.
Scattered anticlinal divisions were also observed but with much less frequency. Cooper and Brink (1945) found the course of seed development in diploid and tetraploid tomatoes the same, one being histologically indistinguishable from the other in kind and se quence of development. This was true also for the early seed de velopment in diploid and tetraploid Physalis pruinosa. Further work should be done on the seed development of these plants since only the young stages were observed in this study. AUTOTETRAPLOID STERILITY
Although autotetraploids have some characteristics which make them desirable for use on a commercial basis, one of their major disadvantages is that they usually have a reduction in the number of seeds as a result of some degree of self sterility
(Demerec 1947» Emsweller and Ruttle 1941» Stebbins 1956). A de creased number of seeds in a plant may be due to several causes which may occur before or after fertilization. Among the former are: differences in flowering time of the parents, differences in the time of maturity of the anther and pistil, non—viable pollen, failure of pollen to germinate on the stigma, not enough pollen . on the stigma, slow growth of the pollen tube, bursting or de formation of the pollen tube in the style, non-viable sperms or
sperms not able to effect fertilization, and disturbances in the development of a normal ovule and embryo sac. If fertilization
does occur, abortion of the zygote, of the endosperm or of both may cause a decrease in seed formation. Since sterility in plants
in general has been adequately reviewed (East 1940, Lewis 1949*
Stout 1938), I will limit this discussion primarily to sterility in autotetraploids of the Solanaceae.
There is still disagreement on the cause of sterility in autotetraploids (Eigsti and Dustin 1955) and various causes have been inferred. Causes commonly associated with autotetraploid
sterility are: irregular chromosome distribution due to unequal
disjunction of raultivalents resulting in abnormal gametes, irregu
lar chromosome distribution due to meiotic abnormalities other than
60 . - V , - ' ■ vC ' ;/v*■?.(■> rV;v.n* \p.. ,;v5cT:;'v.'V;.'if ’.>v‘ • % S".v t:'. ^ f -v-
61 irregular segregation of multivalents, and a genetic-physiological sterility that is independent of meiotic irregularities (Demerec
1947)* The most recent work indicates that autotetraploid sterility is often due to a genetically controlled physiological effect rather than to irregular segregation of chromosomes since disjunction may be nearly regular and sterility still high (Stebbins 1950» Swanson
1957). A study conducted on the self-sterility of autotetraploid
P. pruinosa is reported here.
Observations and Results
The average number of seeds per fruit in the diploid P. pruinosa was 76.9 and in the autotetraploid 20.7, thus the forma tion of a relatively fewer number of seeds typical of autotetra— ploids was present. On the assumption that all the ovules developed into seeds in each case, this could be explained on the basis of a difference in the number, of ovules present in the ovularies of the diploid and tetraploid at the time of anthesis. This was verified when an average of 102.3 ovules in the diploid and 78 ovules in the tetraploid ovary was found when the flowers opened. Further study was made to determine whether in the tetraploid this differ ence was due to abortion of ovules after formation or whether it was due to a difference in number of ovules formed. From general observation it appeared that by the time the mature megaspore mother cells were present, ovule formation from the placenta was nearly complete. Using this time as the basis for counting the number of ovules formed, it was found that apparently there was a 62 difference in the number of ovules formed in the diploid and tetra ploid, the average number of ovules formed in the diploid being
77*3 and in the tetraploid 49»2. The discrepancy between these figures and those of the number of ovules present when the flower opens is probably due to not all ovule formation being complete and the inherent error of counting all the small ovules. Never theless, it appears that in the tetraploid fewer ovules are formed.
In neither the diploid nor the tetraploid was any abortion of dif ferentiating ovules, or ovules with archesporial or megaspore mother cells observed (Table 8).
The magnitude of this apparent difference in the number of ovules formed and present at anthesis will not account for the difference in numbers of seeds in the fruits of the two based on the assumption that each ovule developed into a seed.
