Artificial Triploids in Luffa echinato Roxb. P. K. Agarwal,1 R. P. Roy and D. P. Mishra Department of Botany, University of Patna, Patna-5, India

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1 Cytologia 44: , 1979 Received April 10, 1975 Artificial Triploids in Luffa echinato Roxb. P. K. Agarwal,1 R. P. Roy and D. P. Mishra Department of Botany, University of Patna, Patna-5, India Luffa echinata is a dioecious wild species of the sexually diverse family Cucurbi taceae. It is distributed in Western Himalayas, Peninsular India, Australia and Africa. The plant is economically important. The fruits contain a bitter alkaloid luffein. In indigenous medicine it is used as an emetic and anthelmintic and in the treatment of hiccough, jaundice and phthisis (Chopra et al. 1949). Cytologically this species has not been worked out in detail. Earlier studies by Mishra (1967) and Roy and Ghosh (1971) have revealed the somatic chromosome number of this species to be 26 without any heteromorphic pair of chromosomes. Apparently, the inheritance of sex in this species is genic. Investigations have therefore, been carried out in an attempt to explore the genetic basis of sex expression in this species. The polyploid method as used by Warmke (1942) has been attempted in this species. The present paper deals with the morphological and cytological behaviour of an artificially produced triploid Luffa echinata. Materials and methods Artificial tetraploids in Luffa echinata were raised by colchicine treatment fol lowing the earlier method (Roy and Ghosh 1971). The tetraploid plants thus ob tained were later crossed with diploids to obtain triploids. For mitotic studies tendril tips were pretreated for half an hour with saturated aqueous solution of p-dichlorobenzene at 15 Ž and fixed for 12hrs. in acetic alcohol (1:3) fortified with aqueous ferric chloride solution. For meiotic studies young male flower buds of appropriate size were fixed in acetic alcohol (1:3) for 24 hours. Acetocarmine squash technique was followed. Pollen fertility was determined by the stainability of pollen in acetocarmine solution. Photomicrographs were taken from temporary preparations. Slides were made permanent using n-butyl alcohol series as described by Celarier (1956). Observations Out of a large number of seeds obtained from tetraploid x diploid crosses only few germinated and two male plants (Fig. 1) were obtained. These plants were more like tetraploid males with increased size of different vegetative parts like stem, leaves, and tendrils (Table 1). The stem was thicker more rough and darker green. Leaves were also more rough and developed short spines at the tips. The different tissue cells like palisade and parenchyma were enlarged. Stomata also showed an 1 Present address: Indian Institute of Horticultural Research (ICAR), 255-Upper Palace Orchards, Bangalore , India.

2 740 P. K. Agarwal, R. P. Roy and D. P. Mishra Cytologia 44 Figs , a branch of colchicine induced triploid male Luffa echinata. 2, male flowers of 2n, 3n and 4n L. echinata Triploid Luffa echinata. 3, somatic chromosomes showing 39 chromosomes. 4, PMC at M1 showing 2IV+1III+11II+6I. 5, PMC at M1 showing 4IV+6III +2II+1I. 6, PMC at A1 showing unequal distribution of chromosomes to two poles. 7, pollen grains showing high degree of sterility.

3 1979 Artificial Triploids in Luffa echinata Roxb. 741 Table 1. Comparative study of morphological characters of diploid, t riploid and tetraploid male Luffa echinata Table 2. Chromosome association at M1 in triploid Luffa echinata Table 3. Distribution of chromosomes at anaphase I in triploid Luffa echinata

