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Genetic transformation of Indica rice for sheath blight resistance using chitinase gene
(Punjab Agricultural University, Ludhiana, 2021) Sharma, Manveer; Sandhu, Jagdeep Singh
Trichoderma chitinase enzymes hydrolyze β-1, 4-glycosidic bonds of chitin found in the cell wall of Rhizoctonia solani, the causal agent of rice sheath blight. In this study Tvchit42 gene was introduced into rice using Agrobacterium-mediated genetic transformation. The gene construct, carrying Tvchit42 gene under the control of the CaMV35S promoter and the NOS terminator, was mobilised into two Agrobacterium tumefaciens strains, LBA4404 and EHA105, which were used to agro-infect PR121 and Kitaake rice calli, respectively. In PR121 and Kitaake, a total of 99 and 414 plants were regenerated from transformed calli, respectively. The presence of transgene was confirmed by PCR, revealing amplicon of size 1293 bp corresponding to Tvchit42 with transformation efficiency of 0 % and 0.64 % in PR121 and Kitaake, respectively. Real-Time PCR was used to confirm transgene integration and expression in T0 transformants, revealed 2.94-6.14 fold increase in Tvchit42 expression in six Kitaake plants as compared to non-transgenic controls. Moderate resistance was observed in five transgenic plants after inoculation with RS-1 isolate of R. solani, indicating role of Tvchit42 to provide resistance against rice sheath blight disease.
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Marker assisted introgression of grain size QTL from Aegilops tauschii Coss. to Triticum aestivum L.
(Punjab Agricultural University, Ludhiana, 2020) Preeti Kumari; Chhuneja, Parveen
Hexaploid wheats shows constrained genetic diversity which can be broadened by exploiting the wild relatives of wheat for introgressing the novel genes or alleles. Aegilops tauschii, the diploid Dgenome progenitor of hexaploid wheat, can be used to increase the genetic diversity both for improving grain yield and nutritional quality of bread wheat. Grain size is one of the important variables influencing the grain yield. To achieve higher wheat yields, breeding efficiency needs to increase which can be done through biotechnological interventions. The present study aimed to validate the markers linked to the QTLs for grain weight, grain length and grain width and to use the markers for marker assisted transfer of these traits from Ae. tauschii to hexaploid wheat using T. durum (PBW114) as bridging species. Four hundred and ninety-five wheat- Ae. tauschii advance backcross introgression lines (GWILT) derived form 13 different Ae. tauschii donor accessions (pau9793, pau9809, pau14328, pau14339, pau14158, pau14169, pau14170, pau14200, pau14232, pau3747, pau3752, pau3760, pau9810) and two recipient wheat cultivars PBW550 and PBW621 were evaluated for various agronomic and quality traits for four consecutive crop seasons (2015-16 to 2018-19). In each generation selections were made and phenotypically superior lines were progressed further. Total of two hundred and forty-nine highly promising lines were identified which showed better performance in all the four years. These lines offer a good opportunity to study the genetic loci which may be responsible for variation in grain size and other related traits. The pooled correlation coefficient among traits for wheat-Ae. tauschii AB introgression lines reveals a positive correlation of thousand grain weight (TGW) with grain filling duration (GFD), length (LN), width (WD) and yield per plot (YD) but grain number per spike GNpS showed negative correlation with TGW. Tiller number per meter showed positive correlation with overall yield. TGW depends more on grain width than grain length. Seventeen introgression lines derived from Ae. tauschii accessions pau14169, pau3760, pau9809 showed high level of resistance for stripe rust in the range of 5MR-10MR. In previous study, three Ae. tauschii accessions pau14232, pau14231 and pau9796 which were found to have high grain size were used in present study for transfer of grain size QTLs to hexaploid wheat cv. PBW725 and advance breeding line BWL4478 using T. durum (PBW114) as bridging species. Two molecular markers GS7D and CWI21 which have been reported to be associated with grain size were used in the present study for marker assisted selection at different stages (BC1F1, BC2F1, and BC2F2) and selection of grain size introgressions. One hundred and seventy-one introgression lines in BC2F2 generation were developed and evaluated genotypically as well as phenotypically for grain weight. All these lines showed significant grain weight improvement over the respective recurrent parent PBW725. The selected introgression lines can further be used in wheat improvement programme and mapping yield component traits.
