DEVELOPMENT OF TRANSGENIC KENAF(Hibiscus cannithinus L.) THROUGH A GROBA CTERIUM VECTOR

Abstract

A significant research work about plant regeneration and Agrohacterium-mediated genetic transformation was realized for a Kenaf (Hibiscus cannabinus) variety (HC-2) and an accession of Kenaf (Ace-1665) during the period from July, 2008 to October, 2009 in the Genetic Engineering Laboratory of Genetic Resources and Seed Division, Bangladesh Jute Research Institute (BJRI). Dhalca. The genetic transformation of two gene constructs PGly-1 and PsCIPK (that carry p131121 of 14 K Da plasmid with figlucuronidase p35SGUSINT and plant selectable marker gene nptll) confer both salt and thought tolerance to Kenaf variety HC-2 and Acc-1665 have been undertaken. A cotyledon (with attached petioles) was used as explants for plant regeneration. GUS assay was performed after 30 days of co-cultivation of infected cotyledons. Cotyledons of the both variety and accession (HC-2 and Ace-1665) were cultured on MS medium supplemented with different concentrations of BAP (0, 1, 2, 3, 4, and 5 mg/I) and a constant IAA (0.5mg/I) concentration to induce callus and shoot regeneration. The range of callus induction was found to be 24% to 91 % and shoot regeneration was 16.33% to 91 .33%.The highest callus induction (91%) was found in both HC-2 and Acc-1665 varieties when cultured on MS medium supplemented with 3.0 mg/I BAP and 0.5 mg/I IAA and the highest shoot regeneration (91 .33%) was found in HC-2 variety. GUS histochemical assay was performed for the co-cultivated explants of Kenaf varieties (leaves) regenerated on kanamycin thr the detection of transgenic plants. The result of GUS assay varied according to varieties infected with different strains. Between the varietal response to GUS assay. HC-2 showed 70% and 90 % GUS positive depending upon PGIy-1 and PsCIPK infection. Whereas, Acc-1665 showed 65% and 75% positive for GUS assay depending upon GIy-1 and CIPK infection consecutively. Regeneration of plantlets (putative transformed plantlets) in kanamycin containing medium was found to be the highest for the variety HC-2 (84%) followed by Acc-1665 (79%). In vitro evaluation of explants under various salt concentrations was carried out to detect the tolerance level of transformed plants against different salt concentrations. Transformed plants were able to survive on 125 mM salt concentration while the controlled plants died out at NaCI salt concentration of 75mM. Both the variety and accession survived on (70%) up to 75 mM salt concentrations. However, in case of 125 mM salt concentrations, the survival was 20%. It signals the possibility of the salt tolerant variety development using recombinant DNA technology for cultivation in the saline belt of Bangladesh.