MORPHOLOGY AND EMBRYOLOGY OF GREENE

BY A. S. R. DATHAN AND DALBIR SINGH (Department of Botany, University of Rajasthan, Jaipur) Received March, 20, 1970 (Communicated by Prof. K. S. Thind, r.h.sc.)

ABSTRACT

The ovary is usually tricarpeUary, rarely bicarFellary and covered with spines and glav.dular and eglandular hairs in Marah macrocarpa Greene. Structure and develotzment of spines m~.d two tylces of hairs are described. Ovules are anatropous, bitegmic ar.d crassi~ucellate. The ovular bundle"' ramifies after fertilization. Develo]cment of embryo- sac is of Polygonum tylce. The pollma-tube is rersistent and sometimes dilates at the base of the nr.cellar beak. The er.dosFerm is Nuclear and enucleate cytoplasmic r.odules are formed. The wall formatien is pro- gressive and chalazal endosperm haustorit:m is short and coenocytic. Rarely, it is rudimentary. Embroyogeny is suggestive of Onagrad tyl~e. Hemitriand hemitetracotyledonary embryos are also recorded. The seed.coat develops from the outer integument alone. The mature seedcoat comprises seed epidermis, hypodermis, main scleren- chymatous layer of narrow osteosclereids, (all derived from the outer epidermis of outer integument) aerenchyma and chlorenchyma.

INTRODUCTION Marah Kellogg is a North American genus of the tribe Cyclanthereae of Cucurbitaceao. It includes seven species (Willis, 1966)and provides a rare example of numerical polyploidy in this family. Whitaker (1950) described that the of Marah macroca~Ta Greene (Syn. macrocarpa Greene) sampled in San Diego county, California, are tetraploid. M. macro- carpa, commonly called Chiliclothe or mock , has a large under- ground tuberous system and large spiny fruits dehiscent at the tip. Apart from some observations on the behaviour of endosperm nuclei in developing seeds of E. macrocarpa (= M. macrocarpa) by Scott (1944, 1953) there is no detailed investigation on embryology and development and structure of seed on any species of Marah and thus the present study was undertaken. B4 241 242 A.S.R. DATHAN AND DALBIR SINGH

MATERIAL AND METHODS The material of Marah macrocarpa, fixed in formalin-acetic-alcohol and transferred to 70~ ethanol, was obtained from Dr. Thomas W. Whitaker, LaJolla, California. Mature seeds were obtained from Drs. Whitaker and Howard L. Hyland. The authors sincerely acknowledge their grateftdness to both of them.

Dehydration and embedding were carried out in the usual way. The hairs present on the ovaries were shaved off, when not needed, to permit proper infiltration. Sections cut i0 to 18/z were stained with Heidenhains- iron-alum haematoxylin and safranin and fast green combination. The excised endosperm and embryo were stained with acetocarmine and mounted in glycerine jelly. The spines and the developing seeds were cleared in 10~ KOH solution and lactic acid, washed with water and stained in safranin to study the vasculature. Free hand sections were cut of spines and the mature seedcoat. The latter was also macerated in Jeffrey's solution. Presence of lignin in seedcoat was confirmed by using Phloroglu?inol test.

OBSERVATIONS Ovary, Sp#ws and Epidermal hairs.--The ovary is usually tricarpellary with three parietal placentae (Fig. 1). Rarely, there only are two placentae in M. macrocarpa, the third having suPressed. The ovary in these cases is bicarpel!ary. ~Ihe ovules are borne on the tips of the bifurcated placentae and are arranged in 4 or 6 rows. The inferior ovary is covered with tender spinescent structures which become hard in fruits. The spines are of two types--large and small. Each large spine is surrounded by a nmnber of small spines (Fig. 2). l he spines and the rest of the ovary is clothed with glandular and non-glandular hairs.

Development and structure of' sphws.--A group of hypodermN cells of the ovary wall becomes palisade--like and undergo vigorous anticlinal and periclinal divisions forming a multicellular primordium of the spine. It grows further by cell division and cell enlargement. It is not possible to make any distinction between the growing spines. Usually the tip of each spine termin~tes in a glandular hail which collapses at maturity. Anatomi- cally a large spine at the base consists of an epidermis, hypodermis of 2 or. 3 sclerenchbmatot,s layers and the parenchymatous ground tissue with scattered vascular bundles. The ground tissue decreases from base to tip and the tip of a spine comprises the sclerenchymatous cells o111)'. Morphology and Emb~3'ology of Marah macrocarpa Greene 243