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Genetic and biochemical analysis of bitterness in citrus
(Punjab Agricultural University, Ludhiana, 2020) Priyanka; Sidhu, Gurupkar Sigh
Citrus is the economically important fruit crop belongs to the family Rutaceae. Citrus fruit have abundance of health promoting substances which has various antimicrobial, antiseptic and anticancerous properties. The consumer’s acceptability of citrus juice gets lowered due to the problem of delayed bitterness. Previously identified LGT gene responsible for delayed bitterness was confirmed in the citrus germplasm which was present in all the studied citrus germplasm. The expression of this gene is highest during late fruit development stage and seeds exhibit the highest expression as compared to peel. Among three tissues studied, the highest gene expression was observed in the peel of grapefruit during late fruit development stage. Among studied genotypes, seeds of pummelo had the highest gene expression during late fruit development stage. The expression of this gene increases from early to late fruit developmental stage irrespective of the three tissues studied. The major delayed bitterness causing components are limonoids which occurs in glucoside and aglycone forms were estimated. Among all the commercial recommended scions, Pummelo had highest limonin aglycone content and glucoside content in leaves, peel and seeds. The flavonoid content was also highest in pummelo in all three tissues studied. The effect of citrus rootstocks on morphological, vegetative, physico-chemical, physiological traits and leaf nutrient status in three mandarins (Kinnow, Daisy and W. Murcott) was studied. The total phenolics, antioxidants, vitamin C content increased from early to late fruit development stage. The fruit and vegetative characters also followed the similar trend from early to late fruit development stage. The physiological characters decreased from early to late fruit development stage in all three mandarins irrespective of the rootstock. Leaf nutrients status of three mandarins show variability on different rootstocks studied.
Open Access
Molecular mapping of yellow mosaic virus resistance in bitter gourd (Momordica charantia L.)
(Punjab Agricultural University, Ludhiana, 2021) Gurpreet Kaur; Navraj Kaur
Bitter gourd (Momordica charantia) is a popular cultivated vegetable in India and other countries. The susceptible parent ‘Punjab-14’ and the resistant parent ‘PAUBG-6’ were crossed to obtain F4 mapping population comprising 101 individuals with the objective of mapping yellow mosaic disease resistance in bitter gourd. In the present study, genotypingby-sequencing (GBS) approach was used to develop the genetic linkage map. The map contained 3,144 single nucleotide polymorphism (SNP) markers, consisted of 15 linkage groups and spanned for 2415.2 cM with an average marker distance of 0.7 cM. By adopting the artificial and field inoculation techniques, F4:5 individuals were phenotyped for disease resistance in Nethouse (2019), Rainy (2019) and Spring season (2020). The QTL analysis using the genetic map and phenotyping data identified three QTLs qYMD.pau_3.1, qYMD.pau_4.1 and qYMD.pau_5.1 on chromosome 3, 4 and 5 respectively with phenotypic variation explained (PVE) from 13.5 to 22.1%. In addition, nine horticultural important traits including days to appearance of first female flower, days to appearance of first male flower, days to fruit maturity, node to appearance of first female flower, seed hardness, seed number, fruit length, fruit diameter and fruit weight were evaluated using quantitative data. As a result, twelve QTLs responsible for these traits were also identified. In another experiment, the relative gene expression of begomovirus genes in susceptible and resistant hosts was studied to understand the mechanism of virus resistance. The expression analysis indicated the high expression of AC4 gene in resistant plant provided the information on the efforts of virus to combat the resistance of plant. The information generated in this study is very useful in future for fine-mapping and for marker-assisted selection for these traits.
