Cytological Studies in Two Diploid Mulberry Varieties (Moraceae)
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Chromosome Botany (2014) 9: 35-38 © Copyright 2014 by the International Society of Chromosome Botany Cytological studies in two diploid mulberry varieties (Moraceae) K. H. Venkatesh and Munirajappa Mulberry Breeding and Genetics Laboratory, Department of Sericulture, Bangalore University, Bangalore -560056, India Author for Correspondence: ([email protected]) Received February 10, 2014; accepted March 6, 2014 ABSTRACT. Most of the species of the Morus are diploid having 28 chromosome, but a few species, namely, M. tiliafolia Makino, Morus cathyana and M. nigra L., are higher polyploidy. Chromosome number varies from 2n =28 to 2n = 308 with ploidy level from x to 22x. In the present study two diploid mulberry varieties namely, Kollegal and DD have been analyzed for both mitotic meiotic studies. Based on the somatic chromosome number, ploidy level, chromosome configuration and other meiotic behavior x = 14 have been considered as basic number of the genus. Meiosis showed marginal differences. Various meiotic abnormalities like occurrence of one large chromosome, stickiness and loose association of chromosomes at metaphase I, unequal separation at anaphase I, laggards, precocious movement and unequal separation of chromosomes at anaphase II, have been observed. The low pollen fertility and seed set may be due to irregular chromosomal pairing and separation rather than due to genic or physiological causes. KEYWORDS: Diploids, Meiotic behavior, Mitosis, Morus spp. (Mulberry) For the rearing of silkworms (Bombyx mori) mulberry Mulberry varieties used in the present study are, Kollegal cultivation is the pre requisite. The foliage of the plant is and DD, which are maintained in the germplasm bank used mainly as a unique source of silkworm (Bombyx attached to Department of Sericulture, Bangalore University, mori L.) feed and cultivated in over 40 countries (Machii and Bangalore, India. Somatic preparations were made and Katagiri 1991). Cytogenetical studies, which is from excised root tips of potted plants. Root tips were prerequisite for genetic improvement in plant species. collected at 10.15 A M and pretreated with 0.002 M 8 - Most of the cultivated varieties of mulberry are diploids hydroxy quinoline for 3 hours at 10ºC. After thorough with 2n=28 chromosomes, but a few are ployploids (Datta washing, root tips were hydrolyzed in 1N HCl for seven 1954, Das 1961, Gill and Gupta 1979). The first minutes at 40ºC and then stained with 2% aceto-orcein. cytological research about plants of the genus Morus. L. Squash preparations were made in 45% acetic acid. was done in 1909 by Tahara (Sinoto, 1929) who observed Photographs were made on the same day of preparation to that they generally had a diploid number of chromosomes ascertain the chromosome number. For meiosis, flower equal to 14 (2n=28). Later, in 1920, Osawa who also buds of appropriate stages of development were harvested worked on some species of this kind, observed diploids at 9. 20 A M during sunny days and fixed in 1:3 acetic- and triploids. These two researchers had different opinions alcohol for 24 hours and preserved in 70 % alcohol. Anthers about the size of the chromosomes,Tahara reported the were squashed in 2% aceto-carmine. Photomicrographs presence of four bigger chromosomes and Osawa reported were taken using labomed microscope fitted with Nikon only two bigger chromosomes (Sinoto 1929). Coolfix Digital Camera. MATERIALS AND METHODS RESULTS AND DISCUSSION Datta (1954) studied the meiosis of some species of Morus Kollegal and DD mulberry varieties revealed diploid and confirmed the extreme difference of size between the chromosome number in their both somatic cells and pollen 12 small pairs and two big pairs which are already been mother cells (PMCs) with 2n = 2x = 28. In both the called by Osawa (1920), autosomes and allosomes, varieties one pair of large chromosomes were observed. respectively. Basaviah et al. (1989 and 1990) studied Chromosomes are small with continuous length range and mulberries, M. serrata Roxb. (2n=4x= 84) and M. nigra in most of the cases three types of chromosomes are L. (2n=22x=308) respectively. Review of the literature on observed. Somatic chromosome number and ploidy level mulberry improvement reveals that natural polyploids of chromosomes was also observed (Figs. 1 & 2). Mitosis exist in the Morus and even 22 ploidy was also reported was regular with equal separation of chromatids and (Osawa 1920, Janaki Ammal 1948, Seki 1951, Datta forming normal daughter cells in both the varieties. 1954). Meiotic behavior of a few cultivars has been Meiosis was regular and showed marginal differences. In reported by Datta (1954), Seki (1952), Basaviah et al. metaphase I majority of the PMCs exhibited 14 bivalents (1990), Venkatesh (2012) and Venkatesh et al. (2012) in a scattered in the cytoplasm and of which one pair was few mulberry cultivars. In the present study, an attempt to larger than others (Figs. 3, 4 & 5) respectively. Only few understand the somatic chromosome number, ploidy PMCs exhibited stickiness and clumping 14 bivalents level, chromosome association and meiotic behavior of arranged themselves on equatorial plate without any two diploid varieties of mulberry have been made. additional association (Fig. 6) and some PMCs showed 36 VENKATESH AND MURIRAJAPPA Figs. 1-18. 