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ThesisItem Open Access SCREENING AND SELECTION OF BLB RESISTANT AND DROUGHT TOLERANT RICE LINES DERIVED FROM ANTHER CULTURE(Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.), 2020) Goraguddi, Pradeep; Jha, Zenu; Verma, Sunil; Bhandarkar, S.; Saxena, R. R.The more important factors in producing variability in plants are hybridization, mutation and recombination, hence during experiment rice DH lines developed from cross of Dubraj X RP-Bio 226, Swarna X IR159B as well as mutated rice DH lines of Dubraj X RP-Bio 226-40GY, Swarna X IR159B-20GY were used to screen for BLB and Drought respectively. Because hybridization and Gamma radiation based approach was used for increasing the frequency variation. the mutation in callus developed through anther culture in Dubraj x RP-Bio 226(40GY) and Swarna X IR159B(20GY) cross populations was given to allow a biotechnologist to select cultivars of plants which are more tolerant to biotic and abiotic stresses respectively. In order to identify DH plants, molecular marker are used. In the present investigation, we analyzed resistant double haploid lines with the help of molecular markers i.e. xa5, xa13 for bacterial blight and RM55, RM259 for drought resistance and also we screen the different DH lines for BLB resistance at phenotypic level, whereas screening of DH lines for drought carried out by PEG analysis. In Dubraj x RP-Bio 226 DH lines, after screening on the basis of molecular markers as well as phenotypic performance, we have been found that 10 lines of Dubraj X RP-Bio 226 control viz. DXR–11, 17, 28, 35, 38, 39, , 66, 68, 76 and 9 lines in case of Dubraj x RP-Bio 226 (40GY) viz. DXR(40GY)-34, 35, 36, 41, 44, 47, 48, 51, 59 were found to be resistant for BLB with best performance in case of height/plant (cm), 50% flowering time and aroma score but the yield /plant (g) is not good as compare to the recipient parent Dubraj, so there will be need to re-evaluate the all the selected lines for yield/plant in further generation. During the experiment the screening various rice DH lines for drought tolerance has been carried out by the PEG analysis and molecular characterisation with phenotypic data of rice genotypes for drought tolerance. Molecular markers has potential role in screening of drought tolerant rice lines. Here, among the 25 DH lines of Swarna X IR159B (control), the lines viz. Swarna X IR159B-4, 6, 11 and 15 performed best at phenotypic, PEG and genotypic level. Whereas, within the 30 mutated DH lines of Swarna X IR159B (20GY), the lines viz. Swarna X IR159B (20GY)-7, 12, 18, 24, 25 and 26 also performed good at phenotypic, PEG and genotypic level, therefore all selected DH lines of Swarna X IR159B (20GY) and Swarna X IR159B (control) drought tolerant lines are classified on the basis of phenotypic, PEG, and genotypic basis. The system used in this study would therefore be a quick, rapid and cost-effective method for screening seedling traits for drought tolerance of large set of rice DH lines. Therefore further research with other linked SSR markers and yield related parameters for drought tolerance must be carried out in these rice lines.ThesisItem Open Access CHARACTERIZATION OF EFFECT OF PHOSPHORUS DEFICIENCY AND IT’S INTERACTION WITH NITRATE AND AMMONIUM ION SUPPLEMENTS IN RICE(Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.), 2020) Vahane, Soniya; Banerjee, Shubha,Phosphorus, deficiency is a major constrains for rice production. On the other hand nitrogen deficiency severely affects rice growth and decrease yield. As demand of P and N fertilizers increasing continuously, development of tolerance to P and N nutrient deficiency would be more appropriate solution. Modification in rice root morphology and metabolic process under P deficiency also affects the uptake of other nutrients. Organic acid secreted by the rice root, converts organic P to plant available P (inorganic P) so that plant can easily uptake phosphorus. Activity of PM H+ ATPase increases under P deficiency which is coupled with citrate exudation. Increased activity of PM H+-ATPase is reported to result in increase in net uptake of NO3− and NH4+ (Vinicius et al., 2011). Hence it is important to characterize the interaction effect on rice root morphology and on genes related to PM H+-ATPase pump activity. Field evaluation of selected twenty four diverse rice genotypes indicating significant variation in plant