Cytologia 38: 317-325, 1973

Cytogenetics of charantia and its Polyploids1

R. N. Trivedi2 and R. P. Roy

Department of Botany, Patna University, Patna-5,

Received November 16, 1971

Autotetraploids of M. charantia were raised by colchicine treatment the details of which has already been published (Roy, Thakur and Trivedi 1966). In raising the tetraploid there were mainly two objectives. Firstly, to see whether the , which is an important vegetable, increases in size and thus raise the ; and second ly, to build up material for trisomic lines to locate specific genes on particular chromosome with a view to transfer them to economic varieties for better yield and disease resistance. The triploid M. charantia, was thus raised by hybridisation between the tetraploid and diploid in order to use, this as a breeding material. The present paper deals, with the detailed studies of this artificially produced tri ploid M. charantia. Some interesting results were also obtained in the colchicined M. charantia in addition to polyploid studies in this species. Generally no sex abnormalities have been observed in nature in the strictly monoecious M. charantia . But during the course of present studies several plants, that remained diploid even after colchicine treatment, showed many abnormal features. The detailed behaviour of such plants, especially the hidden genetic make up of sex mechanism that has been exposed, is also presented in this paper.

Material and methods

The cytological and pollination techniques are the same as described by Roy, Thakur and Trivedi (1966) and Trivedi and Roy (1971).

Observations

Triploid M. charantia (Tetraplid x Diploid and reciprocal) Morphology: A) Colchiploid M. charantia was crossed with the diploid to obtain triploid. The crosses were partially successful in both directions. The number of fertile seeds recovered from the crosses were relatively small. Most of the seeds were empty or with poorly developed embryos. The number of crosses made and the germination of the hybrids are summarised in Table 1. The seeds collected were sown for raising the hybrids. The seedlings were weak at early stages, while many of the seeds failed to germinate. Later on they were examined cytologically. 1 This research has been financed in part by a grant made by the Department of Agriculture under PL 480. 2 Department of Botany , Magadh University, Bodh-Gaya, Gaya, Bihar, India. 318 R. N. Trivedi and R. P. Roy Cytologia 38

Table 1. Crosses made between tetraploid x diploid and percentage of seed setting

Figs. 1-3. 1, M. charantia, diploid, triploid and tetraploid (from left to right). 2, M. charantia, leaves of diploid, triploid and tetraploid plants (from left to right). 3, M. charantia, flowers of diploid, triploid and tetraploid plants (from left to right). 1973 Cytogenetics of and Its Polyploids 319

B) The triploid hybrids were vigorous in growth and morphologically inter mediate between the two parents (Fig. 1). The stem was'slightly thicker and stouter than the diploids. The leaflets were also thicker, darker green and broader (Fig. 2) than the diploids. Further the stomatal size was 38.5ƒÊ in length and 19.3ƒÊ in bread th. The number of stomata per unit area was also lesser than that of diploids. Flowering was not delayed in the triploid plants. All the triploid plants were monoecious. In two triploid plants the terminal part of the produced mainly

Table 2. Measurement of different parts of the diploid, tetraploid and triploid M. charantia

pistillate flowers. The number of female flowers per plant was greater than in the tetraploid parent. The flowering period of the triploid plants was about 10 days longer than the diploids but lesser than the tetraploids. The length and breadth of the pedicels, sepals and petals, stamens and pistils showed general enlargement over the diploid parent (Fig. 3). The measurement of the different parts of the triploid plants are given in Table 2. The percentage of pollen fertility as judged by their stainability was about 38.7. The size and shape of the fruit was just like the diploid. No fruit could develop to maturity. The seed setting in triploid plants was practically nil. Cytology: The meiotic plates of the triploid plant showed 33 chromosomes. The chromosomes were small and of the same size as in diploids. There is no report of artificial or natural triploidy in this species. 320 R. N. Trivedi and R. P. Roy Cytologia 38

Table 3. Nature and frequency of chromosome association in different PMCs of triploid M. charantia

Figs. 4-9. 4, M. charantia, PMC of triploid (4n •~ 2n) at MI showing 11 trivalents. •~2000.

