Prashant Govindrao Kawar

Identification of Water Deficit Stress Upregulated Genes in Sugarcane

Sugarcane represents an important renewable source among biofuel crops with high capability to assimilate carbon among the C4 plants. Limited availability of freshwater renders this crop uneconomical, warranting the necessity for the development of varieties with higher water use efficiency and tolerant to water deficiency stress. Sugarcane variety cv. Co740 was subjected to varied levels of water deficiency stress to isolate transcripts differentially expressed to the imposed stress. The leaf relative water content was used as a measure to estimate the stress response. PCR-based cDNA suppression subtractive hybridization technique was applied to construct forward subtracted library for differentially expressed genes under stress. Dot blot-selected 158 clones showing elevated response were sequenced, of which 62% resembled similarity with known functional genes, 12% with hypothetical proteins of plant origin, while 26% represented new unknown sequences. Annotation of these differentially expressed sequence tags (ESTs) in the moderately water deficit stress-tolerant cultivar predicted that most of them encoded proteins involved in cellular organization, protein metabolism, signal transduction, and transcription. Further, semi-quantitative reverse transcriptase PCR carried out for five genes projected the involvement of these ESTs in stress alleviation/tolerance. Results from this study may help in targeting useful genes for improving drought tolerance in sugarcane and other grasses.

Biochemical characterization and identification of differentially expressed candidate genes in salt stressed sugarcane

Sugarcane productivity is worldwide subjected to increasing environmental constraints, predominantly to drought and salinity owing to their high magnitude of impact and wide distribution. The present study provides insights into the knowledge of sugarcane responses at germination, tillering and respective recovery stages to high salinity at physio-biochemical and molecular level. Our results indicated that there was a negative relationship between catalase, and peroxidase activity with lipid peroxidation and SOD activity. Increase in MDA and SOD levels at the earlier stages of stress and later increase in CAT and POD levels on prolonged stress was evidenced. Thus they can be used as indicators of stress for sugarcane plants facing unfavourable environmental conditions. At molecular level, we have identified 137 salinity tolerant candidate cDNAs from sugarcane by cDNA-SSH, representing 20% of which are novel sugarcane genes. These unique sequences, never reported elsewhere to be stress related and might provide further understanding on perception, response and adaptations mechanisms of the non-model plant like sugarcane to salinity stress and will be potential candidates for manipulating salt tolerant trait.

Physio-biochemical analysis and transcript profiling of Saccharum officinarum L. submitted to salt stress

In an attempt to understand the molecular basis of salt-stress response in sugarcane, physio-biochemical assays and cDNA-RAPD-based gene expression studies under high salt (2% NaCl) supply regimes were initiated. The comparative rates of total protein, proline content and lipid peroxidation were found steadily increased, while total chlorophyll content was decreased in leaves of salt-treated over untreated sugarcane plants at corresponding increase in soil electrical conductivity. The comparative transcript responses to salt stress were monitored by ribotyping of both treated and untreated sugarcane plants at early growth stage. Among 335 differentially expressed transcript-derived fragments, 156 up- and 85 down-regulated were reamplified and sequenced. They were functionally categorized as metabolism, DNA/RNA/cellular processes, signal transduction/cell rescue/defense, cell wall modifications, transcriptional regulation, transport/trafficking, retroelements and unknown/hypothetical proteins.

Identification and Isolation of SCGS Phytoplasma-specific Fragments by Riboprofiling and Development of Specific Diagnostic Tool

Sugarcane Grassy Shoot (SCGS) disease caused by phytoplasma, one of the major constraints in sugarcane cultivation, cause severe loss in millable canes. The present study was targeted towards identifying SCGS phytoplasma-specific gene fragments using Arbitrarily Primed-PCR technique, and developing efficient and species-specific detection tools for SCGS phytoplasma. AP-PCR analysis revealed, 306 scorable transcript-derived fragments (TDFs), among them six were up- and 66 were down- regulated. Seventy TDFs were from phytoplasmal origin. Detection efficiency of four potential SCGS specific TDFs Opa11 P3, Opc3P4, Opc6P3 and Opk3P2 was determined. Opa11 P3 was found 13% more efficient than the universal 16S rDNA detection system.