Ovule abortion was studied to determine the quantity of fertile eggs at anthesis. Two and four-tenths per cent of the embryo sacs of the diploid and 12.0 per cent of the embryo sacs of the tetraploid were found to be aborted at this time. Even though embryo sac abortion was higher in the tetraploid, it still is not of sufficient quantity to account for the total reduction of seed set obtained.
Non-viable pollen is often considered a major factor in the sterility of autotetraploids. Seven and four—tenths per cent of 6833 pollen grains of the diploid were ascertained to be non- viable as were 49»4 per cent of 7517 pollen grains of the tetra ploid. In the tetraploid then about one-half of all the pollen that 63 alights on the stigma would "be expected to "be non-viable and thus could account for apparently a large degree of non—fertilization and reduction in seed formation# The amount of decreased seed formation due to this cause can he interpreted in several ways.
Upcott (1 9 3) 5 explained low seed production in tetraploid Lyco— persi cum esculentum hy pollen sterility caused hy non-disjunction of quadravalents. Muntzing (1936) criticized this paper in part on the "basis that the proportion of viable pollen grains would suffice to fertilize all of the viable eggs# When a small number of pollen grains is on the stigma, the chance that there are enough viable ones to result in the fertilization of all the eggs is smaller than when a large number of pollen grains is on the stigma.
So it is the number of viable pollen grains on a stigma that is important when determining sterility on a pollen viability basis#
H. H. Smith (1939) noticed a lack of correlation between pollen fertility and seed formation in that seed formation in autotetra— ploid Nicotians rustics with — 25 per cent pollen abortion was less than in an allotetraploid Nicotiana with — 65 per cent pollen abortion.
To insure an adequate amount of viable pollen on the stigmas of the tetraploid plants, they were self-pollinated by hand. Since the plants were not emasculated, natural self-pollina tion probably also occurred. A minimum of 20 viable pollen grains would be necessary to account for the average number of seeds found in the fruit of the tetraploid. As a result of artificial pollina tion, more than enough viable pollen grains were on the stigma to 64 result in the fertilization of all the viable eggs. Thus pollen sterility per se cannot account for the reduction in tetraploid seed production in this case.
The amount of pollen germination and rate of grovrth of the pollen tube in the style we re also considered. The Niootiana type of sterility (East and Mangelsdorf 1925) is due to a genetic control whereby sterility is due to a slow rate of pollen tube growth which ordinarily does not permit fertilization to occur.
Since the tetraploid has been shown generally to be slower in grovrth and development, this may be true of the pollen tube growth in the style also. The distance the pollen tube of the tetraploid must grow is slightly farther than in the diploid since the tetraploid style is slightly longer, but since this difference is so little it probably is of no significance. The style ordinarily abscises from the ovulary by 48 hours after pollination and thus abscission may occur before the pollen tube reaches the ovulary. By applica tion of a 0 .2 5 per cent naphthaleneacetic acid-lanolin mixture to the stigma, the abscission of the style could be delayed up to 4 weeks. The hormone was applied to one—half of the stigma immediate ly after pollination. Muir (1942) found in Nicotiana tabacum the amount of growth hormone was closely related to the penetration of the pollen tube in the style and P. P. Smith (1942) found in Snap- 1 dragon and Bryophyllum that naphthaleneacetic acid stimulated pollen tube elongation. Eyster (1941) found that by spraying a naphtha- leneacetamide solution on flowers of self-sterile Petunia, raari-^ golds, cabbage and red clover, the plants became self-fertile 65
"because the chemical neutralizes the effect of the ovularian secre tion which diffuses into the style and retards the grovrth of the pollen tube. Lewis (1942) found this 'did not affect the rate of compatible or incompatible pollen tube growth in Primus "but it did delay stylar abscission thus allowing more time for the pollen tube to reach the ovulary#
The average number of seeds in 12 tetraploid fruits thus treated was 15*9 (as compared with an average of 20.7 seeds without the hormone) indicating the hormone in this case, by increasing the stylar persistence and apparently not affecting pollen tube grovrth rate or grovrth inhibition, did not increase the fertility in the tetraploid.
