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INTRODUCTION OF GLYOXALASE I GENE FOR SALT TOLERANCE INTO RICE (Oryza sativa L.) VARIETIES ‘PR118’ AND ‘KITAAKE’ THROUGH PARTICLE BOMBARDMENT
(PAU Ludhiana, 2013) Saroj Kumar Sah; Ajinder Kaur
The present investigation dealing with introduction of glyoxalase I gene for salt tolerance into rice (Oryza sativa L.) through particle bombardment was carried out using two varieties PR118 (indica) and Kitaake (japonica). Calli were induced from both the varieties on MS medium supplemented with 2,4-D (3.0 mgL-1) + BAP (0.25 mgL-1) + proline (600 mgL-1) + maltose (40 gL-1) + phytagel (3 gL-1). Embryogenic calli were subcultured on shoot regeneration MS medium supplemented with BAP (4.0 mgL-1) + NAA (0.2 mgL-1) + sucrose (30 gL-1) + phytagel (2 gL-1) + agar (8 gL-1). An attempt was made to introduce GlyI gene into both the varieties. Mature seed-derived embryogenic calli were used as explants for all the transformation experiments. Success was achieved in both the varieties. Using GlyI gene, in PR118, out of 2600 calli bombarded, 249 putative transgenic plants were regenerated on medium containing hygromycin (30 mgL-1). Likewise, in Kitaake, out of 615 calli bombarded, 469 putative transgenic plants were regenerated on medium containing hygromycin (30 mgL-1). Among the 249 regenerants of PR118, 13 plants were PCR positive and in case of Kitaake, among 615 plants 41 showed PCR positive results. Further, these plants were grown to maturity in transgenic glass house. Presence of copy number of transgene was done by Real Time PCR. In total, a set of 10 PCR positive plants (5 of PR118 and 5 of Kitaake) 10 samples were analyzed, among them sample number four has single copy gene and rest has multiple copy ranging from 2-14 copies. To conclude tissue culture base line has been established in two varieties PR118 and Kitaake. Using this baseline, a total of 54 PCR positive transgenic plants were developed. The protocol developed here is genotype independent and is suitable for japonica as well as indica varieties.
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MOLECULAR PROFILING OF INTROGRESSION LINES DERIVED FROM CROSSES OF O. sativa (L.) X O. longistaminata (A.CHEV. &ROEHR.)
(PAU Ludhiana, 2012) Nguyen Le Van; Kuldeep Singh
Rice (Oryza sativa L.) is staple food for more than a third of the world’s population, and we need to produce 30% more rice to meet the demand in next 25 years. Utilization of wild species for enhancing productivity is one of the several approaches proposed for meeting food requirements. In this study we evaluated a number of BC2F6 introgression lines (ILs) generated from the crosses between O. sativa cv PR114 X O. longistaminata acc IRGC 104301 and using DNA markers identified regions introgressed from the wild species. A set of 45 ILs and three checks were evaluated in a complete randomized block design with three replications for two years. Data were recorded on 11 agronomic traits. Several ILs showed significant increase or decrease over the recurrent parent in yield components like plant height (PH), days to flowering (DF), tiller number (TN), panicle length (PL), spikelet per panicle (SPP), spikelet fertility (SF), grain length (GL), grain width (GW), thousand grains weight (TGW) and plot yield (PY) compared to the recurrent parent PR114. Ten ILs showed significant increase in SPP during both the years over the recurrent parent and the increase ranged from 9.3 – 41.0 per cent. Likewise, five ILs showed significant increase in grain length ranging from 4.95 – 10.1 per cent, two ILs showed significant increase (16.0%) in TGW, and nine ILs showed significant increase (6.4-24.1%) in grain width. Parent polymorphism between PR114 and donor species O. longistaminta was done using a set of 366 SSR markers spanning all the 12 linkage groups and 92 polymorphic SSRs were used for characterizing the ILs. The alien segments introgressed in each of the ILs varied from 1.09% to 13.04%. Alleles which showed positive effect from O. longistaminata were observed in chromosomal regions associated with DF, PL, SPP, GL and GW traits. Alleles associated with negative effects were also observed for PH, TN, SF, PY and grain size traits. The ILs showing significant increase for one or more traits have been identified and these could be used for mapping QTL introgressed for these traits, using bi-parental crosses.