1 and 2. Somatic chromosomes at metaphase plate showing 2n = 28. 3, 4 and 5. Metaphase I showing 13 bivalents and one large bivalent scattered in the cytoplasm. 6. Metaphase I. 14 bivalents align at equatorial plate (stickiness & clumping). 7 and 8. Metaphase I (loose association). 9. Anaphase I (unequal separation). 10. Anaphase I (equal separation). 11. Telophase I. 12. Prophase II. 13 and 14. Metaphase II (stickiness and clumping). 15. Anaphase II (clumping and non separation). 16. Anaphase II (unequal separation). 17. Late anaphase II ( laggard). 18. Telophase II. CYTOLOGY OF TWO DIPLOID MULBERRIES 37 loose association of chromosomes (Figs.7 & 8). Anaphase chromosome number of 2n = 308 (22x) was established disjunction appears normal, while only about 5% of cells by Janaki Ammal (1948) with a polyploidy series of x = showed laggards during anaphasic movement. Likewise 14 to 22x = 308. Seki (1952) and Seki and Oshikane anaphase I showed slightly unequal, lagging, precocious (1960) have also reported 2n = 28 for M. bombyscis. and unequal separation of chromosomes (Fig. 9) and Dandin et al, (1987) have studied karyotype of eleven equal separation of 14 chromatids were clearly discernible mulberry varieties belonging to three species and most of moving to the respective poles (Fig.10). Regular two them are diploids with 2n = 28 somatic chromosomes. daughter nuclei were formed at the end of telophase I The same trend was observed in the present investigation (Fig. 11). Prophase II was also observed (Fig. 12). also. Presence of 14 bivalents in most of the PMCs at As expected, the disturbance in the meiosis I has metaphase I confirm the diploid nature of these varieties. adverse effect on the subsequent stages of meiosis II in One bivalent was found bigger in comparision to other as both the varieties. Degenerating PM Cs and the PMCs also reported by Gill and Gupta (1979) and Trivedi et al. with non synchronous divisional stages were recorded (1986) in Morus by Das (1961) in Morus indica. The frequently. Metaphase II chromosomes were clumping meiotic behavior of diploid varieties indicated the typical and scattered in the cytoplasm with laggards without features met within diploids. In general meiosis in diploid aligning on the equatorial plate (Fig. 14). In early was regular and it was clear that the gametic chromosomes anaphase II was quite abnormal due to the presence of number was n =14 with regular mega chromosomes. The laggards, clumping, non separation, precocious movement high frequency of bivalents is suggestive of a fair degree and unequal separation of chromosomes (Figs. 15 & 16) of homology between the constituent genomes and allo respectively. Late anaphase II showed laggard (Fig. 17). diploid nature of these varieties. Telophase II showed chromosomes grouped at four poles, The present investigation also showed that there were suggesting the action of spindle fibers (Fig. 18). no secondary associations and multivalent in metaphase I The taxa investigated in the present study, Kollegal and in both the varieties studied. Sinoto (1929) while studying DD displayed diploid chromosome number of 2n = 2x = the behaviour of allosomes, except a large pair of 28 and marginal differences in meiosis. Genus Morus has chromosomes in meiosis, has also not reported by any wide range of chromosome number from 2n = 28 to 2n = such secondary associations. Observation made by Datta 308 and ploidy from x to 22x. Tahara (1909) has reported (1954) may be due to the large size of one pair chromosomes 2n = 28 for M. alba and M. indica. Janaki Ammal (1948) which may be mistakes for trivalent/tetravalent. Secondly, has reported 2n = 28 for M. alba, M. bombysis, M. the frequency of secondary association of chromosomes laevigate, M. microphylla and M .rubra. Hence the higher observed by the author is in a very low percentage. 38 VENKATESH AND MURIRAJAPPA Anaphase I & II were quite abnormal due to laggard and LITERATURE CITED unequal numbers of chromosomes segregate to the pole Basaviah, Rajan, M. V., Dandin, S. B., Suryanarayana, N. and which again is attributed to irregularities in basic process Sengupta, K. 1990. Chromosomal association and meiotic behavior of four triploid varieties of mulberry (Morus like chromosome pairing and alignment. spp.) Cytologia 55: 327-333. While considerable chromosomal irregularities are Basaviah, Dandin , S. B. and Mala, V. R. 1989. Microsporogenesis expected in the metaphase I and anaphase I of ploidy level in hexoploid Morus serrata Roxb. Cytologia 54: 747-751. Dandin, S. B., Susheelamma, B. N., Jolly M. S, Mllikarjunappa, such as this it exhibits normal meiosis with good pollen R. S. and Giridhar, K. 1987. Karyomorphological studies fertility. It is also assessed that bivalents were more in few varieties of Mulberry (Morus spp.) Indian Journ.