The amount of pollen germination and pollen tube dis tribution in the style could be determined by staining the style by the method given under "Materials and Methods." As mentioned earlier, the rate of pollen tube grovrth in the styles of the dip—
; loid and tetraploid was similar. There was in the tetraploid a sufficient number of pollen tubes at the bottom of the style at the end of 48 hours to result in the fertilization of most of the eggs ,.
(Figure 34) • An average of 50 pollen tubes were counted at the base of the style 24 hours after pollination and at least 72 after
48 hours. In no instance was a deficiency of pollen tubes noted in the styles. There were no deformed or bursted pollen tubes observed in the styles. Therefore, lack of pollen germination, bursting of pollen tubes, or slow or inhibited growth of pollen tubes in the style does not appear to cause the low frequency of seed set in this autotetraploid. Irfc this study it had "been ■very evident from early studies of the ovulary that there was a definite difference in the number of pollen tubes that could be seen in the ovularies of the diploid and of the tetraploid. There were many pollen tubes visible in the locules of the diploid whereas only a very few could be seen in case of the tetraploid. As a result of the above studies on the pollen tube frequency at the base of the style, it was then con- # eluded that there must be an inhibition of pollen tube growth in the ovulary tissue and that due to this inhibition few pollen tubes would reach the ovules and less fertilization and seed formation
-would result.
To verify this, the number of embryo sacs with pollen tubes in them - and presumably the number of fertilizations - were counted 2 and 5 days after pollination (Table 8). About one-half of the eggs in the tetraploid and one-tenth of the eggs in the dip—
\loid ovules were unfertilized 2 days after pollination. Five days after pollination the number of unfertilized eggs in the tetraploid was slightly over one-half of the 304 examined. By this time the pollen tubes were disintegrated and presence of embryo and endo sperm were used as a basis of deciding whether fertilization had occurred. Unpollinated flowers as well as ovules in parthenocarpic fruits w^re examined and in no case was embryo or endosperm ob served so the assumption that embryo and endosperm are present only o aftere fertilization occurred was substantiated. The ovules con— taining unfertilized eggs were very evident due to having well formed
o O o normal embryo sacs with a visible egg and, in general, well developed Figure 34• Pollen tubes of tetraploid P. pruinosa at base of style of the tetraploid, (l60X).
Figure 35* Pollen tubes of diploid P. pruinosa. at base of style of the diploid, X*200X) • Table 8* Fertilization and pre- and post-fertilization abortion in diploid and tetra ploid Physalis pruinosa.
Diploid______Tetraploid Humber Humber counted * Averagei counted $> Average Megaspore mother cell stage total ovules 232 — - 197 - — aborted ovules 0 0 - 0 0 - When flower opens total ovules 283 — — 216 — — ovules (embryo sacs) aborted 7 2.4 — 26 12 — style length (millimeters) 9 - 3.16 9 - 3.69 pollen counted 68 33 -- 7517 - - pollen aborted 510 7.4 3715 49.4 2 days after self-pollination total ovules 614 — - 423 —- ovules aborted 6 0.9 mm 28 6.6 - ovules with fertilized eggs 541 88.1 - 197 46.5 - ovules with unfertilized eggs 67 1110 - 198 46.9 - pollen tubes in style 10 styles - 75 10 styles - 67.2 style persistence abscised abscised 5 days after self-pollination total ovules 277 - — 304 — — ovules (seeds) aborted 14 5.0 - 52 17.1 - ovules with fertilized eggs 218 78.7 - 91 29.9 — ovules with unfertilized eggs 45 16.3 — 161 53.0 — 69 synergids and fusion nuclei. Thus a major cause of low seed pro duction in the tetraploid apparently is the failure of fertilization as a result of pollen tube inhibition in the ovulary.
