OPTIMIZATION OF AGKOBACTERIUM-MEDIATED TRANSFORMATION METHOD IN SUGARCANE CALLUS

摘要

The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of sugarcane (Saccharum spp. Complex). Embryogenic calli, produced from sugarcane spindles of CP88-1198 were used as target cells for Agrobacterium-mediated transformation. Agrobacterium strain GV3101 harboring vector, pWBVec 10 carrying the hygromycin gene and the GUS (uid A) gene fragment under the control of Ubiquitin promoter, was used for co-cultivation with embryogenic calli. Agrobacterium infection ofcallifor 10 minutes with brief vacuum infiltration, followed by co-cultivation for 48 hrs was found to be optimum for maximum transformation efficiency. During co-cultivation, calli were placed on filter paper discs to avoid direct contact with medium and it helped to check the bacterial overgrowth during co-cultivation. Transient GUS (β-glucronidase) gene expression was used to monitor T-DNA delivery into the target cells. A high frequency of GUS gene expression was obtained following Agrobacterium mediated gene transfer into embryogenic calli and transformation was confirmed through PCR amplification. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of sugarcane with genes of agronomic importance.