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ThesisItem Open Access Molecular marker- assisted gene pyramiding of yellow rust resistance genes Yr5 and Yr10 into agronomically superior and potential cultivar HS 240 and a doubled haploid genotype DH 40(palampur, 2022-08-17) Singh, Kritika; Chaudhary, H.K.The research endeavour entitled “Molecular marker- assisted gene pyramiding of yellow rust resistance genes Yr5 and Yr10 into agronomically superior and potential cultivar HS 240 and a doubled haploid genotype DH 40” was executed in the Department of Genetics and Plant Breeding, CSK HPKV, Palampur during years 2018 to 2022 with the goal to incorporate genes conferring resistance to yellow rust in wheat into single genetic background and develop homozygous lines following doubled haploidy breeding technology. Marker- assisted backcrossing was employed to introgress Yr5 and Yr10 genes into genetic background of HS 240 and DH 40. Donor genotypes used for transfer of resistance genes were near isogenic lines developed using Avocet variety i.e., Avocet-Yr5 and Avocet-Yr10. Molecular markers STS7/8 and Xpsp 3000 were used for selection of Yr5 and Yr10 genes, respectively. Recovery of recurrent parent genome was quantified using polymorphic microsatellites. 448 SSR markers were used for screening of parental genotype, out of which 69 were polymorphic for HS 240 & Avocet-Yr5 and HS 240 & Avocet-Yr10 whereas, 68 markers for DH 40 & Avocet-Yr5 and DH 40 & Avocet-Yr10. For wide hybridization, intercrossed F1s were crossed with Imperata cylindrica to produce haploids which were further treated with colchicine for the development of homozygous doubled haploid plants. In BC1F1 and BC2F1 generation, plants having target genes were subjected to background selection. Background analysis revealed that the extent of recurrent parent genome recovery ranged from 63.07- 84.06 percent, 62.32- 83.33 percent, 60.29- 83.03 percent and 63.97- 83.82 percent for crosses HS 240*2/Avocet Yr5, HS 240*2/Avocet-Yr10, DH 40*2/Avocet-Yr5 and DH 40*2/Avocet-Yr10, respectively in BC1F1 generation. In second backcross, recovery percentage of recurrent parent genome ranged from 84.06- 92.03 percent, 84.78-92.03 percent, 84.56-91.91 percent and 86.76-93.38 percent in crosses HS 240*3/Avocet Yr5, HS 240*3/Avocet-Yr10, DH 40*3/Avocet-Yr5 and DH 40*3/Avocet-Yr10, respectively. Selected individuals having common recurrent parent were intercrossed to produce pyramided lines. The pyramided F1s were further subjected to development of doubled haploids. Various haploidy and doubled haploidy parameters like pseudoseed formation frequency (PFF), embryo formation frequency (EFF), haploid regeneration frequency (HRF), haploid formation frequency (HFF), percent survived plants (PSP) and doubled haploids formation frequency (DHFF) observed in cross (HS 240*3/Avocet-Yr5)//(HS 240*3/Avocet-Yr10) were 55.07 percent, 44.99 percent, 54.54 percent, 21.82 percent, 37.50 percent and 38.89 percent, respectively. While in cross (DH 40*3/Avocet-Yr5)//(DH40*3/Avocet-Yr10), 48.38 percent, 51.17 percent, 51.63 percent, 22.88 percent, 34.29 percent and 41.67 percent of PFF, EFF, HRF, HFF, PSP and DHFF were recorded on wide hybridization with I. cylindrica. Seven doubled haploid plants were developed from F1s of (HS 240*3/Avocet-Yr5)//(HS 240*3/Avocet-Yr10) and five for (DH 40*3/Avocet Yr5)// (DH40*3/Avocet-Yr10). The results of present investigation suggested that marker- assisted backcrossing and doubled haploidy breeding technology hasten the recovery of recurrent parent genome along with screening of target genes at early stage; and reducing number of generation & time for gene introgression in homozygous state thereby accelerating rapid line development.ThesisItem Open Access IDENTIFICATION OF AN EFFICIENT CHROMOSOME DOUBLING AGENT FOR ENHANCING DOUBLED HAPLOID PRODUCTION EFFICIENCY IN WHEAT × Imperata cylindrica DERIVED WHEAT HAPLOIDS AT in vivo AND in vitro LEVEL(CSKHPKV, Palampur, 2017-12-18) Singh, Kritika; Chaudhary, H.K.The research endeavour entitled ―Identification of an efficient chromosome doubling agent for enhancing doubled haploid production efficiency in wheat × Imperata cylindrica derived wheat haploids at in vivo and in vitro level‖ was executed in the Department of Crop Improvement, CSK HPKV, Palampur during the years 2015-16 to 2016-17 with the goal to determine the relative efficiency of colchicine and other potential chemicals for chromosome doubling in I. cylindrica- mediated haploids at in vitro and in vivo level and identify most potential and economically viable chromosome doubling agent capable of inducing significantly high frequency of doubled haploids in wheat. The material for the present investigation comprised of six wheat F1s (DH-100 × DH-5, DH-5 × DH-100, DH-40 × DH-5, DH-5 × DH-40, DH-65 × DH-5 and DH-5 × DH-65) which were utilized as female parents whereas Imperata cylindrica was utilized as pollen