Development of an Efficient Micropropagation-based Agrobacterium-Mediated Genetic Transformation Protocol in Commercial Cultivar of Jute (Corchorus capsularis L.)

Abstract

The present study describes an efficient in vitro culture protocol for direct plantlet regeneration and Agrobacterium- mediated genetic transformation of Corchorus capsularis L. cultivar JRC 517. In vitro morphogenetic capacity of different explants was used as parameter of performance adjudgement. Nodal explants with immature axillary buds when cultured on MS with 0.1 mg l-1 IAA and 0.1 mg l-1 Kin treatment showed maximum in vitro culture response (95%) with plantlets regeneration of 5.67 ± 0.30 %. A. tumefaciens strain LBA4404 harbouring a binary vector pBI121, containing gus A 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 were tested to accomplish utmost transformation competence, which indicates that O.D600. - of Agrobacterium cell suspension: 0.3; one day preculture; vacuum infiltration for 10 min at 600 mm Hg pressure; three days co-cultivation; 100 μM acetosyringone (AS) and 0.001 ml l-1 concentration of non-ionic surfactant (Tween 20) 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 histochimical assay and PCR analysis. The survivor plants were grown under Kan 50 selection pressure, and rooted successfully. Regenerated plantlets were acclimatized, hardened and transplanted to green house. Stable integration of the transgene into the recipient genome was confirmed by PCR using compatible primers of gusA and nptII, and Southern hybridization. The transgenic plants showed normal morphology and most of them followed 3:1 ratio of Mendelian inheritance for single dominant locus. Our findings documenting the in vitro direct shoot regeneration protocol from nodal explants with concurrent transgenic development there from deemed to be successfully involving economically important gene/s and trait enrichment in jute in future.