4 742 P. K. Agarwal, R. P. Roy and D. P. Mishra Cytologia 44 enlargement of guard cells and stomatal aperture, but the number of stomata per field was reduced as compared to diploids. Flowering in these triploids was slightly delayed. The floral parts like calyx, and stamens showed enlargement over the diploids. The petals showed much variation in shape and size, the number of petals varied from 2 to 6 (Fig. 2). The pollen grains of these triploids were bigger in size than the diploids but the fertility was reduced to only 59.12% as against 99.8% in diploids. Number of stamens, however remained constant (Table 1). Cytological examination from tendril tip cells revealed the presence of 39 chro mosomes. All chromosomes were nearly of the same size, no heteromorphy was observed (Fig. 3). Meiosis was highly irregular. Trivalents and univalents were very common at metaphase I, few cells also showed the presence of 2-4 quadrivalents (Figs. 4, 5). The trivalent frequency ranged from 1 to 6 and univalents from 1 to 13 (Table 2). Chromosome distribution at anaphase I and II was also very irregu lar. Chromosomes separated unequally to the two poles at anaphase I (Table 3, Fig. 6). 4% of the cells also showed the presence of laggards at anaphase, leading to a high degree of pollen sterility (Fig. 7). Discussion Artificial triploids in Luffa echinata have been raised during the present in vestigations to find out how the genes are involved in the expression of different sexes in this species. Earlier studies by Mishra (1967) and Roy and Ghosh (1971) did not show any heteromorphy in the chromosomes of this species. Apparently the sex determination here is not chromosomal. The establishment of the hetero gamety of sexes in dioecious plants involves a number of methods. One of the methods is to study the ratio of segregants in the progeny of a cross between the dioecious species with the monoecious or the hermaphrodite species. Since inter specific hybrids between dioecious L. echinata and other related monoecious species like L. graveolens, L. cylindrica and L. acutangula turn out to be completely sterile (Dutt and Roy, 1969, 1971, Roy et al. 1970), it is not possible to carry out further work in this line. The other methods used by Warmke (1942) in case of Silene otites, where also no heteromorphic pair of chromosomes has been observed, have been applied in this case. The method is to raise artificial male and female tetra ploids and triploids, intercross them at different ploidy level, and to study the segregation in the progeny. In case of male heterogamety, a cross between tetra ploid male (AABB) and diploid female (AA) will produce a sex ratio of 5 male: 1 female in the progeny and a cross between diploid male (AB) and tetraploid female will give an equal proportion of males and females. Whereas, if the female is heterogametic, the crosses between tetraploid female (AABB) and diploid male (AA) and reverse will give a ratio of 5 female: 1 male and 50% male: 50% female in the progeny. Further on crossing these triploids with diploids in case of male heterogamety a cross between triploid male and diploid female will produce a higher ratio of males in the progeny. The diploid male on crossing with triploid female will give an equal proportion of males and females. In case of female heterogamety

5 1979 Artificial Triploids in Luffa echinata Roxb. 743 a cross between triploid female and diploid male will give a higher ratio of females in the progeny, where as, diploid females on crossing with triploid males will again give an equal ratio of males and females. The artificially produced male triploids of Luffa echinata were found to be gigas in nature and showed a general increase in size of vegetative and floral parts over the diploid. Pollen grains size was also increased. But there was a marked de crease in the fertility of pollen grains due to highly irregular meiosis associated with the presence of laggards and unequal distribution of chromosomes at anaphase I and II. The number of seeds obtained on crossing tetraploid and diploids were not sufficient enough to study the ratio of segregants. Efforts are further being made to cross this male triploid plant with the diploids and to obtain more female triploids by crossing tetraploid females and diploid males and the reciprocal. These triploids when crossed with diploid will throw some light on the genetic mechanism of sex determination in this species. Summary Artificial triploids were raised in Luffa echinata by crossing colchicine induced tetraploids with diploids. The triploid plants were gigas in nature and had mor phological features intermediate between diploid and tetraploid. Meiosis was characterised by the presence of a high frequency of trivalents and univalents. These triploid plants are being inter crossed with the diploids used in our breeding programme to establish the heterogamety of sexes in this particular species, thereby to explore the genetic basis of dioecism. Acknowledgement Thanks are due to U. G. C. New Delhi (India) for awarding Junior Research Fellowship to one of the authors (P. K. A.). References Celarier, R. P Tertiary butyl alcohol dehydration of chromosome smears. Stain Tech. 31: Chopra, R. N., Badhwar, R. L. and Ghosh, S. L Poisonous plants of India, I. C. A. R. Scientific Monograph No. 17. Dutt, B. and Roy, R. P Cytogenetical studies in the interspecific hybrid of Luffa cylindrica L. and L. graveolens Roxb. Genetica 40: and Cytogenetic investigations in Cucurbitaceae I, Interspecific hybridization in Luffa. Genetica 42: Mishra, A. R Cytogenetic investigation in Cucurbitaceae, Ph. D. Thesis, Patna University. Roy, R. P. and Ghosh, J Experimental polyploids of Luffa echinata Roxb. Nucleus 14: , Mishra, A. R., Thakura, R. and Singh, A. K Interspecific hybridisation in the genus Luffa. Jour. Cytol, Genet. 5: Warmke, H. E A new method for determining the sex heterozygote in species with mor phologically undifferentiated sex chromosomes and its application to Silene otites (Ab stract.). Genetics 27: 174.