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    ThesisItemOpen Access
    Micropropagation of Bamboo (Bambusa balcooa) through tissue culture and fidelity assessment using molecular markers
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2020) Sahu, Ritesh Kumar; Jha, Zenu; Chandel, Girish; Verma, L.S.; Saxena, R.R.
    Bamboo are economically important plants with innumerable uses and many environmental benefits. The bamboo are evergreen perennial flowering plants in the subfamily Bambusoideae of the grass family Poaceae.Bamboo is non timber, forest products and in our country.it is used by several industries like pulp, paper, rayon, mat boards, making baskets, house construction, beds, coffins, bridges, toys, weapons and agricultural implements. Bamboo is a vital resource; therefore, it has an ever-increasing demand but its conventional breeding has not highly successful because of two reasons i.e., non-vegetative phase and irregular flowering. Thus, to overcome and fulfil the requirement of demand in bamboo production, the scientific techniques are also very useful such as in vitro culture techniques (micropropagation). An efficient and reproducible protocol has been established through the technique of forced axillary branching for the propagation of an important bamboo plant species, namely Bambusabalcooa. Among the various surface sterilization treatments deployed on the nodal explant, treatment has the highest aseptic culture performance in 70% ethanol, Tween 20 (15 minutes), Bavistin (30 minutes), Hgcl2 (5 minutes), and PPM culture media. Multiple shoot initiation was achieved on Murashige and Skoog’s (MS) medium supplemented with BAP 4 mg/growth regulator cytokines proved to be better for shoot proliferation in liquid medium between the two cytokinin’s in combination with BAP (4mg/l) and kinetin (2mg/l).MS medium solid shown better performance when supplemented with NAA 5 mg/l .In the present experiment, no variation was reported between in vitro grown seedlings and the mother plant in the band profiles generated by the Inter Simple Sequence Repeats (ISSR) markers. Hence, molecular analysis confirmed that these plants were genetically true to the type and can be used as elite plants.
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    ThesisItemOpen Access
    MICRO PROPAGATION OF CHIRONJI (Bhuchanina lanzan) THROUGH SOMATIC EMBRYOGENESIS
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2020-03) Shori, Savita; Jha, Zenu; Prasad, Archana, S.; Verma, L.S.; Saxena, R.R.
    Buchananialanzan(Chironji) is a deciduous treewhich belongs to Anacardiaceaefamily, produces the seeds known as Chironji. Different species of Buchananialanzanwereabundance in the past, as it was known as the backbone of rural economy of tribal people particularly inChhattisgarh, Madhya Pradesh and Uttar Pradesh. Rapid deforestation,over grazing, mishandling of trees, are main reason behind thedepletion of species populationfrom its natural habitat. The major limitation in the reforestation or domestication of Chironjiis the lowgermination percentage of seedsdue to hard seed coat, less seed viability percentage, fungal contamination during seed storage. Propagation through vegetative methods are less effectivedue to less availability of rootstocks and dependency on seasonal conditions. In therecent years, in tissue culture somatic embryogenesis has emerged as a promising technique to obtaingenetically pure elite plant populations under in vitro conditions. Present study wasconducted in Department of Plant Molecular Biology and Biotechnology, College of Agriculture, IGKV,Raipur. It has been observed that the Sterilization treatments (ST8) for both node and mid rib are very effective with 78.3 and 68.3 percent, respectively. Different explants (Node and mid rib) of Chironjiwereinoculated for induction of somatic embryos treatment (T8) which consist of WPM, PVP and hormonal concentration ( 2,4-D – 1.0mg/l and NAA- 0.6mg/l) for node found to best for induction of somatic embryo with 65 percent followed by mid rib had 58.3 percent response. By using double staining technique selected somatic embryonic callus was confirmed. The cells stain intense bright red with acetocarmine are embryonic cells, whereas cells stain blue with methyl blue are non-embryonic cells. The embryos were cultureon regeneration media treatment (GT7) for greening of somatic embryo considered to be the best with 46 percent.
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    ThesisItemOpen Access
    BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF WATER STRESS RESPONSIVE GENES IN LITTLE MILLET, BARNYARD MILLET AND RICE
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2019) Kathare, Pooja; Chandel, Girish; Prasad, Archana S.; Nair, Sunil; Saxena, R.R.
