71-7393

BAUER, Eleanor Rose, 1930- STUDIES IN THE GESNERIACEAE. DEVELOPMENT OF THE EMBRYO AND THE ENDOSPERM IN AESCHYNANTHUS LOBBIANUS HOOK.

The Ohio State University, Ph.D., 1970 Botany

University Microfilms, Inc., Ann Arbor, Michigan

(£) Copyright by

Eleanor Rose Bauer

I 1971 I

STUDIES IN THE GESNERIACEAE.

DEVELOPMENT OF THE EMBRYO AND THE ENDOSPERM IN

AESCHYNANTHUS LOBBIANUS HOOK.

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Eleanor Rose Bauer, B.A., M.Sc.

*******

The Ohio State University 1970

Approved by

Adviser Department of Botany ACKNOWLEDGMENTS

I wish to express my appreciation to my adviser,

Dr. Glenn W. Blaydes, for his continued encouragement and assistance in the work undertaken.

Thanks are extended to Dr. Richard A. Popham and to

Mildred Stalder who gave much help in photography and to

Dr. Alan S. Heilman for the first two photographs in the paper.

Gratitude is also extended to Drs. Bernard S. Meyer,

Clarence E. Taft, John A. Schmitt, and Joseph N. Miller for reading this manuscript and giving constructive criticism of the work.

ii VITA

June 30, 1930 . . . Born— Cleveland, Ohio

1952 ...... B.A., Case-Western Reserve University, Cleveland, Ohio

1952-1965 ...... Instructor, Science Department, John Marshall High School, Cleveland, Ohio

1958 ...... M.Sc., The Ohio State University, Columbus, Ohio

1965-1966 ..... Muellhaupt Fellowship in Botany, The Ohio State University, Columbus, Ohio

1966-1970 ...... Instructor, Science Department, John Marshall High School, Cleveland, Ohio

FIELD OF STUDY

Major Field: Botany

Studies in Plant Morphology. Professor Glenn W. Blaydes

iii CONTENTS

Page

ACKNOWLEDGMENTS...... ii

VITA ...... iii

FIGURES ...... v

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 5

INVESTIGATION ...... 7

Endosperm Formation ...... 7

Embryo Formation 19

Starch Accumulation ...... 42

Anomalous Embryo Development ...... 46

SUMMARY...... 48

LITERATURE CITED ...... 50

iv FIGURES Figure Page

1 Mature fruits with seed dispersal ...... 3

2 Mature seeds ...... 4

3 Polar nuclei and s p e r m ...... 8

4 First endosperm division ...... 9

5 Bi-nucleate chalazal haustorium, transverse view . . 10

6 Chalazal haustorium ...... 11

7 Second endosperm division, in micropylar cell .... 13

8 Second divisions in micropylar region, endosperm . . 14

9 Two-celled micropylar haustorium, transverse view . . 15

10 Early cellular endosperm ...... 17

11 Fertilization...... 20

12 Z y g o t e ...... 21

13 Proembryo, two-celled stage ...... 22

14 T-shaped proembryo with two-celled embryo proper . . 24

15 Proembryo, quadrant stage ...... 25

16 Proembryo, octant s t a g e ...... 26

17 Globular stage, early ...... 28

18 Globular stage, later development ...... 30

19 Early cotyledon stage ...... 31

20 Late cotyledon stage, complete embryo ...... 34

21 Late cotyledon stage, shoot apex and cotyledons . . . 35

22 Mature stage, shoot apex and cotyledons ...... 36

v FIGURES (continued)

Figure Page

23 Late cotyledon stage, hypocotyl and root apex ..... 37

24 Root apex, late cotyledon stage ...... 38

25 Mature seed ...... •••.•.*••40

26 Late cotyledon stage, hypocotyl level, transverse view ...... 4 1

27 Late cotyledon stage, transverse view with three cotyledons ...... 47

vi INTRODUCTION

Considerable work in embryology has been reported within the

Gesneriaceae. Among the earliest workers in the family, Hielscher studied Streptocarpus in 1883 and Balicka-Iwanowska (1889) the endo­ sperm haustoria in Klugia. The earlier work has been reviewed by

Schnarf in 1929. Davis (1966) has summarized the more recent work.

