In-vitro Factors Affecting Agrobacterium Tumefacience Mediated Genetic Transformation and Regeneration in Gladiolus (Gladiolus Hybrida)

Abstract

Gladiolus (Gladiolus hybrida) is a bulbous ornamental plant, It has a great commercial value in cut flower industry all over the world as well as in India due to its magnificent and colourful spikes. The genus Gladiolus belongs to the family Iridaceae. In-vitro differentiation and regeneration of plantlets in Gladiolus cultivar Sylvia was achieved using cormel shoot bud as an explants. Maximum number of explants producing callus (76%) were observed on MS media supplemented with 2.0 mg/l 2,4-D. Maximum shoot induction was achieved from callus after 28 days of inoculation on MS medium supplemented with 2.0 mg/l BAP, with mean value of shoot number (9.2), shoot length (9.32 cm). For root induction in-vitro propagated micro-shoots transferred to varying concentrations of PGRs, maximum number of root developed were recorded on 2.0 mg/l NAA, after 28 days of inoculation on MS medium as compared to other concentrations. MS medium response was better as compare to B5 medium. Present study deals with the comparative ability of three Agrobacterium tumefaciens strains GV3101, LBA4404 and EHA105 harboring the plasmid pCAMBIA1301 to transform Gladiolus. GUS expression was observed in callus of Gladiolus cv. Sylvia. Callus that developed from control (without Agrobacterium treatment) explants showed no GUS activity while transformed callus stained blue. The putative transgenic calli genomic DNA were subjected to PCR to confirm the presence of uidA and hptII genes in the callus. Maximum number of transformants were found positive by strain LBA4404 followed by EHA105, GV3101. Senescence-Associated Gene 12 (SAG12) of Arabidopsis thaliana was isolated by the polymerase chain reaction (PCR). Isopentenyl transferase gene (ipt) was also amplified by PCR, to constructed the plant-expression vectors (pCAMBIA1300-SAG12-IPT). This vector was used to transform the callus of Gladiolus cultivar Sylvia with the help of the A. tumefaciens system. Calli were cultured in-vitro by selection on medium containing hygromycin B. PCR amplification showed that the target gene was successfully integrated into the transformed calli