CYTOGENETIC STUDIES ON A MULBERRY (MORUS SP.) HYBRID OF A CROSS BETWEEN DIPLOID AND TRIPLOID PARENTS

* R. Ravi Kumara and Basavaiah. Mulberry Breeding and Genetics Laboratory, Department Of Studies in Sericulture Science, University Of Mysore, Manasagangotri , Mysuru -570 006. Karnataka , India . ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

1. In morphological features the F1 hybrid showed intermediate nature between female and male parents. The branching nature, phyllotaxy, stipule nature, leaf base and leaf shape are all similar in the hybrid as well as its parents. The hybrid resembles Bird's foot, its female parent in leaf color, sex expression and rarely occurring lobed leaves. It resembles male parent Suvarna-1 in leaf color, lobation, shape, surface, texture, apex etc. Further, it is intermediate between male and female parents in majority of the characters especially in leaf length, width, leaf size and petiole size. The F1 hybrid and other F1 progenies showed various types of lobed leaves in the initial development of seedlings (Figs.7 -10).
The F1 hybrid is characterized for its reproductive features in comparison with its parent and the data are presented in Table-2. Unlike, its parents it showed stable condition in sex expression with only female inflorescence in all the seasons. On thecontrary its female parent Bird's foot exhibit complete female flowers only during rainy season. Length of its inflorescence was almost equal to that of its parents. The number of flowers was found reduced when compared to its female parent. It resembles its male parent with its increased peduncle length. The style length of hybrid also found increased over its female parent. The fruit size of the hybrid is intermediate between the male and female parent.
The growth parameters of the hybrid are studied with comparison with its parent and data are recorded in Table-3. In all the growth parameters the hybrid has improvement over its female parent. But, it is found inferior to its male parent in almost all growth parameters.
The hybrid also showed good rooting percentage with high percentage of survival and growth characteristics of the saplings (Table-4).
Anatomical studies of the leaf of the hybrid also showed distinctiveness of hybrid over its parents. Its stomata size is significantly higher than its diploid parent but lower than its triploid parent and also stomata frequency is remarkably higher in F1 hybrid. Though, the leaf size of the hybrid is intermediate between the diploid and triploid parents its leaf thickness is lower than both of its parents (Table-5).
To characterize the trisomic hybrid of cross between diploid and a triploid its biochemical characters are also compared with that of its parents. The data on various biochemical parameters studied is presented in Table-6. There was no significant difference between the parents and hybrid in leaf protein content. Similarly, the hybrid doesn't differ from its parents in carbohydrate content of its course leaves. But, there were highly significant differences in tender and medium leaves. The hybrid showed lesser chlorophyll content over its parents and hence, its leaf is light green in color. Moisture content and moisture retention capacity are also found good in the F1 hybrid of the present study.
Cytological studies:-Mitotic studies in mulberry genotype Bird's foot:-Mitotic studies in mulberry genotype Bird's foot revealed that there is sticky nature of chromosomes at their ends (Fig.11). As a result, there was no proper spreading of chromosomes at metaphase. The study with sufficient number of cells of root tips indicated that there are 28 somatic chromosomes (Fig. 12& 13). All the somatic cells studied showed almost normal mitosis. Rarely chromatin bridges are recorded during anaphase separation of chromosomes (Fig. 14).

Meiotic studies in mulberry genotype Suvarna-1:-
Mitotic studies with the shoot tip cells established the chromosome number of this genotype as 2n=3x=42 (Fig. 15). Meiosis during microsporogenesis was studied in Suvarna-1 and it was found irregular. All the pollen mother cells of an anther showed highly synchronized meiotic divisions. During prophase-I PMC's showed early condensation of some bivalents (Fig. 16). At the end of diakinesis there were 14 highly condensed chromosome groups. At the beginning of metaphase-I 14 trivalents were recorded in majority of PMC's. Some PMC's also showed varied number of trivalents along with few bivalents and univalent. The trivalents aligned normally on the equatorial plate ( Fig. 17). Anaphase-I separation was found irregular (Fig. 18). The number of chromosomes moving to opposite poles was unequal in majority of the PMC's studied. The frequency of laggards was also high.

