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Dr. Rajendra Prasad Central Agricultural University, Pusa
In the imperial Gazetteer of India 1878, Pusa was recorded as a government estate of about 1350 acres in Darbhanba. It was acquired by East India Company for running a stud farm to supply better breed of horses mainly for the army. Frequent incidence of glanders disease (swelling of glands), mostly affecting the valuable imported bloodstock made the civil veterinary department to shift the entire stock out of Pusa. A British tobacco concern Beg Sutherland & co. got the estate on lease but it also left in 1897 abandoning the government estate of Pusa.
Lord Mayo, The Viceroy and Governor General, had been repeatedly trying to get through his proposal for setting up a directorate general of Agriculture that would take care of the soil and its productivity, formulate newer techniques of cultivation, improve the quality of seeds and livestock and also arrange for imparting agricultural education. The government of India had invited a British expert. Dr. J. A. Voelcker who had submitted as report on the development of Indian agriculture. As a follow-up action, three experts in different fields were appointed for the first time during 1885 to 1895 namely, agricultural chemist (Dr. J. W. Leafer), cryptogamic botanist (Dr. R. A. Butler) and entomologist (Dr. H. Maxwell Lefroy) with headquarters at Dehradun (U.P.) in the forest Research Institute complex.
Surprisingly, until now Pusa, which was destined to become the centre of agricultural revolution in the country, was lying as before an abandoned government estate. In 1898. Lord Curzon took over as the viceroy. A widely traveled person and an administrator, he salvaged out the earlier proposal and got London’s approval for the appointment of the inspector General of Agriculture to which the first incumbent Mr. J. Mollison (Dy. Director of Agriculture, Bombay) joined in 1901 with headquarters at Nagpur
The then government of Bengal had mooted in 1902 a proposal to the centre for setting up a model cattle farm for improving the dilapidated condition of the livestock at Pusa estate where plenty of land, water and feed would be available, and with Mr. Mollison’s support this was accepted in principle. Around Pusa, there were many British planters and also an indigo research centre Dalsing Sarai (near Pusa). Mr. Mollison’s visits to this mini British kingdom and his strong recommendations. In favour of Pusa as the most ideal place for the Bengal government project obviously caught the attention for the viceroy.
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ThesisItem Open Access Molecular characterization of rice genotypes using salt stress responsive candidate gene based markers(DRPCAU, Pusa, 2020) Agarwal, Ruchi; Sharma, V.K.A study was undertaken for characterization of salt tolerance related response of 18 rice genotypes using morpho-agronomic characters and salt stress responsive 12 candidate gene based newly designed markers. Evaluation of genotypic response to salt stress at seed germination and early seedling stage was performed at 4 dSm-1 and 8 dSm-1 EC levels along with control by adopting a completely randomized design with two replications. Pot experiment was carried out at 8 dSm-1 and 12 dSm-1 EC levels given at vegetative and reproductive stages, respectively, along with control in completely randomized design with two replications. Field evaluation was conducted in randomized block design with two replications under normal and salt affected field conditions. The genotypes were further characterized at molecular level using 12 candidate gene based newly designed 23 genic primers and 21 genic microsatellite primers. Differential genotypic response to salt stress was noticed at germination stage and early seedling stage. Based on response exhibited at early seedling stage, the genotypes CSR-13, CSR- 23, CSR-27, CSR-30, CSR-36, CST7-1 and CSR-2K-262 were characterized with considerably higher level of tolerance to salt stress in comparison to the genotypes NDRK-11-1, NDRK-11-3, NDRK-11-4, NDRK-11-5, NDRK-11-6, NDRK-11-7, CSR-2K- 219 and CSR-2K-242, which were found to be moderately tolerant. The remaining three genotypes, namely, IR-36, IR-64 and Swarna, were found to be susceptible to salt stress. Principal component analysis based spatial distribution pattern of the genotypes in two dimensional projections distinctly separated the genotypes into two broad groups. The first group was further divided into two sub groups consisting of six salt tolerant genotypes and nine closely located moderately tolerant genotypes, whereas the second multi-genotypic group consisted of three salt susceptible genotypes. The basic pattern of differentiation and interrelationships among the genotypes was found to be similar in dendrogram. A comparison of the relative mean values of the genotypes evaluated for 14 morpho-physiological attributes, such as, panicle length, panicles per plant, spikelets per panicle, filled grains per panicle and unfilled grains per panicle, 100-seed weight, biological yield, grain yield per plant, root length, root volume, root dry weight, relative water content, SPAD value and K/Na ratio in pot experiment was made with mean index (MI) value. Based on the results, the genotypes CSR-13, CSR-23, CSR-27, CSR-30, CSR-36 and CST7-1 were found to be salt tolerant, whereas NDRK-11-1, NDRK-11-3, NDRK-11-4, NDRK-11- 5, NDRK-11-6, NDRK-11-7, CSR-2K-219, CSR-2K-242 and CSR-2K-262 were rated as moderately tolerant and genotypes IR-36, IR-64 and Swarna were found susceptible. Spatial distribution pattern of the genotypes in two dimensional ordinations based