Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.)

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - SDS Lauryl sulfate     

Endoplasmic reticulum chaperone protein GRP-78 mediates endocytosis of dentin matrix protein 1

Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant K(D)=85 nm. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development.     

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.     

Advances in development of transgenic plants for remediation of xenobiotic pollutants

Phytoremediation-the use of plants for cleaning up of xenobiotic compounds-has received much attention in the last few years and development of transgenic plants tailored for remediation will further enhance their potential. Although plants have the inherent ability to detoxify some xenobiotic pollutants, they generally lack the catabolic pathway for complete degradation/mineralization of these compounds compared to microorganisms. Hence, transfer of genes involved in xenobiotic degradation from microbes/other eukaryotes to plants will further enhance their potential for remediation of these dangerous groups of compounds. Transgenic plants with enhanced potential for detoxification of xenobiotics such as trichloro ethylene, pentachlorophenol, trinitro toluene, glycerol trinitrate, atrazine, ethylene dibromide, metolachlor and hexahydro-1,3,5-trinitro-1,3,5-triazine are a few successful examples of utilization of transgenic technology. As more genes involved in xenobiotic metabolism in microorganisms/eukaryotes are discovered, it will lead to development of novel transgenic plants with improved potential for degradation of recalcitrant contaminants. Selection of suitable candidate plants, field testing and risk assessment are important considerations to be taken into account while developing transgenic plants for phytoremediation of this group of pollutants. Taking advantage of the advances in biotechnology and 'omic' technologies, development of novel transgenic plants for efficient phytoremediation of xenobiotic pollutants, field testing and commercialization will soon become a reality.     

Photosensitivity in Smith-Lemli-Opitz syndrome: a flux balance analysis of altered metabolism

Ultraviolet A photosensitivity is a debilitating symptom associated with the metabolic disorder Smith-Lemli-Opitz syndrome (SLOS). SLOS is a manifestation of the deficiency of 7-dehydrocholesterol reductase, an enzyme involved in the cholesterol biosynthesis. As a result several abnormal intermediary compounds are formed among which Cholesta 5, 7, 9(11)-trien-3beta-ol is the most likely cause of photosensitivity. The effect of various drugs acting on cholesterol biosynthetic pathway on SLOS is not clear as clinical trials are not available for this rare disorder. A Flux Balance Analysis (FBA) has been carried out using the software CellNetAnalyzer or FluxAnalyzer to gain insight into the probable effects of various drugs acting on cholesterol biosynthetic pathway on photosensitivity in SLOS. The model consisted of 44 metabolites and 40 reactions. The formation flux of Cholesta 5, 7, 9(11)-trien-3beta-ol increased in SLOS and remained unchanged on simulation of the effect of miconazole and SR31747. However zaragozic acid can potentially reduce the flux through the entire pathway. FBA predicts zaragozic acid along with cholesterol supplementation as an effective treatment for photosensitivity in SLOS.     

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.     

Advances in development of transgenic pulse crops

It is three decades since the first transgenic pulse crop has been developed. Todate, genetic transformation has been reported in all the major pulse crops like Vigna species, Cicer arietinum, Cajanus cajan, Phaseolus spp, Lupinus spp, Vicia spp and Pisum sativum, but transgenic pulse crops have not yet been commercially released. Despite the crucial role played by pulse crops in tropical agriculture, transgenic pulse crops have not moved out from laboratories to large farm lands compared to their counterparts - 'cereals' and the closely related leguminous oil crop - 'soybean'. The reason for lack of commercialization of transgenic pulse crops can be attributed to the difficulty in developing transgenics with reproducibility, which in turn is due to lack of competent totipotent cells for transformation, long periods required for developing transgenics and lack of coordinated research efforts by the scientific community and long term funding. With optimization of various factors which influence genetic transformation of pulse crops, it will be possible to develop transgenic plants in this important group of crop species with more precision and reproducibility. A translation of knowledge from information available in genomics and functional genomics in model legumes like Medicago truncatula and Lotus japonicus relating to factors which contribute to enhancing crop yield and ameliorate the negative consequences of biotic and abiotic stress factors may provide novel insights for genetic manipulation to improve the productivity of pulse crops.