In mature fruits from diploid and tetraploid plants various sizes of aborted seeds were observed. Although more abortives were usually found in the tetraploid fruits, they were generally smaller than in the diploid. This may be explained by the large number of ovules with unfertilized eggs in the tetraploid fruit. In unpollinated flowers and in parthenocarpic fruits ovules up to ,25 mm. could be observed and these probably corresponded to the ovules containing unfertilized eggs in the selfed tetraploid for the most part. As a result of a microscopic examination, it was found that the embryo sac was aborted to such a degree that the exact amount of post—fertilization abortion could not be es tablished with any certainty. As evidenced by the increase in the amount of ovule abortion in fruits of both the diploid and tetra— ploid 5 days after pollination, 5»0 per cent and 17.1 per cent, respectively, as compared to 2.4 per cent and 12.0 per cent embryo sac abortion respectively, when the flower opened, there probably is some early post-fertilization abortion in both. The low fre quency of large abortive seeds in the tetraploid probably reflects early abortion due to irregularities in division of the zygote and/ or the endosperm as might be expected in some cases where the gametes or polar nuclei had an abnormal chromosome complement. In the diploid fruits, on the other hand, nearly all of the abortives were large indicating probably abortion at a late stage in develop 70
ment, possibly due to deficient food supplies in all of the de
veloping larger seeds. In the tetraploid, food as a limiting
factor is less likely since there are fewer developing seeds.
Discussion and Summary
In tomatoes Cooper and Brink (1945) found fewer seed produced in the tetraploid than in the diploid, mainly due to
failure of ovules to "become fertile Because of restricted pollen tube growth and in tetraploid Datura (Sansome, Satina and Blakes— lee 1942) a number of embryo sacs with unfertilized eggs were ob
served but a count was not made. In Physalis pruinosa it appears
that due to restricted pollen tube growth in the ovulary, gener ally one-half of all the eggs are not fertilized, thus resulting
in low seed production. This morphological phenomenon has a *» physiological basis which may be due to a substance produced in
the ovulary which inhibits pollen tube growth.
In self-sterile Petunia plants, the selfed pollen tubes are inhibited in the upper part of the style (Eyster 1941)* Pollen from self-sterile plants placed on the stigmas of self-fertile plants germinates and pollen tubes grow all the way down the style.
However, in the latter cross Eyster found that the pollen tubes are
inhibited in the style if ovulary sap from the self-sterile plant
is applied to the stigma before pollination. Yasuda (1931) also found in Petunia that self-incompatibility is due to the ovulary
juice which inhibits pollen tube growth. Since processes in plants are controlled by the heredity of the plants, it can be inferred
that the secretion of this substance has a genetic basis. Appar- 71 ently the genotype of the diploid is such that the amount of this material secreted is not sufficient to inhibit many pollen tubes, at least in quantities that result in a high degree of unfertilized eggs. It is suggested then that as a consequence of doubled chromo some number and thus genic control, the amount of this substance secreted is increased, resulting in an increase in pollen tube growth inhibition. In self—sterile Physalis ixocarpa Pandey found evidence supporting this in that the pollen tubes were inhibited in the ovularies of the diploid and presumably also of the tetra ploid (personal communication).
Although low seed production in autotetraploid P_. pruinosa appears to be due primarily to ovules with unfertilized eggs, other factors are also involved to a lesser extent. Megaspore abortion was not observed and this is similar to what was observed in Datura
(Sansome, Satina and Blakeslee 1942) and so abortion at this stage is apparently not a contributing cause. Abortion of embryo se^os which occurred in approximately 12 per cent of the ovules of the tetraploid would account for some seed reduction. In the diploid this amounted to only about 2 per cent. Pandey (1956b) found auto tetraploid flax sterility was mainly due to the failure of th§ embryo sac to mature. Rick (1946) found in tetraploid Lycopersicum esculentum ovule sterility occurred mostly between the four mega spore and the mature embryo sac stage. Although a cytological study was not made, breakdown of the embryo sac may be due to irregular ities in meiosis as Einset (1940) found to be one cause of partial sterility in autotetraploid lettuce. He also found as other causal o
0 72 factors a reduction in pollen germination and inhibition of growth of pollen tubes in the styles, with the result they never reach tjhe embryo sac.
