Improved methods in 'Agrobacterium'–mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols

Abstract

A protocol for 'Agrobacterium'-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar 'Prunus dulcis' cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 μM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/'pmi' selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a 'pmi' DNA probe. The positive (mannose/'pmi') and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/'pmi' selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/'npt'II selection protocols.