on principal component analysis broadly divided the genotypes into two groups, efficiently separating tolerant and moderately tolerant genotypes from susceptible genotypes. The pattern of genotypic discrimination was similar corroborated by dendrogram. Tolerance indices, such as, TOL, MP, GMP, SSI and STI based principal component analysis and hierarchical cluster analysis also discriminated the susceptible genotypes from moderately tolerant and tolerant genotypes. Relative mean performance of genotypes evaluated under normal and salt affected field conditions for 11 morpho-agronomic characters also reflected that the genotypes CSR- 13, CSR-23, CSR-27, CSR-30, CSR-36, CST7-1 and CSR-2K-262 exhibited tolerant response, whereas NDRK-11-1, NDRK-11-3, NDRK-11-4, NDRK-11-5, NDRK-11-6, NDRK-11-7, CSR-2K-219 and CSR-2K-242 were found moderately tolerant and the genotypes IR-36, IR-64 and Swarna were rated as susceptible. Principal component analysis based spatial distribution pattern separated the genotypes into two broad groups, separating the three susceptible genotypes from moderately tolerant and tolerant genotypes. A similar classification pattern was revealed by average taxonomic distance based dendrogram. Recognizable molecular level genetic polymorphism amongst the 18 genotypes was revealed by amplification of genomic template using 12 candidate gene specific 23 newly designed primer pairs. All primer pairs generated 135 allelic variants with an average of 5.86 alleles per primer. The PIC values exhibited significant variation with the values ranging from 0.308 in primer pair DREB1F-1 to 0.905 in primer pairs SHMT1-1. Considering the level of polymorphism detected, the candidate gene based primer pairs OsHKT2;3-1, OsHKT2; 3-2, SNAC1-1, SNAC1-2, CDMK-1, CDMK-2, DUF6-2 SHMT1- 1, SHMT1-2 and SHMT2-1 appeared to be highly polymorphic and more informative primers for the purpose of discrimination of rice genotypes in relation to salinity tolerance. Principal coordinate analysis and hierarchical classification unambiguously discriminated the tolerant and susceptible genotypes, reflecting the usefulness of these newly designed markers. Using major ion transporter, transcription factor and oxidative metabolic pathway related candidate gene specific newly designed 21 microsatellite primer pairs, ample molecular level genetic polymorphism was detected amongst 18 genotypes. All primer pairs generated 131 allelic variants with 6.23 alleles per primer. The number of unique and shared alleles varied from 0 to 5 and 1-6, respectively. The PIC values ranged from 0.706 to 0.955. Taking into consideration the measures of polymorphism, the primers OsHKT1;5-B, OsHKT1;5-D, OsHKT2; 3-A, OsHKT2; 4–A, DREB1F-C, CDMK-B, DUF6-B, SHMT1- B, and SHMT2-A were found as relatively more polymorphic. Spatial distribution pattern of genotypes based on principal coordinate analysis of the genetic profiles generated by these newly designed primers as well as hierarchical classification unambiguously differentiated the tolerant and susceptible genotypes, validating the usefulness of salt responsive candidate genes based these newly designed microsatellite primer pairs.ThesisItem Open Access Genetic analysis of maize inbreds and hybrids using biochemical and microsatellite markers(DRPCAU, Pusa, 2020) Suman, Sandeep Kumar; Kumar, MithileshCharacterization of inbred lines and hybrids is crucial for release of hybrid and genetic purity analysis. Hence, field evaluation was done at AICRP, Maize, TCA, Dholi farm and lab experiment was done at Molecular Biology Lab, Department of Plant Breeding and Genetics, RPCAU, Pusa for characterization of parental lines and hybrids and genetic purity analysis through biochemical and microsatellite marker. Morphological genetic diversity analysis between thirteen inbreds of maize were done using Mahalanobis D2 statistics for ten characters viz. plant height, ear height, days to 50% tasseling, days to 50% silking, days to 50% Brown husk, ear length/Cob length, ear girth, number of kernels per row, number of kernel rows per cob, grain Yield per Plant. All the thirteen inbred lines under study were grouped in three clusters. Cluster I comprised of eleven inbred lines G18, CLQRC, WLS, HK1, CML490, S8200, S8481, R6429, R4093, VL111 and CLQR. Cluster II and III were found monogenotypic and consisted of CLQ25 and CG18 respectively. The inter-cluster distance between cluster II and cluster III was found maximum 6.67, while lowest inter cluster distance value was found between 4.62 cluster I and cluster II. The maximum value of genetic distance 4.89 was estimated between inbred line WLS and G18, while minimum genetic distance 2.38 was found in between VL111 and S8481. Name of the Student : SANDEEP KUMAR SUMAN Registration No. : D/AB/130/2014-2015 Major Advisor : Dr. MITHILESH KUMAR Degree to be Awarded : Ph.D. (Agricultural Biotechnology) Department : Agricultural Biotechnology and Molecular Biology Major Subject : Agricultural Biotechnology Minor Subject : Plant Breeding and Genetics Year : 2020 Total pages of the Thesis : 144 + xviii (Bibliography) + xxii (Apppendices) Title of Thesis : “Genetic analysis of maize inbreds and hybrids using biochemical and microsatellite markers” The result showed great variability was found in genetic makeup of the inbred lines. Cluster I accommodated eleven inbred lines having maximum mean value for number of kernels row per cob and number of kernels per row. While, minimum value was observed for ear height and plant height. Maximum mean value for grain yield per plant and ear girth was found for inbred line CLQ25 that is present in cluster II. The maximum contribution 26.63% was found for the