Establishment of regeneration and transformation system of sugarcane cultivar GT54-9 (C9)

Abstract

Plant regeneration protocols for sugarcane GT54-9(C9) cultivar were developed for direct organogenesis and indirect somatic embryogenesis, using young leaf segments as explants by studying the influence of different concentrations and types of cytokinin and auxin hormones. For the callus formation from young leaves, a medium containing 4mg/l 2,4-D was found very effective. For embryo formation, MS medium supplemented with 1mg/l Kin and 0.5 mg/l 2,4-D was used. While in the case of direct organogenesis protocol, the medium containing 1mg/l BAP and 2mg/l NAA was the best for direct shoot formation. Data showed that the best shoot regeneration and elongation medium for direct organogenesis and indirect somatic embryogenesis was obtained on medium with 2 mg/l Kin and 0.1 mg/l BAP. Root induction was best performed on 2mg/l NAA and complete plantlets were hardened in the greenhouse before transferring to the field for further evaluation. For transformation, young leaf segments of sugarcane from the cultivar GT54-9(C9) were inoculated and co-cultivated with Agrobacterium tumefaciens strain LB4404 harboring the binary vector pISV2678 with the bar and the gus-intron genes. The obtained putative transgenic plantlets were able to grow under bialaphose containing medium. Stable integration of the bar gene into the plant genomes was tested by PCR and Southern blot hybridization. Histochemical assay and leaf painting analysis were carried out to study the expression of the gus and bar genes in transgenic plants, respectively. The results indicated that the direct organogenesis produced a higher yield of regenerated plants (22% more) within shorter time (4 weeks less). Therefore, this method is recommended for sugarcane regeneration and for further use in genetic transformation via A. tumefaciens with desired genes.