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

The effects of heavy metal cadmium (Cd), alone and in combination with zinc (Zn), on the root growth and activity of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD) in Cucumis sativus L. hairy roots were studied. The purpose was to study the possibilities on using C. sativus hairy roots for phytoremediation of cadmium contamination. The results showed that less than 10 mg/L Cd enhanced the growth of C. sativus hairy roots and increased root diameter only in 5-15 days of root culture. At Cd concentrations above 15 mg/L hairy root growth was gradually inhibited with increasing Cd concentration. The roots formed were shorter with smaller lateral roots. Among all the Cd concentrations tested, except with 10 mg/L Cd, the soluble protein contents in the C. sativus hairy roots cultured with the other Cd concentrations decreased, but the POD and SOD activities increased gradually with time during the culture process. Further tests were conducted using a control culture containing 25 mg/L Zn alone. The addition of 1 mg/L Cd to the 25 mg/L Zn culture stimulated the growth of C. sativus hairy roots after 7-15 days of growth, compared with the control. At all other Cd concentrations the growth of C. sativus hairy roots was inhibited compared to the control. Growth inhibition increased with increasing Cd concentration, and the hairy roots formed fewer, shorter and smaller lateral roots, the tips of which became swollen. After 5 days culture with different concentrations of Cd + 25 mg/L Zn, the root biomass and the activity of POD and SOD were lower than in C. sativus hairy roots cultured without the addition of Zn. However, the soluble protein content was significantly higher when the culture contained 25 mg/L Zn. Our results suggested that C. sativus hairy roots have higher tolerance to heavy metal Cd but higher concentration of Cd inhibited the growth. Cd in combination with Zn would result in more serious Cd-induced growth inhibition.
Biodegradation, Environmental ; Cadmium ; pharmacology ; Cucumis sativus ; enzymology ; growth & development ; Peroxidase ; metabolism ; Plant Roots ; enzymology ; growth & development ; Superoxide Dismutase ; metabolism ; Tissue Culture Techniques ; Zinc ; pharmacology

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.