Studies of cell lineage in early stages of embryo formation have been studied in Klugia notoniana (Arekal, 1961), Rhytidophyllum crenulatum

(Cook, 1907), Chirita lavandulacea (Crfetb, 1949), Didymocarpus

(Thathachar, 1942), Epithema carnosum (Swamy and Padmanabhan, 1961), and other gesneriads. In the instances reported, the embryo was found to be of the Onagrad type. Papers dealing with endosperm formation within the family note a cellular endosperm from the start, with a micropylar and chalazal haustorium. Detailed accounts of the later stages in the embryo development are lacking in the family. There are a few brief observations on the nature of food reserves in the developing seeds (Cook, 1907; Thathachar, 1942, and Glisifc, 1928).

This paper deals with the early stages of embryo formation in

Aeschynanthus Lobbianus Hook, as well as the developmental anatomy in the later embryonic stages. The endosperm pattern of development is correlated with the stages in the embryo formation from fertilization to mature embryo stage. Starch reserves in the developing seed and fruit are also considered. 2

The writer was not able to find information concerning the details of embryo or endosperm formation in Aeschynanthus. However,

chromosome numbers are available for the genus (Lee, 1962). The writer (1958) reported the embryo sac of Aeschynanthus Lobbianus to be of the monosporic 8-nucleate Polygonum type. The fruit is a linear

capsule, two carpels, parietal placentation, with intruded and bifid placentae. The ovulary thus appears quadri-loculate in transverse

section. Often fruits may grow to a length of 42 centimeters and

contain many small seeds (Fig. 1). Each seed has two long, multi-

cellular hairs that develop from the epidermis of the early ovule by

cell division. An aril of thin, spherical cells develops from the

funiculus during seed formation and is conspicuous as a frothy mass

on the mature seed (Fig. 2). Hauss (1927) reported on the seeds of

Aeschynanthus lamponqa in a paper dealing with a study of appendages

found on seeds. Burtt and Woods (1958) described the curled, hairy, juvenile leaves of the seedling stage of Aeschynanthus. There was

some question as to the identity of the species, probably it was

Aeschynanthus maculatus. Clarke was the monographer of the genus in

1883.

Maheshwari (1963) and others have emphasized the importance of embryology in taxonomic considerations. The Gesneriaceae is a dif­

ficult family to separate from the Bignoniaceae, Scrophulariaceae, and Orobanchaceae (Lawrence, 1951). A close relation of gesneriads to

Orobanchaceae is strengthened by the similarity in the early embryo development and endosperm development observed within the two families (Crete, 1955a). This research reinforces the bonds between the two groups.

Fig. 1. Mature fruits with seed dispersal in Aeschynanthus Lobbianus Hook. Fig. 2. Mature seeds of Aeschynanthus; seed with both hairs is 22 mm in length; micropyl hair nearest the aril is about 11 mm long. MATERIALS AND METHODS

From June 1965 to August 1967 fruits of Aeschynanthus Lob­

bianus were collected as available from plants in the botany green­

houses of The Ohio State University. Samples ranged from those showing

syngamy and triple fusion to mature fruit samples 80 days after polli­ nation. Ordinarily, the mature stigma must‘receive pollen from

another flower before fruit is set. Sections of the capsule 5 to 10 millimeters long were fixed in Nawaschin's solution. The samples were dehydrated in an alcohol series and embedded in paraffin. The majority of the longitudinal sections were cut at 10 to 20 microns. The trans­ verse sections of fruit were cut at 5 to 12 microns. Older fruits are

difficult to section. Safranin-Fast Green was used in staining

(Blaydes, 1956; Johansen, 1940). Iodine-Potassium-Iodide solution was used on some preserved sections and fresh material of various

ages. This treatment tended to darken the starch grains. The Biuret

test was used as an indication of the peptide bond in protein, and

Sudan IV stained fat droplets (Meyer, Anderson, and Swanson, 1955).

Under greenhouse conditions the interval between the polli­ nation and fertilization varied from one to three days. Approximately

80 days after fertilization, the capsule dehises and mature seeds are

released. Since there was a variation in the interval between polli­

nation and fertilization, the age indicated for any morphological

5 stage in this paper is the earliest one. As an example, reference to an early cotyledon stage embryo of 50 days denotes the most advanced stage observed 51 days after pollination and at least 50 days after fertilization. INVESTIGATION

Endosperm Formation

The writer has found that Aeschynanthus Lobbianus follows the pattern of endosperm formation most often observed in the Gesneriaceae.