Seed abortion occurs and thus also accounts for some de crease in viable seed formation in the autotetraploid, most of this abortion apparently occurring in young seeds. As indicated before, this is probably due to endosperm and/or zygote abortion which would be expected to result from chromosomal unbalance in the gametes due to irregular segregation. Sansome, Satina and Blakeslee (1942) attribute some sterility in autotetraploid Batura to this. Pandey
(1955) lists endosperm collapse as a major cause of poor seed set in tetraploid Trifolium.
No cytological studies of microsporogenesis were made but it was inferred that the high degree of pollen sterility probably is the result of meiotic irregularities which would cause unequal distribution of chromosome material. Varying size pollen grains, presence of microcytes and more than four microspores in a "tetrad" would support this inference. Lagging chromosomes were also oc casionally observed. A high amount of pollen sterility does not always characterize autotetraploids. Pollen maturation may be generally regular and still seed production may be low as is true in tomatoes (Jorgensen 1928, Lindstrom and Koos 193l)» and maize
(Randolph 1935)• Randolph (l94l) attributes reduced fertility in tetraploid maize largely to a genetical— physiological condition.
Fischer (1941) also agrees that maize sterility is due more to genetical factors than to chromosomal irregularities in meiosis.
o 73
In snapdragon (Sparrow, Ruttle and Rebel 1942) and Lolium (Myers
1945) sterility is not due to multivalents, an explanation often given, but due partly to laggards and other meiotic abnormalities. PAHTHENOCARPY
While naphthaleneacetic acid was used for delaying style abscission in the sterility studies, its possible effects on forma tion of parthenocarpic fruits were also investigated. It is well known that parthenocarpy can he induced in many Solanaceous plants including Lycopersicum. Solanum. Petunia. Datura and others; this has been thoroughly reviewed by Gustafson (1936, 1942)•
A 0.25 per cent naphthaleneacetic acid-lanolin mixture was applied to stigmas of emasculated flowers of diploid and tetra ploid plants which were then allowed to grow to ’’maturity.” The results of this treatment in comparison to self-pollination are summarized in Table 9» Parthenocarpic fruits were formed in both the diploid and tetraploid plants (Figures 36 and 37)* These fruits in both cases averaged only about one-half as large as fruits from self-pollinated flowers. This may be due to the hormone concentra tion per se. or possibly to types of hormones associated with grow ing pollen tubes or developing seeds which are specific to the self pollinated flov/ers. Fruits of both contained an enlarged fleshy placenta and mesooarp resulting in solid and juicy fruits.
Fruits of Physalis are enclosed within the calyx since it enlarges and surrounds the developing ovulary after pollination and fertilization. This also was true of the induced parthenocarpic fruits. This post—fertilization development of the calyx is appar ently due to a hormonal mechanism being an example pf ectogony as the term was proposed by Y/aller (1917) • In selfed flowers hormones produced as a result of the physiology of developing fruits and seeds
74 Table 9* Comparison of size of mature fruits and associated structures in diploid and tetraploid Physalis pruinosa formed as a result of self-pollination and of the application of 0.25 per cent naphthaleneacetic acid on the stigma. Each figure represents an average of 10 measurements.______' Ovulary or Ovulary or Ovule or Style Calyx length Calyx width fruit length fruit width_____seed size_____ persistence Tetra- Tetra- Tetra- Tetra- Tetra- Tetra- Diploid ploid Diploid ploid Diploid ploid Diploid ploid Diploid ploid Diploid ploid
When the flower opens 3.0 mm 3*9 nan 4*0 mm 5*6 mm 1.5 mm 1.9 mm 1.0 mm 2.0 mm 0.20mm 0.25mm present present Mature selfed fruit 2.0 cm 2.2 cm 1.8 cm 2,1 cm 1.0 cm 9*7 mm 1.1 cm 1.1 cm 1.75mm 2.0 mm abscised abscised Mature "hormone” fruit 1.4 cm 1.7 cm 1.2 cm 1.6 cm 5*5 111111 7»5 mm 5»0 mm 6.6 mm O.lSmm 0,21mm present present No pol lination or hor mone 5,0 mm 5.0 mm 3.3 mm 5»3 mm 1.8 mm 1.4 mm 1.4 mm 1.3 mm 0.20mm 0.22mm abscised abscised Figure 36. Fruits of diploid P. pruinosa formed as a result of self-pollination (right") and of hormone (left) placed on the stigma. Scale is in millimeters.
