Evaluation of transgenic tobacco plants expressing a bacterial Co-Ni transporter for acquisition of cobalt.

Phytoremediation is a viable strategy for management of toxic wastes in a large area/volume with low concentrations of toxic elemental pollutants. With increased industrial use of cobalt and its alloys, it has become a major metal contaminant in soils and water bodies surrounding these industries and mining sites with adverse effects on the biota. A bacterial Co-Ni permease was cloned from Rhodopseudomonas palustris and introduced into Nicotiana tabacum to explore its potential for phytoremediation and was found to be specific for cobalt and nickel. The transgenic plants accumulated more cobalt and nickel as compared to control, whereas no significant difference in accumulation of other divalent ions was observed. The transgenic plants were evaluated for cobalt content and showed increased acquisition of cobalt (up to 5 times) as compared to control. The plants were also assessed for accumulation of nickel and found to accumulate up to 2 times more nickel than control. At the same initial concentration of cobalt and nickel, transgenic plant preferentially accumulated cobalt as compared to nickel. The present study is perhaps the first attempt to develop transgenic plants expressing heterologous Co transporter with an improved capacity to uptake cobalt.

Production of camptothecin by hairy roots and regenerated transformed shoots of Ophiorrhiza rugosa var. decumbens.

Camptothecin (CPT), the derivatives of which are used clinically for the treatment of metastatic colon cancer, is isolated from intact plants that can be subjected to environmental fluctuations. In vitro cultures may be an alternate and continuous source for year-round production of CPT. Since CPT production by undifferentiated cell cultures is low, differentiated tissues such as root cultures may be a viable alternate source for CPT production. Hairy roots were induced in Ophiorriza rugosa, a source of CPT, using Agrobacterium rhizogenes strain LBA9402. The hairy roots, when cultured in light, showed spontaneous regeneration of shoots. Analysis of CPT levels in the hairy roots and in vitro-grown transformed shoots revealed 0.009% d.w. and 0.012% d.w., respectively.

Transgenic Nicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper.

The diets of two-thirds of the world's population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T(0) and T(1)) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd(2+) and Zn(2+) did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper.

Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene.

Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of (14)C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T(0) and T(1)) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

Expression of a human cytochrome P4502E1 in Nicotiana tabacum enhances tolerance and remediation of γ-hexachlorocyclohexane.

Lindane (γ-hexachlorocyclohexane), a persistent organo-chlorine insecticide widely used in developing countries, has a negative effect as a polluting agent of soil and surface waters. Plants can be used for remediation of organic pollutants and their efficiency can be enhanced by introduction of heterologous genes. Mammalian cytochrome P4502E1 (CYP2E1), an important monooxygenase is involved in the degradation of a wide range of xenobiotics including environmental pollutants/herbicides and pesticides. Here, we report the development of transgenic tobacco plants expressing human CYP2E1 and the efficacy of plants for remediation of lindane. Transgenic tobacco plants with CYP2E1 showed enhanced tolerance to lindane when grown in hydroponic medium and soil compared to control plants. Remediation of (14)C-labeled lindane from hydroponic medium was higher in transgenic plants compared to that of control plants, with the best performing line showing 25% higher removal of lindane from solution than control plants. Similar results were seen in plants grown in soil spiked with lindane. The present study has shown that transgenic plants expressing CYP2E1 gene have potential use for remediation of lindane from contaminated solutions and soil.

Glutathione transferase from Trichoderma virens enhances cadmium tolerance without enhancing its accumulation in transgenic Nicotiana tabacum.

BACKGROUND: Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. RESULTS: Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. CONCLUSION: The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and in limiting Cd availability in the food chain.

Expression of a Neurospora crassa zinc transporter gene in transgenic Nicotiana tabacum enhances plant zinc accumulation without co-transport of cadmium.