    Water stress, a type of abiotic stress, affects the growth, productivity and quality of agricultural crops in the world. With the recent changes in the rainfall pattern, the occurrence of water stress is becoming regular feature. It is also affecting the yield of the major cereal crop like Rice, which is estimated as 25.4%. Minor millets are the upland crop, show water stress tolerance characteristics. It was found that NAC TFs in minor millet have been characterized to limited extent. The NAC xvi family genes are Osmoregulatory in function and belongs to Trancription factors. The extra ordinary tolerance of millets towards diverse abiotic stresses especially water, heat, salinity, light made them a amenable, influencable and manageable system to investigate and study the genes involved in the management of stress tolerant trait at cellular, molecular and physiological levels. Newly identified and improved varieties ofsmallseded milletsareexpectedtoplay a key roleinthe“New Gren Revolution”-aword coined to denote new requirements which are necessary to deal with complicated challenges in modern world including increasing population and ever declining agriculture land. Identification of genes responsible for particular stress in millets will have great importance in modern agriculture. All these necessitate dissecting the transcript information under different stress conditions to prospect new genes. Transcript analysis of millets after exposure to water stress condition delivers information on genes, that are differentially and specifically regulated on exposure to water stress. Little millet and Barnyard millet are perhaps the least studied crops of millet, there is a need for establishment of genetic map in order to study genome. There is need to extract the transcript information under different stress conditions for the characterization and identification of key for water stress tolerance. The current study was planned to identify some key important genes that would be responsible for water stress tolerant traits, in the Little millet (RLM-37, BL-4, OLM-203, MM-23, BL-8, BL-15-1, MM-10), Barnyard millet (SAWA, VL-29, MELGHAT-1, MELGHAT-3, MM-03) at 5.5 to 6.5 % SMC (Soil moisture content) and Rice (R-RF-127, MOROBEREKAN and MTU-1010) at 15-18 % SMC (Soil moisture content) at vegetative stage. Genotypes belonging to diverse genetic background were grown under control and stress conditions for identification of water stress tolerant characters. Biochemical and Phenological characterization of Minor Millet and Rice genotypes was done under control and water stress conditions and parallely a set of known and unknown water stress responsive genes were selected for expression analysis studies using semi-quantitative RT-PCR analysis. Under Phenological and xvii Biochemical characterization, Leaf rolling, Soil Moisture Content (SMC), Leaf Proline content, Leaf Protein content, Leaf Carbohydrates content and Chlorophyll content (chlorophyll a, chlorophyll b and total chlorophyll content ) were estimated. In this study, among the three crops, the photosynthetic pigments like chlorophyll a (0.400 mg/g f.wt), chlorophyll b (0.203 mg/g f.wt) and total chlorophyll (1.133 mg/gfwt).decreasedandthebiochemial omponentslikeproline(1.2μ10 mole/g f.wt), leaf protein (0.558 mg/g f.wt) and carbohydrate (0.382 mg/g f.wt) increased under water stress respectively. These findings were found to be in correlation with the expression analysis studies carried out with seven water stress responsive gene orthologs (EcNAC 67, SiNAC 29L, OsNAC 29, TaNAC 4, CDPK, U2-SnRNP, Synaptotagmi), which has given a differential expressions under water stress as compared to controlled traits. The result revealed that the all genes were up regulated under water stress treatment in Millets and Rice. Among seven Little millet genotypes, RLM 37 showed a higher level of up regulation for gene SiNAC 29L (4.33 fold), EcNAC67 (3.75 fold), followed by OsNAC29 (2.59 fold), TaNAC4 (2.88 fold) and other genes. Among five Barnyard millet genotypes, Sawa showed a higher level of up regulation for gene SiNAC 29L (103.21 fold), OsNAC29 (24.08), EcNAC 67 (4.29 fold), TaNAC 4 (0.23 fold) and among three Rice genotypes, R-RF-127 showed a higher level of up regulation for gene SiNAC 29L (2.85 fold), OsNAC 29 (2.23 fold), EcNAC 67 (1.27 fold), TaNAC 4 (0.78 fold) and Synaptotagmi (1.19 fold). Based on the expression pattern SiNAC 29L and EcNAC 67 genes was identified as positively up regulated under waters stress in Millets and Rice genotypes. These genes are not expressed in the well watered plants, but only in the stressed genotypes. Biochemical and expression analysis result was used as a basis to select the genes (OsNAC 29 in Little millet, SiNAC 29 L in Barnyard millet and EcNAC67 in Rice genotypes) and genotypes (RLM-37, Sawa and R-RF-127) for the ortholog gene isolation. However positive regulation of these genes may lead to play a key role in identification of different transcription factors which are responsible for different water stress responsive mechanisms or in cross linking different signaling pathways to xviii activate plant defence mechanisms in Little millet, Barnyard millet and Rice. Its attribution under water stress in Little millet, Barnyard millet and Rice may be due to alternate splicing or due to the presence of cis acting elements, suggesting important roles of these genes in combating oxidative stress. These can be taken as base for water stress tolerance response of the crop, which may be useful for further validation studies of the candidate genes for the water stress tolerance in the millet species and other crop plants like Rice.
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    ThesisItemOpen Access
    DECIPHERING THE DIFFERENTIALLY EXPRESSED HEAT STRESS RESPONSIVE CANDIDATE GENES IN RICE (Oryza sativa)
    (2019) Lohitha, Poola; Chandel, Girish; Jha, Zenu; Nair, Sunil; Saxena, R.R.
