Establishment of an in vitro Regeneration System as a Milestone for Genetic Transformation of Sugarcane ( Saccharum officinarum L.) against Ustilago scitaminea

Optimization of stable in vitro regeneration system is indispensible to apply molecular approaches in crops. Due to its profound impact on genetic transformation studies, we established a reproducible and effectual in vitro regeneration system, in two whip smut (Ustilago scitaminea) susceptible genotypes viz., S-2003-us-127 and S-2003-us-371. Twelve callus formation media (CFM) were investigated for callus formation, in which four levels of 2,4-D (1 mg/L, 2 mg/L, 3 mg/L and 4 mg/L), two levels of BAP (0 mg/L and 0.5 mg/L) and two levels of kinetin (0 and 0.1 mg/l) were used in different combinations with basal MS Salt. CFM3 (3 mg/L 2,4-D), CFM4 (4 mg/L 2,4-D), CFM11 (3 mg/L 2,4-D and 0.1 mg/L kinetin) and CFM12 (4 mg/L 2,4-D and 0.1 mg/L kinetin) were proved to be the best for callus formation response in genotype S-2003-us-127. But in case of genotype S-2003-us-371, CFM3, CFM11 and CFM12 showed good response for callus induction. Among good responsive CFM, we selected CFM with low dose of 2,4-D (CFM3 and CFM11) for our regeneration experiment. For regeneration study, four regeneration media (RM) with different plant growth regulators viz., 2,4-D (0.1 mg/L), BAP (0.5 mg/L and 1 mg/L), kinetin (0.25 mg/L) and proline (250 mmg/L) plus MS salt were used. Calli of three (3) different ages, viz., 21 days, 28 days and 35 days from CFM3 and CFM11 were shifted on four regeneration media (RM). Among these four regeneration media, RM2 (0.1 mg/L 2,4-D and 1 mg/L BAP) gave an excellent regeneration response for genotype S-2003-us-127, when 28 days old calli from CFM11 were transferred to this. This combination was selected as combination of choice in genotype S-2003-us-127 for genetic transformation studies. Genotype S-2003-us-371, showed maximum regeneration, when 35 days old calli from CFM11 were kept on RM4 (0.1 mg/L 2,4-D, 1 mg/L BAP, 0.25 mg/L kinetin and 250 mg/L proline). Genetic stability of regenerated plants of selected media combination was confirmed with RAPD (PCR) analysis by using 5 RAPD primers.