height, number of tillers, biological yield and grain yield. Among twenty four genotypes, four genotypes were selected based on their performance under P deficient field condition and grown for 30 days in hydroponic culture under three treatment conditions i.e. P+N+ (N as ammonium sulphate), P-N- (N as ammonium nitrate) and P-N+ (N as ammonium sulphate). In hydroponic condition Sahbhagi Dhan performed well under both P and N ,deficiency stress as well as, under P deficient and N ,sufficient condition,. After 30 days, P was estimated from shoot and root tissue and we found that Sahbhagi Dhan showed maximum shoot P utilization under both P and N deficiency stress ,condition as well as under P deficiency stress,. Whereas maximum root P utilization was observed in Shenong under P-N- stress condition and in RRF-78 under P deficiency stress condition (P-N+). Hence based on hydroponic studies, Sahbhagi Dhan was considered as tolerant for both P and N deficiency stress. Whereas both Sahbhagi Dhan and Shenong were considered as tolerant for P deficiency stress (P-N+). pH was measured and maintained after every two days of interval. Analysis on pH variation showed the treatment effect on genotypes. In Swarna, relatively stable pH was observed at 24th day in P-N+ condition as compared to the control and it showed the minimum effect of P deficiency whereas lower pH was observed at 27th day under P-N- condition. In case of RRF-78, non significant variation was observed in all the three treatment conditions. In Sahbhagi Dhan higher pH was recorded at 27th day under both P and N ,deficiency stress as well as, under P-N+ condition whereas in Shenong, more variation in pH was observed from 24th to 27th day under P-N- condition. Gene expression analysis was conducted with four genes namely OsA3, OsA9, OsSPX1 and OsALMT in all the four genotypes under all the three treatment conditions. Almost all genes were expressed in Sahbhagi Dhan under both P and N deficiency stress as well as under P deficiency stress condition. The gene, OsA3 ,showed differential expression in, all the genotypes. It was not expressed in root tissue of Swarna under P+N+ condition whereas expressed under P and N ,deficiency, stress. OsA9 gene was less expressed in Swarna and RRF-78 under P+N+ condition whereas high expression of the ,gene was, observed under P-N- condition in both shoot and root tissue and multiple bands were observed indicating alternate splicing of the gene under nutrient stress condition. Almost equal expression of OsSPX1 gene was observed in all the four genotypes under all the three treatment condition. OsALMT gene was less expressed in shoot tissue of Shenong under both P and N deficiency stress.ThesisItem Open Access ANALYSIS OF DE NOVO TRANSCRIPTOME SEQUENCE FOR IDENTIFICATION OF PUTATIVE CANDIDATE GENES RELATED TO β- ODAP BIOSYNTHESIS PATHWAY IN LATHYRUS SATIVUS(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2019) Nayak, Jajati Keshari; Banerjee, Shubha; Verulkar, S. B.; Nair, Sunil K.; Saxena, R. R.Grass pea (Lathyrus sativus) is a valuable pulse crop of winter season in terms of low water requirement, tolerance to major abiotic stress, high protein content (~31.6%) and low input cost. It is grown as a fellow crop after rice in tropical region of India, Bangladesh, Nepal, Ethiopia etc. The association of Lathyrus with neurolathyrism, motor degenerative disease caused in human on consumption of L. sativus pulse has led to ignorance of this wonderful pulse crop in main-stream agriculture. The neurological disorder is caused because of presence of a non-protein amino acid β-ODAP in L. sativus seeds which is synthesized as a by-product of by O-acetyl serine metabolism pathway (Malathi et al., 1970). Although the pathway of ODAP biosynthesis was reported long back but the genes which codes the proteins and key enzymes in the pathway are not elucidated yet. Therefore to identify the genes involved in biosynthesis of β-ODAP, RNA sequencing and de-novo transcriptome analysis was done. Leaf tissue transcriptome data of two contrasting ODAP content variety (Mahateora and RLK-1950) was analysed using homology search, protein modelling and enzyme – ligand docking techniques. About 28,000 transcripts were obtained in both the genotypes which were initially annotated using Phaseolus vulgaris gene sequences (Banerjee et al., 2017 unpublished data). NCBI BLAST and LOCAL BLAST was done to classify transcripts based on their