    Global warming is expected to increase the earth’s surface mean temperatures by 1.5 to 4.8 ºC by 2100 (IPCC 2014) and it is reported to reduce the yield of major crop plants including rice by 41% by the end of 21st century. Rice is the most important staple food crop across the world and feeds more than 3 billion people in Asia and Africa. Heat stress affects the growth of rice crop at all stages of growth like vegetative, reproductive and ripening phases but the effect is severe during flowering stage, as it leads to spikelet sterility that causes reduced yields. Improved varieties of rice with heat tolerance are required to meet the complex challenges like increasing population, decreasing arable land and increasing temperatures. Expression analysis of genes responsive to heat stress in different crops will be of great importance in today’s agriculture. Transcriptome sequencing of rice after exposure to high temperature treatments could provide information on genes, that are differentially regulated on exposure to heat stress and its dissection is necessary to identify and characterize key genes responsible for heat tolerance. High temperatures produces the new group of special proteins under stress called as ‘Heat Shock Proteins (HSPs)’, which are categorized into five families based on their molecular weights: 1) small heat-shock proteins (sHSPs), 2) Hsp60, 3) Hsp70, 4) Hsp90 and 5) Hsp100. Studies reported plants synthesize large amounts of sHSPs under heat stress functioning as molecular chaperons, suggesting that they play a major role for enduring thermo-tolerance in plants. Higher plants posses at least 20 sHSPs. Transcription of HSPs is guarded by some regulatory proteins, called heat stress transcription factors (Hsfs). At least 21 Hsfs are seen in plants, with its specific role in regulation. Heat shock proteins perform a major role in signalling, translation, host-defence mechanisms, carbohydrate metabolism and amino acid metabolism by maintaining the functional conformation of proteins, preventing accumulation of non-native proteins and refolding. sHSPs facilitate stabilization of denatured proteins under heat stress. Based on this, the current study was planned to identify some key genes that are responsible for heat tolerance in fourteen rice genotypes (GP-145-103, SL-62, Dagaddeshi, Nagina-22, Swarna, GP-145-55, CGZR-1, Annada, Poornima, Karma mashuri, ARB-6-11, GP-145-40, MTU-1010, RRF-127) at late vegetative stage before panicle initiation. Genotypes belonging to diverse genetic background were grown in two sets under control and stress conditions (42˚C) for identifying heat tolerant traits. Phenological and biochemical characterization of rice genotypes was done under heat stress and in parallel a set of known heat related candidate genes were selected for expression analysis studies using semi-quantitative RT-PCR analysis. Under phenological and biochemical characterization six parameters are taken into consideration. They are membrane stability index (MSI), pollen fertility, spikelet fertility, chlorophyll content, proline and MDA levels. Phenological and biochemical studies on rice resulted in identifying some of the tolerant and susceptible genotypes for heat stress. Genotypes RRF-127 , Nagina-22, Karma mashuri, CGZR-1 and Annada had recorded lower electrolyte leakage of 19.8%, 21.1%, 27.5%, 51.7% and 69.8% respectively under stress when compared to control. Lowest percentage of decrease in pollen fertility under stress conditions was observed in the genotypes RRF-127, Nagina-22, GP-145-103, Annada and CGZR-1 as 14.4%, 18.1%, 24.9%, 28.3% and 37.9% respectively. Percentage of Spikelet fertility decrease under stress was lowest in the genotypes RRF-127, Nagina-22, Annada, Karma mashuri and CGZR-1 as 14%, 19%, 25%, 31% and 42% respectively. When comparing the fold increase in proline content for stress over control, Nagina-22 showed highest fold increase of 20.6 folds followed by CGZR-1 (11.4 fold), RRF-127 (11.2 fold), Annada (10.5 fold), GP-145-103 (7.0 fold ) and Karma mashuri (6.0 fold). In chlorophyll content, Annada has the lowest fold decrease of 1.0 folds for all chla, chlb and total chlorophyll content followed by Karma mashuri (1.0 fold) under stress when compared with control. When comparing the fold increase in MDA content for stress over control. The lowest fold increase in MDA content was shown by RRF-127 with 1.1 fold increase followed by Nagina-22 (1.4 fold), CGZR-1 (1.5 fold), Annada (1.8 fold), GP-145-103 (2.1 fold) and Karma mashuri (2.5 fold). Among the five genes (OsHSP26.7, OsHSP16.9, OsHSP DnaJ, OsHSP18 and 60Kda chaperon), selected for the study, OsHSP DnaJ has shown consistent expression under both control and stress with no significant change. The remaining four genes has shown up-regulation in all the genotypes under stress. OsHSP 26.7 gene has shown strong up-regulation in rice genotype RRF-127 (14.3 fold) followed by Annada (13.9 fold), Karma mashuri (11.5 fold), GP-145-103 (8.6 fold) and CGZR-1 (3.7 fold). OsHSP 16.9 gene has shown up-regulation in RRF-127 (10.0 fold) followed by Annada (3.7 fold), CGZR-1 (3.4 fold), GP-145-103 (3.2 fold) and Karma mashuri (3.1 fold). OsHSP 18 gene showed up-regulation in GP-145-103 (17.7 fold) followed by CGZR-1 (14.1 fold), Annada (13.8 fold), GP-145-55 (11.9 fold) and poornima (8.9 fold). 60Kda chaperon gene has shown up-regulation in almost all the genotypes under heat stress, but the up-regulation was by minimal levels. Among all the fourteen genotypes selected for the study, RRF-127, Annada, Karma mashuri, CGZR-1 and GP-145-103 showed a positive regulation towards heat stress and genotypes like MTU-1010, ARB-6-11 and GP-145-55 showed negative regulation towards heat stress. These findings were observed to be in correlation with the phenological and biochemical characterization and expression analysis studies carried out with five different heat responsive genes belonging to small heat shock proteins family, which had differential expression under heat stress when compared to control conditions. However positive induction of these genes leads to a key role in identification of different transcription factors, that have been responsible for different heat tolerant mechanisms or in cross-linking of different signalling pathways to activate plant defence mechanisms in rice under stress. This can be taken as a base for heat tolerance response of rice crop, which may be useful for further validation studies of the candidate genes responsive for heat stress in rice as well as other crop plants.