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Meiosis-II was also highly irregular in Suvarna-1. At metaphase-II many scattered chromosomes in the cytoplasm are recorded. There was unequal anaphase-II separation and lagging of 6-10 chromosomes. Very few tetrads showed four spore cells of uniform size and majority of them were with unequal sized spores (Fig. 19). Apart from tetrads, dyads, triads, pentads ( Fig.19) with wide range of cell size were also frequently seen. Pollen grains were highly variable in size (Fig. 20). The pollen viability was around 50 per cent indicating high level of sterility in this triploid genotype.
Mitotic studies in F1 hybrid of mulberry:-Mitotic studies in the F1 hybrid of Bird's foot and Suvarna-1 also indicated some degree of stickiness at the ends of chromosomes. As a result there was no proper spreading of chromosomes at metaphase. The mitotic anaphase separation of chromosomes was found normal (Fig.21& 22). The study with sufficient number of cells of root tips indicated that there are 28 somatic chromosomes (Fig. 23). All the somatic cells studied showed almost normal mitosis.

Discussion:-
Cytogenetic information in mulberry is scanty. Even the reports establishing the chromosome number of mulberry genotypes are very few. Also the chromosomes are various germplasm accessions is not determined and documented in the catalogue of mulberry germplasm (Thangaveluet al.,1997). In the present breeding work, two mulberry genotypes-Bird's foot and Suvarna-1 are used as parents. Thisstudy is the first report recording the chromosome number in the genotype Bird's foot which has 2n=28. Further, Suvarna-1 which developed at KSSRDI, Bangalore by crossing M-5 (4x) as female andViswa(2x) as maleparent is reported as triploid(2n=3x=42) (Eswar Rao et al.,2014).
Various workers have studied inheritance of leaf lobation patterns in mulberry (Gray et al.,1987). The hybrid of lobed female and unlobed male is characterized by completely unlobed leaves like its triploid male parent suggesting that the dominance of unlobed condition may be due to triploid genome over diploid genome. Occasionally occurring lobed leaves in the hybrid also indicate the unstable nature of leaf lobation in the hybrid. In this study emphasis is laid to understand the chromosome number of hybrid obtained from a cross between the diploid, Bird's foot and triploid, Suvarna-1. In various crop plants aneuploids have been generated by crossing diploid with triploid. Many studies was established the importance of aneuploids in cytogenetic research.
The mitotic studies of Bird's foot clearly established that, chromosome of this genotypes is 2n= 28 and it also showed normal mitosis. Suvarna-1 is a male parent of the present hybrid and is a triploid with chromosome number 2n=3x=42. The present work also confirms its chromosome number of Suvarna-1 which is characterized by having irregular meiosis. Irregular meiosis leads to reduced fertility and pollen grains of highly variable size. The variable sized pollen grains are resulting from variable chromosome number separated during anaphase movement.
The F1 hybrid of the present study is important from two aspects. Firstly, with regard to the chromosome number and secondly, with regard to inheritance of lobation of leaf. The mitotic studies of the hybrid established its chromosome number as 2n=2x+1=29. Hence, it is a trisomic. Since, various kinds of trisomics carry an extra chromosome, the genetic ratios for the genes which are located on that chromosome are modified in the segregating progenies. In fact, trisomic segregations have been studied extensively in all the genetically well investigated plant species , such as maize, tomato, barly, datura, wheat, etc. primary trisomics provide an excellent cytogenetic tool for testing the independence of linkage groups and for assigning linkage groups to particular chromosomes (Khush, 1973). In this context, the trisomic of the present study is having a lot of importance in mulberry breeding and cytogenetics. However, for further exploitation of this hybrid as trisomic requires the confirmation of its chromosome number with good cytological preparations. The extra chromosome of the compliment can be identified by employing chromosome banding techniques.
The characterization of hybrid indicated that it is having a good combination of leaf yield and quality parameters. Though, the hybrid is showing unlobed leaves like its male parent its morphological, reproductive and growth parameters indicate its intermediate nature between the parents. The anatomical and biochemical parameters of the hybrid also establish its intermediate nature between Bird's foot and Suvarna-1.
The hybrid though not important as improved variety for commercial exploitation, it may serve as breeding stock with a good combination of leaf yield and leaf quality parameters.

Summery &Conclusion:-
The present study established the chromosome number of the hybrid as 2n=2x+1=29, a trisomic. Further, the chromosome numbers of Bird's foot and Suvarna-1 also confirmed through this study. The trisomic identified in this study has lot of importance in mulberry cytogenetics and breeding. However, further confirmation of chromosome number of this hybrid beyond doubt is also required.
In this preliminary characterization, the hybrid appears to be inferior to its male parent Suvarna-1, an improved genotype in relation to leaf yield and quality determining parameters. But, it is having a good combination of characters in relation to leaf yield and leaf quality. The hybrid, though not important as an improved variety for commercial exploitation, it serves as a breeding stock with good combination of leaf yield and leaf quality characters.   Anther Shoot weight (g) Fresh(g) 4 6 5 08 Number of roots 10 8 6