function and localization in cell. ODAP is synthesized in mitochondrial and chloroplast of plants where amino acid transferase and synthase classes of enzymes play important role thus sequence alignment search of about 14000 transcripts for similarity with amino acid transferase or synthase enzymes with localization Mitochondria or chloroplast was done. Further Homology modelling through swiss model workspace (https://swissmodel.expasy.org/interactive) to assess protein structure identity and similariy and Modbase (https://modbase.compbio.ucsf.edu/modweb/) narrowed down to 11 putative candidate transcripts. Docking of the protein corresponding to the candidate transcripts with ligands indicated involvement of two isoforms of candidate gene L-alanine synthase enzymes in the process. Conserve domain search also showed that the candidate genes are involved in cysteine metabolism and have role in amino acid biosynthesis pathway. Pathway mapping was done by BLAST2GO and NCBI biosystem to deduce the involvement of putative candidate genes in various physiological and biochemical pathways. Sequence search and protein modelling led to identification of 11 candidate genes which play key role in ODAP synthesis. Six primers were designed from these potential transcripts and used for expression profiling in leaf tissue of selected L sativus genotypes having different seed ODAP contents. Differential expression of the primers PLPD1 was observed in the high and low seed ODAP containing genotypes. The primers PLPD1 showed two amplicons of length 150 bp and 200 bp, again correlating to the two isoforms found in the sequence analysis. Of these the longer amplicos 250 nt was found to be correlated to high seed ODAP content and the smaller amplicon of 150 nt with low seed ODAP content in Mahateora and Pusa-24. So far, to the best of our knowledge this is the first report of identification of two putative candidate genes encoding L-alanine synthase isoforms in L. sativus. Further sequencing of the amplicons will be done to confirm their role in ODAP biosynthesis and isolate the candidate genes encoding two isoforms of L-alanine synthase in L. sativus.ThesisItem Open Access MOLECULAR CLONING AND CHARACTERIZATION OF WATER STRESS RESPONSIVE NAC FAMILY TRANSCRIPTION FACTOR GENE FROM MINOR MILLET(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) Patil, Arun Hanumantrao; Chandel, Girish; Banerjee, Shubha; Nair, S.K.; Saxena, R.R.Drought stress is one of the abiotic stresses which may alter plant growth, metabolism and yield. Water stress limits the growth, productivity and quality of agricultural crops in the world. Minor millet is known for its great level of tolerance against drought, salinity and diseases. To date, the scope of functional genomics research in minor millet has been quite limited. The NAM, ATAF1/2 and CUC2 (NAC) domain protein family are classic transcription factors (TFs) involved in a variety of abiotic stresses, particularly drought stress. However, the NAC TFs in minor millet have not been characterized. It is necessary to dissect the transcriptome information under different stress conditions to prospect novel genes. Characterization of drought responsive genes in minor millets will provide valuable information on molecular mechanism and dynamics underlying their drought tolerance potential. Important genes identified through such investigation can serve as useful/ novel candidates for improvement of drought tolerance characteristic in other crops as well. With these considerations, the current study was undertaken to characterize drought tolerance mechanism in millets at biochemical and molecular level. In this study, the photosynthetic pigments like chlorophyll a, chlorophyll b and total chlorophyll decreased and the biochemical components like, proline, leaf protein and carbohydrate increased under water stress. Little millet genotype RLM-37 showed minimum decrease in chlorophyll content, relative water content (49%) and maximum increase in proline (17.103 fold), protein (22.36 fold) and carbohydrate content (2.571 fold) when compared to the previously reported tolerant millet genotype. Accumulations of soluble carbohydrates, proline and protein increased under water stress and the genotypes that have more RWC and chlorophyll content are more resistant to drought stress. In this study little millet genotype RLM-37 perform better under water stress condition and showing their ability toward