Abstract
The present research works were conducted on jute using varieties/genotypes belonging to both Corchorus capsularis L. and C. olitorius L. to study important morphological traits and to undertake molecular characterization through RAPD. Optimization of a repeatable in vitro regeneration protocol and development of a protocol for foreign gene insertion through Agrobacterium-mediated genetic transformation in jute have also been studied. Field experiment was conducted at the Jute Agricultural Experimental Station, Manikganj of Bangladesh Jute Research Institute in 2002. RAPD analysis was done at the Biochemistry and Molecular Biology Laboratory, University of Dhaka. Experiments related to optimization of in vitro regeneration and Agrobacterium-mediated genetic transformation was conducted in the Tissue Culture and Genetic Engineering Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh.Through morphological characterization, genetic divergence was estimated among 13 genotypes of each of C. capsularis and C. olitorius on the basis of 13 quantitative characters. The individual species level study grouped the varieties/genotypes into five and six clusters respectively. Clustering pattern revealed that genotypes originating from the same locality did not form a single cluster indicating that there was no relationship between genetic diversity and geographical distribution of the genotypes. This further indicated that the availability of genetic diversity in the types from same geographical origin is possible. On the basis of discriminant function, selection scores were computed for all the genotypes of C. capsularis and C. olitorius and these were ranked at individual species level. At 20 % selection intensity the top ranking three genotypes of C. capsularis were CVE-3, CC-45 and Tri-cap-1 and of C. olitorius were O-4, SDLT-1 and O-72. Results indicated that direct selection based on mean performance for fibre yield would be rewarding for identification of high performance genotypes. These selected genotypes were used in tissue culture and transformation programmes of the present study.In RAPD analysis, of the 40 primers tested, 9 (OPAB-03, OPAB-05, OPAB-06, OPAB-18, OPG-03, OPG-05, OPG-06, OPG-09 and OPQ-17) produced clear and scorable DNA fragments and showed polymorphic fragments on which dendogram was made. The highest (10.9 in C. olitorius 10.5 in C. capsularis) and the lowest (4.0 and 2.6 respectively) percent of polymorphic bands were shown by the primer OPG-05 and OPQ-17 respectively. Squared Euclidean distances indicated that the two species were clearly distinguished by the primers used. Within species some morphologically alike genotypes were clustered closely which had also reflected through morphological grouping. In C. capsularis the genotype Acc. 3695 was grouped alone in the dendogram and this was also grouped as a separate cluster on morphological study. In both the species, there were some genotypes which had close proximity (CVE-3 & BJC-7370, BJC-718 & BJC-2142 of C. capsularis and O-4 & Acc. 1862, brown seeded genotypes of C. olitorius) in the dendogram, the same was also reflected in cluster formation through D² analysis. Despite the strong homology which was exhibited by many of the jute genotypes of the two species, this work demonstrates that it is possible to differentiate between varieties/genotypes using RAPD.Optimization of regeneration protocol indicated that cotton based liquid MS medium containing one per cent sucrose with controlled temperature (26-28º C), light (16/8 h) and the genotype O-4 was the best for healthy seedling production in C. olitorius. Darkness reduced germination. Seedling dry weight was increased in light and it was also increased with increasing sucrose concentrations. In C. capsularis the genotype CVE-3 was the best for healthy seedling production and in vitro regeneration. Seedlings of early age (4-5 days) and cotyledons as a source of explant were better for in vitro regeneration of both Corchorus spp. The highest percentage of root initiation was observed in shoots raised from cotyledon explants (53.05). Minimum days (6) and profuse rooting were observed in medium containing ½ MS + 0.50 mg/l IBA followed by ½ MS + 0.25 mg/l IBA. Coconut dust was found to be the best component for transferring plantlets in soil (95.83 % survivability) followed by sand + coconut dust (79.17%).In Agrobacterium-mediated transformation, growth curve of the tested Agrobacterium LBA4404 (pBI121) was analyzed to identify the log phase and it was between 8-10 hours of culture in induction medium. Following co-cultivation in medium containing acetosyringone, percent shoot generation was found to be higher in hypocotyls than in cotyledons in all genotypes. The highest percent shoot generation was in the genotype Tri-cap-1 followed by CVE-3 and CC-45. But number of shoots produced per explant was higher in cotyledons than in hypocotyls. Irrespective of explants the highest GUS positive was shown by the genotype Tri-cap-1 (75 % in hypocotyls and 65 % in cotyledons). Presence of kanamycin in the selection media greatly affected (albinism and browning) the development of transgenic shoots. This might be due to organogenic regeneration from multiple cells in jute. This was partially overcome by periodical withdrawal of selection pressure. A few of the shoots of all the three genotypes continued to grow for three months and then died.