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

Plant Cell Reports
Sunita A RameshMargaret Sedgley

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 muM 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 ...Continue Reading

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Citations

Jun 14, 2006·Plant Cell Reports·Juliana DegenhardtIris Szankowski
May 2, 2008·Plant Cell Reports·César PetriLorenzo Burgos
Aug 13, 2009·Journal of Experimental Botany·Ana Margarida SantosM Margarida Oliveira
Jun 25, 2015·Frontiers in Plant Science·Vincenza Ilardi, Mario Tavazza
Nov 2, 2016·International Journal of Molecular Sciences·Dongyang WangLi Wang
Mar 10, 2017·Journal of Environmental Science and Health. Part. B, Pesticides, Food Contaminants, and Agricultural Wastes·Jitka HilgertJana Žiarovská
Nov 14, 2012·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Jean-David RochaixMichel Goldschmidt-Clermont
May 7, 2016·Royal Society Open Science·Tomás LuqueSanjay Kumar

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