trait grain yield in manifestation of genetic divergence followed by ear girth (28.47%), ear length (19.23%), number of kernel per row (11.50), number of kernels row per cob (10.04). Out of thirty crosses, eleven crosses manifest positive significant heterobeltiosis for the trait grain yield per plant. Two crosses, CLQ25 × CLQR and WLS × R4093 belongs to high divergent class. It could be deduced that genetic diversity can be used as an authentic parameter to predict heterosis in hybrids. The parental lines and hybrids were characterized with SDS-PAGE using total salt soluble seed proteins. Total salt soluble banding profile identified one hybrid G18 x VL111. The presence of 53 KDa, MW band specific for female parent G18 and 65 KDa, MW band specific for female inbred line confirmed the hybridity and purity of hybrid G18 x VL111. The 27 microsatellite primer amplified a total of 28 loci with an average of 4 alleles per locus. The number of alleles varied from 2 to 8 in case of umc1222 and umc1545 or umc1265 or umc1963 or phi084, respectively. The range of amplification varying from 72-88 in case of umc1327, while in case of phi0116 amplification ranged from 293-345. A total of 112 alleles were found in which 33 were unique and rest 79 were found shared alleles in 13 inbred lines. Based on complementary banding pattern between hybrids and their parents, the six microsatellite markers viz. umc1222, umc1161, umc1196, umc1367, umc1403 and nc133 were found effective marker to distinguish F1 hybrids from its parental lines. These markers were utilized to analyse genetic purity of inbred lines and hybrids.ThesisItem Open Access Characterization and micropropagation of Physalis minima L. in Bihar(DRPCAU, Pusa, Samastipur, 2018) Kumari, Anjani; Kumar, HarshA study was undertaken to characterize populations of Physalis minima L. collected from the seven locations of Bihar. A list of 47 morphological characters were used to score the variations present in the plants. Qualitative and quantitative estimation for the presence of phytochemicals was done to determine the medicinal and nutritive value of the plant. Different populations were also characterized on the basis of their molecular study using ISSR and SSR markers. The superior plant populations were identified on the basis of these studies. The micropropagation of the plants was carried out for their multiplication and maintaining superior characters and also prevent the natural hybridization. The morphological characterization based on 24 qualitative and 13 quantitative characters revealed the presence of variations within and between populations. The variations not reported before were observed, which lead to the identification of new forms of the Physalis minima L. plants in Bihar. The quantitative morphological traits were found to be significantly correlated. The cluster analysis and Principal Component Analysis successfully grouped the populations into three groups. The presence of eight phytochemicals were detected in leaves, stem, fruits and roots of the plants from all the seven populations by using thirteen qualitative tests. The quantitative estimation revealed the presence of a high amount of total soluble sugar, ascorbic acid, protein, phenols, flavonoids and alkaloids in leaves and fruits of the seven populations. The populations showed significant variations in their phytochemical constitution. The dissimilarity coefficient and Principal Component analysis based on quantitative biochemical characters clustered the populations in three groups similar to the groups identified by morphological characterization. The biochemical profile highlighted the fruit and medicinal value of the plant. Three out of the eight SSR markers used in the study failed to achieve amplification in the forty two plants of Physalis minima, suggesting a lack of transferability of these primers. A total of 50 alleles were detected using the five SSR primer pairs. The plants were effectively diversified using the SSR primer based similarity coefficients and Principal Coordinate Analysis. The plants were grouped in three clusters having the plants from different populations. A total of 574 alleles were detected using fifteen ISSR primers. The results of ISSR marker based similarity coefficient and Principal Coordinate Analysis showed corresponding results. The plants were again grouped into three clusters. The similarity coefficients and Principal Coordinate Analysis based on both the markers revealed a similar result, suggesting the applicability of both the markers in scoring the genetic variation present in the Physalis minima L. plants. A high level of genetic heterogeneity was identified in the populations based on molecular studies. The results of the three studies were found to be related suggesting that the variations in morphological and biochemical parameters are a result of the genetic variations. The Selao was identified as the superior population based on the score of biochemical characters. Since the Physalis minima L. populations are highly heterogeneous, there is a need for purification of the populations which can lead to the development of superior genotypes. The tissue culture responses were found to be independent of the effect of population. The shoot apical bud followed by nodal stem were identified as the best explant for most of the tissue culture response except swelling and callogenesis for which leaves were the most effective. MS 30 (1 mg l-1 BAP and 1 mg l-1 2, 4-D) was identified as the best medium for differentiation of multiple shoots in shoot apex and nodal stem while MS 2 (1 mg l-1 BAP) was the best for leaves. MS 7 (1 mg l-1 KIN) was invariably identified