Polar nuclei fuse shortly before fertilization. Antipodals are ephemeral. Prior to triple fusion a sperm may be observed adjacent to the two polar nuclei (Fig. 3). About one to three days after pollination, an embryo sac contains a zygote, endosperm nucleus, and the remnants of the pollen tube. By four days after fertilization, endosperm development is well underway although the zygote itself does not usually start division until about 25 days later. The first division of the endosperm is transverse, resulting in a chalazal cell and a micropylar cell. This first division occurs toward the middle of the embryo sac by about the fifth day (Fig. 4).

The chalazal cell persists and develops into a binucleate haustorium (Fig. 5). By nine days after fertilization the early chalazal haustorium is visible. It continues to enlarge arid is best developed at the time when the embryo is in the globular stage at 35 to 40 days. The later development of the haustorium results in a hook-shaped aspect (Fig. 6). The two endosperm nuclei enlarge during this period. At the peak of development, the tissue surrounding the haustorium is digested, in some cases up to the epidermis of the integument. Later, by about 50 days, the nuclei of the chalazal Fig. 3. Polar nuclei and sperm at time of triple fusion and syngamy in Aeschynanthus. Maximum width of embryo sac, 24 microns; sp, sperm; pn, polar nucleus; es, embryo sac. X 750. 9

Fig. 4. First endosperm division 4 days after syngamy and triple fusion in Aeschynanthus. X 370. Fig. 5. Bi-nucleate chalazal haustorium in transverse section; 46 days after triple fusion in Aeschynanthus. X 330. 11

Fig. 6. Chalazal haustorium, longitudinal section 41 days after triple fusion and syngamy in Aeschynanthus. X 330. 12 haustorium become granular and a general degeneration begins. Small haustorial remnants persist to the mature seed stage. Seed maturity occurs in about 80 days when the capsule dehises.

Meanwhile two cell divisions occur in succession from the original micropylar cell. About five days after syngamy, the micro­ pylar cell divides longitudinally. The mitotic figure is at a slight angle to the longer walls of the embryo sac (Fig. 7). The daughter cells soon divide transversely and there are then a total of four cells in two rows (Fig. 8). The two central cells give rise to the endosperm proper while the two cells nearest the micropyle persist in the enlarging micropylar haustorium (Fig. 13). This micropylar haustorium develops at a faster rate and becomes more extensive than the chalazal haustorium. By nine days the micropylar haustorium is already conspicuous in the developing seed. The two endosperm nuclei remain intact and spindle-shaped throughout the globular and early cotyledon stages of embryo development (Fig. 9). The micropylar haustorium reaches its largest extent by about 40 days. Although it is extensive, the haustorium never does become extra micropylar as in

Didymocarpus tomentosa (Thathachar, 1942). At least in the zygote and two-celled stages of about 30 days, the suspensor appears to be attached to the distal portion of the haustorium (Fig. 13, page 22).

In later stages of embryo formation, from 50 to 80 days, the micro­ pylar haustorium gradually degenerates and enlargement no longer occurs. In some cases the two endosperm nuclei in the haustorium break into four or more pieces. Traces of the micropylar haustorium persist (Fig. 23) until seed maturity. Fig. 7. Second endosperm division in Aeschynanthus» transverse division in the micropylar cell 5 days after triple fusion; cc, chalazal endosperm cell isolated by first endosperm division; me, micropylar cell of endosperm in division; zy, zygote; pt, pollen tube. X 750. Fig. 8. Second cell divisions in micropylar region of endosperm, 5 days after syngamy and triple fusion in Aeschynanthus. X 570. Fig. 9. Tvro-celled micropylar haustorium in transverse section, embryo is 46 days after fertilization at this stage of endosperm development in Aeschynanthus. X 330. I(>

Development of the endosperm proper from the two central ccLls does not begin until the zygote starts its first division about 30 days after fertilization. During the interval from 30 to 40 days, while the proembryo goes through the globular stage, division is frequent in the endosperm and cell numbers increase at a rapid rate.

At first only large, thin-walled cells form (Figs. 10, 12). By 40 days after fertilization these larger cells are seen only towards the center of the main endosperm mass. The outer endosperm layers are much smaller and have a plate-like aspect (Fig. 18). As the globular stage enlarges, by about 45 days after fertilization, the thin-walled endosperm cells nearest the embryo start to break down (Fig. 18).

This continues throughout the later development of the embryo until in the 80-day embryo of the mature stage only one or two layers of endosperm persist (Figs. 24, 25).

