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.
Agrobacterium ; Plant Roots ; growth & development ; Plants, Genetically Modified ; growth & development ; Polyploidy ; Regeneration ; Tobacco ; genetics ; growth & development ; Transformation, Genetic

Abstract: In order to enhance the content of secondary metabolites patchouli alcohol in Pogostemon cablin, we induced polyploid hairy roots and their plant regeneration, and determined the content of patchouli alcohol through artificial chromosome doubling with colchicine. The highest rate of polyploidy induction was more than 40% when hairy roots were treated with 0.05% colchicine for 36 h. The obtained polyploid hairy roots formed adventitious shoots when cultured in an MS medium with 6-BA 0.2 mg/L and NAA 0.1 mg/L for 60 d. Compared with the control diploid plants, the polyploid hairy root-regenerated plants of P. cablin had more developed root systems, thicker stems, shorter internodes and longer, wider and thicker leaves. Observation of the chromosome number in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 128 (4n = 128) chromosomes. The leaves contained around twice as many stomatal guard cells and chloroplasts as the controls, but the stomatal density declined with increasing ploidy. The stomatal density in diploid plants was around 1.67 times of that in polyploid plants. GC-MS analysis shows that the content of patchouli alcholol in the hairy root-derived polyploid plants was about 4.25 mg/g dry weight, which was 2.3 times of that in diploid plants. The present study demonstrates that polyploidization of hairy roots can stimulate the content of patchouli alcholol in medicinal plant of P. cablin.
Colchicine ; Diploidy ; Lamiaceae ; genetics ; growth & development ; Plant Roots ; growth & development ; Plants, Medicinal ; genetics ; growth & development ; Polyploidy ; Regeneration ; Sesquiterpenes ; chemistry

To use hairy roots for producing medicinal ingredients of Phytolacca americana L. we studied the factors influencing the induction and in vitro culture. Hairy roots could be incited from the veins of cut surface (morphological lower) of P. americana L. leaf explants around 18 days after infection with the strain of Agrobacterium rhizogenes ATCC15834. The highest rooting rate, 70%, was obtained when leaf explants were pre-cultured for 1 day, infected for 20 min, and co-cultured for 4 days. The transformation was confirmed by PCR amplification of rolC of Ri plasmid and silica gel thin-layer chromatography of opines from P. americana L. hairy roots. All the hairy root lines could grow rapidly on solid exogenous phytohormone-free MS medium. Among the 9 hairy root lines, the hairy root line 2 had most rapid growth, most branched lateral roots and most intensive root hair; the root surface of some hairy root lines seemed purple or red, while that of the other hairy root line appeared white. Among liquid media MS, 1/2MS, B5 and 6,7-V tested, the best growth for hairy root lines was attained in liquid exogenous phytohormone-free MS medium. Compared with exogenous phytohormone-free MS medium, 6,7-V medium was better for synthesis and accumulation of esculento side A in hairy roots. The established optimal conditions for induction and in vitro culture of P. americana hairy roots had laid an experimental and technological foundation for production of medicinal constituents esculento side A from large scale culture of hairy roots.