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 study the possibilities for improvement of the ornamental character and production of secondary metabolites by using Wedelia trilobata hairy roots, we investigated the induction of W. trilobata L. hairy roots and its consumption changes of carbon resource, nitrogen resource, phosphate and calcium in the medium during liquid culture. The results showed that hairy roots could be incited from the cut edges of leaf explants 7 days after inoculation with Agrobacterium rhizogenes ATCC15834 and could have an autonomous growth on the medium without phytohormones. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes was integrated and expressed into the genome of transformed hairy roots. The hairy root line grew very slowly in 0-7 days, very fast from 7 to 21 days. During the liquid culture of hairy roots, sucrose, NO3(-)-N, PO4(3-) and Ca2+ in the medium could be gradually absorbed and utilized with time. The content of NO3(-)-N in the medium was 5.8% of the initial amount at day 7, while sucrose content was about 50% of the initial amount. At day 35, the NO3(-)-N and sucrose content in the medium was 1.82% and 3.39% of the initial amount, respectively. In combination with Ca2+ consumption, PO4(3-) of the medium was rapidly absorbed and utilized. At day 7, the content of PO4(3-) in the spent medium was only 1.76% of the initial amount; but even at day 35, the content of Ca2+ in the spent medium was still 61.3% of the initial amount. The results presented here had provided the possibilities on improvement the ornamental character and how to prepare optimum medium for large scale cultivation and production of secondary metabolites from W. trilobata L. hairy roots.
Culture Techniques ; methods ; Plant Roots ; cytology ; growth & development ; Plants, Genetically Modified ; genetics ; growth & development ; metabolism ; Rhizobium ; genetics ; physiology ; Transformation, Genetic ; Wedelia ; genetics ; growth & development ; microbiology

To study if Solanum nigrum hairy roots can be used for phytoremediation of Cd contamination, we investigated the effects of cadmium (Cd) alone, and in combination with different concentrations of CaCl2, on growth, activities of superoxide dismutase (SOD) and peroxidase (POD) and Cd absorption by hairy roots of S. nigrum L. var pauciflorum. The results showed that Cd concentrations of lower than 50 micromol/L enhanced the growth of hairy roots, while higher than 100 micromol/L inhibited growth and decreased the number of branched roots, also causing the root tips to become brown and shorter in length. In comparison with a control, the soluble protein content, the activities of SOD and POD in hairy roots cultures showed a trend of first increased and then gradually decreased, while the malondialdehyde (MDA) content significantly increased, when increasing the Cd concentrations. Cd concentration of 100 micromol/L or 300 micromol/L in combination with 10-30 mmol/L CaCl2 resulted in a decreased content of soluble protein and MDA in the hairy roots, but an enhanced SOD activity. The increased POD activities were observed when cultured in 100 micromol/L Cd and 10-30 mmol/L CaCl2 but decreased when cultured in 300 micromol/L Cd and 10-30 mmol/L CaCl2. Atomic Absorption Spectrometry determination showed that the Cd absorbed and adsorbed by the hairy roots increased along with the increase of Cd concentration. The exogenous addition of 10-30 mmol/L CaCl2 could reduce the toxicity of Cd. This was achieved on one hand by reducing the absorption of Cd, on the other hand by decreasing the lipid peroxidation through regulating the activities of antioxidant enzymes SOD and POD in the hairy roots.
Absorption ; Biodegradation, Environmental ; Cadmium ; isolation & purification ; metabolism ; Calcium Chloride ; metabolism ; Peroxidase ; metabolism ; Plant Roots ; growth & development ; physiology ; Soil Pollutants ; isolation & purification ; metabolism ; Solanum nigrum ; enzymology ; growth & development ; physiology ; Superoxide Dismutase ; metabolism

In order to study the physiological mechanism of exogenous calcium on the toxicity of heavy metal cadmium (Cd) to Wedelia trilobata hairy roots, the effects of Cd alone, and in combination with different concentrations of Ca on growth, contents of soluble protein and malondialdehyde (MDA), activities of superoxide dismutase (SOD) and peroxidase (POD), Cd2+ absorption in W. trilobata hairy roots were investigated. Cd concentrations lower than 50 micromol/L enhanced the growth of hairy roots, while concentrations higher than 100 micromol/L inhibited growth, making the branched roots short and small, and also turning the root tips brown, even black. In comparison with the control (0 micromol/L Cd), the soluble protein content in hairy roots was found to increase when cultured with 10-50 micromol/L Cd, and decrease when exposed to a cadmium concentration higher than 100 micromol/L Cd. In addition, the activities of POD and SOD activity and MDA content were significantly higher than the control. Compared to the control (hairy roots cultured without 10-30 mmol/L Ca), 100 micromol/L Cd or 300 micromol/L Cd in combination with 10-30 mmol/L Ca resulted in increased growth, causing the main root and secondary roots thicker and also an increase in soluble protein content. On the contrary, MDA content and POD and SOD activities decreased. Quantitative analysis by Atomic Absorption Spectrophotometry showed that W. trilobata hairy roots can absorb and adsorb heavy metal Cd in the ionic form of Cd2+. The maximum content of Cd2+ absorbed by the hairy roots was obtained with a concentration 100 micromol/L Cd2+ while that of Cd2+ adsorbed by hairy roots was achieved with a concentration of 300 micromol/L Cd2+. The exogenous addition of 10-30 mmol/L Ca2+ was found to reduce the absorption, adsorption of Cd2+ and the toxicity of Cd significantly. This reduction in toxicity was caused by the reduction in the absorption of Cd and decreasing the lipid peroxidation through regulating the activities of antioxidant enzymes SOD and POD in the hairy roots.
Absorption ; Adsorption ; Cadmium ; toxicity ; Calcium Chloride ; pharmacology ; Peroxidase ; metabolism ; Plant Roots ; drug effects ; enzymology ; growth & development ; Stress, Physiological ; drug effects ; Superoxide Dismutase ; metabolism ; Wedelia ; drug effects ; enzymology ; growth & development ; metabolism