Endosperm formation in the Gesneriaceae has been reviewed recently by Cr%t% (1955a). The presence of both a chalazal haustorium and a micropylar haustorium is a feature of the family. The endosperm is cellular from the start of its-development in gesneriads. There are two main patterns of endosperm formation within the family. The usual sequence of events was found present by the writer in

Aeschynanthus and is described above in the endosperm report. In rare cases in the Gesneriaceae, the sequence of the events differs from the pattern just described. The first division of the endosperm is transverse, yielding a chalazal cell and a micropylar cell. The chalazal cell continues as above in Aeschynanthus, but the micropylar cell divides transversely. The central daughter cell isolated divides Fig. 10. Early cellular endosperm, 10 days after triple fusion in Aeschynanthus; ce, chalazal endosperm; ep, endosperm proper; me, micropylar endosperm. X 370. 18 giving rise to the endosperm proper. Meanwhile the daughter cell closest to the micropyle becomes the origin of the micropylar haustorium by a longitudinal division resulting in two cells.

The chief difference in the above two types of endosperm formation is in the second division of the endosperm nucleus. In the first and most frequently observed case outlined, this second division is longitudinal, as in: Aeschynanthus Lobbianus, Klugia notoniana (Arekal, 1961), Haberlea rhodopensis (Glisib, 1928),

Didymocarpus tomentosa (Thathachar, 1942), Rhyncoglossum obliquum

(Thathachar, 1943), Chirita lavandulacea (Crfetb, 1949), and

Alloplectus sanguineus (Crfetfe, 1955b). In the second and more rare case, the second division of the endosperm is transverse as in

Klugia zeylanica (Schnarf, 1921). 19

Embryo Formation

All the gesneriads in which the embryology has been reported have been assigned to the Onagrad type. The zygote undergoes a trans­ verse division and a basal cell and a terminal cell result. The terminal cell of the two-celled stage divides by a longitudinal wall, and the basal cell plays no significant role in the formation of the embryo (Johansen, 1950). In Aeschynanthus the development of the embryo conforms to the Onagrad type.

Early stage

About one to three days after pollination, syngamy and triple fusion occur in the mature embryo sac (Figs. 3, 11). While endosperm division begins a few days after triple fusion, the zygote does not divide until about 25 days later. For the first 10 days after syngamy the zygote is spherical and remains in the enlarging micropylar end of the embryo sac between the two endosperm cells of the developing micropylar haustorium. Fifteen days after fertilization, the zygote elongates and becomes a tubular structure. The nucleus remains at the tip of the tube towards the center of the embryo sac. The tube also starts to grow at the opposite or non-nucleate end to the extent of the micropylar haustorium. The basal end of the zygote is attached to the end of the micropylar haustorium (Figs. 12, 13). A similar pattern of growth has been reported in Didymocarpus (Thathachar, 1942) and other gesneriads.

A transverse division occurs at the nuclear tip of the zygote approximately 30 days after pollination. Nuclei of both the apical Fig. 11. Micropylar end of embryo sac in Aeschynanthus, showing egg and sperm day after polli­ nation; eg, egg; sp, sperm. X 750. 21

Fig. 12. Aeschynanthus zygote 34 days after fertilization, zygote nucleus 14 microns in length; it, integumentary tapetum; ep, endosperm proper; zn, zygote, nucleate tip region; zt, zygote, tubular region. X 370. Fig. 13. Aeschynanthus, 47-day proembryo, 2-celled, entire proembryo 175 microns in length. Stage in above photograph has been observed as early as 30 days after fertilization; ep, endosperm proper; ca, apical cell of proembryo; cb, basal cell of proembryo; mh, micropylar haustorium. X 370. 23

cell and the basal cell remain at the tip end (Fig. 13). The basal

cell is now a long, tubular structure as seen in C’.apsella. However,

in Aeschynanthus the basal cell is not observed to enlarge as it does

in Capsella. Variations occur, but usually a single filament is observed.

After the filamentous stage, the writer will consider in

sequence the globular stage of 40 to 49 days in which a spherically

symmetrical tnass is supported on a suspensor; the early cotyledon

stage of 50 to 55 days in which the cotyledons are initiated on a now l bilaterally symmetrical embryo; the late cotyledon stage from 56 to 77 days in which appressed cotyledons elongate at the end of a hypocotyl; and the mature stage from 78 to 80 days in which the embryo is at the level of development and size as in the ripe seed.

Globular stage

About 40 days after pollination, the apical cell of the two- celled stage starts a swift succession of divisions. The first division is longitudinal and a T-shaped proembryo is observed (Fig. 14).

