Cytogenetical Studies in Some Triploid Mulberry Varieties (Moraceae)

K. H. Venkatesh1, R. Nijagunaiah2* and Munirajappa1

1 Mulberry Breeding and Genetics Laboratory, Department of Sericulture, Bangalore University, Bangalore-560056, India

2 Department of Botany, Bangalore University, Bangalore-560056, India

Received February 12, 2014; accepted May 30, 2014

Summary The mulberry is an economically important crop, cultivated for its foliage to rear the silkworm Bombyx mori L. Its chromosome number varies from 2n = 28 to 2n = 308 with ploidy level from x to 22x. In the present investigation, three triploid mulberry varieties, namely, S41, S1635 and BC2–59, have been analyzed for detailed meiotic studies. Based on the chromosome configuration and other meiotic behaviour, x = 14 has been considered as the basic number of the genus. Meiosis was highly irregular. Various anomalies, such as the occurrence of two nucleolus, association of single trivalent with nucleolus, unequal separation and precocious movement of chromosomes, laggards, and occurrence of multivalents, have been observed. The low pollen fertility and seed set may be due to genic or physiological causes.

Key words Mulberry (Morus spp.), Triploid, Meiotic behaviour, Sericulture.

Triploid mulberry varieties have higher leaf yield and better nutritive qualities from the point of silkworm rearing (Seki and Oshikane 1959, Sugiyama 1959, Abdullae 1962, Mustafaev 1970, Dzhafurov and Alekperova 1978). Triploid varieties are extensively cultivated in Japan, particularly in its northern parts, for their superior quality of leaves and cold and disease resistance (Hamada 1963, Sugiyama 1959). Tripoloids are occasionally found in the natural population (Hamada 1963). Generally, triploids are obtained by crossing tetraploids with diploids, and for this, tetraploids are first induced and then crossed with diploids. In the mulberry, both natural and induced triploids have been reported (Osawa 1920, Seki 1959, Das et al. 1970, Dwivedi et al. 1989). Many triploid varieties of mulberry are considered superior than diploids in yield and nutritive qualities of the leaf (Seki and Oshikane 1959, Dzhafurov and Alekperova 1978). The cytogenetical information available in triploids is fragmentary and only preliminary observations have been made by earlier workers (Tojyo 1966, Agaev and Fedorova 1970). The information on breeding systems and the evolution of different mulberry polyploids are highly useful in evolving elite genotypes required for commercial cultivation. In the present study, an attempt has been made to analyze the meiotic behaviour in three triploid varieties of the mulberry.

Materials and methods

The mulberry varieties used in the present study are S41, S1635, and BC2–59. For meiosis, flower buds of appropriate stages of development were harvested at 9:20 a.m. during sunny days and fixed in 1 : 3 acetic–alcohols for 24 h and preserved in 70% ethanol. Anthers were squashed in 2% aceto-carmine stain. Photo-micrographs were taken using a Labomed microscope fitted with a Nikon

© 2014 The Japan Mendel Society Cytologia 79(3): 365–369

* Corresponding author, e-mail: khvenki1972@gmail.comDOI: 10.1508/cytologia.79.365

366 K. H. Venkatesh et al. Cytologia 79(3)

Coolfix digital camera.

Results and discussion

The taxa investigated in the present study (S41, BC2–59 and S1635) displayed a triploid chromosome number of n = 21. Meiotic behaviour was highly irregular. Apart from the regular occurrence of a single large nucleolus, few pollen mother cells (PMCs) showed two nucleoli during prophase I (Figs. 1 and 2). At diakinesis, 14 trivalents are clearly seen, and in some cells one trivalent is found associated with the nucleolus (Fig. 3). Metaphase I showed various types of chromosomal configurations (Figs. 4–8, Table 1). Depending on the pairing of chromosomes, tetra-, tri- and bivalents showed different configurations. A few PMCs (6.67%) showed the presence of quadrivalents (Fig. 8). The frequency of quadrivalents, trivalents, bivalents and univalents varies from 0-1, 3-13, 0-10 and 0-6, respectively (Table 1). Cells with 13 trivalents were 43.33%. Different types of chromosomal associations found in the present study corroborated the findings of Das et al. (1984) and Basavaiah et al. (1990) in triploids of mulberry. The presence of higher percentages of trivalents in metaphase I is suggestive of a fair degree of homology between the constituent of triploids. In all the varieties, the trivalent frequency was high and various types of chromosomal configurations (‘>-’, ‘---’ and ‘v’) were observed. Bivalent and univalent frequencies were very low, especially in S1635. A majority of PMCs exhibited regular alignment of chromosomes at metaphase plate in S41 and S1635 (Figs. 4–6), but in BC2–59, the chromosomes are scattered in the cytoplasm (Figs. 7 and 8). The alignment of centromeres of trivalents and univalents appears to be random, and hence the assortment of chromosomes was also random. Anaphase I was abnormal due to the presence of laggards (Fig. 9), unequal separation (Figs.10 and 11) and irregular distribution of chromosomes (Fig. 12, Table 2), which were quite common. Out of 30 cells analysed, 6 cells were with equal distribution (21 : 21). The chromosomes in each trivalent separated first and moved to the poles depending on their mode of orientation. The assortment of these chromosomes and the regular univalents appears to be a matter of chance. Hence the majority of the cells showed unequal separation of chromosomes at anaphase I. Chance of equal distribution was meagre and 22/20 and 23/19 distribution were common among the PMCs observed.

