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Electronic Theses and Dissertations
1971
Embryogeny in Haemmanthus Albiflos Jacquin
Ping-Fai David Lo
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BY DAVID, PING-FAI LO
A thesis submitted in partial fulfillment of the requirements for the degree Master of Science, Department of Botany-Biology, South Dakota State University
1971
OU1 H DAKOTA ~ TA UNIVERSITY [fSn __~ EMBRYcx:;ENY IN HAEMANTI-IUS ALBIFLOS, JACQUIN
This thesis is approved as a creditable and independent investigation by a candidate for the degree, Master of Science, and is acceptable as meeting the thesis requirements for this degree. Acceptance of this thesis does not imply that the conclu sions reached by the candidate are necessarily the conclusions of the major department.
Thesis Adviser Date
Head, Botany~Bio ogy/ Department Date ACKNOWLEDGMENTS
The author would like to take this opportunity to extend his sin
cere thanks to his thesis adviser, Prof. C. A. Taylor, for the guidance
· and assistance received during the course of this investigation. He
would also like to thank Dr. C.H. Chen and Dr. G. A. Myers for their
valuable advice and suggestions and other members of the Botany
Biology Department for their encouragement.
PFL TABLE OF CONTENTS
Page
INTRODUCTION .•. 1
LITERATURE REVIEW 2
MATERIALS AND METHODS 6
RESULTS •••. 15
DISCUSS ION 34
RECOfv'UVlENDATION FOR FURTHER STUDY 37
SUMJVlARY . . . . • . • • . • 38
LITERATURE CITED 40 LIST OF TABLES
Table Page I. DEHYDRATION OF FLOWER BUDS AND OVARIES OF HAEMANTHUS ALBIFLOS • . • • • • • • • • • • 11
II. DEHYDRATION OF DEVELOPING SEEDS AND MATURE SEEDS 1 OF HAEMANTI-IUS ALBIFLOS .•••.•••••• . 12
III. FEULGEN REAGENT AND FAST GREEN STAIN FLOW CHART . 14 · IV. THE SUMMARY OF THE DEVELOPMENT OF EMBRYO AND ENDOSPERM OF HAE MA NTHUS ALBIFLOS •.••••• . 31
V. STAINABILITY OF POLLEN GRAIN WITH ACETOCARMINE . 33
VI. EFFECT OF HAND-POLLINATION ON FRUIT-SET •.••• . 33 LIST OF FIGURES
Figure Page
1. Haemanthus albiflos plant with mature fruit •.. 7
Haemanthus albiflos vegetative plant showing bulb 8
3. Haemanthus albiflos, size of ovaries (a) before po 11 ina ti on , ·( b) , ( c ) , ( d) , ( e) , ( f) , at O, 5, 9, 20, and 25 days after hand-pollination and (g), mature fruit .••••. 10
Longitudinal section of ovary, X 24. . . . 16
5. Longitudinal sectio~ of embryo-sac three days after hand-pollination, X 180 ...•• 17
6. Longitudinal sectfon of embryo-sac five days after hand-pollination, X 180 ..••• 18
7. Longitudinal section of embryo-sac seven days after hand-pollination, X 180 .•••• 19
8. Longitudinal section of embryo-sac nine days after hand-pollination, X 180 •...•. 20
9A. Longitudinal section of embryo-sac eleven days after hand-pollination, X 180 .•..•• 22
9B. Longitudinal section of proembryo formed eleven days after hand-pollination, X 180 •••. • . • • • . • 22
lOA. Longitudinal section of embryo-sac fourteen days after hand-pollination, X 24 . . . • . • • • . 23
10B. Longitudinal section of embryo formed fourteen day_s after hand-pollination, X 800 ...... 24-
llA. Longitudinal section of embryo-sac twenty days after hand-pollination, X 24 ...... 25
11B. Transverse section of embryo formed twenty days after hand-pollination, X 500 ...... 26 Figure Page
12A. Longitudinal section of embryo-sac twenty-five days after hand-pollination, X 24 ••.•.•. 28
12B. Transverse section of embryo formed twenty-five days after hand-pollination, X 500 . . • . . • 29
13. Longitudinal section of mature embryo of Haemanthus albiflos . . . • . • • . . • . • . . • • . . • 30
14. Longitudinal section of mature embryo of Allium cepa 30 INffiODUCTION
There has been concern over the controversial position of the genus Allium in the classification scheme. Allium has been put into the Liliaceae because of its superior ovary and into the Amaryllidaceae because of umbels subtended by membranaceous bracts.
