TITLE:
Efficient Regeneration System for Genetic Transformation of Mulberry (Morus indica L. Cultivar S-36) Using in Vitro Derived Shoot Meristems
AUTHORS:
D. S. Vijaya Chitra, Bhaskarrao Chinthapalli, G. Padmaja
KEYWORDS:
Morus indica L. Cultivar S-36; In Vitro Regeneration; Shoot Meristems; Kanamycin; Genetic Transformation
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.5 No.1,
January
3,
2014
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.