The second division is at right angles to the first division. The quadrant stage is now attached to the suspensor (Fig. 15). Each cell in the quadrant now undergoes a transverse division. In the octant stage all cells are approximately the same size (Fig. 16). Periclinal division occurs in the cells of the octant at about 45 days. Deriva­ tives of the octant cells divide repeatedly.

The suspensor consists of a variable number of cells, usually about two to four, attached to the embryo proper at one end and the Fig. 14. Aeschvnanthus. 47-day t-shaped proembryo with 2-celled embryo proper. Length of embryo proper, 15 microns; em, embryo proper; su, suspensor. X 370. Fig. 15. Quadrant stage, 55-day proembryo of Aeschynanthus, length of embryo proper, 24 microns em, embryo proper; su, suspensor. X 370. Fig. 16. Octant stage, 46-day proembryo of Aeschynanthus. Length of octant, 31 microns; oc, octant; su, suspensor. X 330. 2 7 bn.'i.tl c.(>ll at 1110 other end. In most instances the suspensor is uni stir into throughout. Typically the suspensor is attached to the distal portion of the micropylar haustorium (Fig. 13), but in a few cases the suspensor is attached to the wall of the integumentary tapeturn, central to the micropylar haustorium.

In Aeschynanthus the cell directly below the octant is the hypophysis. This cell divides transversely. The two resulting cells are somewhat lens-shaped. As the globular stage continues its development, both of the two lens-shaped cells divide longitudinally.

In most gesneriads the result is two tiers of four cells each (Arekal,

1961). In Aeschynanthus the writer has observed two tiers of cells

(Figs. 17, 19), but is uncertain as to the number of cells in each tier. Cells of the top tier may later divide. The cells differenti­ ate into the root cortex. Derivatives of the lower tier later dif­ ferentiate into the root cap and epidermis of the root.

During the early globular stage the proembryo is a spherical mass of cells subtended by a suspensor. The cells of the main mass are homogeneous and arranged in tiers. All cells of the mass are essentially similar as to size, staining capacity, and shape. Then, while still in the globular stage, the primary meristematic tissues differentiate. The mass of meristematic cells in the sphere progres­ sively changes in its organization (Fig. 17).

The first layer to be distinguished as different is the outer layer of the mass. This outer layer was originally established as a result of the first periclinal divisions of the octant stage. The outer layer is mostly restricted to anticlinal divisions, and the layer Fig. 17. Early globular stage, 46-day proembryo, Aeschynanthus. Length of embryo proper, 30 microns; em, embryo proper; hy, hypophysis; su, suspensor. X 330. 29 remains meristematic to a high degree. Many cells of the outer level in all regions divide during the development of the spherical mass while the internal meristematic cells are also dividing and increasing the volume of the sphere. The outer layer quickly becomes identified as a distinct tissue of uniform cell size and shape (Figs. 18, 19).

After the outer layer is differentiated, the internal mass of cells of the globular stage no longer remains as a tiered arrangement of similar cells. Instead, two distinct, inner regions can be gradually distinguished by the 50-day stage (Fig. 19). There is a more central region of dark-stained, elongated cells. In a surrounding cylinder is a zone of more evenly enlarged, vacuolated cells. When the further development of the embryo is considered, the dense core may be termed the procambium and the outer cylinder, the cortex. As these primary meristematic zones differentiate, cell lineage becomes difficult to trace. In the cortex the cells continue to divide frequently, but most divisions are periclinal. Anticlinal divisions are rare. The cortex is a zone of radial rows of cells that enlarge and become vacuolated. There is no evidence of pith in either the longitudinal or transverse sections at this stage. In contrast, division in the procambium is more often anticlinal rather than periclinal. The cells of the core become elongated and are densely stained. Cells of the procambium are small in transverse section in contrast to those of the cortex or the outer layer. In transverse section cells of the axis form an irregular pattern. The procambium is laid out as a solid block of cells (Fig. 26). Fig. 18. Late globular stage, 49-day proembryo, Aeschynanthus. Embryo proper 60 microns in length; pm, outer layer or protoderm. X 370. Fig. 19. Early cotyledon stage, 52-day embryo, Aeschynanthus. Length of embryo, 85 microns co, cotyledon; pr, procambium; ct, cortex; pm, protoderm; hd, hypophysis, derivatives; su, sus­ pensor. X 370. rz

Orudunlly the spherical mass of the proembryo becomes ovoid

and the top surface somewhat flattened. More divisions now occur in

the portion of the embryo proper farthest from the suspensor. The

distal portion is broader than the portion attached to the suspensor.