Zinc (Zn) is an essential micronutrient required for growth and development of all organisms. Deficiency of Zn in humans is widespread, affecting 25% of world population and efforts are underway to develop crop plants with high levels of Zn in their edible parts. When strategies for enhancing Zn in crop plants are designed, it is essential to exclude cadmium (Cd), a toxic analogue of Zn. In the present work, a high affinity and high specificity zinc transporter gene (tzn1) from Neurospora crassa was cloned and introduced into Nicotiana tabacum with the objective of enhancing the potential of plants for zinc acquisition. When grown in hydroponic medium spiked with (65) Zn, transgenic plants showed enhanced accumulation of Zn (up to 11 times) compared to control plants, which was confirmed further by environmental scanning electron microscopy coupled with Energy Dispersive X-ray analysis. More importantly, no significant difference in uptake of Cd(2+), Fe(2+), Ni(2+), Cu(2+), Mn(2+) and Pb(2+) between the transgenic and control plants was observed. The present studies have shown that Neurospora crassa tzn1 is a potential candidate gene for developing transgenic plants for improving Zn uptake, without co-transport of Cd and may have implications in Zn phytofortification and phytoremediation.

Expression of a fungal endochitinase gene in transgenic tomato and tobacco results in enhanced tolerance to fungal pathogens.

Development of transgenic Nicotiana tabacum and Lycopersicon esculentum expressing an endochitinase (ech42) gene from biocontrol fungus Trichoderma virens using Agrobacterium-mediated genetic transformation is reported in this paper. Integration of transgene in the genome of transgenic plants was demonstrated using polymerase chain reaction and Southern-blot hybridization, while expression was ascertained by reverse transcription polymerase chain reaction. Histochemical analysis confirmed the expression of GUS enzyme in transformed shoots. Levels of endochitinase enzyme in transgenic plants were found to be up to 10 fold higher compared to control plants. Endochitinase enzyme of 42 kDa was also visualized on SDS-PAGE gel using fluorimetric zymogram in transgenic plants. Endochitinase activity was found to be higher in leaf and stem than the root tissue in transgenic tomato plants. Transgenic lines of both plants showed enhanced resistance to fungal pathogens and a strong negative correlation was found between expression level of endochitinase enzyme and size of disease lesions. Inheritance of transgene, expression and resistance to fungal pathogens of T1 transgenic tobacco lines was also analysed. The results of the present studies show that ech42 is a promising candidate gene for developing fungal disease resistance in tomato plants.

Cadmium accumulation and its influence on lipid peroxidation and antioxidative system in an aquatic plant, Bacopa monnieri L.

Bacopa monnieri L. plants exposed to 10, 50, 100 and 200 microM cadmium (Cd) for 48, 96 and 144 h were analysed with reference to the accumulation of metal and its influence on various enzymatic and non-enzymatic antioxidants, thiobarbituric acid reactive substances (TBARS), photosynthetic pigments and protein content. The accumulation of Cd was found to be increased in a concentration and duration dependent manner with more Cd being accumulated in the root. TBARS content of the treated roots and leaves increased with increase in Cd concentration and exposure periods, indicating the occurrence of oxidative stress. Induction in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and guiacol peroxidase (GPX) was recorded in metal treated roots and leaves of B. monnieri. In contrast, a significant reduction in catalase activity in Cd treated B. monnieri was observed. An increase was also noted in the levels of cysteine and non-protein thiol contents of the roots of B. monnieri followed by a decline. However, in leaves, cysteine and non-protein thiol contents were found to be enhanced at all the Cd concentrations and exposure periods. A significant reduction in the level of ascorbic acid was observed in a concentration and duration dependent manner. The total chlorophyll and protein content of B. monnieri decreased with increase in Cd concentration at all the exposure periods. Results suggest that toxic concentrations of Cd caused oxidative damage as evidenced by increased lipid peroxidation and decreased chlorophyll and protein contents. However, B. monnieri is able to combat metal induced oxidative injury involving a mechanism of activation of various enzymatic and non-enzymatic antioxidants.