DEVELOPMENT OF ANTHER AND MALE GAMETOPHYTE L\ CUGURBITAGEAEi

SRIDHAR* AND DALBIR SINGH Department o f Botany, University o f Rajasthan^

-AJBSTRACT Anther development and pollen formation have been studied in 18 species belonging to 14 genera from six of ihr Mven tribes of sub-family Cucurbitoitleae. Development of the anther wall is of (he dicotyledonous type. It comprises besides epidermis, n fibrous cndothecium, 2 ephemeral middle layers and a secretory tapctum cxcepl Luffa (ylindrka in which tapetal peri- plasmodium is forme d. The tapetal cells usually become 2-nucleale, rarely polynuclcate. Micros- poroRcncsis is normal and the simultaneous cytokinesis fonns tetrahedral and isobilateral types of tetrad of tnicrospores. The microspores develop into 2-cclled pollen grains. Enucleate cytoplasmic nodules have been observed in Cucurbita pepo, Trichosmthes hracttata and T. rfWco in developing pollen grains. Rarely, multinucleolate microspore nucleus ha.s been observed in Luffa cylinJrica and Mukia scabnlla, whereas multinucleolate vegetative nucleus has been re­ corded in Dactyliandra wtlwitschii Edgaria darjulinganiis, Luffa ^Imdrictt and Triehesmthis spp.

INTRODUCTION 1932; Wtmderlich 1954; Turala 1958, 1963). In India although a large number Predominantly tropical family Cucur- of investigations have appeared on the bitaceae has 110 genera and 640 species origin and development of female game- (Willis, 1966). Indian flora has 108 tophyte, studies on the development and species belonging to 34 genera (Chakra- structure of anther and the male gameto- varty, 1959). phyte are scanty. The present study was Kirkwood (1907) reported, besides therefore undertaken. epidermis, three wall layers in anthers of MATERIALS AND METHODS Micrompelis and Cyclanthera and four in Cucmnis and FenilUa. Heimlich (1927) All stages, from the youngest bud to observed 3-5 archesporial cells which f>pcn flower, of Citrullus cologinthis (Linn.) gave rise to four parietal layers and Schrad., C. lanatus (Thunb.) Mansf, sporogenous ceils in Cucumis sativus. Cucumis melo L . var. utilissimus, Cucurbita Dzevaltovsky et al. (1973) and Dzeval- pepo L., Lagenaria siceraria (Molina) tovsky and Zhalalov (1976) have also Standi., Luffa

1. Aceepted for publicatioii on May 30, 1985 2. Present address : Department of Botany Govt. Postgraduate College, Sriganganagar-335001 Dactyliandra wdwitschii Hook.f. from Jodh­ Only in case of Cyclanthera pedata the pur, Cyclanthera pedata Schrad., Trichosan- whole androecium is in the form of a thes anguina L., T. bracteala (Lam.) circular disc with a central partition Voighi and T . dioica Roxb. from Dehra- running throughout the disc (Fig. 2). clun, T . lobata Roxb. from Katihar, and Development and structure of anther wall; Sechium edule Sw. and Edgaria darjeelingen- The young anther comprises a homoge­ sii- CJlaike from Darjeeling. neous mass of cells with a procambial The materials were fixed in formalin- strand in the centre. One to three hypo- acetic-alcohol and stored in 70% ethanol. dermal archesporial cells (Fig. 3) diffe­ Customary techniques of dehydration, rentiate at each of the sites destined to infiltration and embedding in paraffin form the pollen sacs. The archesporial was were employed. Microtome .sections cell(s) divide periclinally forming an outer of 8-12 ftm were cut and stained with primary parietal .'ayer (ppl) and an inner ii-on-alum-haematoxylin and safranin-fast sporogenous layer (mmc) (Fig. 4). The green combination. parietal cells undergo periclinal as well as anticlinal divisions forming outer and OBSERVATIONS inner secondary parietal layers (Fig. 5). Anther lobes in the members of The latter enlarges and differentiates to Cucurbitaceae vary from 5(Ctenolepis form the tapetum (Fig. 6), whereas the garcitdi, Edgaria darjeelingensis and Mukia former redivides periclinally (Figs. 5, 6). scabrella) to 7 {Cucurbita pepo), 10 {Dacty- O f the two resulting layers, the outer liandra welwitsckii), 12 (Sechiumedule), 15 undergoes one more periclinal division (Memordica cJiarantia, Trichosanthes spp.) (Fig. 7). Thus the primary perietal layer and 18 (Coccinia grandis), usually distri­ forms, in all, four anther wall la,yers. The buted over three stamens in the ratio of outermost derivative functions as the 2:2:1 (Fig. 1) cxcept in Coccinia grandv! endothecium, the innermost as the and Sechium edule in which distribution, of tapetum and the remaining two as the lobes is 1:1:1 and 2 :1:1, respectively. middle layers (Figs. 7, B, 9, 14). The