as the best medium with the highest number of shoots per explant for all the explants. The leaves were the best explant for callogenesis. The in vitro flowering and fruiting were obtained from shoot apex and nodal stem culture. MS 32 (1.0 mg l-1 BAP and 1.5 mg l-1 2, 4-D) for flowering and MS 30 for fruiting was identified as the best medium for both the cultures. R3 (MS + 1 mg l-1 NAA) was identified as the best medium for rooting. The in vitro studies led to the development of a highly efficient protocol for micropropagation of Physalis minima L.ThesisItem Open Access In vitro morphological and molecular evaluation of rice genotypes and validation of markers for salinity tolerance(DRPCAU, Pusa, Samastipur, 2018) Kumari, Rima; Kumar, HarshA set of thirty rice varieties including two tolerant (Pokkali and CSR-36) and two susceptible (IR-29 and IR-64) checks were screened for assessment of their salt tolerance at early seedling stage on the basis of in vitro seed germination and seedling growth at different salinity levels (0, 4, 8 and 16 dS m-1) created by application of salt mixture consisting of NaCl, CaCl2, Na2SO4 in 7:2:1 ratio. Seed germination and seedling growth were adversely affected with the increase of salt concentrations. The salt tolerance index (STI) of the rice varieties was calculated on the basis of seed germination, seedling shoot and root dry weights at different levels of salt stresses. Phenotypic grouping of the thirty rice varieties for their salt tolerance level on the basis of overall salinity tolerance indices across in vitro seed germination and seedling growth (shoot and root dry weight) under salt stress clearly reflected that seventeen varieties, namely, Pokkali, CSR-36, Mandakini, Kranthi, Jyothi, Bardhan, Pusa Sugandh-2, Duna Sankhi, Sanwal Basmati, Ratnagiri-4, Shanthi, Rajendra Dhan-102, Sahbhagi Dhan, Vaisak, Annada, Badami and Jyotrirmayee were highly tolerant to salt stress. Among the remaining entries, Pusa-834, Sarsa, Govind, Khira, Pusa Sugandh-5, MTU-7029 and Saraswathi were found to be moderately salt tolerant, whereas varieties IR-29, IR-64, Daya, Kalinga-3, Golaka and Shatabdi were highly susceptible to salt stress. Out of thirty rice varieties, eighteen rice varieties, namely, Pokkali, CSR-36, IR-29, IR-64, Mandakini, Pusa Sugandh-2, Saraswathi, Ratnagiri-4, Rajendra Dhan-102, Sahbhagi Dhan, Badami, Sarsa, Jyotrirmayee, MTU-7029, Golaka, Daya, Vaisak, and Shatabdi were further screened with the help of ISSR, SSR, salt stress responsive candidate genes and EST primer pairs for the purpose of their molecular profiling in relation to their salinity tolerance. Genetic profiling of entries with a panel of 14 ISSR markers generated altogether 483 allelic variants including 236 shared and 247 unique alleles with an average of 34.50 alleles per primer, revealing ample extent of genetic differentiation and divergence amongst the entries. Among these markers, 811, 814, 815, 823, 834, 836, 840, 841, 842, 872 were found to be highly polymorphic and informative on the basis of their PIC and PP values. However, using a panel of salt stress response related 24 SSR primer pairs, altogether 205 allelic variants including 114 shared and 91 unique alleles were detected with an average of 8.54 alleles per primer due to length variation of simple sequence repeats. Simple sequence repeat loci with di-nucleotide and tri-nucleotide repeat motifs detected greater number of alleles than the repeat loci with tetra-nucleotide and complex repeat motifs. Additionally, the simple sequence repeat loci with CT, GT, AT, AG and AC di-nucleotide repeat motifs detected greater number of alleles. Contrarily, the loci with GA and CA di-nucleotide repeat motifs appeared to detect relatively lesser number of alleles. Considering the number of alleles generated in conjunction with the level of polymorphism detected in the present study, the primers RM 302, RM 8094, RM 10665, RM 10694, RM 10748 and RM 10825 appeared to be highly polymorphic and comparatively more informative primers. Six SSR primer pairs, namely, RM 140, RM 1287, RM 3412, RM 10745, RM 10764 and RM 10772 were validated on the basis of their efficiency to distinguish salt tolerant varieties from susceptible varieties. These six primers can be utilized for the purpose of genetic differentiation and discrimination in relation to salt stress responsiveness of the rice genotypes. Similarly, microsatellite containing salt stress responsive candidate gene (OsHKT1;5, SNAC1, CDMK, CCC, SHMT1 and SHMT2) and microsatellite lacking salt stress responsive candidate gene (OsHKT1;1, OsHKT1;3, OsHKT2;3 and OsHKT2;4) specific markers based genetic profiling allowed unambiguous discrimination of salt stress responsive and tolerant entries, validating their utility for the purpose of differentiation and discrimination of salt stress sensitive and tolerant varieties. Principal coordinate analysis completely supported the results obtained from hierarchical classification of the varieties. Using a panel of eight salt stress responsive EST-contigs based markers, monomorphic bands amongst all the 18 varieties were recorded for six markers, namely, Contig2 (Ferritin superfamily), Contig54 (Plant peroxidase superfamily), Contig138 (ATPase expression protein), Contig314 (Exonuclease), Contig545 (Major latex protein) and Contig633 (Protein kinase), revealing genetic similarity with respect to primer binding sites and molecular size of targeted genomic regions. Remaining two markers specific to Ferritin superfamily (Contig43) and Microtubule associated protein (Contig215) genes revealed genetic polymorphism in the form of presence or absence of bands. Thus, combining in vitro