tolerant to water stress. Semi quantitative RT PCR was performed to analyze the expression pattern of six NAC TF family genes in minor millet under water stress and control condition. The gene includes, EcNAC67, OsNAC29-1, OsNAC29-2, SiNAC29L, ZmNAC1 and TaNAC4. The result revealed that the all genes are up regulated under water stress treatment in millets. Among seven millet genotypes, little millet genotype RLM 37 showed a higher level of up regulation for gene EcNAC67 (16.4 fold), followed by OsNAC29-1 (7.4 fold), OsNAC29-2 (2.2 fold), TaNAC4 (2.7 fold), ZmNAC 1 (1.8 fold) and SiNAC29 (2.1 fold). Based on the expression pattern EcNAC67 gene was identified as positively up regulated under waters stress in millet genotypes. These genes are not expressed in the well watered plants, but only in the stressed RLM-37 genotype. Biochemical and expression analysis result was used as a basis to select the gene (EcNAC67) and genotype (RLM-37) for the ortholog gene isolation. PsNAC67 gene (orthologous to EcNAC67 gene of Finger millet) have been isolated and sequenced from RLM-37 genotype of little millet. Computational characterization of PsNAC67 gene revealed that the presence of conserved NAM (No apical meristem) protein domain between the 29-153 amino acid position and motif in the predicted protein of PsNAC67 gene sequence. The identified genes showed high level sequence homology to known NAC TF genes. The presence of these features unique to NAC TF genes confirms them as true and functional NAC TF genes from little millet. Further the identified PsNAC67 gene was cloned into plant expression vector pCAMBIA1301. The recombinant/ positive clone were screened and confirmed for PsNAC67 insert by colony PCR and restriction digestion of recombinant plasmid. Our result provides useful information for the functional characterization of minor millet NAC TF genes and rich resource and opportunity for understanding little millet drought stress tolerance mechanism. Plant transformation and over expression studies using this gene construct will be useful for development of water stress tolerant transgenic in other related crops.ThesisItem Open Access IDENTIFICATION AND DEVELOPMENT OF ALLELE SPECIFIC SNP / INDEL MARKERS FOR YIELD RELATED GENES USING WHOLE GENOME RE-SEQUENCING APPROACH IN RICE(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) Thombare, Devidas Tulsiram; Verulkar, S. B.; Prasad, Archana S.; Sarawagi, A.K.; Saxena, R.R.Rice (Oryza sativa L.) is one of the major staple foods of developing countries of Asia.. The world population is projected to increase 9 billion by 2050 years, and therefore the world’s rice production has to increase by 25% or more to meet demands imposed by this projected population growth. To achieve the targeted yield gains, all the approaches of genetic improvement has to be used, including the genomic tools which has a potential of increasing the precision of selection and increasing genetic gains. In this study, 147 germplasm lines were evaluated during kharif season 2016 and 2017 under irrigated and drought stress condition and phenological data for 17 yield related traits were recorded. On the basis of mean performance, significant variation was observed in all the traits under irrigated and drought stress condition. Based on the overall mean performance of 17 phenological traits, with particular emphasis on grain yield, 5 genotypes were selected and used for Sanger sequencing and primer walking, for the identification of SNPs / Indel markers. These markers or FNPs (Functional Nucleotide Polymorphic) they in turn facilitate the identification of novel/superior alleles which may have fixed during the evolutionary process in local germplasm lines. In the selected rice germplasm collection of Chhattisgarh region along with parents, donors and advance breeding lines sequencing of 9 yields related genes (Gn1a, SPL14, Ghd7, SCM2, GS5, GIF1, GS3 and DEP1) was carried out in Sanger sequencing and primer walking through out sourcing (Scigenome Pvt. Ltd. Hyderabad). DNA polymorphisms were obtained by amplicons sequencing data to identify the yield related genes containing putative FNPs. The Gn1a gene from a promoter 5’ UTR region showed that the presence of 7 SNPs and 4Indel in Swarna and 17 SNPs and 2 Indels in R-RF-78 genotype on chromosome #1. From a promoter region of SPL14 genes 14 SNPs and 4Indels in swarna, while 1 SNP in E1703 and followed by 2SNPs in E1827 genotypes on