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    ThesisItemOpen Access
    SEQUENCE VARIABILITY AND DIFFERENTIAL EXPRESSION PATTERN OF OsNRT2.3 NITRATE TRANSPORTER IN RICE
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2019) Yadav, Ankit; Verulkar, S. B.; Banerjee, Shubha; Guhey, Arti; Saxena, R.R.
    Rice (Oryza sativaL.) is very important food crop in whole world which provides food to nearly half of the world’s population. The rice is mainly grown in puddled condition, requires 4000-5000 litres of water to produce 1 kg of rice. In the present day climatic condition the scenario has been changed due to increase in drought frequencies. The availability of water is not adequate and also decreasing day by day. So the conventional method for growing rice will not be possible in near future. The aerobic rice cultivation is the possible and logical way to tackle this problem. The availability of nutrients is greatly affected by growth condition. Nitrogen is major limiting nutrient for rice production. The nitrogen is available in nitrate form under aerobic condition and in ammonium form under anaerobic condition.Absorption of these different ionic forms of nitrogen changes the pH of cell cytoplasm and consequently affect the rhizospheric soil pH. One of the spliced form ofOsNRT2.3 nitrate transporter has the capacity to maintain the cytosolic pH by differential expression. The understanding of chemistry behind uptake of nitrogen forms and pH homeostasis is prerequisite for development of genotype with high nitrogen use efficiency under aerobic condition. Considering these facts and problem the present study was formulated to understand the relationship between OsNRT2.3, water stress and nitrate absorption, with an overall goal to identify more effective allele of this gene which can impart drought tolerance and enhance nitrogen use efficiency simultaneously. Four rice genotype viz. ISD-1 (low NUE), Swarna (high NUE), Danteshwari (Moderate NUE), Dagad Deshi (Low NUE) with three nitrogen treatment (nitrate, ammonium and control) was grown in 4.5” wide pots under aerobic and anaerobic condition. Along with the pot experiment a hydroponics culture with these genotype were also conducted to understand the change in pH due to plant root exudates. The pH from the rhizospheric soil was estimated and expression of OsNRT2.3was measured by Quantitative Real Time PCR by using gene specific primer. The results of pH estimation revealed that under aerobic condition the pH of soil was higher than anaerobic condition. In ammonium treatment the pH of soil was significantly lower than the nitrate as well as control condition. The correlation between pH and nitrogen percent in shoot was significantly negative for nitrate and control treatment while in ammonium treatment it was revers. The pH values of hydroponics solution shows that rice plant prefer nitrate form of nitrogen in early stage of growth and development and after a particular stage they switches into absorbing ammonium form of nitrogen. The expression of OsNRT2.3 nitrate transportershowed the differential expression pattern under aerobic soil condition but under anaerobic condition the expression was not much fluctuate as in case of aerobic. This was also correlated with pH of soil for genotype Danteshwari under aerobic soil condition. To understand the DNA sequence variability the full length OsNRT2.3 gene was amplified by using PCR and sequenced for two genotype (Danteshwari and Dagad Deshi). The sequenced result was 6.77% different from each other. The sequence of Danteshwari was 29.72% different from OsNRT2.3 gene sequence present in database from rice genotype Nipponbare while for genotype Dagad Deshi it was 37.63%.
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    ThesisItemOpen Access
    IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF POTENTIAL MICROBIAL STRAINS OF PSEUDOMONAS SP. FOR PLANT GROWTH PROMOTING ACTIVITY IN VEGETABLE CROPS
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2019) Shinde, Nikhil Dhanaji; Verulkar, S. B.; Kotasthane, A.S.; Banerjee, Shubha; Saxena, R.R.
    The present investigation entitled“Identification and molecular characterization of potential microbial strains of pseudomonas sp. For plant growth promoting activity in vegetable crops.”carried out in Microbial Biotechnology Laboratory, Department of Plant Molecular Biology and Biotechnology, College of Agriculture, Indira Gandhi Agricultural University, Raipur (C.G). The plant roots surrounds thin layer of soil, which contains thousands of soil microbes. Some of them actively colonizes plant roots and improve plant growth by several growth promoting mechanisms. Such beneficial group of microbes is known as Plant Growth Promoting Rhizobacteria(Kloepper & Schroth, 1981). Among several well-known PGPRs Pseudomonas is ubiquitous bacteria in agricultural soils with several contrasting characters. In present study, we tested 31 Fluorescent Pseudomonas isolates for their growth promoting activity in different vegetable crops. The biochemical and morphological characterization classified isolates into major groups’ P. aeruginosa (22.50%), P. putida (38.70%), P. fluorescence (19.35%) and unidentified fluorescent Pseudomonas (19.35%). The isolates screened for their growth promoting potentials, 19 isolates (62%) found positive for hydroxyquinoline mediated siderophore production, all isolates found positive for hydroxymate but they were negative for catechol type of siderophore production.The siderophore quantificationwas done by using chrome azurolsulfonate (CAS) assay(Schwyn & Neilands, 1987), showed siderophore production ranging from 10.21% to 95.24%. The IAA quantification assay (Bric, Bostock, & Silverstone, 1991) showed indole production ranging from 0.43 to 51.69 μg/ml in DF minimal medium in presence of L- tryptophan. All the isolates exhibited different rates of growth inhibitions of R. solaniand S. rolfsiiranging from 1.124 to 77.528% and 1.123 to 55.056% respectively, Therefore it can used as effective biocontrol of phytopathogens. The isolates P12, P21, P22, and P23 in tomato; P3, P11, P22 and P23 in brinjal; P13, P17, P19, P23, P141 and P229 in fenugreek; P1, P2, P7, P29, P229 and P260 in spinach showed superior germination over control. The top five isolates P19 (19.13 cm), P18 (16.5 cm), P22 (13.33 cm), P12 (13.25 cm) and P16 (13.25 cm) of replication 1 while P14 (18.48 cm), P7 (16.9 cm), P12 (15.86 cm), P10 (15.13 cm) and P21 (15 cm) of replication 2 showed superior plant height in tomato. The top five isolates