Agrobacterium pRi TL-DNA rolB and TR-DNA Opine Genes Transferred to the Spiny Amaranth (Amaranthus spinosus L.), A Nutraceutical Crop

Ajantaa Pal^1,2, Swasti S. Swain^1ψ, Arup K. Mukherjee^2# and Pradeep K. Chand<sup>1∗

1</sup>Plant Cell and Tissue Culture Facility, Post-Graduate Department of Botany, Utkal University, Bhubaneswar-751004, Orissa, India
^ψPresent address: Department of Botany, Ravenshaw University, Cuttack-753003, Orissa, India
²DNA Fingerprinting Laboratory, Division of Plant Biotechnology, Regional Plant Resource Centre, Bhubaneswar-751015, Orissa, India
^#Present address: Division of Crop Protection, Central Institute for Cotton Research, Shankarnagar, Nagpur-440010, India

Article history:
Received June 30, 2011
Accepted February 3, 2012

Key words:
Amaranthus spinosus L., Agrobacterium rhizogenes, rhizoclones, PCR amplification, opine assay

Summary:                                                                                                                                                                                 
In vitro rhizogenesis occurred with a characteristic pattern typical of transformed roots following explant (internode/leaf) inoculation of Amaranthus spinosus L. with four different wild type Agrobacterium rhizogenes strains. The extent of rhizogenesis varied considerably with the explant type and source, and with the Agrobacterium strains employed; internodal segments performed better than leaves. Of the strains employed for cocultivation, A. rhizogenes LBA 9402 carrying pRi 1855 was the most virulent and infectious, causing hairy root induction in the maximum number of explants regardless of their type. Individual root clones (rhizoclones) were maintained on Murashige and Skoog's basal medium without growth regulators. The physical presence of the rolB gene in the TL-DNA segment of the Ri plasmid of the infecting Agrobacterium in leaf tissues of plants regenerated from selected rhizoclones was confirmed by a positive PCR amplification. The ability of the genetically transformed plants to harbour and express TR-DNA specific opine synthase genes (man2 and ags) was substantiated by PCR and opine assay respectively, demonstrating the production of characteristic opines. Such findings are implicated in the context of pharmaceutical exploitation of transformed root cultures of A. spinosus and also towards protecting this nutraceutically important crop, amaranth, against biotic stress challenges via transgenic manipulations.   

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