The first purpose of this investigation is to study the embryo genesis and subsequent embryogeny of Haemanthus to evaluate affinities between Allium and Haemanthus. Since Allium has stalked cotyledons, it was thought that we could appeal to Haemanthus embryogenesis for evidence as to its relationships, since it is a genus which has been considered both primitive with the Amaryllidaceae and related to
Allium. If Haemanthus is shown to have stalked cotyledons, this would tend to support the position that Al_lium may be closely related to the
Amaryllidaceae. If not, this would tend to discourage adoption of the hypothesis that Allium is related to the Amaryllidaceae.
Another phenomenon recognized in Haemanthus is the difficulty of obtaining high percentage of fruit-set. In an inflorescence of about
100 flowers only about 10 fruits are produced. Each fruit contains one or t wo seeds and very rarely three seeds. The second purpose is J . to study (1) pollen grain viability; (2} developmental sta~es i n micro- and megasporogenesis; and (3) the effects of hand-pollination on fruit-set. 2
REVIEW OF LITERATIJRE
Stenar (1925) in the study of Haemanthus albiflos, mentioned that
the embryo was small as compared with the large endosperm with no sus
pensor. No detailed account of the development of the embryo was given.
Stenar (1951) investigated Haemanthus Katherinae and found that
the endosperm was probably of the Helobial type. In the later post
fertilization stages two chambers were always found in the embryo-sac.
The three antipodals were persistent and remained visible for a long
time. He had also observed a basal endosperm cell in Haemanthus
albiflos.
Poster (1936) studied the embryo development of Alliurn rnutabile
and found it had a cotyledonary slit and the stern tip was formed be
tween the root which developed from a cell cut off from the suspensor
. and the cotyledon.
Soueges (1926) found that after several divisions, the zygote of
Alliurn ursinurn formed a young proernbryo which was differentiated into
three regions: the upper quadrant, which through, longitudinal and
oblique divisions, resulted in the cotyled~n; the middle (two-celled)
which gave rise to the hypocotyl; the basal (several celled) which
developed -the initial cells of the cortex, root and root-cap. No sus
pensor was observed.
Guerin (1933) studied the embryo development of Erythron-ium dens
canis. He found after the early divisions, the zygote gave rise to a
mass of cells, the micropylar end of which differentiated into a
several-celled suspensor, while the inner end, which was derived from 3
the lower cell of the first division of the zygote furnished t~e embryo. Polyembryony was very common in Erythronium dens-canis.
Maheshwari and Kapil (1963) investigated the development of endo sperm and that of the embryo of Lemna pauciostata and found that the primary endosperm nucleus divided earlier than the zygote. The first division was followed by a transverse wall which divided the embryo-sac into a larger micropylar and a narro~er chalazal chamber. The zygote and a persis~ent and a degenerated synergid were present in the mi cropylar chamber. The chalazal chamber might have sometimes contained the remnants of the antipodal cells. The second division was also transverse so that a longitudinal row of four cells was formed. The next two divisions occurred in a vertical plane and the- walls formed perpendicularly giving rise to a 16-celled endosperm arranged in four tiers of four cells each. The endosperm was not of the Helobial type.
The first division of the zygote occurred when the endosperm was 4- celled. A transverse wall segmented it into a terminal cell and a basal cell. The latter divided transversely into cells. The apica l cell underwent two longitudinal divisions at right angles to each other forming a tetrad of four cells. In the meantime, the derivatives of the basal cell also divided longitudinally giving rise to a proernbr yo . of eight cells. The four cells of the quadrant divided transversely resulting in an octet composed of two tiers. Frequently two cells of the quadrant divided transversely while the other two divided by peri clinal walls, the pairs of cells following one or the other course of 4
division being oriented diagonally. This arrangement differed from the typical octant formed in angiosperms and a parallel in some abnor mal instances reported in Ottelia (Hacius, 1952) and Stratiotes (Haude,
1956) could be cited.
