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Agrobacterium Tumefaciens-mediated Genetic Transformation And Regeneration Of Transgenic Plants From Leaf Explants Of Jatropha Curcas: A Candidate Biodiesel Plant | 12218
ISSN: 2155-952X
Journal of Biotechnology & Biomaterials
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Agrobacterium tumefaciens-mediated genetic transformation and regeneration of transgenic plants from leaf explants of Jatropha curcas: A candidate biodiesel plant
Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple
and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas
using leaf explants. Agrobacterium strain LBA 4404harbouringthe binary vector pCAMBIA 1304 having sense-dehydration
responsive element binding (S-DREB2A), β-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used
for gene transfer. A number of parameters such as preculture of explants, wounding of leaf explants, Agrobacterium growth
phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized
transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf
explants infected with Agrobacterium culture corresponding to OD600=0.6 for 20 min, followed by co-cultivation for 4 days in a cocultivation
medium containing 100 μM acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige
and Skoog (MS) medium supplemented with 2.27 μM thidiazuron (TDZ) for regeneration of shoot buds, followed by selection
on same medium with 5 μg ml-1 hygromycin. Selected shoot buds were transferred to MS medium containing 10 μM kinetin (Kn),
4.5 μM6-benzyl aminopurine (BA), and 5.5 μMα-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were
elongated on MS medium supplemented with 2.25 μM BA and 8.5 μM indole-3-acetic acid (IAA). The elongated shoots were
rooted on half strength MS medium supplemented with 15 μMindole-3-butyric acid (IBA), 5.7 μM IAA, 5.5 μM NAA, and 0.25
mg l-1 activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR
and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was
achieved for leaf explants using this protocol.
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