Figs 1-16 : Development of anther wall. Fig. 1. Momordica charantia. T. s. anthers showing 13 lobes in a ratio of 2:2:1. Fig. 2 Cyclanthera pedata. T. 8. of male flower showing annular pollen chamber with a partition. Fig. 3. M . charantia. A portion showing two archesporial cells. Fig. 4 Citrullus coloiynthes. Showing differentiation of microspore mother cells and begin­ ning of division in primary parietal layer cells. Figs. 5-7 M . charantia. T.s. anther lobe showing formation of wall layers and microspore mother cclls. Fig. 8 Lagenaria siceraria. Showing completion of development of anther wall layers. Fig. 9 M . charantia. T.s. pan of anther. Note the degeneration of the inner middle layer and 2 or 3 nucleate tapetal cells. Fig. 10, Coccinia grandis. T s part of mature anther wall showing persistent epidermi.s and fibrous endothecium. Fig. 11 Cuaarbitapepo. T.s. part of mature anther wall. Note that besides the endothe­ cium the persistent outer middle layer has developed fibrous thickening. Fig. 12 Edgaria darjeelingensis. T.s. part of mature anther wall. Note peristent epidermis, fibrous endothecium and persistant cells of middle layer. Fig, 13 Coccinia grandis.T.n. part o f anther wall. Note the enormous radial enlargement in tapetal cells. Fig. 14. Citrullus Umatus. T.s. part of anther showing wall layers. Note large nucleus formed by furion of nuclei in tapetal cclls and degeneration of inner middle layer. Figs. 15, 16 Ltfffa tylindrica. Fig. 15, T.s. part of anther wall showing degenerating middle layers and 1 or 2 nucleated tapetal cells. Fig. 16, same showing in situ tapetal periplasmodium. {ac, archesporium; en, endothccium;«/), epidermis; ispl, iraier secondary parietal layer; ml, middle layer; mmc, microsporc mother cell; ospl, outer secondary parital layer; ppl, primary parietal layer; t, tapetUQi). 15 16 25 M" 17-23.28 ,

T'igs. 17-31, Microsporogenesis and microgametogcnesis. Fig. 17. Cucurbita pepo. T.s. anther showing two microspore mother cells. Figs. 18-24 Momordica charmtia. Stages in microsporegeneisis. Note tetrahedral and isobilateral arrangement of microspores in figures 23 and 24 respectively. Fig. 25 C itnllus lanatus. One celled pollen grain. Fig. 26 Showing multinucleolate nucleus of microspore. Fig. 21 Lagmariassceraria. 2-celled pollen grain. Fig. 28 Luffa tylindma. 2-celled pollen grain showing multinucleolate vegetative cell. Fig. 29 C /jepo. Uninucleate pollen grain showing enucleate cytoplasmic nodules. Figs. 30, 31 TrichosetUh*s braeteala and T. didiea respectively. Two'Celled pollen grains showing enucleate cytoolasmic nodules. (cn—cytoplasmic nodule') development of the anther wfall conforms (Figs. 3,4). The cells stretch tangentially to the dicotyledonous type of Davis and finally become compressed with (1966). scanty cytoplasmic contents (Figs. 11, 12). Initially the epidermal cells are Next to the epidermis lies the cndothe- rectangular and rich in the cytoplasmic cium. Its cells undergo pronoimced en­ contents and divide only anticlinally to largement and develop fibrous thickenings keep pace with the growing anther (Figs. 10, 11, 12). These thickenings are observed to extend to 2 or 3 layers on the each other. Cytokinesis is simultaneous connective side in all the members studied and the microspores aie arranged presently. tetrahedrally or isobilaterally in tetrads The middle layers are ephemeral, (Figs. 23, 24). the inner one degenerates first (Figs. 9, Mictogam«U>gtnesis : The young 14), followed by the outer. Rarely, at nucrospore is ovate or spherical, richly places some cells of the outer middle cytoplasmic and has a prominent, centrally layer persist in C. lanatus, C. melo var. situated nucleus. It grows in size and utilissimus, C. pepo (Fig. 11), C. pedata and simultaneously a large vacuole develops E. darjtelinginsis (Fig. 12). The persis­ ui the centre and the microspore wall tent cells acquire tibrous thickcniugs iii differentiates into intine and exine. The C. pepo alone (Fig. 11). microspore nucleus is pushetl near the The innermost wall layer I'orms the wall after the formation of the vacuolc glandular tapetuin witich ib o i dual origin. and is usually