Gahukar, Dr. S.J.SURBHAIYYA, SHOBHA DEVIDAS.2024-01-112024-01-112021-08-27SURBHAIYYA, SHOBHA DEVIDAS. (2021). Molecular and biochemical responses of wheat genotypes (Triticum aestivum) under water stress. Department of Agricultural Botany, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola. Ph.D. (2021). Print.xxvii,259p. (unpublished).https://krishikosh.egranth.ac.in/handle/1/5810206152The present investigation entitled “Molecular and biochemical responses of wheat genotypes (Triticum aestivum) under water stress” was carried out at the Biotechnology Centre, Department of Agriculture Botany, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola during 2016-2019. In the present study, the objectives were accomplished at two phases of environment, physiological screening under in-vitro (lab) condition and morpho-physiological, biochemical, molecular screening under in-vivo (green house and field) condition. The experiments comprised of eight genotypes with two check viz., AKAW-3717 (tolerant) and AKAW-3722 (susceptible). In first phase of experiment, osmotic stress imposed under laboratory condition by using different concentration of PEG- 6000 viz., 0, 15 %, and 25 % and performances of various genotypes were monitored against a control. Seedling traits such as germination percentage, seedling vigor index, shoot length, root length, shoot fresh/dry weight and root fresh/dry weight were studied in unstressed and stressed condition. The result indicated that increase in osmotic stress caused a significant decreased in above seedlings parameters. Correlation coefficient studies revealed considerable and positive correlation among seedling traits.The result indicated that decrease in one trait may cause simultaneous decrease in other traits; hence, selection for any of these seedling attributes will lead to develop water stress tolerant wheat genotypes. Based on these observations, most water stress tolerant and susceptible genotypes were selected. (Check tolerant) AKAW-3717, AKAW-4842, and AKAW-5017 were recorded as the most water stress tolerant genotypes, whereas, (Check susceptible) AKAW-3722, AKAW-4925, and AKDW-5012 were recorded as susceptible genotypes. In second phase of experiments (under green house and field condition), the extent of yield reduction with water deficit depends not only on the magnitude of water deficit but also on the stage of plant growth at which it develops. Under green house condition, a pot study water stress treatments were created by using different concentration of PEG-6000 (viz., -3 bar and -6 bar PEG-6000) and water withholding. Under field condition, water stress were created by only withholding of water to assess the effect of induced water stress on performance of ten wheat genotypes at two critical growth stages (tillering and flowering). After seven to fourteen days, plants exhibited visible effects of stress. Thus plant sample were collected for further analysis and data collection. Screening of wheat genotypes for water stress tolerance on the basis of morphological, physiological, biochemical, and molecular level. The results showed that water stress significantly reduced in morphological and physiological traits like plant height, total tillers, spike length, number of spikes per plant, number of grains per spikes, 1000 grain weight, yield per plant, relative water content, and chlorophyll content. The biochemical analysis revealed increased total soluble sugar, total soluble protein, and proline significantly with increasing water stress. Proline content in stressed tolerant plants is found to be very higher as compared to that in unstressed susceptible plants suggesting its key role in water stress tolerance in plant. The activity of CAT was found to be highest in AKAW-3717 at both tillering and flowering growth stages which was found to a tolerant genotype in prior morphological and biochemical screening. The activity of POD was found maximum in all tolerant genotypes viz., AKAW-3717, AKAW-4842, and AKAW-5017 than in susceptible genotypes viz., AKAW-3722, AKAW-5010, and AKAW-4926. Superoxide dismutase activity was found to be highest in AKAW-5017. Water stress preferentially enhanced the activities of enzymatic antioxidant and osmolytes. Differential transcriptome analysis using cDNA based start codon targeted polymorphism (SCoT marker) and oligodecamer (RAPD marker) were accomplished To identify differentially expressed TDFs. TDFs (cDNA-SCoT profiling) were produced in unstressed and stressed plant at tillering and flowering stages. At tillering stage, 186/215 (green house/field) TDFs were found differentially regulated out of 191/218 TDFs while 171/228 TDFs were found differentially regulated out of 177/229 TDFs at flowering stage. cDNA-SCoT profiling revealed that marker SCoT 03, SCoT 05, SCoT 11, SCoT 13, SCoT 14, and SCoT 18/SCoT 01, SCoT 04, SCoT 11, SCoT 14, SCoT 18, and SCoT 20 showed 100 % polymorphism at both stages. Similarly, TDFs (cDNA-RAPD profiling) were produced in unstressed and stressed plant at tillering and flowering stages. At tillering stage, 381 TDFs were found differentially regulated out of 398 TDFs while 373 TDFs were found differentially regulated out of 384 TDFs at flowering stage. AT both stages, cDNA-RAPD profiling revealed that marker OPF 7, OPF 14, OPH 16, OPB 10, OPI 16, OPI 13, OPI 2, and OPH 12 showed 100 % polymorphism. Further, gene expression studies were carried out using the four contrasting genotypes AKAW-3717 (check tolerant), AKAW-3722 (check susceptible), AKAW-4842, AKAW-4925, and AKAW-5017. Under green house as well as field condition there are five water stress specific markers (WDHN 13, α-Tubulin, WPIP, WTIP 11, and DREB 1A) were used for presence and expression of gene which can confer water stress resistance to a genotype. RNA was extracted by TriZol method. Then first strand cDNA synthesis was done by using Himedia cDNA synthesis kit. PCR was carried out by using the cDNA. PCR products were separated on polyacrylamide gel and visualized under gel doc system. AKAW-3717 followed by AKAW-5017 and AKAW-4842 performed best at various stress levels for morpho-physiological and biochemical parameters. However performance of AKAW-3722, AKAW-5010 and AKAW-4926 was poor. The gene expression, results indicated that, α-Tubulin showed their expression in all genotypes grown under controlled as well as water stressed conditions. While, WDHN 13 showed their expression in AKAW-3717, AKAW-5017, and AKAW-4842. None of the other gene expression was recorded in any other genotypes. It can be concluded that water stress levels had substantial effects on germination and seedling growth. Morphological, biochemical, physiological and molecular analysis revealed that adequate genetic difference for water stress tolerance existed in wheat genotypes tested AKAW-3717 and AKAW-5017 may prove a promising parent material for breeding water stress tolerant wheat genotype. Further molecular investigations are suggested to assess the genetic basis of water stress tolerance. AKAW-3717 and AKAW-5017 may be considered better genotypes for low rainfall drought prone areas. The present study can provide clues in identifying candidate genes for further functional analysis to delineate their precise role in abiotic stress response. As key genes are identified, efficiency increase and opportunities for genetic engineering are realized. This is a fundamental aspect of research into abiotic stress tolerance, and discoveries of abiotic stress tolerance genes, which is explored in the present study.EnglishThesis