Each g-oup of spines receives the vascular supply from one or more bundles of the ovary wall. "l-he vascular traces divert to the base of the spines, ramify and form a net-work before entering the spines. Usually a small spine receives one trace while a large one gets 6 to 12 branches (Fig. 2). The bundles in the spines branch and become feeble during their upward course. Development of Glandular hairs.--Any epidermal cell of the ovary wall may function as the hair initial. It elongates, becomes protoplasmically rich and divides transversely (Fig. 3). The basal cell undergoes a transverse division (Fig. 4) while the terminal cell enlarges. More transverse divisions in the derivatives of the hair initial form a row of 5 or 6 cells (Fig. 5). Vertical divisions now follow in the terminal or the upper two or three tiers (Figs. 6, 7). Further anticlinal and periclinal divisions in these cells produce a glandular head (Figs. 8-11). Simultaneously, the remaining cells undergo a few transverse divisions and elongate forming a long hair stalk (Fig. i2). During fruit development the hair stalk collapses and finally it dMntegrates. Non-glandular hairs.-It also arises as an extension of the epidermal cell of the ovary wall or its spine. The development of these hairs is similar to that of the glandular hairs but only transverse divisions take place in its formation (Figs. 13, 14). Megasporogenesis and Megagametogenesis.--The earliest stage observed in megasporogenesis is a deep-seated megaspore mother cell (Fig. 15). It divides forming a dyad (Fig. 16) which finally produces a linear or a T-shaped tetrad of megaspores (Figs. 17, 18, 21). The chalazal megaspore functions to form the embryo-sac but in more than 50 per cent tetrads one or two additional megaspores remained healthy (Figs. 18, 21, 22). The first mitotic division in the functional megaspore (Fig. 19) results in a 2-nucleate embryo-sac (Figs, :~0, 31). The two nuclei divide simulta- neously (Fig. 22) forming a 4-nacleate embryo-sac. The organised embryo- sac has an egg-apparatus, 2 polar nuclei and 3 ephemeral antipodal cells (Fig. 23). The antipodal cells closely simulate the egg-apparatus. Fertilization and pollen-tube.--The pollen-tube is porogamous (Fig. 24) and crushes the cells of the nucellus that come in its way. Syngamy and triple fusion are not observed. The pollen tube is persistent and it either remains uniformly broad or dilates in the nucellar beak as described by Singh, D. (1963)in Cyclanthera pedata, Dicoelospermum ritehiei, Edgaria darjeelineensis, Herpetosperrnum peduneulosum and Melothria maderaspatana, 244 A.S.R. DATHAN AND DALBIR SINGII

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Endosperm. -The endosperm development is Nuclear. Successivemitotic divisions produce a large number of free nuclei which become distributed along the periphery of a central vacuole (Fig. 25). At this stage, the coeno- cytic endosperm shows repeated formation of enucleate cytoplasmic nodules which fuse with each other and finally merge in the general cytoplasm as described by Singh, D. (1964 b). Wall formation in the endosperm is progressive and at the globular stage of the embryo, endosperm proper comprises two regions, i.e., the micropylar region of small and the chalazal of large cells (Fig. 26). This distinction gradually disappears during further growth of the endosperm.

The endosperm in M. macrocarpa possesses a small, tubular and coeno- cytic chalazal haustorium (Figs. 35, 26). Rarely, it is broad (Fig. 27) and in a few cases the embryo-sac is only pointed at the chalazal end (Fig. 28). The chalazal endosperm haustorium usually varies from 630 to 1,328 t~ and remains coenocytic throughout its life. It collapses after the differentiation of cotyledons in the embryo.

During advanced stages of seed development, the endosperm cells and their nuclei undergo changes as described in detail by Scott (1944, 1953). Initially the endosperm cells are small and the'.'r nuclei are 5 to 7 t~, round or oval and 1-3 nucleolate (Fig. 29). The cells and their nuclei enlarge, vacuolate and become irregular in shape (Figs. 30-33). The nucleoli develop intra nucleolar vacuoles and become amoebiform from which the segments break off making the nucleus multinucleolate (Figs. 31-33). Finally, most of the endosperm is consumed by the growing embryo and in the mature seed its remnants occur only between the cotyledons which show a characteristic groove in their distal part (Fig. 43 enp). Embryo.--The zygote divides by a transverse wall forming ca and cb (Fig. 34). Cb divides transversely forming ci and m while the division in ca is vertical (Fig. 35). Figures 36 and 37 show the pattern of division in q and m but further steps in the embryogeny of M. macrocarpa could not be closely followed on account of the paucity of material. The globular embryo does not show any suspensor (Fig. 38). After the embryo becomes cordate (Fig. 39), the cotyledonary primordia grow very rapidly and consume the surrounding endosperm while the remainder is engulfed between them (Fig. 40). One or both the cotyledons in some of the embryos developed a notch at the apex (Fig. 41). The notch deepens during further growth and results in the formation of hemitrihor hemitetracotyledonary embryos (Fig. 42). The mature embryo is erect with a small hypocotyledonary root 246 A.S.R. DATHAN AND DALBIR SINGH