These changes occur before the cotyledons appear.

Early cotyledon stage

The early cotyledon stage starts at about 50 days when the

embryo proper has a length of approximately 80 microns. The stage

continues through about the 55th day. As the surrounding cells

become vacuolated, the shoot apex, a zone of meristematic cells can be observed. On either side of this shoot apex the cotyledons develop.

There are periclinal divisions in the sub-surface layers. One of the

cotyledons in Aeschynanthus is longer than the other one. This dif­

ference in size is established at the early cotyledon stage and is maintained through to the mature seed. Cells of the outer layer of

the cotyledons continue to divide anticlinally. Cells in the sub­

layers divide in various planes in the early stages. The outer layer

is still distinguishable as a layer of light-stained cells of similar

size. The embryo is no longer radially symmetrical. Instead there is

an approach to a bilaterally symmetrical form (Fig. 19).

Late cotyledon stage

From about 56 to 77 days the cotyledons are somewhat elongated

at the end of the hypocotyl. Cotyledons are ovoid structures that

become pressed close to each other. They are flattened on the surface where they meet. Orientation of cells indicates that the outer layer 33 of cells in the cotyledon undergoes anticlinal division. Elongation is mostly by transverse division in the inner cells of the cotyledon.

Back from the tips of the cotyledons the cortex and procambium appear as continuous with the same tissues in the rest of the embryo

(Figs. 20, 21).

The shoot apex remains as a narrow, flat region between the cotyledons. Usually there are anticlinal divisions in the outer layer of the shoot apex, but periclinal divisions also occur (Fig. 21).

The hypocotyl continues to increase in length and width. There is no evidence of a pith, and the procambium is a solid core of elongated cells. It is possible to trace the procambium from the hypocotyl into each of the cotyledons as two continuous branched arms (Fig. 22).

In the cases observed, the procambium of a cotyledon was never dis­ continuous with the procambium of the hypocotyl of the embryo.

The root tip organization is established during the very late cotyledon stage. In the region of the hypophysial derivatives, the protoderm arises from the initials coming from the distal tier of cells. By approximately 60 days, periclinal division in this outer layer initiates the root cap (Figs. 23, 24). Derivatives of the inner tier of cells from the hypophysis differentiate into the cortex of the root tip. In most cases the suspensor degenerates by the late coty­ ledon stage, but it is possible to follow its course through the endosperm. Even in the mature seed vestiges of the suspensor persist. Fig. 20. Late cotyledon stage, 56-day embryo of Aeschynanthus. Embryo length, 300 microns co, cotyledon; pr, procambium; ct, cortex; pm, proto derm; en, endosperm; su, suspensor remnant. X 370. Fig. 21. Late cotyledon stage, 56-day embryo, Aeschynanthus. Length of longer cotyledon, 70 microns; pd, periclinal division in outer layer. X 330. Fig. 22. Portion of Aeschynanthus 78-day embryo, mature stage, shoot apex and cotyledons. Longer cotyledon, 160 microns in length. X 370. 37

Fig. 23. Portion of Aeschynanthus 77-day embryo, late cotyledon stage, hypocotyl and root apex; en, endosperm; ep, epidermis; pr, procambium; ct, cortex; rci, root cap initial; mhr, micropylar haustorium remnants. X 370. 37

Fig. 23. Portion of Aeschynanthus 77-day embryo, late cotyledon stage, hypocotyl and root apex; en, endosperm; ep, epidermis; pr, procambium; ct, cortex; rci, root cap initial; mhr, micropylar haustorium remnants. X 370. Fig. 24. Root apex of 75-day Aeschynanthus embryo, late cotyledon stage; pr, procambium; ct, cortex; rci, root cap initial. X 370. 39

Mature stage

In the mature seed the embryo occupies about two-thirds the

length of the seed. The cotyledons extend about 170 microns and the mature embryo is approximately 480 microns long. The hypocotyl con­ tinues to be longer than the cotyledons as in the previous two

stages. The shoot meristem remains flat in the mature seed. There is as yet no mound of cells that might indicate an active shoot meristem. In the root meristem region only a few initials of the root cap are formed by this time. There is no pith (Fig. 25).

