Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

Handle URI:
http://hdl.handle.net/10754/577054
Title:
Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants
Authors:
Kumar, Nitish; Vijay Anand, K.G.; Pamidimarri, D.V.N. Sudheer; Sarkar, Tanmoy; Reddy, Muppala P.; Radhakrishnan, T.; Kaul, Tanushri; Reddy, M.K.; Sopori, Sudhir K.
Abstract:
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 explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, 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 OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M 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. (C) 2010 Elsevier B.V. All rights reserved.
KAUST Department:
Center for Desert Agriculture
Publisher:
Elsevier BV
Journal:
Industrial Crops and Products
Issue Date:
Jul-2010
DOI:
10.1016/j.indcrop.2010.03.002
Type:
Article
ISSN:
0926-6690
Sponsors:
The authors gratefully acknowledge the Council of Scientific and Industrial Research, New Delhi, India for financial assistance.
Appears in Collections:
Articles; Center for Desert Agriculture

Full metadata record

DC FieldValue Language
dc.contributor.authorKumar, Nitishen
dc.contributor.authorVijay Anand, K.G.en
dc.contributor.authorPamidimarri, D.V.N. Sudheeren
dc.contributor.authorSarkar, Tanmoyen
dc.contributor.authorReddy, Muppala P.en
dc.contributor.authorRadhakrishnan, T.en
dc.contributor.authorKaul, Tanushrien
dc.contributor.authorReddy, M.K.en
dc.contributor.authorSopori, Sudhir K.en
dc.date.accessioned2015-09-10T09:27:53Zen
dc.date.available2015-09-10T09:27:53Zen
dc.date.issued2010-07en
dc.identifier.issn0926-6690en
dc.identifier.doi10.1016/j.indcrop.2010.03.002en
dc.identifier.urihttp://hdl.handle.net/10754/577054en
dc.description.abstractJatropha 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 explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, 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 OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M 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. (C) 2010 Elsevier B.V. All rights reserved.en
dc.description.sponsorshipThe authors gratefully acknowledge the Council of Scientific and Industrial Research, New Delhi, India for financial assistance.en
dc.publisherElsevier BVen
dc.titleStable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explantsen
dc.typeArticleen
dc.contributor.departmentCenter for Desert Agricultureen
dc.identifier.journalIndustrial Crops and Productsen
dc.contributor.institutionCent Salt & Marine Chem Res Inst, Council Sci & Ind Res, Discipline Wasteland Res, Bhavnagar 364002, Gujarat, Indiaen
dc.contributor.institutionNatl Res Ctr Groundnut, Junagadh, Gujarat, Indiaen
dc.contributor.institutionInt Ctr Genet Engn & Biotechnol, Plant Mol Biol Div, New Delhi 110067, Indiaen
kaust.authorReddy, Muppala P.en
All Items in KAUST are protected by copyright, with all rights reserved, unless otherwise indicated.