5, M. charantia, PMC of triploid 4n •~ 2n at M1. •~2000. 6, M. charantia, PMC of triploid (4n•~

2n) at MI showing uni, bi and trivalents. •~2430. 7, M. charantia, PMC of triploid (4n•~2n) at A1 showing unequal separation. •~800. 8, M. charantia, PMC of triploid (4n•~2n) at Al

showing laggards. •~1000. 9, M. charantia, pollen grains of triploid (4n•~2n). •~125. 1973 Cytogenetics of Momordica charantia and Its Polyploids 321

The complete conjugation into eleven sets of trivalents was observed very frequently (Figs. 4 & 5). In many cases some of the trivalents were replaced by bivalents and univalents (Fig. 6). All cases of chromosome combinations are tabu lated in Table 3. Among the trivalent con figuration the chain of three, V and Y shaped chromosomes, were most frequent. The triple arch was not found. At anaphase I the trivalents segregate at random. Con sequently, the resultant gametes possessed varying number of chromosome ranging from thirteen to twenty (Fig. 7). The distribution was ex tremely irregular being charac terised by a large number of laggards (Fig. 8). Even when the chromosomes at the poles entered the telophase stage lag gards (univalents) were found to remain at the equater. At anaphase II, also the chromosome distribution was unequal. Some of the laggards were left in the cytoplasm rather than being included into one of the nuclei. Micro nuclei ranging from 1-4 were also observed in a tetrad. The stainable pollen average is 38.7% (Fig. 9). The frequency of various types of chromosome associations and chaismata are Figs. 10-12. 10, a twig of the colchicine treated di summarised in Table 4. ploid M. charantia showing gynoecious condition. 11, a hermaphrodite flower in the colchicine treated Abnormalities in colchicine in diploid M. charantia. 12, a cluster of male flowers in the colchicine treated diploid M. charantia. duced diploid M. charantia These plants were easily distinguishable from normal ones by their sex expression and pollen sterility. The sex types that appeared were as follows: A) Gynoecious plants: Two gynoecious types were observed, among the plants treated with colchicine. Gynoecious type of sex forms are defined as the condition, when the plant bears only pistillate flowers. In these plants the pistillate flower appeared singly in leaf axils in a series (Fig. 10). But most of them did not 322 R. N. Trivedi and R. P. Roy Cytologia 38

open. Their flowering period was very short (only 30 days). These plants were crossed with control ones. In the next generation all plants were monoecious type and like the diploid ones. B) Andromonoecious type: After colchicine treatment three plants were found to produce hermaphrodite flowers in place of pistillate ones resulting into conditions termed as andromonoecious. The pollen of the hermaphrodite flowers (Fig. 11) were 80 to 88% sterile, and hence, selfing produced no fruit. However, crossing with the male flowers of the same plant resulted into , but with very less number of fertile seeds. The pollen stainability of the staminate flowers was 90 to 95%. Only three plants which could be raised in the next generation were monoecious diploids.

Table 4. Chromosome pairing and chiasma frequency in the diploid, tetraploid and triploid M. charantia at metaphase I

C) Trimonoeciousplants: Among the aberrant diploid plants, obtained from colchicine treatment, five showed trimonoecious condition (i.e., the plants bearing staminate, pistillate and perfect flowers). The perfect flowers (Fig. 11) could not develop into mature fruits. However, some fruits developed from the pistillate flowers. But the plants of the next generation reverted back to normal monoecious condition. D) Monoecious type with slight variation: Four plants produced staminate flowers in clusters in contrast to the solitary axillary condition in normal untreated plants (Fig. 12). Here the number of female flowers was very low. In the next generation this abnormality disappeared. 1973 Cytogenetics of Momordica charanita and Its Polyploids 323

E) Monoecious type with low fertility: There were twelve plants which have pollen fertility as low as 14 to 40 %. The seed setting was also very poor. However, it changed to normal condition in the next generation.

Discussion Morphologically the triploid plants showed intermediate characters between diploid and tetraploid. The meiosis in the triploid was highly irregular. It showed 0-6 bivalents, 0-2 rod bivalents, 0-5 ring bivalents and 5-11 trivalents with a corres ponding mean of 1.06, 0.22, 0.57 and 10.18 per cell. It is obvious that the number of bivalents is significantly lower than trivalents. The high frequency of the occur rence of trivalents may indicate in general the homologous nature of the three geno mes, i.e., this is strictly autotriploid. Besides, Univalents are also three. Therefore , at anaphase unequal number of chromosomes segregate to the two poles. The PMC showed laggards at first and second divisions. The resulting tetrads, there fore, is aberrant and unbalanced. The pollen fertility is accordingly much reduced and is only 38.7%. The fruit fails to develop in the triploids showing that meiosis in the female side is also irregular. In nature the wild and cultivated varieties of M. charantia are strictly mono ecious. Therefore, no clue to the genetic basis of monoecism in this species could be established. The appearance of different types of sex forms in colchicined M. charantia which could not be converted into polyploid is very revealing. The treated plants instead of remaining monoecious, produced different types of sex forms in different seedling-some became gynoecious, some androecious, some trimono ecious and some variants from monoecious, etc. Thus, here we have an array of the same type of sex forms as we find in interspecific and intervarietal hybrids in . It would be worthwhile recapitulating the various sex froms and their genotypes in Luffa (Singh, Ramanujam and Pal 1948, Richharia 1948, Singh 1958, Choudhury and Thakur 1966, Roy and Mishra, 1967). According to these authors principal ly two genes are involved in the production of various sex forms, as follows: Monoecious - AAGG Hermaphrodite - aagg Androecious - AAgg or Aagg Gynoecious - aaGG or aagG Gynomonoecious - aagG Monoecious - AaGG Monoecious - AAGg Trimonoecious - AaGg Roy and Mishra (1967) have discussed in detail how the various heterozygotes give rise to variant sex forms. However, different gene combinations giving rise to intermediate sex forms are unstable and rarely found in nature in Luffa. Such a situation does not exist in Drosophila where the eye colour gene has several multiple alleles, but the most stable genotypes are white and red. The other genes of the series like apricott, eosin, blood, buff etc. are found only in the culture, but do not 324 R. N. Trivedi and R. P. Roy Cytolgoia 38