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Agrobacterium mediated genetic transformation of pigeon pea (Cajanus cajan L. Millsp.) for resistance to spotted pod borer Maruca vitrata
(Punjab Agricultural University, Ludhiana, 2020) Manjinder Singh; Ajinder Kaur
The present investigation dealing with introduction of cry1Ab gene for resistance to Maruca vitrata into pigeonpea (Cajanus cajan L. Millsp.) through Agrobacterium-mediated in planta transformation was carried out using three pigeonpea genotypes i.e. AL 15, AL 201 and PAU 881. Two methods of in planta transformation were used for generating transgenic plants, including inoculation of pricked embryo axes and floral dip transformation. Out of seven experiments on in planta transformation method involving inoculation of pricked embryo axes, only two (OD600 of Agrobacterium broth = 0.6 - 0.7 containing 100 mM acetosyringone, 1 h dipping of 2-day old pricked seedlings) gave positive results. A total of 5,022 half seeds were treated that gave rise to 4059 plants, out of which 15 (representing AL 15 and AL 201 genotypes) were PCR-positive with an overall transformation frequency of 0.36 %. Out of 15 primary transformants, only 11 plants showed transcript accumulation by semi-quantitative RT-PCR. The primary transformants were also analyzed for chimerism by ELISA-based protein accumulation in selected branches. Out of a total of 182 branches of 10 RT – PCR positive plants analyzed, 57, 43 and 49 branches had Cry1Ab protein content equal to or more than 1.08 µg/g [Positive calibrator, (PC) 5.0 ppb], 0.58 µg/g (PC, 2.5 ppb) and 0.08 µg/g (PC, 0.5 ppb), respectively. Besides, there were 33 such branches which did not show accumulation of Cry protein indicating the presence of chimerism in primary transformants. The putative transgenics were advanced to T1 generation, out of 904 plants analyzed, 97 were PCR-positive, thus exhibiting transformation efficiency of 11.70 % (with genotype AL 201) and 10.24 % (with genotype AL 15). Twenty three T1 plants were randomly taken for determination of Cry1Ab protein content, out of which 12 showed high protein content of more than 0.72 µg/g. These plants showed positive results in PCR and RT-PCR, and further used for determining the efficacy of cry1Ab gene against second instar larva of Maruca vitrata using insect bioassay. Both flowers and pods of these 12 T1 pigeonpea plants were used for bioassay experiments. All 12 T1 plants showed restricted increase in larval weight as compared to nontransgenic (control) plant. No insect mortality was observed, but larvae fed on two transgenic plants i.e. 201-344 and 15-537 showed no adult emergence as compared to other transgenic and control plants where adult emergence was normally observed. Both of these two plants (201-344 and 15-537) had high Cry protein content (0.88 µg/g) as compared to other transgenic plants, which established clearcut positive correlation between amount of protein accumulated and inhibition of M. vitrata larval growth. Further, T1 plants were advanced to T2 generation that were maintained in transgenic glasshouse. In case of floral dip transformation, a total of 139 flower buds were treated and 454 seeds were obtained. Ten out of 454 putative T1 plants were observed to be PCR-positive with a transformation frequency of 2.2 %.
Open Access
Mapping and transfer of GENES/QTLs for nematode (Meloidogyne graminicola) resistance from Oryza glaberrima into Oryza sativa L.
(Punjab Agricultural University, Ludhiana, 2020) Gurwinder Kaur; Vikal, Yogesh
Rice root knot nematode (Meloidogyne graminicola) is one of the emerging constraints for rice production, causes about 50% yield losses in field conditions and 98% yield losses in pot experiments. The identification and exploitation of resistant rice genotypes is one of the economic and sustainable option to minimize the yield losses caused by M. graminicola infestations. The present investigation was undertaken to identify the QTLs associated with rice root knot nematode resistance as well as the transfer of resistance to elite rice cultivars. A total of 42 accessions of O. glaberrima along with O. sativa cultivar PR121 were screened in triplicates during kharif 2016 and kharif 2017 in nematode infested sick plot at initial nematode population density of one juvenile per gram of soil. Reproduction factor of rice root knot nematode among O. glaberrima accessions was found to be <1 while in O. sativa cultivar PR121, reproduction factor was >1. Out of 42 accessions, three accessions (IRGC102196, IRGC102538 and IRGC102557) were found to be highly resistant, thirty-three accessions were resistant, and six accessions were moderately resistant. Data on different morphological traits (plant height, root length, fresh shoot weight, fresh root weight, dry shoot weight and dry root weight) revealed that no statistically significant differences were found among O. glaberrima accessions whereas PR121 exhibited significant reduction in all growth parameters in nematode infested sick plot as compared to controlled conditions. The BC1F1 population derived from the cross of O. glaberrima acc. IRGC102206 × PR121 was used for mapping of rice root knot nematode resistance. The BC1F1 plants were screened against M. graminicola in triplicates and genotyped using 84 polymorphic SSR markers. Both phenotypic data and genotypic data was analyzed and a total of 13 QTLs associated with gall number, gall index and different morphological traits were mapped on chromosomes 1, 3, 4, 5, 6 and 8. Out of 13 QTLs, three QTLs associated with gall number were identified; two QTLs designated as qGN4.1and qGN4.2 were mapped on chromosome 4 and one QTL, qGN6.1, was mapped on chromosome 6. QTL associated with gall index (qGI6.1) was found to be co-localized with qGN6.1. Similarly, qGN6.1 QTL was co-localized with plant height and root length. Due to less coverage by SSR markers, we simultaneously performed BSA-QTLseq (Bulked segregant analysis coupled with QTL-seq approach) analysis and identified QTLs for nematode resistance on chromosomes 1, 2, 3, 4, 5, 6, 11 and 12. The genomic interval of all QTLs was narrowed down to 1–2 Mb using QTL-seq analysis. The development of SNP based molecular markers from identified QTL regions will further help to saturate the linkage map and to identify closely linked markers to rice root knot nematode resistance. The identified markers will further fasten the improvement of genotypes for rice root knot nematode resistance through marker assisted breeding approach. Based on introgressed genomic regions from O. glaberrima carrying QTLs for nematode resistance the BC1F1 resistant plants were selected and backcrossed to generate BC2F1 and subsequently BC3F1 progenies for transfer of nematode resistance in the background of PR121. The data generated from this study can serve as valuable genomic resources for rice breeding programmes.
