N Sai | Gitam University,Visakhapatnam,India (original) (raw)

Papers by N Sai

Production of bioethanol from lignocellulosic biomass is gaining much research interest because o... more Production of bioethanol from lignocellulosic biomass is gaining much research interest because of its abundant supply. Saccharomyces cerevisiae is The commonly used industrial yeast for ethanol production but it lacks the property to ferment pentose sugars mainly xylose present in lignocellulosic materials. In this study, the hybrid yeast strains are developed by genome shuffling process between the xylose sugar fermenting yeast Pichia stipitis and hexose sugar fermenting yeast Saccharomyces cerevisiae. Lyticase enzyme is used to establish viable protoplasts from both the parental yeast strains. Factors affecting physic chemical properties viz., protoplast isolation, enzyme concentration and incubation time were investigated. The optimal parameter for the protoplast release of S. cerevisiae and P. stipitis include 700 µg/µl Lyticase for 60 min and 700 µg/µL Lyticase for 120 min respectively. The maximum protoplast formation ratios were 98.75% and 82.24% for S. cerevisiae and P. stipitis respectively. The frequency of the protoplast fused hybrids was carried out by using Polyethylene Glycol (PEG) as fusogen. Fused hybrids were produced by use of 35% (w/v) PEG 4000, optimized conditions of protoplasts fusion of S.cerevisiae and P. stipitis genome shuffling was achieved at a rate of 80.90% with fusion of 20 min. Further, these hybrids produced through genome shuffling will be further evaluated for production of bioethanol using lignocellulosic materials.

Lignocellulosic biomass consist of inedible parts of woody grass plants, stalk of sweet sorghum a... more Lignocellulosic biomass consist of inedible parts of woody grass plants, stalk of sweet sorghum and agricultural residues, are the sources for the 2 nd generation biofuels. Lignocellulosic biomass comprises of many different polysaccharides cellulose, hemicelluloses, phenolic polymer lignin and proteins. The problem of the 2 nd generation feed stock is the extraction of the sugars located inside the lignin and cellulose structure. To convert lignocellulosic biomass to biofuels the complex polysaccharides and lignin must be broken down or hydrolysed into simple sugars. This process of bioconversion of cellulose to ethanol involves pre-treatment, saccharification and fermentation. The other challenges include types of the biomass and their availability round the year, logistics, and production technologies. These challenges take in identification and improvement of energy crops like sweet sorghum, switch grass, miscanthus, alfa alfa, etc., by biotechnological approaches by generating feed stocks with low lignin content and modified traits which can tolerate biotic and abiotic stresses with improved cellulose content. Down regulation of key enzymes involved in lignin biosynthetic pathway may be a promising approach to decrease or alter the hard lignin content in lignocellulosic feed stock materials. With the advent of genetic engineering and crop improvement strategies in energy crops may provide continuous supply of feed stock for the production of biofuels as an alternative source of energy. To benefit the environment and also to meet the global demand of fossil fuels, the biofuels produced from lignocellulosic biomaterial by biotechnological route may help in decreasing the emission of green house gasses and also save our food crops.

Sorghum is the fifth most important cereal crop in the world. It is largely grown on marginal soi... more Sorghum is the fifth most important cereal crop in the world. It is largely grown on marginal soils with residual moisture where other major cereals cannot be grown due to inadequate water. Sorghum is a multipurpose crop and the species shows great diversity. For a large part of Asia and Africa, sorghum's grain is used as food and its stalk as fodder and feed. In rest of the world, sorghum is considered as forage crop and also as stock for ethanol production. Sorghum yield has been substantially increased through conventional breeding in India. However, resistance to abiotic stresses and biotic stresses such as shoot fly, stem borer, grain mold, and charcoal rot is limited due to inadequate genetic resources that can be readily used in crop improvement programs. Therefore, genetic engineering technology can assist the production of agronomically desirable crops that exhibit increased resistance to pests, pathogens, and environmental stress and enhancement of nutritional qualities.

To select agronomically useful transgenic plants, a large number of transgenic events are initial... more To select agronomically useful transgenic plants, a large number of transgenic events are initially produced, gene transfer confirmed, and advanced to obtain homozygous lines for testing in field trials. Direct in planta assays for identifying the transgene carriers in the segregating progeny are based on the activity of selectable marker gene and are easy, simple and inexpensive. For this purpose, expression of bar gene as measured by tolerance to damage by glufosinate ammonium, the active ingredient in the herbicide BASTA, was investigated. Dose damage curves were generated by leaf paint tests with BASTA on four genotypes of sorghum. Transgenic plants were characterized in terms of sensitivity to the concentration of glufosinate ammonium. In transgenics, symptoms of BASTA swab tests at different growth stages and PCR analysis for cry1B were carried out and correlated. Germination tests could not be employed for large scale evaluation of transgenic progeny because of mortality of tolerant seedlings after transplantation to soil. Based on the above findings, a simple, inexpensive, time-saving, twostep scheme for effective evaluation of transgenics and their progeny containing bar gene as selection marker using BASTA swab tests is described.

Transgenic sorghum plants were produced through particle bombardment and Agrobacterium methods in... more Transgenic sorghum plants were produced through particle bombardment and Agrobacterium methods in two elite, but recalcitrant genotypes of Sorghum bicolor L. Moench. Use of target cells from developing tissues (immature embryos and multiple shoot buds), pre-culture of target tissue, small size of target tissue (2-3 mm), and regular subculture improved the selection and regeneration efficiencies. Addition of amino acid L -cysteine during co-cultivation and blotting sheet interface was helpful for complete decontamination of Agrobacterium tumefaciens and regeneration of transgenic plants. We demonstrated production of transgenic sorghum plants expressing a Bacillus thuringiensis lepidopteran toxin, through tailored in vitro protocols. Our results showed that decontamination of agrobacteria employing subtle treatments aided recovery of transgenic plants in recalcitrant genotypes. We generated 14 independent transgenic lines carrying different classes of B. thuringiensis toxin genes, cry1Aa and cry1B. Many single copy events were generated in two elite parental lines, CS3541 and 296B. Accumulation of the B. thuringiensis protein in leaves during the susceptible period of plant growth ranged from 35 to 500 ng/g fresh leaf tissue. Comprehensive insect bioassays for tolerance to spotted stem borer (Chilo partellus ) were conducted through leaf disk and whole plant assays. Transgenic progeny plants showed 20-30% of damage as compared to 70-80% in nontransformed controls.