uninucleolate (Fig. 25). On the conncctivc side the tapctum is In L . cylindrica and M ukia scabrella rarely tbriuei by the cells lining the pollen sac. tlic multinucleolate condition is observed It is usually 1-layered but in M . charantia, (Fig. 26). C. grundii and C. pepo it beconics 2-layered ;\n interesting phenomenon, hitlierto at places (Fig. 13). I'o begin with, the unreported in pollen grains, is tlie forma­ tapctal cells are uninucleate and isodia- tion of enucleate cytoplasmic nodixles metric and full ol dense cytoplasmic observed in the pollen grains of Cucurbila contents but laier on, ;is the naicrospore pepo, Trichosanthes bracteata and T . dioica. mother cells enter mciosis, these cells The nodules are observed as small balloon­ enlarge radially and become usually binu- like outgrowtlis from the inner lining ol' cJcatc (Fig. 14), rarely polynucleatc in the cytoplasm into the large central M. charantia (Fig. 9) only. The radial vacuole (Figs. 29, 30, 31). clongatioji ol' the tapctal cells is more The nucleus of tlie luicrosporc pronounced in C. pedala, C. grandis (Fig. divides to form a large vegetative and a 13) and C. lanalus (Fig. 14). The lapetal small lenticular generative cell (Fig. 27). nuclei fuse to form large multinucleolatc The generative cell separates i'rom tlie nuclei (Fig. 14). The tapetum is usually wall and lies free in tl»e cytoplasm of the of tlie Secretory type except in L. cylindrica pollen grains. Tiie polleii is usually shed wherein the cells lose cell wall and form a at thisstage. The vegetative nucleus which icontiiiuous homogeneous pcriplasuiodiuin is usually uninucleolate (Fig. 27), becomes ^Figs. 15, 16). multinucleolate during the itiaturation Mtcrosporogentsis: The rnicrosporo- of pollen grains in L. cylindrica (Fig. 28), genesis follows a common pattern and, D. wtlwitschii, E . dargeeUrigenesis, T . dioica thereibre, diagrams of M . charantia alone and M . scabrella. The ntmiber (2-5) and are provided. The sporogenous tissue the shape of nucleoli is variable. undergoes a few mitotic divisions before DISCUSSION their protoplasts round off (Figs. 6, 17). A mucilaginous wall is secreted around The male archesporium is usually each ceil. The microspore mother cells 1 to 3-celled but Heimlich (1927) haj> undergo normal meiotic division (Figs. reported 3-5 archesporial cells in each 18-24). During Meiosis II, the spindles lobe in Cucumis sativus. In all the investi­ may lie parallel or at right angles to gated species including the anther wall is composed of an epi­ indica have recorded uni-, bi-or poly- dermis, fibrous endothecium, two ephe­ nucleate cells in tapetum. Turala (1958), meral middle layers and a layer of in a detailed study on endomitosis in secretory tapetum. The inner middle the tapetal cells in C. pepo, has described layer degenerates soon after its formation. that they remain uninucleate throughout This may account for the observation of and further differentiation is attained only 3 wall layers in Cyclanthera and Micro- through endomitosis. In another paper mpelis by Kirkwood (1907). However, in covering 6 species of the Cucurbitaccae, Cucumis and Fevillea (Kirkwood, 1907) Turala (1963) has asserted that the tape- and in Cucumis sativus (Hemilich, 1927) tum remains uninucleate throughout in a 4-layered wall besides the epidermis, Cucumis sativus, Cyclanthera pedata, 'Schino- has been recorded. cysiis lobata, Momordica charantia and Sicyes I’hc epidermis is persistant but its angulata; and only in Biyonia dioica cells are thin-walled and almost com­ besides iminucleate, binucleate cells have pletely depleted of contents at maturity. been observed. According to her, further Heimlich (1927) has reported the disap­ differentiation is through endomitosis in pearance of the epidermis in dehisced all of them. This is hi contrast to our anthers in Cucumis sativus. The endotlie- observations which have clearly shown cium develops the characteristic fibrous that the cells become usually 2-nucleate Uiickenings which extend to 2 or in all the taxa including C. melo var. layers on the connective side. utilissimus, C. pedata and M . charantia. Periaswaniy and Swamy (1966) have Turala (1963) has not figured the tapetal reported that the tapetum is of dual cells for the above plants and thus, ii origin in angiosperms. In this study the close comparison