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Flos, 34-54 Morphology and Embryology of Marah macrocarpa Greene 247 axis and massive cotyledons. The latter have a characteristic groove enclosing the remnants of endosperm (Fig. 43 enp). Ovule and development of seedcoat.--The young ovule is straight and the two integuments appear simultaneously. It curves (Fig. 44)through 180 ° and finally becomes anatropous (Fig. 46). The mature ovules are bitegmic, crassimucellate, anatropous and erect. The nucellus is flask-shaped and the nucellar beak reaches the tip of the micropylar canal. The outer integument comprises 8 to 10 layers of parenchymatous cells while the inner one is 2 to 4-layered (Fig. 48). Each ovule receives a single vascular supply which traverses the inner layers of the outer integument (Figs. 45, 46). It branches profusely in fertilized ovules. The ramifications are confined to the inner layers and fuse with each other forming a reticulate supply (Fig. 47). Changes in the nucellus.--The nucellar cells which are initially small, polygonal and full of dense protoplasmic contents in unfertilized ovules enlarge enormously and vacuolate after fertilization. The cells of the chalazal region undergo repeated periclinal divisions. The nucellus is completely absorbed in mature seeds. Changes in the integuments.--The outer integument alone forms the seedcoat whereas the inner one degenerates. It starts disintegrating in the neck region (Fig. 24) and is completely lost in seeds showing pear-shaped embryo. The outer integument undergoes changes as described for seeds of Echinocystis wrightii (Singh, B., 1952). ]-he epidermal cells, which are radially elongated in mature ovules, by two successive periclinal divisions form three layers designated as e, e" and e' from outside to inside (Fig. 49). The cells of e' divide only anticlinally to keep pace with the growing seeds, elongate radially (Fig, 50) and undergo sclerosis forming narrow palisade-like osteosclereids (Figs. 51, 52, 53). The cells of e and e" divide anticlinally as periclinally forming 10 to 12 layers (Fig. 50). The outermost layer forms the seed epidermis and the rest, the seed hypodermis. The epidermis and the hypodermal layers become lignified in mature seedcoat (Fig. 51) and accumulate pigmented cell contents. Concurrently, rapid divisions take place in the ovular hypodermis form- ing 3 to 4 layers on the sides and more layers in the micropylar and chalazal regions. Periclinal divisions in other layers of the integument have also 248 A.S.R. DATHAN AND DALBIR SINGH been observed. The aerenchyma cells are stellate with prominent air spaces (Fig. 54) whereas the remaining layers become chlorenchymatous.

The mature seed is large, ovate, gray to tan colour and smooth. Anatomically it consists of the massive embryo and the seedcoat (Fig. 43). The remnants of endosperm occur only in between the cotyledons. The seedcoat comprises 5 zones--seed epidermis, hypodermis, main scleren- chymatous layer of narrow osteosclereids, aerenchyma and chlorenchyma.

DISCUSSION

Marah macrocarpa Greene was earlier included under Echinocystis Torr. et Gray. Though only scanty observations are available on Echino- eystis species, a comparison is made between them and the present observa- tions on M. macrocarpa. The occasional suppression of one of the placentae in tricarpellary ovary of M. macrocarpa is reported in Echinocystis lobata by Barber (1909) and Kratzer (1918). Singh, B. (1952)recorded the failure of ovule development on one or two placentae in E. wrightii. The ovules are anatropous, bitegmic and cressinucellate in the investigated species of Marah and Eehinocystis but the ovular supply is branched in M. macrocarpa (present study) and E. lobata (Barber, 1909; Kratzer, 1918) but unbranched in E. wrightii Singh, B., 1952). The development of embryo-sac conforms to the Polygonum type in M. macrocarpa as well as E. wrightii (Chopra and Basu, 1965).

Marah macrocarpa (present study) and E. wrightii (Singh, D., 1964 a; Chopra and Basu, 1965)are characterised by short coenocytic chalazal endosperm haustoria. M. macrocaiTa is rather peculiar since some of the endosperms have rudimentary haustorium.

The seedcoat develops from the outer integument in Marah (present study) and Echinoeystis (Singh, B., 1952)whereas the inner integument degenerates. The mature seedcoat is built on a common pattern comprising seed epidermis, hypodermis, main sclerenchymatous layer, aerenchyma and chlorenchyma in both the genera. The main sclerenchymatous layer consists of narrow osteosclereids in M. macrocarpa (present study), E. lobata (Barber, 1909; Kratzer, 1918) and E. wrightii(Singh, B., 1952). The epidermal cells are small, horizontally oriented, homogeneous, thick- wailed and lignified in M. macrocarpa but they are heterogeneous of two types, small and elongated, the latter thin-walled in E. lobata (Barber, 1909; Kratzer, 1918) and E. wrightii (Singh, B., 1952). Morphology ond Embl'yology of Marah macrocarpa Greene 249

ACKNOWLEDGEMENTS The authors are thankful to Prof. B. Tiagi, Head of the Botany Depart- ment, University of Rajasthan for facilities of work and to Drs. Thomas W. Whitaker and Howard L. Hyland for the materials received from them. One of us (A. S. R.) is also thankful to the University Grants Commission for the award of a Research Training Scholarship.

