Induction of polyploid in hairy roots of Nicotiana tabacum and its plant regeneration

Abstract:

By genetic transformation with Agrobacterum rhizogenes and artificial chromosome doubling techniques, we studied the induction of hairy roots and their polyploidization, and subsequent plant regeneration and nicotine determination to enhance the content of nicotine in Nicotiana tabacum. The results show that hairy roots could be induced from the basal surface of leaf explants of N. tabacum 8 days after inoculation with Agrobacterium rhizogenes ATCC15834. The percentage of the rooting leaf explants was 100% 15 days after inoculation. The hairy roots could grow rapidly and autonomously on solid or liquid phytohormones-free MS medium.The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and paper electrophoresis of opines from N. tabacum hairy roots. The highest rate of polyploidy induction, more than 64.71%, was obtained after treatment of hairy roots with 0.1% colchicine for 36 h. The optimum medium for plant regeneration from polyploid hairy roots was MS+2.0 mg/L 6-BA +0.2 mg/L NAA. Compared with the control diploid plants, the hairy roots-regenerated plants had weak apical dominance, more axillary buds and more narrow leaves; whereas the polyploid hairy root-regenerated plants had thicker stems, shorter internodes and the colour, width and thickness of leaves were significantly higher than that of the control. Observation of the number of chromosomes in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 96 (4n=96) chromosomes. Pot-grown experiments showed compared to the control, the flowering was delayed by 21 days in diploid hairy roots-regenerated plants and polyploid hairy root-regenerated plants. GC-MS detection shows that the content of nicotine in polyploid plants was about 6.90 and 4.57 times the control and the diploid hairy roots-regenerated plants, respectively.