morphological and molecular assessment, seventeen varieties were considered as salt tolerant varieties which can be used as parental donor in rice breeding programme to develop salt tolerant rice varieties.ThesisItem Open Access Molecular dissection of quantitative trait locus in relation to drought tolerance in rice(DRPCAU, Pusa, Samastipur, 2019) Kumar, Pankaj; Sharma, V.K.Thirty-one genotypes of rice were evaluated for various morpho-physiological characters and to study the microsatellite markers based polymorphism for their characterization and genetic diversity among these rice genotypes. Identification of putative gene within QTL (qDTY1.1) and their expression profiling for enhance the drought tolerance. Experimental materials were estimated in randomized block design with three replications during two consecutive years. Molecular characterization was done of the genomic DNA using a set of 23 microsatellite primer pairs. Gene expression response of drought tolerant (Nagina22 and Sahbhagi dhan) and drought sensitive (IR64) rice genotypes to 15 days of drought stress in reproductive stage. Statistical methods and parameters used for deriving inference were analysis of variance, range, mean values, pooled analysis, relative mean performance, and drought tolerance indices. An analysis of variance revealed significant differences among the genotypes for all the characters evaluated during present study. On the basis of the relative mean performance of 15 genotypes, namely, Sahbhagi, Nagina22, MTU1010, Silhatidhan, RAU1417-11-2, Rajendra Bhagwati, RAU1415-7-1, Anandidhan, Rajendra Nilam, Aus257, IR7343-71-1, Chengari2, Kali-Aus, Dangar and Dular out of 31 genotypes were appeared to be relatively more drought tolerant than other genotypes under evaluation. Four tolerance indices viz. TOL, SSI, DTE and STI were used. On the basis of stress tolerance level, stress susceptible index, stress tolerance index and drought tolerance efficiency, 18 genotypes, namely, IR7343-71-1, Chengari2, Aus257, MTU1010, Dangar, Anandidhan, Silhatidhan, Dular, Kali-Aus, Nagina22, Sahbhagidhan, Rasi, Vandana, R.Nilam, R.Bhagwati, RAU1421-12-4, RAU1417-11-2 and RAU1415-7-1 out of 31 genotypes were identified as relatively more tolerant to drought stress. A total 179 and 73 unique allelic variants were detected among the eighteen rice genotypes with an average of 7.8 alleles per primer. The primers RM 5359, RM 7025, RM 10772 and RM 11008, appeared to be highly polymorphic and comparatively more informative primers. Analysis of divergence pattern based on microsatellite markers allowed differentiation and classification of rice genotypes into two groups. The first multi-genotypic group consisted of ten genotypes, namely, IR7343-71-1, Chengar2, Aus257, MTU1010, Dangar, Anandidhan, Silhatidhan, Dular, Kali-Aus and Nagina22 whereas the second multi-genotypic group consisted of eight genotypes, namely, RAU1401-18-5, RAU1428-54-5, RAU1421-15-7, RAU1421-12-4, RAU1417-9-3, Sahbhagidhan, RAU1397-18-7 and Rasi. The magnitude of similarity coefficient between Anandidhan and Dangar (0.739) was found to be the maximum which indicating that these were more closely related. A set of microsatellite markers used for molecular characterization in the present study showed very high degree of efficiency in discrimination of genotypes in relation to drought tolerance. In this study we have identified 73 genes within QTL (qDTY1.1) using sequence information available in rice genome database and bioinformatics tools. The expression of identified genes were analysed. Our studies showed that two genes namely LOC_OS01g66520 and LOC_01g 66170 were upregulated consistently in the time point after drought imposed. Most of the genes were in downregulated.ThesisItem Open Access Biotechnological development of Panama wilt resistance in local cultivars of banana(RAJENDRA AGRICULTURAL UNIVERSITY, BIHAR PUSA (SAMASTIPUR), 2017) Kumari, Anita; Kumar, HarshBiotechnological development of resistance to Panama wilt, the most important disease caused by Fusarium oxysporum f.sp. cubense, was explored in four important local cultivars of banana namely Robusta, Alpan, Malbhog and Kothia employing tissue culture studies and genetic transformation. Using tissue culture, efficient micropropagation protocol and disease resistance utilizing somaclonal variation were developed. Biological control and management of the disease was done utilizing endophytic association of nonpathogenic strain of Fusarium oxysporum. The culture of shoot apices and male flower buds resulted in their establishment and swelling, and callogenesis, caulogenesis, differentiation of shoots and roots from them. Elongation of existing shoots was observed from shoot apices and somatic embryogenesis from floral buds only. The best medium for caulogenesis was BM19 (MS+1.14 µM IAA+19.97 µM BAP) for cultured shoot apices and medium BM8 (MS+22.19 µM BAP) for cultured male flower buds. The best medium for somatic embryogenesis was BM20 (MS+18.10 µM 2,4-D+5.37 µM NAA+ 5.71 µM IAA+ 1mg Biotin). The cultivar Robusta was the best for all tissue culture responses except somatic embryogenesis followed by either cultivar Alpan or cultivar Malbhog. The cultivar Kothia showed the least tissue culture responses. However, the best response for somatic embryogenesis was observed in cultivar Kothia followed by cultivars Malbhog, Alpan and Robusta respectively. The subculture of in vitro developed multiple shoots on medium BM19 resulted in cent percent shoot multiplication with a high multiplication rate. An efficient micropropagation protocol was developed for banana cultivars. The Panama wilt resistance was developed in two susceptible