In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro View Full Text


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Article Info

DATE

2008-10

AUTHORS

K. Sankara Rao, Rohini Sreevathsa, Pinakee D. Sharma, E. Keshamma, M. Udaya Kumar

ABSTRACT

Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans' AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22-25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes. More... »

PAGES

321-328

References to SciGraph publications

  • 1993-12. Plant regeneration from leaf discs of peanut and pigeonpea: Influence of benzyladenine, indoleacetic acid and indoleacetic acid-amino acid conjugates in PLANT CELL, TISSUE AND ORGAN CULTURE (PCTOC)
  • 2006-03. Agrobacterium-mediated production of transgenic pigeonpea (Cajanus cajan L. Millsp.) expressing the synthetic BT cry1Ab gene in IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY - PLANT
  • 1996-05. Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens in PLANT CELL REPORTS
  • 1994-04. Organogenesis and embryogenesis from diverse explants in pigeonpea (Cajanus cajan L.) in PLANT CELL REPORTS
  • 2003-03. Expression of hemagglutinin protein of Rinderpest virus in transgenic pigeon pea [Cajanus cajan (L.) Millsp.] plants in PLANT CELL REPORTS
  • 2006-09. Ubiquitous presence of β-glucuronidase (GUS) in plants and its regulation in some model plants in PLANTA
  • 2003-02. In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp] in MOLECULAR BREEDING
  • 1990-01. Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium in PLANT MOLECULAR BIOLOGY
  • 2003-07. An efficient protocol for shoot regeneration and genetic transformation of pigeonpea [Cajanus cajan (L.) Millsp.] using leaf explants in PLANT CELL REPORTS
  • 1994-08. Regeneration of pigeonpea (Cajanus cajan) from cotyledonary node via multiple shoot formation in PLANT CELL REPORTS
  • 1998-06. Plant regeneration in pigeonpea [Cajanus cajan (L.) Millsp.] by organogenesis in PLANT CELL REPORTS
  • 1998-06. Thidiazuron-induced shoot regeneration in pigeonpea (Cajanus cajan L.) in PLANT CELL, TISSUE AND ORGAN CULTURE (PCTOC)
  • 1995-01. Genotype-Dependent morphogenetic potentiality of various explants of a food legume, the pigeon pea (Cajanus cajan L.) in IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY - PLANT
  • 1983-09. A plant DNA minipreparation: Version II in PLANT MOLECULAR BIOLOGY REPORTER
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s12298-008-0030-2

    DOI

    http://dx.doi.org/10.1007/s12298-008-0030-2

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/23572898


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    29 schema:description Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans' AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22-25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes.
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