Although there is no evidence of mature xylem or phloem by

the mature seed stage, there are some indications of potential xylem and phloem in the older embryonic stages. Esau (1965) distinguishes between phloic and xylary regions of the procambium. In comparison to the potential xylem, the phloic areas have narrower, more strongly stained cells (Fig. 26). The cell arrangement may not be as orderly in the potential xylem as in the phloem. Mature xylem is present in the seedling stage in Aeschynanthus. A xylem element is termed mature by Esau (1965) if the secondary wall is lignified and the nucleus and cytoplasm are no longer present in the cell. Esau considers a phloem element mature if it lacks a nucleus and if the cytoplasm is lightly stained (1965). Fig. 25. Mature seed of Aeschynanthus with 80-day embryo. Length of main portion of seed, without 2 hairs, 640 microns; length of embryo, 480 microns. X 140. Fig. 26. Aeschynanthus, 58-day embryo in transverse section, late cotyledon stage. Width of embryo, 150 microns. X 370. 42

Starch Accumulation

Visible food reserves in the ovulary, ovule, fruit, and seed are traced from the time of pollination to seed maturity. During this interval while seed formation occurs, it would seem that there must be a supply of nutrients within the growing seed. Patterns of accumulation and depletion of starch deposits in the developing fruit itself are somewhat variable in individual fruits of the same stage of development. For example, three days after pollination the usual pattern of starch deposition is one more or less limited to cells immediately adjacent to the vascular bundles of the outer ovulary wall. However in other samples of the same age, starch grains may be abundant throughout all of the cells in the outer wall of the ovulary. In rare cases almost no evidence of starch accumulation is observed in the ovulary three days after pollination. Starch depo­ sition patterns within the ovulary may vary from flower to flower.

The time of day that samples are collected may be involved with the variations observed. In general, starch deposits in the fruit are observed in moderate amounts either in cells adjacent to the vascular supply or scattered in all cells of the outer wall of the fruit during the first 35 days of development. Later, while the embryo is de­ veloping in the globular and early cotyledon stage, there is often a greater deposit of starch, again either in all of the outer wall cells of the fruit or cells adjacent to a vascular supply. Much later, by

70 to 80 days, starch deposit is scant in the fruit as seeds approach maturity. 43

Within the ovule itself the accumulation and depletion of starch deposits exhibit less variation. At pollination there is an abundant deposit of starch grains in the cells surrounding the embryo sac. Usually this deposition is not equally distributed. Only cells adjacent to the half of the embryo sac nearest the micropyle show this heavy starch accumulation. In samples after pollination, but just prior to the first division of the endosperm, starch grains are found within the embryo sac itself. Often the deposition is large in the micropylar end and in the central portions near the zygote. In some samples of the same stage there is only a scant supply of starch as small grains mostly towards the center of the embryo sac. In any case, the integumentary tapetum typically has an abundant deposit in the area nearest the micropylar end^. Five days after fertilization, as the early divisions of endosperm occur, starch deposition is abundant in the early cellular endosperm, especially in the chalazal portion, near the zygote, and at the micropylar end. Tapetum deposits continue as in the early samples. Ten days after fertilization there is a scant accumulation of starch within the endosperm proper. At

10 days the micropylar haustorium is prominent and the integumentary cells adjacent to it have become dense with starch grains. Slight deposits are visible in all cells of the integument by the 10-day stage.

The visible food reserve pattern changes somewhat in the seed as the early cotyledon stage of 40 days develops. The endosperm now has a more abundant starch deposition. Throughout the endosperm proper, in the larger, thin-walled cells, starch grains are deposited 44 in dense clusters. The accumulation is especially heavy near the embryo at the micropylar end of the developing seed. In later samples from late cotyledon stage to the mature seed the starch grains continue to be present in the integument and the endosperm, at least in small supply.

Other food reserves also begin to accumulate, starting with the early cotyledon stage, inside the embryo. The cells of the embryo become packed with more or less uniform granules that give a negative test for starch. The biuret test is positive in these areas and also in the endosperm in the later stages. Protein may be a food reserve deposited in the embryo. In addition, Sudan IV gave a vermillion red color in some areas of the embryo when fresh material was tested. A definite starch accumulation was not observed within the embryos during seed formation. However, starch grains are found in cells adjacent to the vascular supply of the hypocotyl during seed germination.

Dahlgren (1939) reviewed the problem of starch deposition in relation to embryology in angiosperms. Within the Gesneriaceae the writer has observed in Aeschynanthus the major starch deposits to be: the deposition in the integument, the starch in the embryo sac itself, and the starch supply of the endosperm proper. Cook (1907) reported starch in the embryo sac of another gesneriad, Rhytidophyllum, at the time of the primary division of the endosperm nucleus. Starch was also found in the integumentary cells around the micropyle. Thathachar

(1942) reported large starch deposits in the endosperm proper of the mature seed of Didymocarpus and deposits in the integument and later 45 in the tapetum in Rhyncoglossum (1943). In Haberlea Glisifc (1928) observed starch grains in the micropylar region of the ovule and within the embryo sac just prior to fertilization and through to early endosperm divisions.