cultivars Alpan and Malbhog exploiting somaclonal variation. Endophytic nonpathogenic Fusarium oxysporum strains were found effective in biocontrol of wilt disease in banana. Genetic transformation of callus and regenerating shoots was done using GUS and eGFP marker gene constructs and it was confirmed using histochemical GUS assay and fluorescence microscopy respectively, and by molecular validation using specific SSR primer pairs. Thus, the work established a suitable protocol of the genetic transformation, which can be used for the development of Panama wilt resistance in local cultivars of bananas.ThesisItem Open Access Microsatellite markers based assessment of genetic diversity and construction of heterotic groups in maize(Dr. Rajendra Prasad Central Agricultural University, Pusa (Samastipur), 2016) Punya; Sharma, V. K.A study was undertaken to elucidate the nature and extent of differentiation and divergence among eighteen inbred lines of maize and to separate them into heterotic groups based on the analysis of targeted microsatellite sites. Experimental materials of the present study comprised fifteen inbred lines, three testers and forty five single cross experimental hybrids, which were evaluated in randomized block design with three replications under rabi and kharif seasons. Statistical analyses to derive inferences regarding divergence pattern of parental lines and heterosis manifestation in experimental hybrids were carried out using the data recorded on days to tasseling, days to silking, days to brown husk, plant height, ear height, ear length, number of kernels per ear and grain yield per plant. Numerical taxonomic approach of classification using these eight quantitative characters of agronomic importance based average taxonomic distance was done to discriminate the inbred lines into different clusters. Inbred lines were also raised in pots for extraction of genomic DNA from the young seedlings. Amplification of the genomic DNA was carried out using a panel of twenty eight microsatellite sites specific primer pairs covering all the chromosomes existing in the genome. Microsatellites based allelic diversity data were utilized for classification and formation of heterotic groups of the inbred lines. Analysis of variance revealed the existence of significant differences among the inbred lines and hybrids for all the characters recorded under both the seasons. Mean performance of parents and hybrids significantly varied for the eight quantitative characters under study. Among the inbred lines, CML467 recorded significantly higher grain yield per plant followed by tester CML165, which was statistically at par to inbred lines LM13, HKI586, CML465, HKI162 and HKI323B over the environments. Sixteen crosses recorded significantly higher mean performance and significantly positive heterosis for grain yield per plant. Among these sixteen crosses, the highest yielding cross HKI162 × CML161 was statistically at par to five crosses, namely, CML468 × CML161, CML471 ×CML161, HKI323B × CML161, CML469 × CML165 and CML471 × CML165 for grain yield per plant. Taxonomic distance based cluster analysis revealed ample genetic diversity among inbred lines, which occupied different clusters during rabi and kharif season and across the seasons. The results indicated a close correspondence with respect to clustering pattern of inbred lines during rabi and kharif seasons. A perusal of dendrogram generated on the basis of eight quantitative traits indicated that the inbred lines CML467, CML470 and DH2012 were relatively more diverse from the remaining inbred lines under evaluation. Altogether 296 allelic variants were detected amongst amplified products generated with 28 primer pairs. A total of 49 loci were assigned to 28 primer pairs with an average of 6.04 alleles per locus The number of alleles ranged from 6 in the cases of umc1136, phi072 and umc1179 to 16 in case of phi053 with a range between 56 to 352 bp. Considering the number of alleles generated by different primer pairs in conjunction with the level of polymorphism, the primers umc1297, phi053, umc1266, phi093, bnlg118, phi034, phi115, phi065 and phi084 appeared to be more informative primers. Among the inbred lines under molecular characterization, CML163 and CML467 appeared as the most diverse genotypes. A remarkably higher level of genetic polymorphism was revealed by the use of 28 microsatellite markers. Both quantitative and molecular data based cluster analyses revealed that inbred lines CML 468 and CML469 are closely related to each other. Remarkably greater extent of similarity was also noticed between inbred lines HKI323B and HKI586. All the three testers, namely, CML161, CML165 and CML163, were included in same cluster in both the cases and seemed to be closely related. However cluster analysis using quantitative characters and microsatellite markers based data did not exhibit a complete correspondence. Parental genetic distance based on quantitative data or genetic similarity based on molecular data did not exhibit significant association with grain yield heterosis in hybrids. Results clearly indicated that a high grain yield is not always obtained from hybrids that have high parental genetic distance value, but generally from those with moderate to high genetic distance. A comparative assessment of heterotic groups formed by using microsatellite markers based genetic similarity, hybrid index value and hybrid mean value, indicated that CML471 and CML115 or HKI323B and HKI586 or DH2012 and LM13 belonged to same heterotic group in all the cases. The distribution pattern of the entries into different heterotic groups formed on the basis of hybrid index value