Key players associated with tuberization in potato: potential candidates for genetic engineering

Abstract Tuberization in potato (Solanum tuberosum L.) is a complex biological phenomenon which is affected by several environmental cues, genetic factors and plant nutrition. Understanding the regulation of tuber induction is essential to devise strategies to improve tuber yield and quality. It is well established that short-day photoperiods promote tuberization, whereas long days and high-temperatures inhibit or delay tuberization. Worldwide research on this complex biological process has yielded information on the important bio-molecules (proteins, RNAs, plant growth regulators) associated with the tuberization process in potato. Key proteins involved in the regulation of tuberization include StSP6A, POTH1, StBEL5, StPHYB, StCONSTANS, Sucrose transporter StSUT4, StSP5G, etc. Biomolecules that become transported from “source to sink” have also been suggested to be important signaling candidates regulating the tuberization process in potatos. Four molecules, namely StSP6A protein, StBEL5 RNA, miR172 and GAs, have been found to be the main candidates acting as mobile signals for tuberization. These biomolecules can be manipulated (overexpressed/inhibited) for improving the tuberization in commercial varieties/cultivars of potato. In this review, information about the genes/proteins and their mechanism of action associated with the tuberization process is discussed.

Functional characterization and expression study of sugarcane MYB transcription factor gene PEaMYBAS1 promoter from Erianthus arundinaceus that confers abiotic stress tolerance in tobacco

Sugarcane is a glycophyte which has to confront various biotic and abiotic stresses while standing in fields. These stresses ultimately affect the growth and sucrose contents, causing heavy losses to farmers. A genetic approach through transgenic technology offers promising avenues to counter stresses and overcome the losses in production. In this study, PEaMYBAS1 promoter from Erianthus arundinaceus, a wild relative of sugarcane, was isolated to reveal its stress tolerance mechanism at the transcriptional level. A series of PEaMYBAS1 promoter deletions constructed from the transcription start sites F1 (−161 bp), F2 (−282 bp), F3 (−554 bp), F4 (−598 bp), F5 (−714 bp), F6 (−841 bp), and F0 (−1032 bp) were fused to the uidA reporter gene (GUS) separately, and each construct was analyzed by agroinfiltration in tobacco leaves subjected independently to drought, cold, salinity and wounding. Deletion analysis of the PEaMYBAS1 promoter revealed that the F3 (−554 bp) region was required for basal expression. Interestingly, full length deletion fragment F0 (−1032 bp) showed the highest GUS activity in drought (4.9 fold), among the other abiotic stresses such as cold (3.89 fold), salinity (3.87 fold) and wounding (3.06 fold). GUS induction characterization of the promoter revealed the enhanced stress tolerance capacity against abiotic stresses in the model plant Nicotiana tabacum. Thus, the full length deletion fragment F0 (−1032) of the inducible promoter PEaMYBAS1 can be advocated as an important genetic engineering tool to develop stress tolerant plants.

Differential filtration approach for isolation and enrichment of Sugarcane Grassy Shoot phytoplasma

The phytoplasma-associated grassy shoot disease is one of the major threats in sugarcane cultivation. A rapid method for enrichment and isolation of Sugarcane Grassy Shoot (SCGS) phytoplasma from the infected plants was developed. Differential filtration approach was used to isolate and enrich the SCGS phytoplasma and its genomic DNA that was detected by PCR analysis for phytoplasmal 16S rDNA. Ratio of pathogen to host plant DNA was found in the order of 103 and 105 in infected tissue and enriched fraction respectively, offering 148-fold increase in sensitivity for their detection.

Enriched Potato for Mitigating Hidden Hunger

Potato (Solanum tuberosum L.) is the most important non-grain food crop in the world, ranking third in terms of total production after rice and wheat and is one of main commercial crops grown in the country. Being a short duration crop, it produces more quantity of dry matter, edible energy, and edible protein in lesser duration of time than cereals like rice and wheat. Hence, potato may prove to be a useful tool to achieve the nutritional security of the nation. It can also be fitted suitably into different cropping systems. It is a highly nutritious, easily digestible, wholesome food containing carbohydrates, proteins, minerals, vitamins, and high-quality dietary fiber. It is a very popular food source for human health with potato protein “patatin” having high biological value than proteins of cereals and even better than that of milk and can be substituted for meat and milk products with improving taste, lowering energy intake, and reducing food cost. Boiled or baked potatoes are virtually fat-free and palatable and contain about 60–80 % of the fatty acid which is composed of unsaturated fatty acids that increases the nutritive value of the potato fat. Consuming potatoes just once or twice each day lowers high blood pressure almost the same as oats without resulting in increase in weight. Potato protein has 18–20 essential amino acids in varying quantities along with various important minerals and trace elements. In addition, potato is used for various value-added products, viz., chips, French fries, cubes, granules, and canned products with high resistant starch and nutritional value. Its nutritional value can be greatly enhanced with diverse agricultural practices including the modern tools of biotechnology. This paper gives an insight on the role of potato – food for future – in the food security of developing nations.