Open Access
Identification and characterization of high temperature stress responsive genes in maize (Zea mays L.)
(Punjab Agricultural University, Ludhiana, 2020) Ashok Babadev Jagtap; Vikal, Yogesh
Heat stress due to climate change is an emerging issue for maize breeders as it largely affects the yield. The present study focuses to elucidate molecular mechanisms and underlying genes, or QTLs associated with heat stress tolerance in maize. The transcriptional studies of maize leaves, pollens and ovules using heat stress susceptible and tolerant inbred lines, namely LM11 and CML25, respectively revealed a total of 2,164 (1127 up-regulated and 1037 down-regulated) differentially expressed genes (DEGs) between LM11 (HS) and CML25 (HT) samples, with 1151, 451 and 562 DEGs were identified in comparisons of corresponding leaf, pollen and ovule samples, respectively. Functional annotations of DEGs showed that many of them were related to transcription factors (TFs) viz. AP2, MYB, WRKY, PsbP, bZIP and NAM, heat shock proteins (HSP20, HSP70 and HSP101/ClpB), as well as genes related to photosynthesis (PsaD and PsaN), antioxidation (APX and CAT) and polyamines (Spd and Spm). KEGG pathways analyses explicated that metabolic overview pathway and secondary metabolites biosynthesis pathway, with involvement of 264 and 146 genes, respectively were highly enriched in response to heat stress. A bioinformatics pipeline was used to call and type SNPs from RNA-seq reads and applied it to transcriptomic data of LM11 and CML25. A total of 554,423, 410,698 and 596,868 polymorphic SNPs were identified respectively among leaf, pollen and ovule of the LM11 and CML25. A total of 100 genome-wide SNP based KASP assay markers were developed and validated as well as subsequently genotyped on 90 F2 individuals derived from the cross of LM11 × CML25. The success of SNP conversion rate was 71%. In addition, F2 population and their parental inbreds were genotyped using 94 polymorphic SSR markers. The 175 F2:3 families during late March (Spring 2017) were evaluated for heat stress under field and glass house conditions. Heat stress significantly affected all the morpho-physiological and yield contributing traits. Grain yield was positively associated with ear weight, number of kernels per ear, pollen viability, pollen shedding durations and chlorophyll content. Furthermore, secondary traits like membrane thermostability, days to anthesis and silking, anthesis-silking interval, canopy temperature, leaf firing and tassel blast showed significant negative impact on grain yield in both field and glass house conditions under heat stress. Both genotyping and mean phenotypic data of each component trait was analyzed for single marker analysis (SMA) and composite interval mapping (CIM) using WinQTL Cartographer. A linkage map of 1857.1 cM in total length was constructed by applying both SSR and SNP markers. A total of 11 QTLs were detected for 7 traits on chromosomes 1, 3, 4, 6, 7 and 9 with phenotypic variance ranged from 8.67 to 29.62 per cent. Four of these QTLs, qKPE6.1, qPV6.1, qCC9.2 and qLF4.1, accounted for above 15 per cent of phenotypic variation, and might be considered as major QTLs for heat tolerance. The data generated in present investigation laid the foundation for future work to uncover genes and mechanisms critical for the development of heat-resilient maize using genetic and biotechnological approaches.