After syngamy the zygote underwent a period of rest which varied with species and to some extent was dependent on environmental con dition. Cheesman (1927) found in Theobroma cacao that the primary endosperm nucleus divided 4-5 days after fertilization and the zygote
14-15 days after fertiliza~ion. The zygotes of fertile banana, "Rodoe
Clamp" (White, 1928) Carya illinoensis (Mckay, 1947) and Epidendrum primatocarpium (Swamy, 1948) remained undivided for about six weeks after fertilization. Heiman-Winawer (1919) found in Colchicum autumnale that fertilization took place in autumn and the endosperm nuclei were forme d soon after, but the zygote remained dormant for a period of 4-5 months, during the winter. The shortest resting period occurred in the Compositae and Gramineae. Gerasimova (1933) found in
Crepis capillaris the primary endosperm nucleus underwent its first division within 4-7 hours after pollination and its second division
1-3 hours after pollination. Noguchi (1929) found in Oryza sativa the/ first div~sion of the zygote occurred about six hours after fert i l i- · zation and eighteen hours later the embryo consisted of 4-7 cells.
After four days it began to differentiate into the various body regions and in ten days it was completely mature . Pope (1937) gave a detailed sequence of development from the time of pollination to the first division of the zygote in Hordeum distichon palmella. Dahlgren (1928) 5 found in Alisma, Damasonium the first division of the zygote and that of the primary endosperm nucleus occurred at approximately the same time. Less frequently the zygote might have divided before the pri mary endosperm nucleus, as in Cynornoriurn (Steindle, 1945) Restio
(Brown et al. 1949) and some species of Allium (Weber, 1929). In any case, eventually the development of the endosperm proceeded at a quicker rate and surpassed that of the embryo.
Jackson (1966) suggested that the blooming of Haemanthus Kather inae could be induced by d~ought.
Mole-Bajer and Bajer (1963) recommended that the flowers of
Haemanthus Katherinae be hand-pollinated and the fruit-set could be increased. Jackson (1966) reported increase from 10 per cent to 80 per cent. 6
MATERIALS AND METHODS
The African Blood Lily, Haemanthus albiflos Jacquin (Figure 1), a
member of the family Amaryllidaceae, is a native of South Africa. Now
adays it is not common in South Africa. In Europe it is readily avail
able commercia 11 y. _ It is not common in the United States. It is a
perennial. It has a bulb (Figure 2) which is somewhat rotund, scarcely
compressed with an inch thick tunica. It has several broad, mostly
obtuse leaves somewhat acute, entire, fleshy, ciliate on the borders
toward the tip, elsewhere glabrous and dotted, very faintly striate,
15 cm. long, from 2-5 cm. wide or more, at the very first. The scape
begins to bloom springing upright, erect, then bending forward. The
scape is borne at the side of the leaves, a little compressed, hairy,
thick like a cane, declined, green somewhat stiff about 25 cm. long.
The inflorescence is an umbel with about 100 flowers, erect and .dense.
The flowers are ivory and pedicellate. The fruit is a golden yellow
berry with one to three seeds. The seeds are white, globose and hair
less. Inside the seed the embryo is green. Haemanthus albiflos grows well in a rather light well-drained soil. It needs little care. It usually blooms in September. Jacquin found that it blooms in October and November at the Cape of Good Hope. The fruit-set is poor.
In January, 1970 10 bulbs of Haernanthus albiflos were seperated.
The wounds were dusted with sulphur powder to avoid rotting. The bulbs were laid under fluorescent light overnight so as to induce cork
formation at the wounds. The bulbs were then planted separately in 7
Figure 1. Haemanthus albiflos plant with mature fruits. 8
Figure 2. Haemanthus albiflos vegetative plant showing bulb. 9
6-inch pots. Several plants were left pot~bound in clusters of about
four bulbs. All plants were watered regularly until April. Begin
ning from April the plants were watered rather infrequently about
once every 2 or more weeks. In late May, 1970, inflorescences began
to appear in the pot-bound plants only. One inflorescence was hand
pollinated between 8-9 a.m. with a camel's hair brush. A second in
florescence was not hand-pollinated but left to set fruit naturally.
Flower buds were collected as soon as the inflorescence emerged from
the leaf-axil for microsporogenesis and megasporogenesis studies.
