Development of an efficient Micropropagation based Agrobacterium-mediated Genetic Transformation Protocol in Commercial cultivar of Jute (Corchorus capsularis L.)
Raju Mondal, Kanti Meena, P.G. Karmakar, Mrinal K. Maiti1, Satyahari Dey1 and Asit B. Mandal*
1Depertment of Biotechnology, Indian Institute of Technology, Kharaghpur 721 302, India
Biotechnology Unit, Division of Crop Improvement, ICAR-Central Research Institute for Jute and Allied Fibres (CRIJAF), Barrackpore, Kolkata 700 120, India Email: email@example.com
The present study describes an efficient in vitro culture protocol for direct plantlet regeneration and A grobacterium- mediated genetic transformation of Corchorus capsularis L. cultivar JRC 517. In vitro morphogenetic capacity of different explants was evaluated. Nodal explants with immature axillary buds showed maximum in vitro culture response (95%) with plantlets induction when cultured on MS with 0.1 mg l-1 IAA and 0.1 mg l-1 Kin. A . tumefaciens strain LBA4404 harbouring a binary vector pBI121, containing gusA reporter gene under the transcriptional control of Cauliflower Mosaic Virus (CaMV) 35S promoter and NOS terminator was used in addition with neomycin phosphotransferase (npt-II) as plant selection marker gene. Different parameters viz. O.D600. of A grobacterium cell suspension: 0.3; one day preculture; one min explants dipping, vacuum infiltration for 10 min at 600 mm Hg pressure; 0.001 ml l-1 concentration of non-ionic surfactant (Tween 20) and two days cocultivation with 100 μM acetosyringone (AS) were found to be optimum treatment to achieve maximum number of stable genetic transformants (~3.6%). The putative transformants were screened on MS medium supplemented with 50 mg l-1 kanamycin (Kan50) and their transient expression was confirmed through GUS histochemical assay of the reporter gene and PCR analysis. The survivor plants were grown under Kan50 selection pressure, and rooted successfully. Regenerated plantlets were acclimatized, hardened and transplanted to glass house. Stable integration of the transgene into the recipient genome was confirmed by PCR using compatible primers of gusA and nptII, and through Southern hybridization. The transgenic plants showed normal morphology and most of them followed 3:1 ratio of Mendelian inheritance for a single dominant transgene In vitro direct shoot regeneration protocol from nodal explants with concurrent transgenic development deemed to be successfully involving economically important gene/s and trait enrichment in jute.