Abstract
Genetic transformation of guava (Psidium guajava L.) was developed for the first time using in vitro grown shoot tip explant co-cultivated with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pIIHR-JBMch with endochitinase and nptII genes. The highest transformation efficiency was achieved by wounding explants with tungsten particles (0.5 µm) through particle acceleration system, followed by infection for 45 minutes with A. tumefaciens, grown overnight with 100 µM acetosyringone, corresponding to OD600=0.5 followed by co-cultivation for 72 hours under dark condition on co-cultivation medium (MS+100 µM acetosyringone+100 mg L-1 L-Cystein). Putative transformed explants regenerated shoots on selection medium stressed with 200 mg L-1 kanamycin for 12 weeks. Molecular analysis of putative transformants by PCR confirmed the integration of endochitinase and nptII gene in the plant nuclear genome.
Highlights
Guava (Psidium guajava L.), an important fruit crop belonging to family Myrtaceae, is cultivated in many tropical and subtropical countries of the world
Genetic transformation of guava (Psidium guajava L.) was developed for the first time using in vitro grown shoot tip explant cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pIIHR-JBMch with endochitinase and nptII genes
The highest transformation efficiency was achieved by wounding explants with tungsten particles (0.5 μm) through particle acceleration system, followed by infection for 45 minutes with A. tumefaciens, grown overnight with 100 μM acetosyringone, corresponding to OD600=0.5 followed by co-cultivation for 72 hours under dark condition on co-cultivation medium (MS+100 μM acetosyringone+100 mg L-1 L-Cystein)
Summary
Guava (Psidium guajava L.), an important fruit crop belonging to family Myrtaceae, is cultivated in many tropical and subtropical countries of the world. Many products are based on the juice and pulp of the fruit. Production of guava has been affected worldwide by the wilt disease. This soil borne disease was first reported by Das Gupta and Rai (1947). Alternate approach to control the wilt disease is to develop a transgenic guava plant expressing the endochitinase gene isolated from Trichoderma harzianum (Saiprasad et al 2009). The technique of expressing the endochitinase gene in the plant system to confer resistance against fungal diseases has been successfully demonstrated in apple (Bolar et al 2000), cotton (Emami et al 2003, Cheng et al 2005), broccoli (Mora and Earle 2001), lemon (Gentile et al 2007) and rice (Lu et al 2004, Shah et al 2009). In this study we describe the development of an efficient transformation system from in vitro grown shoot tips of guava
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