from brinjal trial namely P13 (40 cm), P7 (35.5 cm), P30 (33 cm), P6 (32.8 cm), P24 (32.3 cm) of replication 1 while P14 (42 cm), P19 (39 cm), P5 (37.5 cm), P15 (36 cm), P7 (35.3 cm) of replication 2showed superior plant height over control. Root morphology analysis of fenugreek revealed highest root length (357.552 cm), surface area (22.876 cm2), root volume (0.116 cm3), number of tips (4386) and number of forks (4575) was scored by isolate P18 while highest avg. diameter (0.330 mm) was scored by isolate P30. The tomato pot trial per plant yield obtained by top five isolates P11 (431 gm), P15 (211.67 gm), P7 (162 gm), P3 (132 gm) and P17 (128.67 gm) of replication 1; P19 (246 gm), P5 (128 gm), P22 (125.5 gm), P10 (116.25 gm) and P14 (110.75 gm) of replication 2 were superior than control (99.33 gm). The Rep-PCR fingerprinting with ERIC, BOX and REP primers revealed high level of polymorphism among isolates.The entirepopulation is divided into three major groups Group 1 contains P66 and P141; Group 2 contains P1, P3, P4, P5, P2, P6 and P7 ; Group 3 contain rest of the 22 isolates.
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    ThesisItemOpen Access
    DOUBLED HAPLOID PRODUCTION FOR DEVELOPMENT OF BLB RESISTANT RICE THROUGH ANTHER CULTURE TECHNIQUE
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) Prabodhrao, Pusadkar Pratik; Jha, Zenu; Verulkar, S.B.; Bhandarkar, S.; Saxena, R.R.
    Rice considered as most important crops in the world a staple food for half of the world population. As to meet demand of ever increasing populationthe production of rice must continuously be increase on sustainable basis. In order to relive and resolve the contradiction between food supply and consumer demand we need to produce new cultivars which will minimize losses due to biotic diseases such as BLB and BLAST. As there are no effective chemical agents to control bacterial blight pathogen the only way to produce resistant varieties combating BLB disease. So for development of new varieties by conventional breeding it willrequire another 7-8 years of time interval which can be get done in 2-3 years with anther culture process. Anther culture known as a process of obtaining doubled haploid (DH) lines andit provides opportunities for shortening breeding cycles in which homozygosity achieved through doubling of haploid set of chromosomes. In our study for anther culture process individual plants of F3 population derived from three parent’s i.eSafri-17, Dubraj (Susceptible Parent) and RP-Bio226 (Resistant Parent) crosses S x R, D x R, S x P and double crosses S x R x P and D x R x P were evaluated for field infection to bacterial blight (BLB) during Kharif 2016. The panicles were collected from only those plants which showed resistant to BLB based on infection seen on clipped leaves. The pre-treatment of 10 ˚C for 10 days given to fix the meiotic division in pollens which were dusted on N6 medium containing 2,4-D. After 30 days of inoculation callus induction percentage calculated in each cross as it wasfound 11.48 % in S x R, 5.1 % in D x R, 8.12 % in S x P, 6.45 % in S x R x P and 4.88% in D x R x P. The variable callus induction may be due to different genetic constitution of genotypes as different genotype respond differently under in-vitro conditions. The ploidy level of calluses obtained with anther culture process was checked through flow cytometry (FCM). In FCM analysis we have observed 90-95% haploid cells in S x R cross callus while in SRP and DRP cross it is showing 89.4 % and 92.5 % of haploid cells which proved that callus generated were haploid.The calluses were transferred to greening medium. After 15-20 days green callus observations were taken in which S x R was having highest greening percentage of 24.12%, D X R is having 13.06 % while S X P, SRP, DRP are having 9.55%, 1.55% and 3.71% green callus respectively. In another experiment different colchicine treatment given in media in which treatment containing 100 mg/l of colchicine in regeneration media followed by 48 hrs of incubation has given highest green callusing percentage of 23.13 % which recorded 1.5-2 fold increase in greening percentage of calluses. The green calli were transferred for shooting after shoot initiation shoot elongation done with higher concentration of BAP. These shoots were transferred to plain MS medium for rooting purpose. The primary hardening of plantswas done with hydroponics process in which YOSHIDA medium was used for providing nutrients to plants. After transfer of haploid plants in field at tillering stage of plant colchicine injection were given for chromosome doubling. The seeds collected from each plant were considered as separate double haploid (DH) line. Several double haploid lines are produced in which cross S x R produced 179 lines while other crosses D x R, S x P, SRP and DRP produced 69, 6, 14 and 8 lines respectively. These seeds were sown in field for further genotypic and morphological studies. The lines obtained were found homozygous in nature phenotypically from which DNA isolated. DNA used for further screening of these lines for (Xa 21, xa13, xa5R) resistant genes for BLB and (Pi1 and Pi2) resistant genes for BLAST. In S x R DH lines after screening with markers we have found that 34 lines were found to be resistant for xa21, 61 lines were resistant for xa5R and 4 lines were found resistant for xa13. Out of 179 DH lines some lines are found with two resistant genes in which 12 DH lines were found positive for (Xa21 and xa5) combination of genes, 3 DH lines were found positive for (Xa 21 and xa 13) genes. Besides DH line number 23 showed a combination of two genes (xa5R and xa13). In D x R DH lines out of 69 lines we have found that 39 lines are resistant to xa5R gene. In cross S x P we have found that 4 lines were showing resistant to both BLB and BLAST. In DRP line no 7 was showing resistance to both BLB and BLAST while line no. 6 was showing resistance to both Pi1 and Pi2 BLAST resistant genes. In cross SRP we have found that 10 lineswhich showed resistance for both BLB and BLAST. Also in line no. 14 both Pi1 and Pi2 genes were present on the basis of marker data.The resistant lines were further evaluated for six yield and its contributing traitson the basis of data collected DH line are found superior than parents as height found reduced also number of tillers/panicles increased in DH lines. The superior DH lines (S x R 20, 26, 52, 72, DRP5 and DRP7) on the basis of molecular and morphological data were selected and seeds given for state multilocation trials.