Figure 37* Fruits of tetraploid P. pruinosa formed as a result of self-pollination (right)' and of hormone (left) placed on the stigma. Scale is in millimeters. 77 must be involved. As was true of the fruit size, the calyx of the parthenocarpic fruit was not as large as the calyx of the self pollinated plants, reflecting possible differences in hormone con centration or kind.
There were no seeds present in any of the parthenocarpic fruits. Aborted ovules were present, and their size was smaller than at the time of flower opening. Microscopic observation of these ovules showed only an aborted embryo sac with the large in tegumentary tapetum surrounding it. Van Overbeek, Conklin and
Blakeslee (l94l) by injecting a 0.1 per cent solution of naphtha leneacetic acid in the ovulary of 2n and 4n Baturas obtained parthe nocarpic fruits with enlarged ovules and seed coats which often contained a mass of several hundred cells they called a "pseudo embryo." It was not a true parthenogenetic embryo since it was formed as a result of the proliferation of the integument cells,
Fo seeds were formed in tomato, petunia, or eggplant parthenocarpic fruits (Gustafson 1936) or in parthenocarpic Capsicum annuum (Kormos
1947).
The parthenocarpic fruits and calyx of the tetraploid were larger than those of the diploid. This might be expected since the ovulary and calyx are larger in the tetraploid than the diploid at anthesis and the same amount of hormone has apparently relatively similar growth effects on each, thus resulting in a cor responding larger size at maturity. Similar fruit size in selfed diploid and tetraploid plants are the result of fewer seeds and the apparently resultant effect of less hormone production in the de— 78 veloping fruit of the tetraploid in comparison with the diploid, thus accounting for the fruit of the selfed tetraploid not "being larger than the fruit of the selfed diploid (Figures 36 and 37)*
In all cases, non-hormone treated as well as unpollinated emasculated flowers aborted generally in 3 to 12 days after the flower opened in the diploid and tetraploid plants. There might be a slight enlargement of calyx and ovulary before abortion but this is followed by a yellowing and drying of the flower at time of abortion. Neither fruits nor ovules developed without auxin or pollination in Datura (Yah Overbeek, Conklin and Blakeslee
1941)* In eggplant, fruit is formed only when pollinated; emasculated flowers abort in 4 days (Magtang 1936).
As in the case of other Solanaceous plants (Gustafson
1942), parthenocarpy can be induced in Physalis pruinosa. This plant may afford good experimental material for further work which should be conducted on parthenocarpy and parthenogenesis. o
SUMMARY
1. Autotetraploid. plants of Physalis pruinosa L, in comparison with the diploid have larger, thicker and greener leaves, larger guard cells and stoma-es, smaller stomatal frequency per unit area, larger pollen, flowers and seed. The larger leaves are due to larger cell size and not to more cell layers. Increased number of chloroplasts accounted for the darker green color. The pollen is more variable in size and in number of pores, and has a higher degree of abortion.
