P. S. Srivastava

Plant biotechnology and molecular markers

In Vitro Androgenesis: Events Preceding Its Cytological Manifestation.- Doubled Haploids: A Powerful Biotechnological Tool for Genetic Enhancement in Oilseed Brassicas.- Double Fertilisation in vitro and Transgene Technology.- Polymorphism of Sexual and Somatic Embryos as Manifestation of Their Developmental Parallelism under Natural Conditions and in Tissue Culture.- Molecular Biology and Genetic Engineering of Polyamines in Plants.- Biotechnological Approaches Towards Improvement of Medicinal Plants.- Production of Phytochemicals in Plant Cell Bioreactors.- Development of Biotechnology for Commiphora wightii: A Potent Source of Natural Hypolipidemic and Hypocholesterolemic Drug.- Biotechnology in Quality Improvement of Oilseed Brassicas.- Role of Biotechnology for Incorporating White Rust Resistance in Brassica Species.- Current Trends in Forest Tree Biotechnology.- Cloning Forestry Species.- Micropropagation of Woody Plants.- Biotechnology in Mulberry (Morus spp.) Crop Improvement: Research Directions and Priorities.- Development of High Efficiency Micropropagation Protocol of an Adult Tree-Wrightia tomentosa.- In Vitro Regeneration and Improvement in Tropical Fruit Trees: An Assessment.- Tissue Culture of Cashewnut.- Changing Scenarios in Indian Horticulture.- Cryopreservation: A Potential Tool for Long-term Conservation of Medicinal Plants.- Molecular Mapping and Marker Assisted Selection of Traits for Crop Improvement.- Studies on Male Meiosis in Cultivated and Wild Vigna Species.- Transgenic Crops for Abiotic Stress Tolerance.- Cell Differentiation in Shoot Meristem: A Molecular Perspective.

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.

Comparative transcriptome analysis of contrasting foxtail millet cultivars in response to short-term salinity stress.

Soil salinity represents a major abiotic stress that adversely affects crop growth and productivity. In this study, 21-day-old seedlings of two foxtail millet (Setaria italica) cultivars differing in salt tolerance were found to also differ in lipid peroxidation, ion balance and activity of antioxidative enzymes (glutathione reductase and catalase) under short-term salinity stress (250 mM NaCl for 1-48 h). With the aim of better understanding the molecular mechanisms underlying plant responses to short-term salinity stress, two suppression subtractive hybridization cDNA libraries (forward and reverse) were constructed of these cultivars. A total of 249 non-redundant ESTs was identified by random EST sequencing and grouped into 11 functional categories. Macroarray analysis of these clones showed that 159 (63.9%) were differentially expressed (≥ 2-fold) in response to salinity stress, with 115 (72.3%) up and 44 (27.7%) down-regulated. A data search of transcriptional profiling under salinity stress in other species revealed that 81 (51%) of the 159 differentially expressed transcripts found in foxtail millet have not been reported in previous studies. Hence, these new transcripts may represent untapped gene sources allowing specific responses to short-term salt-stress in an orphan crop known to possess a natural adaptation capacity to abiotic stress. Quantitative real-time PCR of 21 highly up-regulated (≥ 2.5-fold) transcripts showed temporal variation in expression in both cultivars under salinity. Among them, several transcription factors and signalling genes were preferentially expressed in the tolerant cultivar. These results suggest that the tolerant cultivar possesses more effective signal-perception mechanisms for metabolic adjustments in plants under harsh saline conditions. Our findings provide evidence that the unknown genes identified in this study, in addition to several known genes, may play important roles in stress tolerance mechanisms present in foxtail millet.