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    ThesisItemOpen Access
    CHARACTERIZATION OF LATHYRUS TRANSCRIPTOME FOR IDENTIFICATION OF PUTATIVE CANDIDATE GENES RELATED TO β- ODAP BIOSYNTHESIS
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) BHARIYA, SANJAY KUMAR; Banerjee, Shubha; Verulkar, S.B.; Sahu, Mayuri; Saxena, R.R.
    Grass pea (Lathyrus sativus L.) is an important crop in India. Its vital importance in the Indian agriculture emanates from its resistance to drought, salinity, waterlogging and low soil fertility. Lathyrus contains energy 362.3 cal; protein 31.6%; fat 2.7%; nitrogen-free extract 51.8%; crude fibre 1.1% and ash 2.2%. However, low levels of the amino acids methionine and tryptophan and the presence of the neurotoxin β-N-oxalyl-L-2,3-diaminopropanoic acid (β-ODAP) in the seeds are the major limitations of the crop. β-ODAP can cause lathyrism in humans and animals after over consumption of the seeds of certain Lathyrusspecies. A set of 30 genotypes including germplasm lines and varieties of grass pea available at IGKV, Raipur were characterized for morphological and β-ODAP contents. High variation was observed among genotypes regarding all the studied traits. The β-ODAP content was ranged from 0.07 and 0.57 mg g-1 with an average of 0.29 mg/g. Further, the correlations between the traits were also investigated and the strongest positive correlations were observed between Pod weight was highly significant and positively correlated with Number of seeds, Seed weight, Seed Pigmentation and Per gram seed. whileNumber of seed significant and positively correlated with Seed weight, Seed pigmentation, Per gram seed and Harvest index. It was also found to be correlations were observed between ODAP content significant and positively correlated with Leaf length and Seed weight. Homlogous gene based method is most important tool for validation of neurotoxin non-amino acid β-N-oxalyl-L-2,3 diaminopropanoic acid responsive gene in lathyrus sativus. CoA transferase III gene family i.e. Formyl CoA Transferase, Carnitine Dehydratase and Acyl CoA tranferase used to identifiy gene which act for transfer of CoA group from 2-3 diamino propanoic acid to β-N-oxalyl-L-2,3-diaminopropanoic acid. The total RNA was isolated from Lathyrus lines and synthesized cDNA from Lathyrusleaves. The method yielded similar results on all types of lines but low expression of CoA transferase family III showed in Prateek,Mahateora, Pusa 24, compare to RLK1950 and SEL2156. RNA sequencing tools are more advanced and effective for identification and characterization of putative candidate gene at particular stage.RNA sequene based approaches were done to identify the candidate gene that is involved in ODAP biosynthesis pathway in which 26 CoA associated genes was differentially expressed in Mahateora (201M) and RLK 1950 (202R) genotypes.
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    ThesisItemOpen Access
    EXPRESSION ANALYSIS OF GENES UNDER DROUGHT AND DIFFERENT FORMS OF NITROGEN IN RICE (oryza sativa L.)