chromosome 8 were detected and Sequence analysis showed that Swarna genotypes had 5SNPs and 1Indel followed by 25 SNPs / 1SNP in wild rice1 and 1Indel in E1827 genotypes in 3’- exon region on same chromosome. Swarna and Lalmati genotypes were amplified in SCM2 gene from a promoter region. Sequence analysis of SCM2 gene showed the presence of 3SNPs/2Indels in Swarna followed by 24SNP/9 Indels in Lalamati genotypes on chromosome 6. Sequence analysis of SCM2 gene from promoter region (3.2-1.9 kb) showed that the presence of 28SNPs/7Indels in Swarna. For Ghd7 gene from a 1’-exon region showed that the 12SNPs/4Indels in E1703 and 9SNP/4 Indels in E1827 genotypes on chromosome 7 were present while on same gene from a 2’-exon region exhibited the presence of 4SNPs/1Indels in Swarna, 3SNPs followed by 2SNPs/1Indel in E18027 genotype on same chromosome. The GS5 gene from a promoter region showed that the swarna genotypes were found 12SNPs/13Indels, 9SNPs/2Indels in wild rice1, 25SNPs/19Indel in E1703, 16SNPs/9Indels in E1827 followed by 24SNPs/19Indels in M2260 genotype on chromosome 5. The sequence analysis of GIF1 gene showed the presence of 11SNPs/12Indels in swarna, 13SNPs/1Indel in B6 and 12SNPs/ 5Indels in E1703 genotypes on chromosome 4. Sequence analysis of GS3 gene showed that the presence of 8SNPs/2Indels in swarna, 26 SNPs/6Indel in wild rice1 and 11SNPs/2Indels in wild rice116 on chromosome 3. In DEP1 gene, five genotypes were amplify from a 5th’-exon region which were sequenced by Sanger sequencing. Sequence analysis of DEP1gene showed that the presence 4SNPs in swarna, 53SNPs in wild rice1, 2SNPs in kalokuchi223, 3SNPs in E1703 and 2 SNPs in M2260 genotypes on chromosome 9. Based on sequence analysis, we developed 24 SNP/Indel markers for 9 yield enhancing genes, including allele determination of target genes in the rice germplasm panel/mapping population. For rapid improvement of genetic yield potential in rice, we need to evaluate and use the previously identified yield enhancing genes in local germplasm accession. The allele specific markers for 9 yield enhancing genes will be helpful for MAS breeding of yield enhancing traits/genes. The Tetra primer ARM-PCR method was used for allele specific primer designing. These newly developed and synthesized markers (7) along with 13 already reported markers were used for the amplification of DNA from a panel of germplasm lines a mapping population of Danteshwari/Dagaddeshi (F16 generation with 124 lines) for cross validation. Single marker analysis (SMA) used for this study, though the simplest associated with single markers of the phenotypic data based on genotype at each marker traits associated. We identified marker-trait association for the 12 agronomic traits include DTF, PH, FLL, FLW, TNT, NFSPP, NSSP, SF, GW, LB, GY and HI under drought condition on 2016. Marker Gn1a-17 SNP on chromosome 1had the greatest effect on the traits HI and GY up to 0.0385 and 0.001. Similarly Gn1a-Indel3 on chromosome number 1 had the significant association with PH, LB, GY and HI up to 0.0407, 0.0491, 0.0581 and 0.0352 % respectively.ThesisItem Open Access IDENTIFICATION OF POTENTIAL FLUORESCENT PSEUDOMONAS ISOLATES INDUCING DROUGHT TOLERANCE IN RICE AND WHEAT(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) AKASH, P.; VERULKAR, SATISH B.; JHA, ZENU; KOTASTHANE, ANIL S.; SAXENA, R.R.The present investigation was carried out in Microbial Biotechnology Laboratory at the Department of Plant Molecular Biology Laboratory, College of Agriculture, Indira Gandhi Agricultural University, Raipur, Chhattisgarh. Roots are the important part of a plant which were unnoticed in the circle of crop production of cereals. Roots which grow into the complex soil system have to face a lot of obstacles, both abiotic and biotic.The role of microbes in management of biotic and abiotic stresses is gaining importance and they are one of the major parts of soil which shows tremendous different activities in relation with crops. Beneficial microorganisms colonize the rhizosphere / endo-rhizosphere of plants and promote growth of the plants through various direct and indirect mechanisms. The development of stress tolerant crop varieties through genetic engineering and plant breeding is essential but a long drawn process, whereas microbial inoculation to alleviate stresses in plants could be a more cost effective environmental friendly option which could be available in a shorter time