REFERENCES

Barber, K. G. .. "Comparative histology of fruits and seed of certain species of ," Bot. Gaz., 1909, 47, 263-310. Chopra, R. N. and Basu, B. .. "Female gametophyte and endosperm of some members of the Cucurbitaceae," Phytomorph., 1965, 15, 217-23. Kratzer, I. .. "Die Verwandtschaftlichen Beziehungen der Cucurbitaceen auf Grund ihrer samenentwicklung mit spezieller Beruch Sichtigung der Caricaceen, Passifloraceen, Aristolochiaceen und Loasaceen," Flora, 1918, 110, 275-343. Scott, F. M. .. "Cytology and mierochemistry of nuclei in developing seed of Echinocystis macroearpa," Bot. Gaz., 1944, 105, 329-38. .. "The physical consistency of the endosperm nucleus of gchino- cystis macrocarpa." Phytomorph., 1953, 3, 66-76. Singh, B. "Studies on the structure and development of seed in Cucurbitaceae. I. Seed of Echinocystis wrightii Cogn.," Ibid., 1952, 2, 201-09. Singh, D. .. "Studies on the persistent pollen-tubes on the Cucurbitaceae," d. Indian bot. Soe., 1963, 42, 208-13. .. "A further contribution to the endosperm of the Cucurbi- taceae,', Proe. Ind. Acad. Sci., 1964a, 60B, 399--413. .. "Cytoplasmic nodules in the endosperm of angiosperms," Bull. Torrey hot. CI., 1964 b, 91, 86--94. Whitaker, T. W. .. "Polyploidy in Echinocystis," Madrono, 1950, 5, 209-11. Willis, J'. .. Dictionary of Flowering Plants and Ferns, VII Ed., Cam- bridge University Press, 1966.

]~XPLANATION OF FIGURES

FIGS. 1-33. Marah macrocarpa. Fig. 1. T.S. of ovary showing T-shaped parietal placentae. Fig. 2. A group of spines cleared to show their vasculature. Figs. 3-7. Stages in the develop- ment of glandular hairs. Figs. 8-11. Transections of developing glandular heads. Fig. 12. Fully formed glandular hair. Figs. 13, 14. Development of eglandular hairs. Fig. 15. L.S. part of nucellus showing deep-seated megaspore mother cell. Figs. 16-23. Stages in the mega- sporogenesis and megagametogenesis. Fig. 24. L.S. part of micropylar region of the ovule showing persistent pollen-tube. Fig. 25. L.S. coenocytic endosperm. Note the tubtllar chalaza haustorium and enucleate cytoplasmic nodules in the endosperm proper. Figs. 26-28. Excised BS, 250 A. S. R. DATHAN AND DALBIR SINGH endosperms showing various conditions of chaiazal haustorium. Figs. 29-33. Endosperm nuclei showing their behaviour during the absorption of endosperm. (cn, cytoplasmic endosperm nodules; pt, persistent pollen tube.) FiGs. 34-54. M. macrocarpa. Figs. 34-42. Stages in embryogeny, for explanation ~'ee text. Fig. 43. Semidiagrammatic longisection of mature seed. Fig. 44. L.S. ofyoung ovule. Figs. 45, 46. T.S. and L.S. of mature ovule respectively. Fig. 47. Semidiagrammatic represen- tation of whole mount of cleared developing seed showing the branched and reticulate vascular supply. Fig. 48. T.S. part of outer and inner integuments. Fig. 49. L.S. outer part of outer iIitegument showing perielinal divisions in the epidermis. Fig. 50. T.S. part of the developing seedcoat, note the radially elongated cells of e'. Fig. 51. T.S. part of mature seed-coat. Fig. 52. Narrow palisade-like osteosclereids from macerations. Fig. 53. Same in surface view. Fig. 54. Aerenchyma cells in surface view. (e, e," e,' layers of cells formed by periclinal divisions of (he outer epidermis of outer integument; em, embryo; en, endosperm; enp, pouch for the remnants of endosperm in the distal part of the cotyledons; eps, seed epidermis; h~, seed hypodermis; i/, inner integument; oi, outer integument; sol, sclerenchymatous layer; us, vascular supply.)