    (Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) Wallalwar, Mayur Ravindra; Verulkar, Satish; Banerjee, Shubha; Saxena, Ritu R. Saxena
    Rice (Oryza sativa L.) is the staple food for nearly half of the world’s population, particularly in different parts of Asia. Rice production is affected by various stresses among which drought is the one that critically limits crop production by reducing water availability along with low nutrient availability to the plant. Among nutrients, Nitrogen (N) is essential macronutrient, whose availability play important role in plant growth & development and also nitrogen use efficiency (NUE) of rice is ~33% which is poorest among cereals. Most available form of nitrogen in aerobic soils/rainfed condition is nitrate (NO3-) while ammonium (NH4+) predominate in anaerobic soils/irrigated condition. Unlike other crops, rice prefers to utilize NH4+ over NO3- as it is well adapted and evolved under irrigated/anaerobic conditions. Development of cultivars with drought tolerance along with improved nitrogen (NO3-) use efficiency by exploiting variability for drought tolerance and NUE traits could be an integrated approach. Considering whole scenario, the present study was undertaken to understand, the genetic variability and expression pattern of the important genes along with enzymes assay, involved in nitrogen absorption and assimilation along with drought tolerance. A set of four hundred eighty seven and thirty two recombinant inbred lines (RILs) of IR84984-83-15-862-B × IR90019-17-159-B and Danteshwari × Dagaddeshi respectively were evaluated under Rainfed, Irrigated and TSD condition with three nitrogen treatments (two form of nitrogen and one without nitrogen) at research cum instructional farm of COA, IGKV, Raipur, for generation of phenotypic data during wet season 2015-16. Performance under rainout shelter and field condition for leaf rolling score, set of selected genotypes was again evaluated under rainout shelter. The higher mean performance of yield related traits observed in irrigated condition while under rainfed and TSD condition where these trait values were reduces significantly due to stress. On the basis of yield, genotype G-206 (Rainfed), G-154 (Irrigated) and G-387 (TSD) performed well. The population of 24 genotypes (consist of 12 resistant and 12 susceptible genotypes) were selected on various criteria such as grain yield, green pigmentation and leaf rolling score under rainout shelter. The 32 genotype (24 selected along with popular checks) were evaluated in factorial RCBD design with two replications each under three nitrogen treatments (two form of nitrogen and one without nitrogen). Three factor ANOVA showed high level of significance for variance individual components (G, N, E) along with their interactions effects (GXN, GXE, NXE, EXNXG) for yield, Nitrogen use efficiency (NUE) traits and yield related traits. Mean performance of genotypes under NH4+ treatment showed higher values for agronomically important traits i.e. yield and NUE as compared to NO3- and control treatment under all environments. Highest NUE was observed in genotype G-31 (IBD-1) under irrigated condition is 18.5gg-1N (ammonical treatment) and 13.58 gg-1N (Nitrate treatment). The genotype G-27 (Dagaddeshi) also had higher NUE under rainfed condition is 9.42 gg-1N (Ammonical treatment) and 9.91 gg-1N (Nitrate treatment). Grain yield and NUE had significant and positive association under all sets of environments. So the set of 10 genotypes (consist of 5 high NUE and 5 with low NUE) were selected. The set of 48 genotypes were made with selected genotypes from both populations along with germplasm lines were evaluated under irrigated and rainfed condition with different form of nitrogen during wet season 2016. Three factor ANOVA showed high level of significance for variance individual components (G, N, E) along with their interactions effects (GXN, GXE, EXNXG) for yield. Genotype G-16 (E-1702) manifest higher yield in control and ammonical treatment while G-14 (GP-145-42) in nitrate treatment under irrigated condition. Genotype G-43 (12.1(487)-283) manifest higher yield in nitrate and ammonical treatment and G-18 (RKVY-104) in control treatment under rainfed condition. Grain yield showed significant and positive association under all sets of environments with biological yield only. In order to develop genotype with drought tolerance along with nitrogen use efficiency, the enzyme involved in nitrogen assimilation were used for enzyme assay. In set of 48 genotypes, assay of four enzymes carried out at Department of Plant Molecular Biology and Biotechnology, IGKV, Raipur. Genotype G-33 (12.1(487)-126) and G-39 (12.1(487)-184) showed higher values for GS enzyme activity while Genotype G-6 (DxD(124)-121), G-11 (IR 64), G-15 (WR 36), G-17 (Azucina), G-26 (ARB6) showed higher values for GOGAT activity in all treatment under rainfed condition. Grain yield has non-significant and positive correlation with GS enzyme activity while it has non-significant and negative correlation with GOGAT activity. The set of 12 genotypes (G-5, G19, G-24, G25, G-28, G-34, G-36, G-39, G-41, G-43, G-45 and G-46 ) selected on basis of grain yield, green pigmentation and enzyme assay were again analyzed with different form of nitrogen (ammonium nitrate, calcium nitrate, ammonium sulphate, ammonium bicarbonate and half concentration of ammonium nitrate) under hydroponics condition. Under all set of treatments, Genotype G-11 (12.1(487)-324) manifest overall higher GS activity, G-6 (12.1(487)-166) showed overall higher performance for GOGAT activity, G-3 (Abhaya) had overall higher Nitrate reductase activity and Nitrite reductase activity was higher in G-5 (12.1(487)-50). Analysis of four enzyme activity showed overall performance of genotype G-3 (Abhaya) and G-6 (12.1(487)-166) were higher under ammonium sulphate treatment, G-8 (12.1(487)-184) under calcium nitrate treatment, G-11 (12.1(487)-324) under ammonium nitrate treatment, G-4 (Safri-17) under ammonium bicarbonate treatment and G-2 (BAM 5926) & G-4 (Abhaya) under half concentration of ammonium nitrate treatment. Correlation analysis showed GS enzyme activity has significant and positive correlation with GOGAT activity (0.676*). In order to explore more variability set of 40 