2. The mature fruit size is dependent in part on the included number of seeds, thus accounting for the similarity in size of the fruits of diploid and tetraploid plants.
3. The rate of seed germination and of subsequent growth and time of flowering, are generally slower in the tetraploid. Two tetraploid plants, although they initially grew slower than the dip
loids, later grew faster. Differences in growth physiology due to differences in environment and genotype are suggested as an explanation. The tetraploid had fewer branches than the diploid.
4» The developmental sequence of megaspore, megagameto- phyte, and pollen formation is similar in the diploid and tetra ploid. The hypodermal archesporial cell functions as the megaspore mother cell in both. A linear tetrad of megaspores is formed, three disintegrate, the chalzal one giving rise to a normal (Poly gonum) type 8-nucleate embryo sac in both. Fusion of the polar nuclei is slower in the tetraploid. . The mature embryo sac lies within a conspicuous integumentary tape turn, the nucellus having dis-
79 * ‘ ° o
* 80
integrated* The size of the tetraploid embryo sac and included
nuclei is larger than of the diploid. In both the development of
pollen in the anther is earlier than development of the embryo sac
in the ovule. Mature pollen is 2-nucleate and wall formation is
simultaneous in both.
5. Fertilization is porogamous and occurs primarily 24
to 48 hours after pollination in both the diploid and tetraploid.
6. Endosperm formation is initiated before division of
the zygote and is cellular. The first division of the endosperm
nucleus is transverse. Subsequent divisions vary in the plane of
wall formation. The tetraploid endosperm initially appears to de
velop slower than the diploid.
7. In both, the zygote divides, after a time lag follow
ing fertilization, resulting in the formation of first a 2-celled
and then a 4-celled linear embryo. In the diploid, four types of
8-celled embryo development and in the tetraploid three and possibly
the fourth type as classified by Bhaduri (1936) were found. In
addition one other variation in the configuration of the 8-celled
embryo in the diploid and two others in the tetraploid were ob
served. The tetraploid embryo initially appeared to develop slower
than the diploid.
8. The young seed coat of both enlarges due to many peri-
clinal divisions and fewer anticlinal ones throughout. Crushed in
tegument cells are first observed behind the tapetum, apparently due
o to the enlarging embryo and endosperm.
o o o 81
9. The autotetraploid is characterized by 73 per cent
fewer seeds than the diploid. This is due primarily to ovules
containing unfertilized eggs since there is an inhibition of pollen
tubes in the ovulary tissue. Embryo sac abortion and post
fertilization abortion account for a smaller amount.
10. Parthenocarpic fruits from diploid and tetraploid plants were obtained by applying a 0 .2 5 per cent naphthaleneacetic— acid-lanolin mixture on the stigma. In both, these fruits were only about one-half as large as fruits resulting from self-pollina tion and those of the tetraploid were larger than the diploid.
Enlargement of the calyx also occurred in both. Parthenogenesis was observed in neither.
11. The morphology of Physalis pruinosa as found in this study is generally similar to that found to be characteristic of other members of the Solanaceae. LITERATURE CITED
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AUTOBIOGRAPHY
I, Robert D. Henry, was born in Columbus, Ohio, August 25,
1927. I received my elementary and high school education in the public schools at Marysville, Ohio. After high school graduation
in 1945 I enlisted in the Army and served for one and one-half years in the Air Corps. After being discharged, I enrolled in Ohio
Wesleyan University for my freshman and sophomore years. I trans ferred to The Ohio State University for my junior and senior years, graduating with the Bachelor of Science degree in Agriculture in
1951* Prom the University of Illinois I received the Master of
Science degree in 1953* Y/hile in residence there I held research and teaching assistantships. While completing the requirements for the degree Doctor of Philosophy in the Department of Botany and
Plant Pathology at The Ohio State University, I held appointments of research assistant, graduate assistant, assistant, and assistant instructor. I have been appointed Instructor in Botany at Ohio
Wesleyan University starting in September, 1958.
91