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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 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.ThesisItem Open Access Microsatellite markers assisted characterization of rice genotypes in relation to salt-tolerance.(Rajendra Agricultural University, Pusa (Samastipur), 2015) Rani, Bibha; Sharma, V. K.A study was done for the screening and molecular characterization of 18 rice genotypes with diverse genetic background for their different adaptation to salt tolerance. Separate experiments were conducted for the screening of genotypes at seedling stage (petriplate experiment in CRD with two replication) with different salinity level (0, 2, 4, 6 and 8 dSm-1), screening at vegetative, reproductive and maturity stage (pot experiment in CRD with two replications) with two set ups- normal and salinized. Salinization was done at different growth stages with different salinity levels (4, 8 and 12 dSm-1 at vegetative, reproductive and maturity stage respectively) and screening under field condition (field experiment in RBD at two sites in kharif season of 2013: South pangabdi for normal condition with pH 7.74, EC 0.41 dSm-1, SAR 11.32 and ESP 14.32 and Harpur jhilli for salt stress condition with pH 9.62, EC 0.37 dSm-1, SAR 9.86 and ESP 12.70). For screening at molecular level, four primers (RM140, RM10665, RM10772 and RM 10864) were selected out of 44 SSR primers used for molecular characterization study. At seedling stage screening, genotypes were evaluated on the basis of modified SES (1-9 scale of salt tolerance), germination percent, shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight and K/Na ratio. On the basis of SES at 8 dSm-1 the genotypes were categorized into highly tolerant with score 1 (CSR36, CSR27-192, CST7-1, CSR2K-242, PNL4-35-20-4-1-4, NDRK11-1 and CSR2K-262), tolerant with score 3 (NDRK11-3 and CR2814-2-4-3-1-1-1), moderately tolerant with score 5 (CR2218-64-1-327-4-1, NDRK11-7, CSR2K-219, KALANAMAK, NDRK11-5, NDRK11-6, NDRK11-4 and RAU1-1648) and susceptible one with score 9 (IR64). Increasing level of salt (NaCl) adversely affected all the morpho-physiological characters over control at seedling stage. IR64 was found to be least tolerant while the genotype CSR2K-262 showed superior performance than tolerant check CSR36. Screening at vegetative, reproductive and maturity stage confirmed the differential response of genotypes at different growth stages and different salt concentrations. The characters studied under pot experiment were chlorophyll content, relative water content, flag leaf area, K/Na ratio, panicle length, tiller number per plant, root length, root volume, root dry weight, grain yield, biological yield, spikelet number per panicle, number of filled and unfilled grain. The agro-morphological and physiological parameters measured at all the growth stages the most tolerant genotypes were CSR36, CSR2K-262, CST7-1 and PNL4-35-20-4-1-4, while the most susceptible genotype was IR64. Under field condition, all the genotypes observed reduction over control for all the morphological characters due to salt stress (sodic soil condition). The characters studied under field experiment were chlorophyll content, days to 50% flowering, plant height, flag leaf area, tiller number per plant, panicle length, biological yield, number of panicle per plant, number of spikelet per panicle, number of filled and unfilled grain per panicle, 100 seed weight, grain yield per plant, root length, root biomass and root volume. Five tolerance indices were studied in the present investigation under pot and field experiment for grain yield per plant viz. TOL, SSI, MP, GMP and STI. According to these tolerance indices the genotypes CSR36 and CSR2K-262 showed best yield performance under stress condition and least for IR64. The characters MP, GMP, STI, tiller/plant, root biomass, 100 seed weight, root volume, plant height were significantly correlated with grain yield/plant under stress. For molecular characterization study, a total of 44 SSR primers were taken covering 8 chromosomes ( chromosome number 1,6,7,8,9,10,11,12) out of 12 chromosomes in rice. The average number of alleles per locus was 9.3 indicating greater magnitude of diversity among plant materials. The average PIC value 0.767 confirmed that the markers used were highly informative. The cluster analysis grouped the 18 genotypes into three major clusters. Cluster I was the largest with 9 genotypes (CSR36, CSR27-192, CST7-1, CSR2K-242, PNL4-35-20-4-1-4, NDRK11-1, NDRK11-3, CR2814-2-4-3-1-1-1, CSR2K-262) with all highly tolerant and tolerant genotypes, Cluster II comprised 8 genotypes (CR2218-64-1-327-4-1, NDRK11-7, CSR2K-219, KALANAMAK, NDRK11-5, NDRK11-6, NDRK11-4, RAU1-1648) with all tolerant and moderately tolerant and Cluster III comprised of 1 genotype (IR64) identified as susceptible one. The maximum similarity value of 0.85 was observed between the genotypes i.e. genotype CSR2K-242 and genotype PNL4-35-20-4-1-4 indicating that these were more closely related. The minimum similarity value of 0.75 was observed between genotypes IR64 and NDRK11-1, IR64 and CR2218-64-1-327-4-1 and IR64 and NDRK11-7 indicating that these two genotypes were most diverse. Further, four SSR markers were selected on the basis of polymorphic nature and clear-cut differentiation to screen the genotypes for salt tolerance. The number of alleles per locus was 4.75. The PIC values for the four SSR markers varied from 0.593 to 0.741 with an average PIC of 0.676. All the genotypes had similar banding pattern with CSR36 (tolerant check) considered as tolerant, while IR64 identified as susceptible.ThesisItem Open Access Biotechnological development of Panama wilt resistance in local cultivars of banana(Rajendra Agricultural University, Pusa (Samastipur), 2015) 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.
