Rapid in vitro Multiplication of Sugarcane Elite Genotypes and Detection of Sugarcane Mosaic Virus through Two Steps RT- PCR

A voluminous body of research has reported the establishment of efficient protocols for sugarcane multiplication through tissue culture. However, a reliable and reproducible in vitro plant production system remains obscured. Furthermore, validation of virus free nature of in vitro plants using molecular techniques is the most challenging one. Considering the need for high yielding cultivars due to land and constraints, this study was devised for mass multiplication of high yielding elite cultivars of sugarcane viz. HSF-240, YT-55 and YT-53. Use of 100% Clorox for surface sterilization of apical and lateral buds, and of cefotaxime (500 mg L-1) for controlling bacterial contaminants revealed complete sterilization of field grown explants. Culture initiation was dependent upon plant growth regulators (PGRs), genotype and type of explants. The highest shoot initiation frequency of 96% was obtained with combination of four plant growth regulators (0.1 mg L-1 BAP), (0.1 mg L-1 NAA), (0.1 mg L-1 Kn) and (0.1 mg L-1 GA3). Maximum shoot number (17.4) was exhibited by HSF-240 on MS media when the concentrations of BAP, Kn and GA3 were increased to 1 mg L-1 in combination with NAA (0.25 mg L-1) indicating preference for higher concentrations of PGRs. Half-strength MS media with 6% sucrose resulted in increased root length (9.2 cm) and root number (20.5) Hardening efficiency of 98.6% was achievable in sandy clay loam soil. Two steps reverse transcription PCR (RT-PCR) was successfully employed for detection of sugarcane mosaic virus (SCMV) in in vitro plants. These results have implications for understanding optimum conditions for in vitro mass production of sugarcane plants, molecular detection of SCMV in in vitro raised plants, and stable genetic transformation studies. [copy 2012 Friends Science Publishers