is not possible. tapetum is found to difl'erentiate all rhe microsporogeixesis and micro- round but only on the outerside it i.s of gametogenesis are normal and pollen perietal origin whereas on the inside and grains are shed at 2-celled stage. The in lateral positions the cells lining the abnormalities in nieiosis and the theca form the tapetum. Kirkwood development of the male gametophyte (1907) and Heimlich (1927) have both reported by Dzevaltovsky (1970, a, b) stated that the innermost parietal layer and Dzevaltovsky and Sankin (1971) in develops into a part of the tapetuni. triploid watermelon and tetraploid melon Although Heimlich (1927) has made no respectively have not been observed mention oi' dual origin of tapetum in C. during this study. An interesting and sativus, hi.s figures (Heimlich Fig. nos. hitherto unrecorded feature is the forma­ 29, 30), clearly show this fact. tion of enucleate cytoplasmic nodules in The tapetum is secretory and in L. the uninucleate pollen grains in C. pepo cylindrica alone it forms a periplasmo- and Trichosanthes spp. The nodules arise dium. The nuclei in the tapetal cells fuse from the inner lining layer of tlie cytoplasm and the fusion nuclei show variation in and bulge into the large central vacuole size and number of nucleoli. Kirkwood of the uninucleate pollen grains. Such (1907) in Favillea indica, Heimlich (1927) cytoplasmic nodules have been recorded iji Cucumis sativus, Thomas (1931), cited in the endosperm of many plants belong­ from Wunderlich (1954), in Biyonia dioica, ing to dicotyledons and monocotyledons iVsana and Sutaria (1932) in Luffa tugyp- (see Singh, 1964; Singh & Dathan, 1973). tiaca and Chakravorty (1947) in Coccinia The significance of the cytoplasmic no­ dules even in endosperm development formation in experimental polyploid water­ remains disputed. Singh (1955, 1964) m elon. Cytol. & Of rut. 4 : 411-416 (Rus.si.ui, and Singh & Dathan (1973) consider it Eng. Summ.). Dzbvaltovsky, a. K. and L. S. Sankin 1971. an active method of endosperm growth Experimental synthesis of the fetraijloid form helping in the obliteration of the central in melon and study of ils male geinelophyte vacuole. Whether it plays tlie same role development. Ukr. J . Hol. 2 8 i4 1 -4 U . in micro-spore also, needs further investi­ Dzkvaltovskv, a. K. 1973. Some data on simly gation. of the male gcneralive spore in CtKumin an- guria (Pr. S. E. .Africa). Collected scientific Another interesting feature in pollen articles : Publishing llou.se ol Kies Siuir grains is the multinuclcolate condition of Univ., Kiev. the vegetative nucleus in L. cylindrical OzliVAlTOVSKY, \. K. /v.vn ti. /,HAI,A1.0V 1971). D. welwilschii, E. darjeelingensis and Tricho- On embryology of Mtlothria scahra N aud, (!h . saathes spp., Chaidian (1970) has reported J. Hot. 33 ! 287-290. (Russian, Eng. Suniin.). Hi.iMUGit, L. F. 1927. 1 he development iind atia- variations in the uuuiber and size of toiny of the staminate flower of the cucumber. nucleoli of microspore nucleus in Lufja Am. y . Bat. 14 : 227-237. echinala. During iJiis study th<; inicrospore KiRKwoot), J. E. 1907. Some ieaturc-s ul polKu nucleus was found to be uniiiucleolatc formation in the Gucurbitacca<'. Hull, 'lorrev all the laxa except L. cylindrica and M. bol. Club. 34 :2 2 i-'H 2 . Passmorb, S. 1930. Microsporogom-is in the tUmn - scabrella in which it oHcn Ijecame niulti- bitaceae. Bot. Gaz. 90 : 213-222. nucleolatc. I’euiaswamy, K . and U. (i. 1- Swamy 1‘Uib, M or­ phology of the anther tapetum of anjfiosperins. tVurr. ici. 35 : 427-130. r e f e r Ei\ c;es Singh, D. 195,'). Cytoplasmic nodule* In the i-ndos- •\sANA, J . J . AND R . N. S u ta ri* 1932. M icrospo- perm of some Cucurbitareae. Salure. 176 : sporogenesis in LufJa aegyptiaca Mill. J . Indian 607-608. hot. Soc. II : 1U1-I87. SiNOH, D. 1964. C ytoplasm ic nodules in the endos- (jAstejtkh, E. E. 1920. Cylok>gical sludics in ilu' ])erm of angiosperni'.. Hnll. Totra. hut. Club Cucurbitac(;ae ; Microsporogcnesis in Cuciirbitu 91 : 86-94. maxima. Am. J . Hoi. 13 : I-10. SiNc’.n, I). ANii A. .S. K. Dath.s': I'>7.1. I'nrtlni ( .‘hakravarty, H. I.. 1959. Monograph on Indian siudies in the enctosperni nl (incnrbilaciMr, Cucurbitaceac, Rec. Bol. Surv, India. 17 : I-23'! . /V. Sci. 5 : 4 2 -5 1. Chakravorty, a. 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