Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment

The phytopathogenic bacteria Agrobacterium rhizogenes genetically transforms plants by transferring a portion of the resident Ri plasmid, the T-DNA to the plant. Plant species differ in their temporal competence for transformation. But various physical and chemical methods are found to enhance transformation frequency. Agrobacterium rhizogenes mediated transformation efficiency was assessed under the influence of sonication, calcium treatment, acetosyringone and macerating enzymes in suitable combinations in Nicotiana tabacum as a model system. Manual wounding resulted in 21 percent transformation frequency. Where as sonication resulted in 2.2 fold increase, followed by sonication with CaCl2 treatment resulted in 2.5 fold increase and sonication with acetosyringone treatment resulted in 4.1 fold increase in transformation frequency. However, sonication with macerating enzyme treatment resulted in 1.5 to 5.25-fold decrease in transformation frequency. Micro wounding through sonication followed by acetosyringone treatment enhanced transformation frequency substantially. The results of this study may be very useful in genetic manipulation of plants by Agrobacterium rhizogenes mediated gene delivery to higher plants, which are recalcitrant to A. tumefaciens mediated genetic manipulation.

Peroxidase production from hairy root cultures of red beet (Beta vulgaris)

The genetically transformed roots of red beet have been shown, for the first time, to produce very high levels of peroxidase (POD; EC 1.11.1.7) accounting for 1.21 x 10(6) Units L-1. Of the ten clones established using different strains of Agrobacterium rhizogenes, one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods. Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L-1) as well as POD (ranging from 8000-9000 U g-1 FW and 1.18 x 10(6) U L-1 with a specific activity of 600 U mg-1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10(6) U L-1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of Rm 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at Rm 0.06, 0.16, 0.25 and one extra band of Rm 0.575 in the spent medium where isozymes of Rm 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.