Open Access
MARKER ASSISTED INTROGRESSION OF THE opaque2 (o2) GENE INTO ELITE MAIZE (Zea mays L.) INBRED LINES
(PAU Ludhiana, 2012) Ravneet Kaur; Yogesh, Vikal
Maize (Zea mays L.) is deficient in the essential amino acids, lysine and tryptophan. The low nutritive value of maize endosperm protein is genetically corrected in Quality Protein Maize (QPM), which contains the opaque 2 (o2) gene along with numerous modifiers for kernel hardness. Two normal inbred lines viz. LM12 and LM13 were targeted for conversion into high quality protein versions using gene based SSR markers located within o2 using two generation marker-based backcross breeding program. DMR7 and CML165 were used as the QPM donor parents. In BC2F1 foreground selection for o2 gene was done using SSR marker phi057 in cross 1 (LM12/DMR7//2*LM12) and with SSR marker umc1066 in cross 2 (LM13/CML165//2*LM13). Out of the total 188 BC2F1 plants of cross 1, 87 heterozygous plants were obtained and similarly in cross 2, 114 heterozygous individuals were obtained out of 206 plants. The BC2F1 plants having o2 allele in heterozygous form were further screened using o2 gene flanking SSR marker and the plants either single or double recombinants, were identified. Whole genome background selection on the single or double recombinants using 104 SSR markers identified three plants with 83.7 to 91.0% recurrent parent genome content in each cross and were selfed to generate BC2F2 population. The three BC2F2 families were subjected to foreground selection and phenotypic selection for kernel modification. Fifty six plants in cross 1 and thirty nine plants in cross 2 having o2 allele in homozygous condition were selfed to generate BC2F3 progenies. The kernels from BC2F2 ears were segregated for hardness of endosperm and showed different levels of modification. In BC2F2 kernels the tryptophan concentration ranged from 0.56% to 0.97% for cross 1 whereas for cross 2 it ranged from 0.58% to 0.91%. 50% opaque BC2F3 lines were evaluated for agronomic traits for selection of single plant progenies to generate BC2F4 progenies.
Open Access
CYTOGENETIC CHARACTERIZATION AND MOLECULAR TAGGING OF LEAF RUST RESISTANCE GENE TRANSFERRED FROM Aegilops peregrina INTO HEXAPLOID WHEAT
(PAU Ludhiana, 2012) Deepika Narang; Satinder Kaur
Leaf rust is the most damaging disease of wheat worldwide. Wild germplasm of wheat is known to be a rich source of different useful genes. Ae. peregrina acc. pau 3519, a non-progenitor tetraploid species with UUSS genome, was found to be an excellent source of resistance for various diseases. In the present investigation, WL711-Ae. peregrina introgression lines (ILs) were characterized through Genomic in situ hybridization and no hybridization signal was detected in any of the IL. F2 population derived from the cross of introgression line 973 with WL711, was tested at seedling stage and segregated in a ratio of 191 resistant:63 susceptible plants with 2=0.0052(3:1). At the adult plant stage, the population segregated into 185 resistant: 65 susceptible with 2=0.0053(3:1). The progeny testing in F3 confirmed the transfer of a single gene for leaf rust resistance. Molecular characterization of the IL973 with the SSR markers indicated Ae. peregrina specific introgression on homoeologous groups 1, 2, 5 and 7. Bulked segregant analysis was done using 79 polymorphic markers and one of the SSR marker Xcfd50, detected IL973 specific allele in resistant bulk. This marker has already been reported to be linked with known gene Lr58 from Ae. triuncialis on long arm of chromosome 2B. STS marker Xncw-Lr58 amplified on whole F2 population and it was mapped at a distance of 1.7 cM from leaf rust resistance gene using computer software MapDisto. Nullitetrasomic analysis confirmed the introgression of Ae. peregrina leaf rust resistance gene (LrAP) on the wheat chromosome 2D. LrAP gene studied during the present investigation is either a new gene or an allele of the already known gene Lr58 will be established through allelic tests in subsequent studies.