source. During rabi 2015-16 and 2016-17, wheat x I. cylindrica (from last week of March to April) hybridization was carried out followed by treatment of haploids at in vitro and in vivo levels with six chromosome doubling agents namely, colchicine, pronamide, amiprophos methyl, caffeine, oryzalin and trifluralin. To assess the efficiency of different chromosome doubling agents at in vitro level, the embryos of each genotype were cultured in media consisting each of chemicals at three different concentrations and four different durations. At in vivo level, the plants were treated with each of the six chemicals at 3-4 leaf stage for three concentrations and four durations. The response of various chromosome doubling agents applied for different concentration and varied duration revealed significant difference for various haploid induction and doubled haploidy parameters. The statistical analysis of data generated over two years regarding various parameters showed that chromosome doubling was reported at 1500 ppm and 2000 ppm colchicine for 72 hours, pronamide 2 ppm and 3 ppm for 96 hours, amiprophos methyl 5 μM for 96 hours and oryzalin 10 μM for 48 hours and 96 hours at in vitro and 750 ppm and 1500 ppm colchicne for 4 hours & pronamide 2 ppm and 4 ppm for 8 hours and 3 ppm for 16 hours at in vivo level. The chromosome doubling agents which induced doubled haploid production exhibited similar efficiency at in vitro and in vivo level. Pronamide was found to be most economically viable chemical for DH production. DH-100 × DH-5 and DH-5 × DH-100 performed better than other genotypes with respect to various haploid induction as well as doubled haploidy parameters. Use of different chromosome doubling agents to find most potential chemical at in vitro and in vivo level for induction of doubled haploids has opened new vistas in bread wheat improvement programme.ThesisItem Open Access IDENTIFICATION OF AN EFFICIENT CHROMOSOME DOUBLING AGENT FOR ENHANCING DOUBLED HAPLOID PRODUCTION EFFICIENCY IN WHEAT × Imperata cylindrica DERIVED WHEAT HAPLOIDS AT in vivo AND in vitro LEVEL(CSKHPKV, Palampur, 2017-12-18) Singh, Kritika; Chaudhary, H.K.The research endeavour entitled ―Identification of an efficient chromosome doubling agent for enhancing doubled haploid production efficiency in wheat × Imperata cylindrica derived wheat haploids at in vivo and in vitro level‖ was executed in the Department of Crop Improvement, CSK HPKV, Palampur during the years 2015-16 to 2016-17 with the goal to determine the relative efficiency of colchicine and other potential chemicals for chromosome doubling in I. cylindrica- mediated haploids at in vitro and in vivo level and identify most potential and economically viable chromosome doubling agent capable of inducing significantly high frequency of doubled haploids in wheat. The material for the present investigation comprised of six wheat F1s (DH-100 × DH-5, DH-5 × DH-100, DH-40 × DH-5, DH-5 × DH-40, DH-65 × DH-5 and DH-5 × DH-65) which were utilized as female parents whereas Imperata cylindrica was utilized as pollen source. During rabi 2015-16 and 2016-17, wheat x I. cylindrica (from last week of March to April) hybridization was carried out followed by treatment of haploids at in vitro and in vivo levels with six chromosome doubling agents namely, colchicine, pronamide, amiprophos methyl, caffeine, oryzalin and trifluralin. To assess the efficiency of different chromosome doubling agents at in vitro level, the embryos of each genotype were cultured in media consisting each of chemicals at three different concentrations and four different durations. At in vivo level, the plants were treated with each of the six chemicals at 3-4 leaf stage for three concentrations and four durations. The response of various chromosome doubling agents applied for different concentration and varied duration revealed significant difference for various haploid induction and doubled haploidy parameters. The statistical analysis of data generated over two years regarding various parameters showed that chromosome doubling was reported at 1500 ppm and 2000 ppm colchicine for 72 hours, pronamide 2 ppm and 3 ppm for 96 hours, amiprophos methyl 5 μM for 96 hours and oryzalin 10 μM for 48 hours and 96 hours at in vitro and 750 ppm and 1500 ppm colchicne for 4 hours & pronamide 2 ppm and 4 ppm for 8 hours and 3 ppm for 16 hours at in vivo level. The chromosome doubling agents which induced doubled haploid production exhibited similar efficiency at in vitro and in vivo level. Pronamide was found to be most economically viable chemical for DH production. DH-100 × DH-5 and DH-5 × DH-100 performed better than other genotypes with respect to various haploid induction as well as doubled haploidy parameters. Use of different chromosome doubling agents to find most potential chemical at in vitro and in vivo level for induction of doubled haploids has opened new vistas in bread wheat improvement programme.