Fully opened flowers wer~ hand-pollinated with a camel's hair brush
between 8-9 a.rn. Each flower was marked with a paper tag bearing the
time of pollination so that the time of pollination could be used as
the reference standard. Ovaries and developing seeds were collected at various intervals following pollination from plants for a period of
twenty-five. days. (See Figure 3). The flower buds as well as ovaries
collected at 3, 5, 7, 9, 11 and 14 days after hand-pollination were
killed and fixed in GRAF solution. Developing seeds removed from
the ovaries of flowers which had been hand-pollinated after twenty and
twenty-five days. The'testa and part of the endosperm of developing
seeds and mature seeds were trimmed off for easy penetration before
being killed and fixed in GRAF solution. · The fixed materials were dehydrated with a series of tertiary butyl alcohol and infiltrated and embedded in paraffin for sectioning. The schedule for dehydration is recorded in Tables I and II. All serial longitudinal sections of 10
Figure 3. Haemanthus albiflos, size of ovaries a. before pollination b, c, d, e, f, at O, 5, 9, 20, and 25 days after hand-pollination and g. mature fruit. 12
TABLE II
DEHYDRATION OF DEVELOPING AND MATUR E SEEDS
OF HAEMANTHUS ALBIFLOS
Step Treatment Time Interval
1 3056 Ethanol + 70% Water 4 hours
2 50% Ethanol + 50% Water 4 hours
3 40% Ethanol+ 10% Butanol ~ 50?'6 Water 4 hours
4 50% Ethanol+ 20% Butanol + 30% Water 4 hours
5 50J6 Ethanol+ 35% Butanol + 15% Water 4 hours
6 40% Ethanol+ 55% Butanol + 5% Water 4 hours
7 25% Ethanol+ 75% Butanol 4 hours
8 100% Butanol 4 hours
9 100% Butanol 4 hours
10 100% Butanol 4 hours 13
flower buds, ovaries, developing seeds and mature seeds were made on a rotary microtome . The sections were 10 microns thick. All sections used in this s tudy were stained by Feulgen reagent and counterstained with Fast Green. The staining and dehydration procedures are recorded in Table III. Al l drawings we re made with the aid of a camera lucida.
2 5 G7 L1. S
·souTH DAKOTA STATE UNIVERSITY LIBRARY 14
TABLE III
FEULGEN REAGENT AND FAST GREEN STAIN FLOW CHART
100% Xylol 100% Xylol
2-5 minutes 2 minutes -.l, 1- 50-50 Xylol & Ethanol 100% Xylol 2 minutes 2 minutes .J, '1- 100% Ethanol 100% Xylol 2 minutes 2 minutes t 1- 95% Ethanol Clove Oil 2 minutes 2 minutes -l, -1' 70% Ethanol 100% Ethanol 2 minutes 2 minutes .J- 1' 50% Ethanol 95% Ethanol 2 minutes 2 minutes .J, 30% Ethanol 1% Fast"' Green in 95% Ethanol 2 minutes 5 seconds -,!, 't Dist. Water 95% Ethanol 2 minutes 2 minutes t 1 N HClJ.(Cold) 70% Ethanol 2 minutes 2 minutes.,. 1 N HC1~(60 degrees C.) 50% Ethanol 7 minutes 2 minutes.,. 1 N HCl J, ( Cold) 30Jl Ethanol 2 minutes 2 minutes.,. Feulgen ""Reagent Dist. Water 3 hours 2 minutes 't Sulphitt W~shing Fluid Sulphite Washing Fluid 10 minutes 10 minutes 15
RESULTS
I. Embryonic Development
In a longitudinal section of an ovary it was found that in each locule there was a single bitegumental orthotropous ovule. The micro pyle was formed from both integuments. In a mature embryo-sac there were one egg and two synergids at the micropylar end, three antipodal cells at the chalazal end and two polar nuclei in the center (Figure 4).