Although starch grains are no direct indication of the utilization or movement of nutrients, the pattern of localized deposits may give indirect evidence. In the early cotyledon stage the suspensor remains intact. Often the suspensor is considered as an absorbing structure (Maheshwari, 1950) that supplies food to the embryo. Haustoria of endosperm origin have long been considered as absorbing structures (Crete, 1951) in the developing seed. There is a conspicuous build-up of starch in the integument of Aeschynanthus surrounding the micropylar haustorium, the region of suspensor attach­ ment. This may suggest that nutrients are absorbed from the integumentary supply into the micropylar haustorium and then to the embryo via the suspensor. In the later stages of embryo formation the suspensor degenerates; it no longer appears active. The con­ spicuous starch reserve is now in the endosperm cells nearest the embryo. It is possible that in later stages the nutrients may be absorbed from the endosperm proper. 46

Anomalous Embryo Development

Variations in types of embryos were observed in Aeschynanthus

Lobbianus. The most frequent pattern seen involved a situation in

which the seed coats developed and seeds enlarged to full size, but

the embryo remained at an early globular stage. In these cases, even

80 days after pollination, the small proembryo had no differentiation

of procambium or cortex. Both the chalazal and micropylar haustorium

remained prominent and showed no indications of disintegration. The

integumentary tapetum also remained well-developed. In contrast, the

endosperm was poorly formed in these cases and consisted of a reduced

number of thin-walled cells. In one case a double globular embryo

developed which lacked differentiation of procambium or cortex.

A less frequent form of anomalous development is the formation

of embryos with three cotyledons of about equal size. Only one seed

out of 100 developed the three cotyledons. Seeds with embryos con­

taining the three cotyledons germinate readily and continue to grow

as a seedling. Embryos with three cotyledons and globular stages without tissue differentiation have also been reported in

Rhytidophyllum (Cook, 1907) (Fig. 27). Fig. 27. Ae9chynanthu8 embryo, 74 days, late cotyledon stage, transverse section with three cotyledons. X 370. SUMMARY

Endosperm is cellular from the start in Aeschynanthus

Lobbianus. A bi-nucleate chalazal haustorium and a two-celled micro­

pylar haustorium develop by the tenth day. Both haustoria become

aggressive by the time the proembryo is at the globular stage.

Embryo development conforms to the Onagrad type. The tubular nature of the zygote was continued in the basal cell on the suspensor.

Early divisions culminated in a globular mass of similar tiered cells

subtended by a suspensor. The outer layer was the first to become distinguished and later the procambium and cortex differentiated.

Cotyledons arise at about 50 days. The shoot apex remains relatively

inactive through to the mature seed stage while the cotyledons and hypocotyl are elongating. In the embryo of the mature seed the pro­ cambium is continuous throughout the embryo from the hypocotyl to

the cotyledons and also from the hypocotyl to the root meristem. The

procambium remains a solid core through to the final stage. Starting with the late cotyledon stage, potential xylem and phloem may be

identified within the procambial core.

The largest localized starch deposits are found in the integu­ mentary cells adjacent to the micropylar haustorium in the earlier

stages and in the endosperm cells in the later stages in embryo

formation.

Anomalous embryos include those with three cotyledons.

48 SUMMARY OF EMBRYO DEVELOPMENT, ENDOSPERM DEVELOPMENT, AND STARCH DEPOSITS DURING THE DAYS FROM POLLINATION TO MATURE SEED FORMATION IN AESCHYNANTHUS

Days 0 18 27 36 45 54 63 72 81

E zygote zygote tubular linear globular early late cotyledon mature M spherical stage stage coty- stage stage B ledon R Y suspensor intact suspensor degenerating 0 differentiation of tissues

E first endosperm proper many celled break down of endosperm proper N divi­ 2 cells endosperm D sions 0 start of chalazal aggressive degenerating chalazal haustorium S haustorium haustorium P E start of micropylar maximum degenerating micropylar haustorium R haustorium haustorium size M

S embryo endosperm deposits sparce deposits in endosperm T sac abundant A deposits R C micropylar region H of integument, abundant sparce deposits in integument LITERATURE CITED

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