G.B. Pant University of Agriculture and Technology Pantnagar -263145, INDIA
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Extracts of leaf tissues of experimental hybrids and their parents in Brassica juncea were separated by electrophoresis in polyacrylamide gels and stained for esterase isozymes to investigate the possibility of utilizing isozyme variation to determine the purity of hybrids. In general, the isozyme bands of hybrids represented the sum bands in the respective parental lines. However, some of the bands were missing and some new bands were also seen. Occurrence of additional bands in hybrids may be due to product of sub unit reassociation or occurrence of hybrid bands due to heterozygosity effects while missing bands may attributed due to heterozygosity inhibition or presence of mosaic genes. Isozyme exhibited limited variation thus it is not found very suitable for characterization of hybrids. On the other hand DNA marker may be a suitable alternative for hybrid characterization.
KEYWORDS : Esterase isozymes, hybrid characterization, Indian mustard.
Brassica juncea is a leading oil crop in India. Prospects of hybrid technology in Indian mustard are promising to brake the yield varriers. Brassica species is polymorphic for many isozyme systems and it is also highly variable within and between the cultivars. The objective of this study was to characterise the hybrids and their parents by esterase isozyme electrophoresis.
Hybrids alongwith parents were grown at G.B Pant Univ. of Agric. and Tech., Pantnagar in 1996-97. Enzyme was extracted from the blades of fresh and healthy young leaves of 107, 108 and 109 days old plants. One gram of leaf for each genotype was homogenized in a pre-chilled mortar and pestal with one ml of chilled 50 mM Tris-HCl buffer (pH=7.6). The extracts were centrifuged at 14000 rpm for 30 minutes at 40C. Immediately 20 μl of each samples were loaded in the wells for electrophoresis. Gel was run at 36 mA for 4 hours.
Gel was removed carefully in a tray and stained in α-napthyl acetate and fast blue RR salt solution for 30 minutes in dark, and destained in 7% acetic acid. The gel was visualized on a white light box illuminator and photographed (Tanksley and Orton, 1983).
Entire genetic materials were grouped into three sets. Each set consisted of five hybrids and their respective parents (Fig.1-3). Lane 1 in each Figure shows maintainer line while male sterile line that was common to all five hybrids of concerned set shown in lane 2. The hybrid genotype profiles are in lanes 3, 5, 7, 9 and 11 while their respective pollen parents are shown in lane 4, 6, 8, 10 and 12.
The isozyme marker is a co-dominant marker. Thus isozyme bands of hybrids could be regarded as summation of the parent derived bands through some of the bands present in respective parents were missing in hybrids. Similarly, some new bands were observed in hybrids which were missing in the parents. The missing bands in hybrids may be attributed due to some allelic interaction rather than showing co-dominance, or heterozygosity inhibition, or presence of mosaic gene. On the other hand, occurrence of new bands in hybrids may be due to subunit reassociation, or occurrence of hybrids bands due to heterozygosity (Arus et al. 1985, Vaughan et al. 1970, Kato and Tokumasu, 1979, Simonsen and Heneen, 1995). The variation in occurrence of bands in hybrids with respect to their parents reveals the fact of differential gene expression in hybrids.
While, considering the similarity of hybrids among different genotypes of a particular set, the highest similar bands were observed in genotypes of set 1 followed by set 3 and 2. There were few bands very much specific to specific genotypes. Variation among different genotypes are expected due to fact that these genotypes are from different source and location hence likely to have different gene pool.
In conclusion isozyme exhibited limited variation as many of the lines and hybrids were indistinguishable based on the esterase isozyme pattern. Thus it was not found very suitable for characterization of hybrids for complete fingerprinting. On the otherhand DNA marker may be a more suitable alternative for hybrid characterization in Indian mustard.
The authors are grateful to the CSIR, New Delhi for providing financial assistance.
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