Plant regeneration from decapitated mature embryo axis and Agrobacterium mediated genetic transformation of pigeonpea

• Published in: Biologia plantarum Vol. 46; no. 4; pp. 519 - 527
• Main Authors: Mohan, M.L, Krishnamurthy, K.V. (National Chemical Lab., Pune (India). Plant Tissue Culture Div.)
• Format: Journal Article
• Language: English
• Published: 01.12.2003
• Subjects: AGROBACTERIUM TUMEFACIENS; AIA; ARTIFICIAL REGENERATION; BROTES; CAJANUS CAJAN; CAL; CALLO; CALLUS; CULTIVO IN VITRO; CULTURE IN VITRO; CULTURE MEDIA; DOSAGE; DOSE; DOSIFICACIÓN; EXPLANT; EXPLANTES; EXPLANTS; FLUORESCENCE; FLUORESCENCIA; GENES; GENETIC TRANSFORMATION; GUISANTE DE ANGOLA; GÈNE; IAA; IN VITRO CULTURE; MEDIO DE CULTIVO; MILIEU DE CULTURE; PIGEON PEAS; POIS PIGEON; POUSSE; PROTEINS; PROTEÍNAS; PROTÉINE; REGENERACIÓN ARTIFICIAL; RÉGÉNÉRATION ARTIFICIELLE; SHOOTS; SUPERVIVENCIA; SURVIE; SURVIVAL; TRANSFORMACIÓN GENÉTICA; TRANSFORMATION GÉNÉTIQUE
• ISSN: 0006-3134; 1573-8264
• DOI: 10.1023/a:1024803325682

The aim of the investigation was to find an efficient method of direct organogenesis from decapitated mature embryo axes (DCMEA) explants and its suitability for genetic transformation studies of pigeonpea using GUS and GFP as reporter genes and nptll gene as selectable marker. 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 microM gibberellic acid. Elongated shoots were rooted with 80-85 % efficiency on half strength MS medium with 0.5 microM indole-3-butyric acid. The DCMEA explants were treated independently with two Agrobacterium tumefaciens strains harbouring a binary vector carrying the green fluorescent protein (GFP) and beta-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 microM BAP and 1 microM IAA. 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.