Molecular, phenotypic and biosynthetic stability in Dioscorea floribunda plants derived from cryopreserved shoot tips

Abstract Shoot tips of Dioscorea floribunda , a medicinal species of yam, were cryopreserved using the vitrification technique, resulting in 87% survival and 30% plant regeneration. Genetic stability of plants derived from cryopreserved shoot tips was evaluated using molecular, morphological and biochemical methods. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 64 clear reproducible bands, with the amplification products being monomorphic for all the plantlets tested except 1. A total of 5120 bands obtained from this study exhibited no aberration in RAPD banding except 1 being polymorphic. The morphological analysis of plants, after regrowing in green house, revealed no significant difference between cryopreserved-derived and control plants. Eigtheen important morphological characters (based on descriptor list) were examined including length/breath ratio of leaf, number of primary stems, petiole length, internodal length and lamina/petiole ratio. The diosgenin contents of cryopreserved-derived plants, analyzed using high performance liquid chromatography, were found to be same as those of control plants. Thus, with the experimental conditions tested, the D. floribunda plants derived after cryopreservation were found to be genetically stable at the molecular, phenotypic and biosynthetic levels.

Characterization of stress and methylglyoxal inducible triose phosphate isomerase (OscTPI) from rice

As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min−1mg−1 which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG.

Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli.

BackgroundE. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products.ResultsThe availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield.ConclusionsThe E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.

Molecular markers in medicinal plant biotechnology: past and present

Plant based medicines have gained popularity worldwide due to their almost negligible side effects. In India, the three traditional medicinal systems, namely homeopathy, Ayurveda and Siddha rely heavily on plants for medicinal formulations. To prevent the indiscriminate collection of these valuable medicinal plants and for their proper authentication and conservation, it is imperative to go for sustained efforts towards proper germplasm cataloguing and devising conservation strategies. For this purpose, molecular markers have a significant role, as they provide information ranging from diversity at nucleotide level (single nucleotide polymorphisms) to gene and allele frequencies (genotype information), the extent and distribution of genetic diversity, and population structure. Over the past twenty years, the molecular marker field has completely transformed the meaning of conservation genetics which has emerged from a theory-based field of population biology to a full-fledged pragmatic discipline. In this review, we have explored the transition and transformation of molecular marker technologies throughout these years.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

A reliable protocol for transformation of Catharanthus roseus through Agrobacterium tumefaciens.

Proliferation of axillary shoot buds and multiple shoot formation in Catharanthus roseus was obtained in 96 % explants on MS medium (3 % sucrose) containing NAA + BA. 2,4-D induced callusing in both, the nodal as well as in leaf segments. Leaf-derived callus was used for transformation with Agrobacterium tumefaciens LBA4404/pBI-S1. Bacterial cell concentration, duration of co-cultivation and acetosyringone concentration influenced transformation efficiency. Under optimal co-cultivation conditions, 98 % of the explants showed GUS expression. PCR based amplification of the transformed and subsequently selected callus tissue indicated the presence of uidA, Gly I and nptII genes.

Physical mapping, expression analysis and polymorphism survey of resistance gene analogues on chromosome 11 of rice

Rice is the first cereal genome with a finished sequence and a model crop that has important syntenic relationships with other cereal species. The objectives of our study were to identify resistance gene analogue (RGA) sequences from chromosome 11 of rice, understand their expression in other cereals and dicots by in silico analysis, determine their presence on other rice chromosomes, and evaluate the extent of polymorphism and actual expression in a set of rice genotypes. A total of 195 RGAs were predicted and physically localised. Of these, 91.79% expressed in rice, and 51.28% expressed in wheat, which was the highest among other cereals. Among monocots, sugarcane showed the highest (78.92%) expression, while among dicots, RGAs were maximally expressed in Arabidopsis (11.79%). Interestingly, two of the chromosome 11-specific RGAs were found to be expressing in all the organisms studied. Eighty RGAs of chromosome 11 had significant homology with chromosome 12, which was the maximum among all the rice chromosomes. Thirty-one per cent of the RGAs used in polymerase chain reaction (PCR) amplification showed polymorphism in a set of rice genotypes. Actual gene expression analysis revealed post-inoculation induction of one RGA in the rice line IRBB-4 carrying the bacterial blight resistance gene Xa-4. Our results have implications for the development of sequence-based markers and functional validation of specific RGAs in rice.