It was either a monosporic 8-nucleate embryo-sac of the Polygonum type or a bisporic 8-nucleate embryo-sac of the Allium type. Since early stages of megasporogenesis were not available, more accurate determin ations were not possible. However, it would follow logically that it would be the Polygonum type because that type was found in Haemanthus
Katherinae (Stenar, 1925). Three days after hand-pollination (Figure
5) the egg was very much larger than the synergids. The zygote was about 70 microns in diameter but had not divided. The polar nuclei re mained unfused and the antipodal cells persisted. Five days after hand-pollination (Figure 6) the synergids, polar nuclei and antipodal cells still persisted. The zygote remaincJ undivided but it grew in size. It was about 80 microns in diameter. Seven days after hand pollinatio~ (Figure 7), the synergids began to disintegrate. The polar nuclei had fused and had become the primary endosperm nucleus. The antipodal cells still persisted. The zygote had not divided yet but its diameter was about 100 microns. Nine days after hand-pollination
(Figures), the synergids we re disintegrating. The antipodal cells per sisted. The primary endosperm nucleus and the zygote remained 16
b ------
------a
Figure 4 . Longitudinal section of ovary, X24 . a. perianth b . style c. ovule 17
b - -· ------a
0 0- - - - - C
d- - - - -
Figure 5. Longitudinal section of embryo-sac three days after hand-pollination, Xl80. a. Zygote b. Synergid c. Polar Nucleus d. Antipodal Cell 18
b ------a
(')0_ - - - - - C
------d
Figure 6. Longitudinal section of embryo-sac five days after hand-pollination, Xl80. a. Zygote b. Synergid c. Polar Nucleus d. Antipodal Cell 19
b ------a
- - - C
Figure 7. Longitudinal section of embryo-sac seven days after hand-pollination, Xl80. a. Zygote b. Synergid c. Primary Endosperm Nucleus d. Antipodal Cell 20
b ------a
0 ------C
------d
Figure 8. Longitudinal section of embryo-sac nine days after hand-pollination, X 180. a. Zygote b. Disintegrating Synergid c. Primary Endosperm Nucleus d. Antipodal Cells 21 undivided. The zygote was about 120 micrcns in diameter. Eleven days after hand-pollination (Figure 9), only two antipodal cells persisted.
The third one had disintegrated. The primary endosperm nucleus had divided, producing many endosperm nuclei each containing one nucleolus.
They were arranged at the periphery of the embryo-sac by the formation of a large central vacuole. The endosperm was therefore of the nuclear type. The zygote had divided many times. The proembryo was about 130 microns in diameter and was now distinctly axiate. The dermatogen was very well defined. Three ~egions were differentiated in the proembryo.
Region A would develop into the root and the root-cap, region B would develop into the cotyledon, and region C would develop into the hypo cotyl. No suspenso~ was observed in the proembryo.
Fourteen days after hand-pollination (Figure 10 A & B), the anti podal cells had disintegrated. The cytoplasm was highly vacuolate at the base of the free nuclear endosperm. The free nuclei at the base were 22 microns in diameter while those at the periphery were 9 icrons in diameter. The embryo was about 140 microns in diameter but no fur ther differentiation in histogens was recognized.
Twenty da ys after hand-pollination (Figure 11 A & B), a longitudinl al section _of the embryo-sac showed that the embryo was about 200 mi crons in diameter. The endosperm area was large compared to the embryo.
Cellular endosperm was apparently initiated between the 15th and the
19th day after hand-pollination. It was difficult to say at which end cellularization began. 22
B
b - - - - -
- - - - -a
- - C
Figure 9~ A longitudinal section of embryo-sac eleven days after hand-pollination, X 180. a. Endosperm Nu cleus b. Vacuole c. Antipodal Cells
B. The Proembryo , X 180. 23
Figure 10A. Longitudinal section of embryo-sac fourteen days after hand-pollination , X 24. a. Embryo b. Endosperm Nucleus 24
Figure l0B. Longitudinal section of enbryo fourteen days after hand-pollination, X 800 . 25
- - - - - a
Figure 11 A. Longi tudina 1 section of embryo-sac. twenty days after hand-pollination , X 24. a. Embryo 26
Figure llB. Transverse section of embryo twenty days after hand-pollination, X 500. 27
Twenty-five days after hand-pollination (Figure 12 A & B), a
longitudinal section of the developing seed showed the embryo was
about 220 microns in diameter.
Approximately two months after hand-pollination, the photosynthet
ic embryo became mature. In a longitudinal section of a seed (Figure
· 13) it was found that the embryo consisted of a short primary root,
hypocotyl and a long cotyledon. The shoot apex was lateral and it
emerged through a slit. No intercalary meristem was observed at the
base of the cotyledon. The food reserve in the mature endosperm tis
sue was starch. The peripheral endosperm had heavy starch concentra
tion while in the central .region, starch was much less concentrated.
The embryo had high starch concentration, owing to its photosynthetic capability. The endosperm in the mature seed was cellular throughout.