frame. Finding the effective isolate which can induce water stress tolerance in host plants requires both laboratory and on field studies. In the present study 31 fluorescent pseudomonas isolates(P1, P2, P3, P4, P5, P6, P7, P8, P10, P11, P12, P13, P14, P16, P17, P18, P19, P21, P22, P23, P24, P25, P27, P28, P29, P30, P66, P141, P200, P229, P260) were tested under different aspects like responds to different biochemical tests, PCR based molecular analysis and actual field performance. PCR based DNA finger printing revealed polymorphism between 31 isolates of fluorescent pseudomonas. All cultures responds variably towards biochemical tests and P5, P7, P8 and P10 were identified as the most potential isolate which can induce water stress tolerance to rice and wheat. These potential isolates can be further used for gene expression studies in host crops and can explore different array of changes.ThesisItem Open Access “STANDARDIZATION OF IN-VITRO PROPAGATION PROTOCOL FOR CHIRONJI (BUCHANANIA LANZAN SPRENG)”(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) PATEL, INDRAPAL; (SMT) JHA, ZENU; KOTHESTHANE, A.S.; SHARMA, DHANANJAY; SAXENA, R.R.BuchananialanzanSpreng is a tree which belongs to Anacardiaceae family, which produces seeds commonly known as Chironji. The species was in abundance in past, as vit was the backbone of rural economy of tribals particularly in Chhattisgarh, Madhya Pradesh and Uttar Pradesh.The major problem in the reforestation or domestication of Chironjiis the low germination percentage of seeds. Vegetative propagation methods are less effective due to less availability of rootstocks and dependency on seasonal conditions. In the recent years, tissue culture has emerged as a promising technique to obtain genetically pure elite plant populations under in-vitro conditions. Present study was conducted in Department of Plant Molecular Biology and Biotechnology, IGKV, Raipur. Different explants (E-1, E-2, E-3 and E-4) of Chironji were inoculated in different media compositions with various combinations of growth hormones for multiple shoot initiation and multiplication (S-1 to S-4) and root initiation (R-1 to R-6).Shoottipof1-2cmsizewereexcisedfromthe1-2 year old plants. The sterilization procedureapplied for all types of explants. Sterilizedexplantswere inoculatedinto the tubes and culture bottlesand5-6weeksafter micro shoots are sub culturing. The maximum shoot initiation percentage was gained with E1S1(28.33) Maximum average number of shoot multiplication E1S1was(5.5%). The maximum root initiation 25 % under the R6 treatments. Shoot tip when used a explant showed best response over leaf, root and node explant and maximum shoot initiation %was obtain in the treatment S1- ½ WPM + BAP (2.0 mg/L) + GA3 (0.5 mg/L) + kn 1.0mg/l. In-vitro propagation techniques may further be improved for commercial production of planting materials from elite high yielding lines.ThesisItem Open Access “γ -IRRADIATION FOR GENERATION OF ADDITIONAL VARIATION IN ANTHER CULTURED RICE PLANTS”(Indira Gandhi Krishi Vishwavidyalaya, Raipur, 2018) NAWRANG, SATISH KUMAR; (SMT) JHA, ZENU; VERULKAR, S.B.; BHANDARKAR, S.K.; SAXENA, R.R.γ -irradiation is one of the physical mutagens that are widely used for mutational breeding. Anther culture plays an important role in rapid development of homozygous lines and genetic manipulation, especially in isolation of desirable individuals from segregating population in the early generation. The production of new varieties via anther culture shortens the breeding cycle, raises the efficiency of selection, and saves space and labor in the experimental field. For these reasons, intensive research on rice anther culture has been carried out since long time. In vitro irradiation is considered as a powerful method for rice improvement because γ-ray at suitable dose not only stimulates callus initiation and plantlet regeneration but also induces mutation. The present work was carried out to study the effects of γ-irradiation on growth of anther cultured rice plants. Callus obtained from anther culture of indica rice cross Swarna sub-1 x IR 90019-17-159-B, MTU1010 x Dagaddeshi, Safri-17 x RP bio-226 x PR-122, Dubraj x RP Bio, Safri-17 x RP Bio-226, Safri-17 x PR-122, DRP-263 and DRP-271 were subjected to radiation with γ rays at 0 (control), 20, 40, 60 and 80 Gy respectively. A gradual increase in maximum green callus was observed when irradiation level was increased and it reached