Successful expression of the synthetic merBps gene in tobacco.

Organomercury is the most toxic biomagnifiable state of mercury, and to date, no natural organomercurial detoxification mechanism is encountered in plants. Bacterial merB gene encoding organomercury lyase show low expression in transgenic plants. For ideal expression, a synthetic merBps gene possessing143 out of 213 codons discrete from native merB gene from Escherichia. coli was fabricated based on codon usage in tobacco. Through Agrobacterium-mediated transformation, the merBps gene got successfully integrated into tobacco. Of several putative merBps transformants selected with 200 µg ml-1 kanamycin, only ∼45% were PCR positive for both nptII and merBps genes. Healthy and vigorously growing shoots of few PCR-positive putative transgenic lines were multiplied and rooted. After transplantation and acclimatization, the resultant plants flowered and fruited in pots. Southern analysis revealed the presence of a single copy of the merBps gene in four lines. RT-PCR and Western investigations established successful transcription and translation of the merBps gene in these transgenic lines, respectively. Fabrication of fully functional organomercury lyase in merBps transgenic lines was established based on the potential of their (i) seeds to germinate; (ii) shoots to grow and multiply; and (iii) leaf disc to remain green, even in the presence of 4 nmole ml-1 phenylmercuryacetate (PMA) while the wild type was susceptible to even 1 nmole ml-1 PMA. These findings confirmed that the synthetic merBps gene could be effectively expressed in plants and exploited for remediation of organomercurial contaminated sites.

Studies on root anatomy, morphology and physiology of rice grown under aerobic and anaerobic conditions.

With the changing climate and rainfall abrasions, there is a gradual shift in the system of rice cultivation from traditional transplanted anaerobic to aerobic system. Studies on the root anatomical and morpho-physiological traits provide insights about the adaptation under aerobic conditions. We investigated the root anatomical and morpho-physiological traits in anaerobic (BPT 5204) and aerobic (CR Dhan 202) adapted rice genotypes grown under anaerobic and aerobic conditions. It was observed that the formation of fewer aerenchyma, thickened root and larger xylem area were critical anatomical traits associated with aerobic adaptation as compared to anaerobic conditions. The root length of CR Dhan 202 significantly increased under aerobic condition which may be attributed to its aerobic adaptation in terms of water acquisition. The photosynthetic rate was significantly higher in CR Dhan 202 as compared to that of BPT 5204 under the aerobic condition. The morpho-physiological results showed that the root length, total dry weight and photosynthetic rate are the key parameters for imparting aerobic adaptation. These root anatomical and morpho-physiological traits associated with the adaptation can be used as screening criteria for phenotyping and selection of genotypes suitable for aerobic system of cultivation. Such study is expected to expedite the development of rice aerobic varieties in aerobic breeding programmes.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera.

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80-100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25-80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Development and characterization of a high temperature stress responsive subtractive cDNA library in Pearl Millet Pennisetum glaucum (L.) R.Br.

Pearl millet (Pennisetum glaucum L. R. Br.) is an important cereal crop grown mainly in the arid and semi-arid regions of India known to possess the natural ability to withstand thermal stress. To elucidate the molecular basis of high temperature response in pearl millet, 12 days old seedlings of P. glaucum cv. 841A were subjected to heat stress at 46 degrees C for different time durations ( 30 min, 2, 4, 8, 12 and 24 h) and a forward subtractive cDNA library was constructed from pooled RNA of heat stressed seedlings. A total of 331 high quality Expressed Sequence Tags (ESTs) were obtained from randomly selected 1050 clones. Sequences were assembled into 103 unique sequences consisting of 37 contigs and 66 singletons. Of these, 92 unique sequences were submitted to NCBI dbEST database. Gene Ontology through RGAP data base and BLASTx analysis revealed that about 18% of the ESTs showed homology to genes for "response to abiotic and biotic stimulus". About 2% of the ESTs showed no homology with genes in dbEST, indicating the presence of uncharacterized candidate genes involved in heat stress response in P. glaucum. Differential expression of selected genes (hsp101 and CRT) from the SSH library were validated by qRT-PCR analysis. The ESTs thus generated are a rich source of heat stress responsive genes, which can be utilized in improving thermotolerance of other food crops.

Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.).

Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. The amplification of required size fragment of 750 bp with npt II primers and 901 bp with cry1AcF gene primers confirmed the integration of the gene. The expression of the cry gene was ascertained by ELISA in T2 generation, and the maximum concentration of cry protein in transgenic plants reached approximately 0.82 µg/g FW. Further, Southern blot analysis of ten T2 transgenic plants proved that transgene had been integrated in the genome of all the plants and Northern analysis of the same plants demonstrated the active expression of cry1AcF gene. The highest mean % larval mortalities 80.0 and 85.0 with an average mean % larval mortalities 16.25 (n = 369) and 26.0 (n = 80) were recorded in T1 and T2 generations, respectively. Segregation analysis of the selected lines in the T3 generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T2 generation, the cry1AcF gene was effective against S. litura only to some extent.

Metabolomics in agriculture.

Metabolome refers to the complete set of metabolites synthesized through a series of multiple enzymatic steps from various biochemical pathways processing the information encrypted in the plant genome. Knowledge about synthesis and regulation of various plant metabolic substances has improved substantially with availability of Omics data originating from sequencing of plant genomes. Metabolic profiling of crops is increasingly becoming popular in assessing plant phenotypes and genetic diversity. Metabolic compositional changes vividly reflect the changes occurring during plant growth, development, and in response to stress. Hence, study of plant metabolic pathways, the interconnections between them in context of systems biology is increasingly becoming popular in identification of candidate genes. The present article reviews recent developments in analysis of plant metabolomics, available bioinformatics techniques and databases employed for comparative pathway analysis, metabolic QTLs, and their application in plants.

Bioinsecticidal activity of Murraya koenigii miraculin-like protein against Helicoverpa armigera and Spodoptera litura.

Miraculin-like proteins, belonging to the Kunitz superfamily, are natural plant defense agents against pests and predators, and therefore are potential biopesticides for incorporation into pest-resistant crops. Here, a miraculin-like protein from Murraya koenigii was assessed for its in vitro and in vivo effects against two polyphagous lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura. M. koenigii miraculin-like protein (MKMLP) inhibited the trypsin-like activity and total protease activity of H. armigera gut proteinases (HGP) by 78.5 and 40%, respectively, and S.litura gut proteinases (SGP) by 81 and 48%, respectively. The inhibitor was stable and actively inhibited the proteolysis of both HGP and SGP enzymes for up to 72 h. Incorporation of MKMLP into artificial diet adversely affected the growth and development of pests in a dose-dependent manner. After 10 days of feeding on diets containing 200 µM MKMLP, larval weight was reduced to 69 and 44.8% and larval mortality was increased to 40 and 43.3% for H. armigera and S litura, respectively. The LC(50) of MKMLP was 0.34 and 0.22% of the diet for H.armigera and S. litura, respectively. These results demonstrate the efficacy of MKMLP as a potential plant defense agent against H. armigera and S. litura.

Cloning of an ovule specific promoter from Arabidopsis thaliana and expression of beta-glucuronidase.

Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.