The vascular system did not appear in the integument. It was found at the funicular base only. In a longitudinal section of a mature seed of Allium cepa (Figure 14) it was found that the embryo consisted of a primary root, a hypocotyl and a very long cotyledon. The shoot apex was lateral and emerged through a slit. In the region marked A in
Figure 14 there was an intercalary meristem. Through the activity of this region .the cotyledon later increas~d in length. The embryo and the endosperm of Allium cepa did not contain starch. The development of the embryo and that of the endosperm of Haemanthus albiflos were summarized in Table IV. 28
- - - - a
Figure 12A. Longitudinal section of embryo-sac twenty five days after hand-pollination , X 24 . a. Embryo · 29
Figure 12B. Transverse section of embryo twenty five days after hand-pollination, X 500. 30
Figure 13. Longitudinal section of mature embryo, Haemanthus albiflos. a. primary root, b. hypocotyl, c. cotyledon.
Figure 14. Longitudinal section of ma ture embryo, Allium cepa. a. primary root, b. hypocotyl, c. cotyledon, A. inlercalary meristem. 31
TABLE IV THE SUMMARY OF lliE DEVELOPMENT OF EMBRYO AND ENDOSPERM OF HAEMANTIIUS ALBIFLOS
Time elapsed Development of Embryo Development of Endosperm after pollination
3 days The zygote was 70 The polar nuclei were microns in diameter. not fused.
5 days The zygote was 80 The polar nuclei re microns in diameter. mained unfused.
7 days The zygote was 100 The polar nuclei had microns in diameter. fused and the primary endosperm nucleus was formed.
9 days The proembryo was 130 The primary endosperm microns in diameter. nucleus had not divid ed yet.
11 days The proembryo was 130 The endosperm consisted microns in diameter. of many endosperm nu clei each of which con tained one nucleolus. They were arranged in the periphery of the embryo-sac by formati on of a large vacuole.
14 days The proembryo was 140 The endosperm nuclei at microns in diameter. the base were 22 microns in diameter and those at the periphery were 9 mi crons in diameter.
20 days The embryo was 200 mi- The endosperm was cellu- crons in diameter. lar throughout.
25 days The embryo was 220 The endosperm was cellu- microns in diameter. lar throughout.
2 months The embryo consisted The endosperm contained of a radicle, hypocotyl, more starch at the peri- and cotyledon. There phery than in the cen- was no cotyledonary tral region. stalk. 32
II. General Information
When the plants were subjected to drought they bloomed earlier
than usual. They began to bloom in late May, 1970. In the past
years it had been observed that the plants bloomed in September. It was found that all the bulbs which were separated and repotted in 6-
inch pots in January, 1970, did not bloom this year. Each bulb could
only produce one inflorescence arising from the leaf axil.
Pollen grains were stained with acetocarmine to determine their viability. There were 2564 pollen grains observed and 2503 were
stainable, 61 were non-stainable, and their.viability percentage is shown in Table v. The longitudinal sections of flower buds collected as soon as the inflorescence emerged showed that the microspores and
the embryo-sac were fully developed.
One inflorescence, containing 98 flowers which were not hand pollinated, was left to set fruits naturally. Only 10 fruits were produced. Another inflorescence, containing 95 flowers which were hand-pollinated when they opened, produced 51 fruits. The results are shown in Table VI. 33
TABLE V
STAINABILI1Y OF POLLEN GRAIN Wllli ACETOCARMINE
Stained Not Stained Total
of No. No. No. /0
2503 97.6 61 2.4 2564 100
TABLE VI
EFFECT OF HAND -POLLINATION ON FRUIT-SET
Fruit-Set
No. Pollinated No.
Hand-pollination 95 51 51.6
Self-pollination 98 10 10.2 34
DISCUSSION
Stenar (1925) reported that Haemanthus Katherinae had an anatro- ·
pous ovule but in Haemanthus albiflos an orthotropous ovule was found.
It is an exception to the general rule that two different kinds of
ovules would not be found in the same genus.
The zygote of Haemanthus albiflos had a resting period until at
least the ninth day after hand-pollination. It has been reported that
Colchicum autumnale in the Liliaceae (Heiman-Winawer, 1919) and Epi
dendrum primatocarpium in the Orchidaceae (Swamy, 1948) had a resting
period of 4-5 months and six weeks respectively.
Stenar (1951) reported that Haemanthus Katherinae had a Helobial
type of endosperm but the present investigation showed that Haemanthus albiflos had a nuclear type of endosperm. Helobial endosperm is con sidered intermediate between the cellular and nuclear types
(Maheshwari, 1950). There is no evidence which of the types is more primitive (Eames, 1961). Therefore, it is difficult to attach any significance to endosperm developmental differences in Haemanthus
Katherinae and Haemanthus albiflos.
