Agrobacterium-mediated genetic transformation of Nagpur mandarin (Citrus reticulata Blanco).

摘要

Genetic transformation protocol was developed for Nagpur mandarin (Citrus reticulata Blanco), a choicest citrus variety grown in India and South East Asia. Cotyledon segments from mature seeds were co-cultivated with Agrobacterium tumefaciens for two days and cultured on an adventitive embryo induction medium containing MS basal medium supplemented with 8.87 micro M BAP+2.32 micro M kinetin+5.37 micro M NAA with 60 micro g/ml kanamycin and 300 micro g/ml cefotaxime. The putatively transformed adventitive embryos that regenerated on the cotyledon segment were transferred for complete plantlet development on auxin-rich medium having MS basal salts fortified with 9.80 micro M IBA and 100 mg 1-1 activated charcoal. The transgenic plants were then hardened in glass jars filled with soilrite and perlite (1:1). GUS activity was detected in putatively transformed plants and maximum positive GUS assay frequency 34.5% was noted. Genetic transformation was confirmed by PCR analysis and Southern hybridization with alpha -32P-labelled riboprobes of the gus gene..