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Molecular cytogenetic characterization of wheat- Aegilops umbellulata leaf rust and stripe rust resistant introgression lines
(PAU Ludhiana, 2011) Mitaly Bansal; Parveen, Chhuneja
Leaf rust and stripe rust are the most damaging diseases of wheat. Ae. umbellulata, was found to be an excellent source of resistance for various diseases. In the present investigation, characterization of the WL711-Ae. umbellulata introgression lines (ILs) through Genomic in situ hybridization to determine the size of the alien segment and Bulked Segregant Analysis (BSA) for tagging of stripe rust and leaf rust resistance genes introgressed from Ae. umbellulata into Triticum aestivum were undertaken. GISH showed the presence of complete Ae. umbellulata chromosome in ILs T311-5 and T315-5 whereas no hybridization signal was detected in other ILs. BC2F4 population derived from the cross of introgression line T393-4 with PBW343 segregated for a leaf rust (103R:66H:77S; 2=4.102) and a stripe rust resistance gene (108R:64H:74S; 2=6.401). The progeny testing in BC2F5 confirmed the transfer of a single gene for leaf rust resistance. BSA using 31 polymorphic markers indicated the association of short arm of chromosome 5D with rust resistance. The 5DS mapped SSR markers Xgwm190 and Xwmc805 detected parental IL specific alleles in resistant bulk. In order to distinguish the leaf rust and stripe rust resistance gene transferred from Ae. umbellulata (LrU and YrU) from previously mapped Lr57/Yr40 at same location, the Lr57/Yr40 CAPS primer was amplified and the amplicons were sequenced. A number of SNPs were detected which could distinguish two introgressions carrying Lr57/Yr40 and LrU/YrU. Linkage mapping using computer software MapDisto mapped leaf rust gene on 5DS with Lr57/Yr40 CAPS marker at a distance of 2.5cM. Stripe rust resistance gene mapped towards the distal end of 5DS at a distance of 4.1cM from the LrU. LrU/YrU carrying alien introgression was further distinguished from Lr57/Yr40 introgression, through the identification of SNPs in the grain softness protein specific amplicons. The LrU and YrU are proposed to be putatively new genes which provide complete resistance against leaf rust and stripe rust, respectively.
Open Access
MOLECULAR CYTOGENETIC CHARACTERIZATION OF WHEAT- Aegilops umbellulata LEAF RUST AND STRIPE RUST RESISTANT INTROGRESSION LINES
(PAU Ludhiana, 2011) Mitaly Bansal; Parveen, Chhuneja
Leaf rust and stripe rust are the most damaging diseases of wheat. Ae. umbellulata, was found to be an excellent source of resistance for various diseases. In the present investigation, characterization of the WL711-Ae. umbellulata introgression lines (ILs) through Genomic in situ hybridization to determine the size of the alien segment and Bulked Segregant Analysis (BSA) for tagging of stripe rust and leaf rust resistance genes introgressed from Ae. umbellulata into Triticum aestivum were undertaken. GISH showed the presence of complete Ae. umbellulata chromosome in ILs T311-5 and T315-5 whereas no hybridization signal was detected in other ILs. BC2F4 population derived from the cross of introgression line T393-4 with PBW343 segregated for a leaf rust (103R:66H:77S; 2=4.102) and a stripe rust resistance gene (108R:64H:74S; 2=6.401). The progeny testing in BC2F5 confirmed the transfer of a single gene for leaf rust resistance. BSA using 31 polymorphic markers indicated the association of short arm of chromosome 5D with rust resistance. The 5DS mapped SSR markers Xgwm190 and Xwmc805 detected parental IL specific alleles in resistant bulk. In order to distinguish the leaf rust and stripe rust resistance gene transferred from Ae. umbellulata (LrU and YrU) from previously mapped Lr57/Yr40 at same location, the Lr57/Yr40 CAPS primer was amplified and the amplicons were sequenced. A number of SNPs were detected which could distinguish two introgressions carrying Lr57/Yr40 and LrU/YrU. Linkage mapping using computer software MapDisto mapped leaf rust gene on 5DS with Lr57/Yr40 CAPS marker at a distance of 2.5cM. Stripe rust resistance gene mapped towards the distal end of 5DS at a distance of 4.1cM from the LrU. LrU/YrU carrying alien introgression was further distinguished from Lr57/Yr40 introgression, through the identification of SNPs in the grain softness protein specific amplicons. The LrU and YrU are proposed to be putatively new genes which provide complete resistance against leaf rust and stripe rust, respectively.