survive in nature under pressure of natural selection. On the same analogy the monoecism, dioecism and hermaphroditism are most stable types in the Luffa. But other sex forms resulting from different combinations of the genes A and G are unstable and are not usually recovered in natural population. They, however, do appear as soon as hybrid progenies are raised and in the segregating population various recombinants are scored. In the same way it may be visualised that most stable genotype, and the sex form phenotype, in M. charantia is monoecism. The other intermediate types do not survive under pressure of natural selection. But the colchicined plant has given exciting result as it has exposed the hidden genetic basis of sex mechanism in this species also, which is similar to those in Luffa. It is unfortunate that these plants were sterile, but the experimental result has opened up great possibilities not only from the fundamental standpoint, but also from economic breeding aspect in that by suitable treatment it may be possible to raise fertile gynoecious, androecious and trimonoecious types of sex forms, thus increasing economic possibilities. One may hope to raise gynoecious and the hermaphrodite types which would prove a great boon in the release of improved varieties. For the present it will suffice to mention that even simple expreiment with colchicine has proved fruitful in that it has exposed the hidden genetic basis of sex form in this important vegetable. It is also important in the sense that the basis of genetic mechanism in the genus Luffa and M. charantia appears to be the same. Cucumis melo (Pool and Grimball 1939) provided the first clue to the number and methods of expression of sex forms. Later, this mechanism was discussed in Luffa and now it is possible to visualise the mechanisms in M. charantia on the same basis.

Summary

The triploid M. charantia raised by hybridisation between the tetraploid and the diploid show highly irregular meiosis with 5 to 11 bivalents and 0 to 6 uni valents. The pollen fertility is 38.7%. The triploid did not set any fruit. The appearance of various sex forms obtained by colchicine treatment of the monoecious diploid M. charantia has indicated the possibilities of understanding the sex mechanism in this species and related species. The resulting variants from the treated materials which remained diploid showed three types of sex expression, namely, Andromonoecious, Gynoecious and Trimonoecious. These intermediate sex forms are phenotypically, similar to those in the genus Luffa and are likewise unstable.

References

Choudhury, B. and Thakur, M. R. 1965. Inherintance of sex forms in Luffa. Ind. J. Genet. 25(2): 188-197. Poole, C. F. and Grimball, P. C. 1939. Inheritance of new sex forms in Cucumis melo L. J. Hered. 30: 21-25. Richharia, R. H. 1948. Sex inheritance in , Curr. Sci. 17(12): 359. 1973 Cytogenetics of Momordica charantia and Its Polyploids 325

Roy, R. P. and Mishra, A. R. 1967. Cytogenetic investigations in Ph. D. thesis. - , Thakur, V. and Trivedi, R. N. 1966. Cytogenetical studies in Momordica L. J. Cytol. and Genet. 1: 30-40. Singh, H. B., Ramanujam, S. and Pal, B. P. 1948. Inheritance of sex forms in Luffa acutangula Roxb. Nature 161: 775. Singh, S. N. 1958. Studies in the sex expression and sex ratio in Luffa species. Ind. J. Hort. 15: 66-75. Thakur, M. R. and Choudhury, B. 1966. Inheritance of some qualitative characters in Luffa species. Ind. J. Genet. 26(1): 78-86. Trivedi, R. N. and Roy, R. P. 1971. Interspecific hybridisation and amphidiploid studies in the genus Luffa. (in press). - 1972. Cytological studies in the genus Momoridica. Genetica 43: 282-291.