Efficient Regeneration System for Genetic Transformation of Mulberry (<i>Morus indica</i> L. Cultivar S-36) Using <i>in Vitro</i> Derived Shoot Meristems

Abstract

Shoot meristems used for the study were exercised from the in vitro regenerated shoots cultured on MS medium supplemented with 0.5 mg/L of BAP for multiplication. The sensitivity of the in vitro regenerated was studied using shoot meristems of 0.5 cm. Shoot meristems were cultured on medium containing 10-100 mg/l kanamycin to determine the concentration that was lethal for multiple shoot induction and root induction. The response of shoot multiplication decreased (66.2%-6.2%) as the concentration of kanamycin increased (10.0-70.0 mg/L) with complete inhibition of shoot proliferation at 100 mg/L kanamycin. The rooting phase was very sensitive to kanamycin compared to shoot multiplication. The percentage of shoots that rooted decreased (53.8%-4.8%) with increase in the concentration of kanamycin (10.0-70.0 mg/l) on IBA and 2,4-D supplemented medium. For transformation studies, the shoot tips that were infected with Agrobacterium strain were placed on selection medium containing MS medium with 0.5 mg/L BAP and 100 mg/L kanamycin and scored for the putative transformed shoots. An average of 62.2% of shoot tips developed shoot buds from the base and the shoots reached a length of 0.5-1.0 cm at the end of 30 days of culture on the selective medium in comparison to control which showed no response. An average of 66.7% of the regenerated plants showed GUS expression on selection medium where 43.2% and 65% of GUS expression was recorded in the leaves and callus. Leaves and callus induced from the controls did not show GUS activity. Stable integration of nptII gene with the genomic DNA from these transformed plants was confirmed through PCR analysis. Our result presents an efficient regeneration system using in vitro derived shoot meristems for Agrobacterium mediated gene transfer.