Open Access
INTRODUCTION AND EXPRESSION OF ANTIFUNGAL CHITINASE AND β-1, 3-GLUCANASE GENES IN ARBOREUM COTTON TO INDUCE RESISTANCE AGAINST Fusarium oxysporum
(PAU Ludhiana, 2013) Aradhna Sonik; Jagdeep Singh, Sandhu
In the present study, immature embryos of diploid cotton (Gossypium arboreum L. cv. RG8) were transformed with three recombinant plasmids viz., H1Z, H2Z and pBI121 containing -1, 3-glucanase, chitinase and GUS gene cassettes respectively, under the control of CaMV 35S promoter and NOS terminator, with an objective to induce resistance against Fusarium oxysporum. The immature embryos were excised from 18-day old cotton bolls obtained during early, mid and late flowering seasons of the diploid cotton plants, cultured on germination medium (GM) [basal MS medium containing proline (560mg/l), BAP (2mg/l), GA3 (40mg/l), activated charcoal (0.20%), omnatax (500ppm) and casein hydrolysate (500mg/l)] followed by incubation under dark at 28±2oC. After five days of culture establishment, the germinating embryos were transferred under 16 hr(s) photoperiod during which they elongated into shoots and developed true leaves. Root multiplication was induced by placing elongated shoots on root induction medium (1/2 strength MS medium fortified with 0.20% activated charcoal). The plantlet formation efficiency of the embryos cultured during the mid flowering season was found to be significantly higher (37.67%) compared to the embryos excised during late (32.80%) flowering season and at par with the efficiency of embryos excised during the early season (36.28%). The three recombinant plasmids–H1Z, H2Z and pBI121, after being confirmed for the presence of gene cassettes by PCR and restriction digestion were introduced in the immature embryos using particle bombardment. The integration of gene cassettes was analysed in transient histochemical GUS assay and PCR, 48 hr(s) post bombardment. The results revealed 91.6% bombarded embryos expressing GUS gene, whereas 66.6% embryos had the co-integration of H1Z and H2Z plasmids containing -1, 3-glucanase and chitinase coding region respectively. The bombarded embryos were cultured on media GM supplemented with 210mg/l ampicillin for five selection cycles of 20 days each. After five selection cycles, 12 plantlets survived and the plantlet formation efficiency of 0.65% was obtained, which was significantly higher than the efficiency of non-bombarded embryos (0%). The PCR analysis, 100 days post bombardment, showed co-integration of -1, 3-glucanase and chitinase genes in eight plantlets with a transformation efficiency of 0.43%, while the remaining 4 plantlets showed integration of either of the genes. These putative transgenic plantlets are currently growing in the glass-house.