and hybrid mean value corresponded with the microsatellite markers based heterotic groups to the extent of 75% and 67% in the first heterotic group, 40% and 29% in the second heterotic group and 33% and 40% in the third heterotic group, respectively. Overall coincidences of inbred lines in heterotic groups formed on the basis of microsatellite markers with the hybrid index value and hybrid mean value based heterotic groups were considerably higher. Results, therefore, indicated that microsatellite markers may be effectively and efficiently utilized to assign the inbred lines to heterotic groups for grain yield and to establish heterotic groups of maize inbred lines.ThesisItem Open Access Genetic Diversity Assessment In Relation To Submergence Tolerance In Rice Using Microsatellite Markers(DR. Rajendra Prasad Central Agricultural University, Pusa (Samastipur), 2016) Prakash, Nilmani; Kumar, MithileshA study was undertaken to initially characterize and evaluate 184 submergence tolerance related varieties of rice, but 38 were taken for further analysis in the present study for five submergence related characters. To investigate the SSR marker based polymorphism for characterization and differentiation, nineteen rice varieties were selected on the basis of maximum plant percent survival to estimate the extent of genetic diversity among these rice varieties using morphological characters and SSR markers. The experimental materials were evaluated in randomized complete block design with four replications at the research farm of Dr. Rajendra Prasad Central Agricultural University, Pusa during Kharif, 2014-15. Using standard formula for calculation of different submergence related traits in rice observations were recorded. The materials were also grown in pots for extraction of genomic DNA from the young seedlings and then targeted amplification of the genomic DNA using a panel of thirty four SSR primer pairs covering especially the chromosome 9 of rice. All molecular studies were conducted in the Rice Molecular Breeding Laboratory, Deptt. of PBG at Pusa. The statistical methods and parameters used for deriving inference were analysis of variance, range, mean values, coefficient of variation, taxonomic distance and similarity coefficient based dendrogram and numerical taxonomic analysis of divergence. High heritability values were observed for all the five submergence related characters percent plant survival, tolerance score, leaf senescence, total shoot elongation and relative shoot elongation. Considerable extent of variability existed for the values of genetic advance expressed as per cent of mean was observed to be high for all five characters relative shoot elongation, per cent plant survival, total shoot elongation, leaf senescence and tolerance score. By considering mean performance in conjunction with clustering pattern of entries based on quantitative attributes, hybridization involving CR 1009 with AnhHsungSeln (CI 1), Karnal Local and Suraha with Swarna, Jadhan and HsungTeing with Moroberekan, FR 13B and S-150 with Paiam, Zobgui and Rom Runtik with IR-36, Kala Bunde with AI Chih AI K Who 2, Swarna Sub-1 with Lunishreeappeared as superior combinations for their further utilization in genetic enhancement programme. A panel of thirty four primers generated 410 allelic variants ranging from four in the cases of RM 23843 to twenty seven in the cases of RM 23662. Altogether 410 allelic variants were detected at 42 SSR loci with an average of 9.76 alleles per locus. Out of 410 alleles, 177 were shared and 233 were unique alleles. The primer pairs RM 8300, RM 23901, RM 23958, RM 215, RM 257, RM 316 and RM 23662 generated amplified products due to amplification of more than one locus. In general, the SSR loci with di-nucleotide repeat motifs tended to detect greater number of alleles than the locus with tri-nucleotide, tetra-nucleotide or penta-nucleotide repeat sequence. The marker with a GA or AG repeat motif showed greater variability than marker with a CT or TG or TA repeat motif among the loci with perfect di-nucleotide repeat motifs. The number of unique allelic variants per primer ranged from 0 out of 5 amplified products in the case of SC 3 to 13 out of 15 amplified products in the case of RM 23901. The primer pairs RM 23901, RM 910, RM 23770, RM 23928, RM 23958, RM 24103 RM 23662, RM 23915 and RM 24005 generated considerably greater percentage of unique alleles. Considering the number of alleles in conjunction with the level of polymorphism detected, the primers RM 23662, RM 23958, RM 257, RM 23901, RM 8300, RM 215, RM 23928, RM 23915, RM 24005, RM 23902, RM 23679, RM 8303, RM 23770 and RM 23788 appeared to be more informative primers. Occurrence of null alleles for a particular repeat locus was noticed with primer pairs RM 23843, RM 23902, RM 23922, RM 24103, and RM 23865 in some of the varieties. The total repeat count of the di-nucleotide SSR loci seemed to be associated with the number of alleles, revealing that the larger the repeat number involved in the SSR locus, the larger was the number of identified alleles. Presence of stutter bands was detected generally for di-nucleotide SSR motif amplified by primer pairs RM 8303, RM 23770, RM 23805, RM 23831, RM 8300, RM 23901, RM 23915, RM 23917, RM 23922, RM 23928, RM 23958, RM 23996, RM 24103, RM 105, RM 215, RM 316, RM 23662, RM 24013, RM 910, RM 7175, ART 5, SC3, Sub 1BC2 and RM 23865 with generation of minor products. Among the rice varieties evaluated in the present study, AnhHsung Seln (CI 1) and HsungTeing exhibited maximum similarity in the divergence analysis using quantitative characters as well as SSR markers. Analysis of divergence pattern based on