Starch grains were found in both the endosperm and the embryo of
Haemanthus albiflos but no starch grains were found in Allium. This indicates that Haemanthus albiflos still possesses the primitive character with reference to reserve food. Eleven days after hand-pollination the proembryo of Haemanthus albiflos had a well defined dermatogen and general regions for the 35
development of root, hypocotyl and cotyledon could be distinguished.
This is comparable with Luzula in the Juncaceae (Soueges, 1923) which
had extremely precocious differentiation of dermatogen which was cut
off immediately after the quadrant stage. This early differentiation
as depicted by Luzula and Haemanthus albiflos is a rare occurrence
among angiosperms (Maheshwari, 1950).
Stenar (1925) reported no suspensor was found in Haemanthus
albiflos. This investigation supports his observation.
The mature embryo of Haemanthus albiflos was very similar to that
of Allium cepa except that in Haemanthus albiflos there was no inter
calary meristematic region at the base of the cotyledon but there was
an intercalary rneristematic region at the base of the cotyledon in
Alliurn cepa. Thus this does not support the hypothesis that Allium is
related to Amaryllidaceae. In Haemanthus albiflos hand-pollination increased the fruit-set
from 10.2 per cent to 51.6 per cent over natural pollination. Jae son
(1966) reported that by hand-pollination the fruit-set of Haemanthus
Katherinae could be increased from 10 per cent to 80 per cent. The
· effectiveness of hand-pollination on fruit-set was very important be
cause by mean_s of hand-pollination material for the study of embryo
geny was much more accessible. Blooming of Haernanthus albiflos could
be quickened by drought. Jackson (1966) reported the effectiveness of
drought on the blooming of Haemanthus Katherinae. In this investigation
the plants were watered very infrequently (once every two to three weeks
when severe wilting appeared), and as a result the plants bloomed three 36
months earlier than usual. This would become a means by which material for study for a longer period in the year could be obtained. At the time when the inflorescence emerged from the leaf-axil the micro spores and the embryo-sac were fully developed. Therefore for ,the study of microsporogenesis and megasporogenesis, the flower buds should be collected when the .inflorescence is still deeply seated in the bulb.
The staining of 97 .6 per cent of ·pollen grains shows that ·the poor natural fruit-set in Haemanthus albiflos is not due to pollen sterility. 37
RECOMMENDATIONS FOR FURIBER STUDY
1. Although fertilization took place within three days after hand
pollination, the exact time of fertilization was not known. Further
investigation on the exact time of fertilization could be made by
sampling the material every two hours for the first three days
after hand-pollination.
2. It has been found that Haernanthus albiflos might have the first
division of the zygote taking place on the tenth day after hand
pollination. It is taxonomically important to find out the first
and the subsequent divisions of the zygote so that the appropri
ate type of embryo could be assigned to Haemanthus albiflos.
3. It has been found that drought could quicken blooming. Further
investigation could be made on the degree of dryness of the soil
so that blooming conditions could be defined more precisely.
4. A survey is needed in both the Amaryllidaceae and the Liliaceae
to determine the frequency of occurrence of the intercalary
meristem at the cotyledonary base and the reserve food ·type in
the endosperm tissue. 38
SUMMARY
1. The ovule was orthotropous and the micropyle was bitegumental. -
2. Fertilization took place within three days after hand-pollination.
3. The zygote had a resting period until the ninth or tenth day after
hand-pollination.
4. The synergids persisted until the seventh day after hand-pollin
ation.
5. The antipodal cells persisted until the fourteenth day after
hand-pollination.
6. The polar nuclei fused seven days after hand-pollination.
7. The primary endosperm nucleus started to divide at least nine
days after hand-pollination.
8. The endosperm was of the nuclear type. The endosperm nuclei at
the base of the embryo-sac were two and a half times larger than
those at the periphery.
9. No suspensor was observed.
10. No intercalary meristematic region at the base of the cotyledon
appeared in the mature embryo.
11. In the mature seed the endosperm was cellular throughout and it
contained starch as food reserve. 12. The mature embryo was photosynthetic and contained starch.
13. Blooming was quickened by drought.
14. Fruit-set was increased from 10.2 per cent to 51.6 per cent by
hand-pollination. 39
15. The microspores and the embryo-sac were fully developed at the
time the inflorescence emerged from the leaf-axil.
16. Acetocarmine stained 97.6 per cent of the pollen grains, showing
that they were viable. 40
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