Open Access
MEAN AND VARIANCES AMONG F4 PROGENIES AND THEIR PREDICTION FROMPARENTAL MEANS AND GENETIC DISTANCES BASED ON MOLECULAR MARKERS IN Gossypium arboreum L.
(Punjab Agricultural University, Ludhiana –, 2011) Jamwal, Navdeep Singh; Gumber, R.K.
The objective of the present study was to see whether genetic distance based on SSR markers (GDSSR) and Mahalanobis D2 statistics (GDDy), parental means ( P1 + P2 /2) and absolute difference of parental means (| P1 - P2 |) can be used for predicting the means and variances of F4 populations derived from 52 crosses of Gossypium arboreum L. Fifty-two F4 populations along with 17 parents were evaluated in a randomized complete block design using three replications during Kharif 2009. Data were recorded for seed cotton yield (g), number of bolls, boll weight (g), seed index (g), halo length (mm), ginning out turn (%) and plant height (cm). Fifty-seven SSR primers belonging to BNL, MUSS, MUCS, MUSB, CIR, NAU and MGHES series detected 74 alleles in 17 arboreum lines. Total number of bands ranged from 1-3 with an average of 1.29 bands per primer. Polymorphism information content (PIC) ranged from 0.00 to 0.88 with a mean of 0.12 for all 57 primers. Maximum genetic distance based on SSR markers was observed between LD866 and AH11(0.059), whereas minimum genetic distance recorded was 0.00 between lines LD327 and MDL2643; LD694 and DLSa1001. The genetic similarity coefficient among 17 genotypes ranged from 1.00 to 0.94 with an average genetic similarity of 0.97. The maximum genetic distance (280.63) based on Mahalanobis D2 statistics was observed between LD694 and RG395, whereas minimum of 2.49 between lines LD866 and RG8. No association was found between parentage and geographical origin of parental lines with genetic distance among parental lines revealed by both the estimates of genetic divergence. Poor correlation (r=0.06) between both estimates of genetic distance GDSSR and GDDy was observed. The means of F4 populations can be predicted successfully from the means of the parents for seed cotton yield (r=0.40) and halo length (r=0.273). Likewise, | P1 - P2 | and GDSSR proved to be good predictor of F4 population means for boll weight (r=0.273) and ginning out turn (r=0.290), respectively. However, the prediction of F4 variance for most of characters except plant height (r=0.277) from different properties of parental lines still remains an unsolved problem.
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GENETIC DIVERSITY AMONG Gossypium arboreum L. GERMPLASM LINES AS DETERMINED BY SSR MARKERS AND MORPHOLOGICAL CHARACTERISTICS
(Punjab Agricultural University, Ludhiana –, 2013) Lovepreet Singh; Pathak, Dharminder
The present study was undertaken to study the genetic diversity among Gossypium arboreum germplasm lines using molecular and morphological markers. Sixty four germplasm lines from diverse eco-geographical regions of India were taken for evaluation. Forty SSR primer pairs belonging 11 of the 13 A-genome chromosomes were employed to assess diversity at the molecular level. LD 694 and RAC 024 were observed to be quite diverse from each other. G. hirsutum accession TM1 and G. herbaceum accession RAHS 14 formed separate clusters. PIC value of the markers ranged from 0.11 to 0.79. The dissimilarity coefficient for G. arboreum ranged from 0.08 to 0.29. Mahalanobis D2 distances statistics was used to cluster the genotypes into six groups. Cluster I was the largest with thirty three genotypes followed by cluster II which consisted of 25 genotypes. Cluster III and IV both included two genotypes, whereas, cluster V and VI were having one genotype each. The maximum inter-cluster distance was 97.85 (cluster IV & V). The maximum intra-cluster distance was 30.68 (cluster I). Both the estimates of genetic distances did not find any relationship between genetic diversity and eco-geographical distribution. Several morphological characters such as plant body colour, flower colour, leaf shape, presence/ absence of leaf nectar did not have any impact on the clustering pattern of the genotypes

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