As expected, the disturbance in meiosis I has an adverse effect on the subsequent stages of meiosis II in all the three varieties. Metaphase II showed many chromosomes scattered in the cytoplasm without aligning on the equatorial plate, and an unequal number of chromosomes in the daughter cells (Figs. 13 and 14). This has lead to an unequal anaphase II separation (Fig. 15). Unequal separation of chromosomes in anaphases I and II lead to the formation of pollen grains with an unequal number of chromosomes.

In the mulberry, triploids, arising spontaneously in diploid lines (Osawa 1920, Seki 1959) as well as induced by crossing diploid with tetraploid (Das et al. 1970) and also by diploidizing the female gamete cell (Dwivedi et al. 1989), have been reported. Triploid mulberries are proved to be superior to diploids and tetraploids in yield and quality of leaves (Dzhafurov and Alekperova 1978). However, triploidy appears to represent an optional level of ploidy in the mulberry from a sericultural point of view.

The meiotic behaviour of triploid cultivars of the present study indicated the typical features of met within triploids. In general, meiosis in the triploid is highly irregular. PMCs at early prophase stages showed two similar-sized nucleoli. This indicates that two homologous nucleolar organizers of the diploid complement organize a large nucleolus, while the other organizer of the third genome forms an extra nucleolus. The number of nucleoli in plants has usually been correlated with the number of secondary constrictions present in the complement and also the ploidy level (Darvey and Driscoll 1972, Thomas and Kaltsikes 1977). The high frequency of trivalents is suggestive of a fair degree of homology between constituent genomes and the auto triploid nature of these varieties.

2014 Cytogenetical Studies in Some Triploid Mulberry Varieties (Moraceae) 367

The same condition is observed in some natural triploid varieties of mulberry by Seki (1959) and Das et al. (1984). At anaphase, unequal numbers of chromosomes segregate to the poles, which again is attributed to irregularities in basic processes like chromosome pairing and alignment. Meiotic abnormalities such as unequal separation, laggards, and irregular distribution of chromosomes to different poles have resulted in the size variation of pollen and their low fertility compared to diploids. The reduced pollen fertility in triploids invariably results in loss of chromatin materials

Figs. 1–15. Chromosome association and behaviour during meiosis I and II in triploid varieties of mulberry (S41, BC2–59 and S1635). 1, prophase I. 2, prophase I with two nucleoli. 3, diakinesis̶one trivalent associated with nucleolus. 4–8, metaphase I showing different types of chromosomal configurations. 4, 12III+2II+2I, 5, 13III+1II+1I, 6, 13III+1II+1I, 7, 12III+2II+2I, 8, 1IIII+10III+3II+2II, 9, anaphase I (laggards). 10, 11, anaphase I (unequal separation). 12, anaphase I (irregular distribution of chromosomes). 13, 14, metaphase II (irregular alignment of chromosomes at equatorial plate). metaphase II (unequal number of chromosomes in daughter cells). 15, early anaphase II (unequal separation).

368 K. H. Venkatesh et al. Cytologia 79(3)

(Darlington 1965, Gottschalk 1978).

Conclusion

The taxa studied (S41, BC2–59 and S1635) displayed n = 21 chromosomes in dividing PMCs. Meiotic behaviour in all three varieties were highly irregular. Two nucleoli were observed at prophase I. Trivalent, bivalent and univalent were observed at diakinesis and metaphase I. Separation of chromosomes during anaphase was highly irregular. Precocious movements of chromosomes were also recorded during metaphase and anaphase II. Due to the non-disjunction of chromosomes, viable gamete formation was prevented, leading to the development of aberrant pollen grains and sterility. However, all the taxa studied carry commercial attributes in them, such as high yielding ability coupled with nutritious foliage production. These are multiplied through vegetative propagation mainly using hard wood stem cuttings.

References

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Table 1. Chromosome association at metaphase I in triploid.

Sl. No Chromosome association No. of cells Percentage

1 12III+2II+2I 02 6.662 13III+1II+1I 11 36.663 13III+2I+1I 06 20.004 12III+2II+2I 04 13.335 1V+10III+3II+2I 01 3.336 12III+2II+2I 04 13.337 11III+3II+3I 02 6.66

Total 30 99.97

Table 2. Chromosome distribution at anaphase I in triploid.

Sl. No.Distribution of chromosomes B = Lagging bivalents U = Lagging univalents

No. of cells Percentage

1 21 : 21 06 20.002 22 : 20 06 20.003 20 : 20+2U 05 16.664 23 : 19 04 13.335 18 : 20+4U 03 10.006 21 : 19+1B 03 10.007 18 : 21+2B+1U 03 10.00

Total 30 99.99

2014 Cytogenetical Studies in Some Triploid Mulberry Varieties (Moraceae) 369

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