and further decreases at 60 and 80 Gy. The green callus and plantregeneration frequency was significantly stimulated by irradiation of theγ-rays compared with the control. The maximum callus greening percentage 19.35 % (20 Gy) was generated. Maximum number of plant regenerated found in DxR 40 Gy(910). Irradiated callus was transfer to different mediaT11 and T15 along with control for regeneration. T15 was found to be best as it has greeningof 12.44 % of callus. The M-0 plants were raised in field and M-1 seeds were obtained. The seed colour, seed size and widththisis showing variability. The M-1 seed were sown to check the viability obtained of γ-irradiation. To differentiate haploid and diploids plants flow cytometrywas used.The analysis difference shows that the percentage of haploid in calluses SxIR is 89.4, DxRxP263 is 92.5 and SxRxP77.9the analysis confirms the produce of haploid callus through anther culture.ThesisItem Open Access MOLECULAR CHARACTERISATION OF GENETIC DIVERSITY IN AROMATIC RICE GENOTYPES OF CHATTISGARH REGION(Indira Gandhi Krishi Vishwavidhyalaya, Raipur, 2017) Perween, Sabiha; Prasad, Archana Shashank; Jha, Zenu; Chaudhari, Prabha Rani; Saxena, R.R.Demand of aromatic rice is increasing globally and trending upward. The present study carried out with 131 small and medium grained aromatic rice genotypes for deciphering the existing diversity resulted in wide variations among the genotypes for morphological and yield related traits. Maximum variation was observed in biological yield and total number of filled grains. Panicle length was positively correlated with total number of filled grains. Grain yield was recorded maximum and minimum in Bindli and Basmati 213. 100 seed weight was found to vary from 1.79 gm to 4.40 gm in Kanikhabhog and Nepalijoha respectively showing wide diversity. The existing diversity can very well contribute to the gene-pool of aromatic rice cultivars being superior in panicle length, number of filled grains, and test weight. Aroma profiling performed resulted in 4 groups starting from negligible aroma to strong aroma, where Dubraj Selection1 ,Mungfalli, Dhania B2, Chinikapoor was found to strongly aromatic and Tarunbhog Selction 1, Badshahbhog Selection 1 along with 42 other genotypes were found moderately aromatic. Genotyping with a set of 27 functional/gene tagged markers located on chromosome #3, 4, 8 resulted in characterisation of all genotypes as aromatic. The average number of allele was 2.11 ranging from 1 in ARSSR3, SCU 01, RG28L AND FMbadh2 E2a to 4 in 10LO3_FW. The average PIC value of 0.13 indicating the homozygosity due self-pollinated nature of rice plant.RM 5474, RM 5633, RM 282, RM 223, R, 42, RM 3459, BO2_127.8, SCU-RICE SSR-1, BADEX7-5,ARSSR3, RG28L amplified in all genotypes. Badex7-5 and CP04133 classified Tarunbhog Selection1 and Badshahbhog Selection 1 as non-aromatic and Dubraj Selection one as aromatic thereby supporting the hypothesis of existence of new allele or locus for aroma in them. HRM results for detecting SNP for markers RG28L and SCU 01 could not classify the Chhattisgarh core collection distinct groups, indicating the presence of Class 4 SNP which can be confirmed by sequencing. For ARSSR3 the Chhattisgarh core collection was distinctly classified into 7 variants with Tm difference greater than 0.5 ºC Class 1 SNP. HRM results also supported the results obtained from genotyping using the 27 markers and classified Tarubhog Selection 1 with Swarna as same variant. The results of Powermarker, for diversity analysis grouped the 131 small and medium grained aromatic rice genotypes in 3 major groups. IET22787, separated out from rest of the genotypes. Second group consisted of genotypes including Pant Sugandh Dhan 15, Tedesi, Chambalte Basmati etc. whereas the rest were group in a major class. Among the major class, three sub groups originated, in which Dubraj Selection 1 separated out with Rest of the Basmati rice genotypes, Tanrubhg Selection1 separated grouped with Badshahbhog Selection1 and Swarna (negative control) and Jeeradhan was in third sub group with Lohondi and Lehsunhog. The results indicate presence of some other locus or allele among the 131 genotypes that can be the candidate gene for aroma and it can be further validated by sequencing, also the results obtained predict existing of SNPs that could be validated and used for breeding programme in development of elite varieties with improved aroma content.