SSR markers allowed differentiation and classification of rice varieties into eight clusters. Use of SSR markers appeared more efficient in achieving unique and unambiguous characterization and differentiation of varieties used in the present study.The SSR analysis also revealed unique or variety specific allele, which could be useful as DNA fingerprints in the identification and preservation of rice varieties.ThesisItem Open Access Genetic and Molecular analysis with respect to Yellow Vein Mosaic Virus Resistance in Mungbean (Vigna radiata)(Rajendra Agricultural University, Pusa (Samastipur), 2015) Suman, Sugandh; Shahi, V. K.Mungbean, [Vigna radiata (L.) Wilczek], is one of the most important legume crop of Asiatic region. The production of mungbean is severely hampered by mungbean yellow mosaic virus (MYMV) caused by begmoviruses transmitted by white fly, Bemesia tabaci. Use of disease resistant crop varieties is regarded as an economical and durable method of controlling viral diseases. The advancements in the field of biotechnology and molecular biology such as marker assisted selection can be utilized in developing MYMV resistance mungbean genotypes. Taking into consideration the above aspects, the present study was aimed to evaluate mungbean genotypes for resistance to MYMV disease, to characterize mungbean genotypes based on molecular markers and to identify useful molecular markers in relation to MYMV resistance. A total of 35 genotypes of mungbean were screened for resistance against MYMV disease under field conditions and forced inoculation methods. The genotypes were scored on the basis of the degree of incidence of MYMV disease by using 1-9 rating scale and percent disease incidence for their classification into different infection categories. Based on overall response across the seasons, the genotypes IPM 02-14 and PDM 139 were rated as resistant. Highly susceptible response was observed in only one entry, LGG 450. Rest of the genotypes showed moderately resistant to highly susceptible reactions against MYMV. In general, the disease was found to be more severe during kharif season in comparison to summer season. The reaction pattern of the mungbean genotypes based on forced inoculation method was similar to that deduced on the basis of screening of the genotypes against MYMV disease incidence under natural conditions. Eighteen genotypes, namely, IPM 02-14, PDM 139, Pant Mung 4, HUM 12, HUM 1, TMB 37, Pusa 9531, Meha, HUM 16, IPM 99-1-6, Pusa 105, AKM 8803, AKM 9910, Pusa 031, TARM 2, LGG 407, T 44 and LGG 450, were included in the SSR markers based assessment of genetic differentiation and divergence as they showed extremities for MYMV reactions under natural field conditions. Seeds were grown and young seedlings were used for genomic DNA amplification. A panel of 24 microsatellite based primer pairs covering 12 chromosomes in the genome of mungbean were used for genomic DNA amplification. The statistical methods and parameters used for deriving inference were polymorphism information content, similarity coefficient and numerical taxonomic analysis of divergence. Altogether, 183 allelic variants including 112 shared and 71unique allelic variants were generated at 37 loci among the 18 mungbean entries with an average of 4.95 alleles per locus. The primer pairs CEDG 008, CEDG 068 and CEDG 154 among the total primer pairs used in the present study were found to be highly informative. The microsatellite loci with di-nucleotide repeat motifs, in general, tended to detect relatively greater number of alleles than the repeat loci with tri- nucleotide repeat motifs and complex repeat motifs. Appearance of more than one band in the same genotype was noticed revealing the existence of the duplicated region in the genome of mungbean. Presence of stutter bands in the primer pairs, namely, CEDGAG 001, CEDG 037, CEDG 066, CEDG 091, CEDG 092, CEDG 127, CEDG 154 and CEDG 172 indicated the presence of minor amplified products that had lower intensity than the main allele and normally lacked or had extra repeat units. The magnitude of similarity coefficient between LGG 407 and T 44 was found to be the maximum amongst pair-wise combinations of entries. The SSR primer based analysis revealed unique or genotype specific allele which could be useful as DNA fingerprints in the identification and preservation of mungbean genotypes. The use of 24 microsatellite markers in the analysis exhibited a remarkably higher level of genetic polymorphism, which allowed unique and unambiguous genotyping of 18 entries included in the analysis. Cross combinations involving the highly susceptible genotype LGG 450 in combinations with the resistant genotypes IPM 02-14 and PDM 139 were made for the development of F2’s (IPM 02-14 x LGG 450 and PDM 139 x LGG 450) and BC1’s (IPM 02-14 x LGG 450 x IPM 02-14 and PDM 139 x LGG 450 x PDM 139) as the mapping populations. Amongst these three genotypes, LGG 450 was agronomically superior and high yielding but highly susceptible to MYMV, while IPM 02-14 and PDM 139 were low yielders but resistant to MYMV. Out of the seven disease response associated SSR primers used, only the primer pair CEDG 008 was validated and found to be suitable for discrimination of resistant and susceptible types. The present study led to identification and validation of MYMV response related marker, CEDG 008, which may be further utilized for generating superior genotypes with durable MYMV-resistance and hopefully aid in the development of resistant cultivars in relatively shorter time span, in addition to selection of MYMV resistant lines, genetic diagnostics of MYMV and isolation of the gene responsible for resistance to MYMV.