genotypes were made including 12 selected genotypes with 16 genotypes (from ISD-1 and Swarna cross) and 12 germplasm lines. The ammonium bicarbonate treatment was removed because it wasn’t much effective as ammonium sulphate. Under all four treatments in leaf tissue, Genotype G-24 (ISD x Swarna 620-414) manifest overall higher GS activity, G-16 (ISD x Swarna 620-243) showed higher overall performance for GOGAT activity, G-14 (ISD x Swarna 620-217), G-18 (ISD x Swarna 620-259) and G-22 (ISD x Swarna 620-370) had overall higher nitrate reductase activity and nitrite reductase activity had overall higher values in genotype G-14 (ISD x Swarna 620-217), G-24 (ISD x Swarna 620-414) and G-27 (ISD x Swarna 620-593). In root tissue under all treatment, genotype G-24 (ISD x Swarna 620-414) manifest overall higher GS activity, G-20 (ISD x Swarna 620-317) showed overall higher performance for GOGAT activity and genotype G-4 (Safri-17) had overall higher nitrate reductase activity. Under particular treatment, overall performance of genotype for four enzyme activity were analyzed, genotype G-11 (12.1 (487)-324) showed higher performance under half (half concentration of ammonium nitrate) treatment in leaf tissue while G-12 (12.1 (487)-363) in root tissue, G-12 (12.1 (487)-363) and G-16 (ISD x Swarna 620-243) had higher values under ammonium nitrate treatment in leaf tissue and G-7 (12.1 (487)-170) in root tissue, G-17 (ISD x Swarna 620-248) and G-36 (IR 64) shows higher activity under calcium nitrate treatment in leaf tissue and G-30 (Swarna) in root tissue, G-7 (12.1 (487)-170) under ammonium sulphate treatment in leaf tissue and G-20 (ISD x Swarna 620-317) in root tissue. Correlation analysis between treatments under different tissue showed positive and significant correlation between GS activity in root with GOGAT activity in shoot (0.177*), between GOGAT activity in shoot and root tissue (0.348**), between GOGAT activity and NR activity in shoot (0.192*) and between NR activity and NiR activity in shoot (0.200*) while negative and significant correlation were observed between NR activity in shoot and root tissue (-0.312**) and between GS activity and NiR activity in shoot (-0.160*). Gene involved in nitrogen uptake and assimilation along with drought tolerance were investigated in four parent genotypes which have different morphological and biochemical behavior with different form of nitrogen and drought stress. Gene under study was member of Nitrate transporters (NRT), Glutamine Synthetase (GS), Nitrate reductase (NR) and Glutamate Synthase (GOGAT), DREB family and root development gene family. The expression pattern of each gene using gene-specific primer was analyzed in rice seedlings (35 days old) by quantitative real time PCR which showed a distinct expression pattern of those genes in four selected genotypes. Nitrate transporter gene, NRT2.3b showed shoot preferential expression while NRT2.3a showed root preferential expression. NRT2.3b gene expression was highly down regulated in Dagaddeshi compared to other genotypes in both root and shoot tissue under all the treatment, because Dagaddeshi is drought tolerant variety but low grain yielding as researcher reported that NRT2.3b involved in increasing grain yield. Glutamine synthetase, Gln2 exhibit strong root preferential expression while Gln1.2 has neither root nor shoot, but express equally in both. Gln1.2 has strong expression under ammonium sulphate and ammonium nitrate treatment compared to other treatment (because of presence ofammonicalform of nitrogen) in both root and leaf tissue which indicates Gln1.2 is ammonical form responsive gene. Gln2 gene expression was higher in Swarna and Dagaddeshi under all treatment in leaf tissue, shows that both genotypes were responsive to different form of nitrogen for glutamine synthetase activity. In root tissue, individual form of nitrogen much effective in genotypes ISD-1 and Swarna for Gln2 gene expression compared to mixed and its half concentration treatment. Researcher reported that GS activity is one of the parameter for selecting high yielding variety as its higher expression in Swarna proves it. Swarna is the potential genotype which had higher glutamine synthetase activity which correlates with NUE that reflects in terms of grain yield. Glutamate synthase gene, Glt1 was strongly expressed in Swarna and Dagaddeshi in shoot and Danteshwari in root under all treatment. Significant and positive correlation between GS and GOGAT enzyme activity supported by GS and GOGAT gene expression in Swarna which ultimately results in high nitrogen assimilation rate in terms of grain yield, which was supported by NUE of Swarna. Nitrate reductase gene, NR manifest shoot preferential expression. Effect of nitrate form of nitrogen were observed as calcium nitrate treatment manifest higher NR gene expression compared to mixed and ammonical form of nitrogen. Nitrate reductase expression under calcium nitrate was higher in ISD-1 and Swarna in root and shoot respectively explains their efficiency for nitrate absorption and utilization. Drought tolerant gene, DREB manifest shoot specific expression compared to root. In Dagaddeshi, DREB gene was strongly expressed under all the treatment compare to other genotypes in shoot and root tissue which explains drought tolerance ability of Dagaddeshi. The DREB gene expression responsive to stresses condition as treatment containing less concentration of mixed form of nitrogen had lower gene expression compared to individual form of nitrogen in leaf tissue. Root development gene, both GLR3.1 and OsIAA23 had higher gene expression in all treatments except under half treatment due to low concentration of both form of nitrogen. Both genes were expressed strongly in ISD-1 under all treatment except under half treatment. ISD-1 also had higher DRO1 gene expression in calcium nitrate and ammonium nitrate treatment which correlates with root length under those treatment, while DRO1 gene expression contradict with root length phenotype in ISD-1 under half treatment. There no effect of different form of nitrogen on expression of OsAPY gene. ISD-1 manifest overall higher root development gene expression under all treatment except under half treatment which correlates with healthy root structure under hydroponics condition.