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QTL Mapping For Terminal Heat Stress Tolerance In Synthetic Hexaploid Wheat Derived Population
(Punjab Agricultural University, Ludhiana-, 2020) Amandeep Kaur; Satinder Kau
Common bread wheat (2n=AABBDD) ranks among the primary cereal crop throughout the world and is an allohexaploid evolved from two spontaneous hybridization events followed by spontaneous chromosome doubling. The D-genome of this polyploidy is the least diverse among the three constituting wheat genomes and is unarguably less diverse than that of diploid progenitor Aegilops tauschii (2n=DD). Due to this considerable similarity, Ae. tauschii is extensively used in wheat improvement programs introducing new genetic diversity for various biotic and abiotic traits. A large number for Ae. tauschii accessions are available at Punjab Agricultural University, Ludhiana. Seven of these accessions were used to generate wheat synthetics targeting heat stress tolerance. All of these SHWs performed better than elite cultivars under terminal heat tolerance. Two of these synthetics, Syn14128 and Syn14170 selected on the basis of their better and stable performance under heat stress. These two synthetic wheat were crossed with stripe rust resistant version of two elite wheat varieties PBW343 and HD2967 to generate four nested population of chromosomal segmental substitution lines (CSSLs). CSSLs thus generated, were evaluated for terminal heat tolerance and yield associated traits under timely and late sown conditions. A majority of CSSLs out-performed the checks for different traits like number of effective tillers, ear length, TGW and harvest index, as compared to checks and recurrent parents used in the study. The genotypic performance evaluated using Heat Susceptibility Index (HSI) further suggested that almost 50 percent CSSLs from each set were either highly heat tolerant or moderately heat tolerant. Further, to assess the extent of alien introgressions these lines, genotype-by-sequencing was outsourced and unique SNP from SHW and derived populations were identified. A total of 724 synthetic wheat specific SNPs found in syn14170xHD2967 derived CSSLs while 1546 synthetic specific SNPs were identified in syn14128xPBW343 derived CSSLs. The graphical genotype of SNPs derived from syn14170xHD2967, identified four potential introgressions on chr 2A, 3D, 6D and 7A. Based on yield per plot, harvest index and TGW, 18 out-performing lines had one or more of these introgressions. In CSSLs derived from syn14170xPBW343, two potential introgressions on chr 1B and 5B were found. Based on yield per plot, harvest index and TGW, 15 outperforming lines had introgressions either on chr 1B or 5B. Further to explore the seedling stage heat tolerance in the selected SHW lines, a 35°C heat shock for 12- and 20-hr was given to 4 day old seedlings. A significant variation was observed in the content of MDA, DPPH, total phenols and total flavanols, total sugars and amylase activity along with antioxidant scavenging activities and synthetics better activities of most of these parameters compared to elite and durum wheats. One of the synthetic, Syn14128 had higher amylase activity and better recovery even after a heat shock of 20hrs which was confirmed by the gene expression analysis of α-amylase gene. A better performance of synthetics under both seedling stage and terminal stage heat stress makes them the potential source for the improvement of heat tolerance in the present day wheat cultivars. However, a deep exploration the exotic introgressions in the synthetic derived lines will also help to refine useful exotic chromosome segments for improving various traits and increasing D-genome diversity among cultivated varieties.
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Transformation of antifungal gene β-1, 3-glucanase in rice (Oryza sativa L.) and its expression analysis for sheath blight resistance
(Punjab Agricultural University, Ludhiana-, 2020) Pathania, Shivali; Sandhu, Jagdeep Singh
Trichoderma cell wall degrading β-1, 3-glucanase enzymes hydrolyse β-1, 3-glycosidic linkages of β-glucan rich cell wall of Rhizoctonia solani, a causal agent of rice sheath blight. To this date, no cultivar is known to provide true resistance to the phytopathogen. In this study, β1, 3-glucanase gene, encoding antifungal protein, was introduced into rice through Agrobacterium-mediated genetic transformation. Gene construct was generated in pRI 101-ON binary vector, carrying β-1, 3-glucanase under the control of CaMV35S promoter and NOS terminator and was mobilized into two Agrobacterium tumefaciens strains, LBA4404 and EHA105 that were used to agro-infect Kitaake and PR124 rice calli. A total of 434 and 388 regenerated plants were obtained in Kitaake and PR124, respectively. The presence of transgene was confirmed by PCR, revealing amplicon of size 2,307 bp corresponding to β-1, 3glucanase with transformation efficiency of 0.76 % and 0.46 % in Kitaake and PR124, respectively. The integration and expression of transgene in primary transformants was verified by Real-Time PCR, depicting up to 5.13- and 3.81-fold increase in β-1, 3-glucanase expression in transformed Kitaake and PR124 plants, respectively than non-transgenic control. Moderate resistance to RS-1 isolate of R. solani was found in transgenic plants, indicating potential use of β-1, 3-glucanase from Trichoderma spp. in rice to confer resistance to sheath blight disease.

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