Biologia plantarum 46:519-527, 2003 | DOI: 10.1023/A:1024803325682

Plant Regeneration from Decapitated Mature Embryo Axis and Agrobacterium Mediated Genetic Transformation of Pigeonpea

M.L. Mohan1, K.V. Krishnamurthy1
1 Plant Tissue Culture Division, National Chemical Laboratory, Pune-, India

A reliable method of plant regeneration has been achieved from decapitated mature embryo axes (DCMEA) explants. Shoots appear directly from explants of genotype T-15-15 when cultured on Maheswaran and Williams (EC6) basal medium supplemented with N6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) at various combinations. The shoots elongated on half strength Murashige and Skoog (MS) medium fortified with 3 μM gibberellic acid. Elongated shoots were rooted with 80 - 85 % efficiency on half strength MS medium with 0.5 μM indole-3-butyric acid. Survival of plants in the pots was 75 - 80 %. This protocol was used in Agrobacterium mediated transformation. The DCMEA explants were treated independently with two A. tumefaciens (LBA 4404) strains harbouring a binary vector carrying the green fluorescent protein (GFP) and β-glucuronidase (GUS) reporter genes, respectively. Both the strains contained neomycin phosphotransferase selectable marker gene. After co-cultivation, the explants were cultured on EC6 basal medium supplemented with 5 μM BAP and 1 μM IAA. The selection of putative transformants was on a medium containing 50 mg dm-3 kanamycin. Expression of GUS and GFP gene was confirmed by histochemical assay and fluorescence microscopy, respectively. The elongated shoots expressing GFP reporter gene were rooted and transferred to pots for hardening. The integration of GFP gene into the genome of putative transformants was confirmed by Southern blotting.

Keywords: Cajanus cajan; EC6 medium; β-glucuronidase gene; green fluorescent protein
Subjects: auxins; Cajanus cajan; cytokinins; embryo axis, plant regeneration, β-glucuronidase gene; β-glucuronidase gene; green fluorescent protein, plant regeneration; in vitro culture, nutrient media modification; in vitro culture, plant regeneration, organogenesis; pigeonpea, plant regeneration, growth regulators

Prepublished online: May 1, 2003; Published: December 1, 2003Show citation

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Mohan, M.L., & Krishnamurthy, K.V. (2003). Plant Regeneration from Decapitated Mature Embryo Axis and Agrobacterium Mediated Genetic Transformation of Pigeonpea. Biologia plantarum, 46(4), 519-527. doi: 10.1023/A:1024803325682.
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