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Morphological, biochemical and molecular characterization of available germplasm and induced mutants in garlic (Allium sativum L.)
(Punjab Agricultural University, 2023) Karanveer Kaur; Sharma, Madhu
Garlic (Allium sativum L.) a bulbous crop propagated through vegetative means, has high therapeutic value due to abundance in sulphur containing compounds. In spite of clonal propagation, garlic has shown tremendous genetic variation. Present study was planned with the vision to explore magnitude of variation at genetic level among 139 garlic clones. Significant variation at morphological, biochemical and molecular level was witnessed among the garlic clones. Mahalanobis D2 categorized them into 8 clusters and PCA compressed 15 vectors to 7 components with 92.44% variation. Genotyping analysis represented that the PIC value ranged from 0.041 - 0.888, with average value of 0.583. Jaccard’s dissimilarity coefficient constructed dendrogram into two major clusters, where garlic clones belonging to Allium ampeloprasum formed cluster-I and rest Allium sativum subsp sativum and Allium sativum subsp ophioscorodon formed cluster-II. Extensive variation in biochemical attributes among various genotypes exhibited huge variation, where, dry matter ranged from 26.68 to 53.96%, pyruvic content 119.99 to 443.19mg/100g, allicin content 0.12 to 0.42%, total soluble solid content (TSS) 23.70 to 49.63ºB, total phenol content 0.32 to 1.39mg/100g, ortho-dihydroxy phenol 0.02 to 0.18mg/100g and Flavonol content 0.04 to 0.30 mg/100g. Screening of germplasm against stemphylium blight under natural conditions categorized it into- moderately resistant (28), moderately susceptible (72), susceptible (33) and highly susceptible (6), whereas, no genotype was found to be immune and resistant. Assessment of available germplasm has its own constraints, as it is limited to studying the existing variations and lacks the ability to generate novel genetic alterations. Due to sexual sterility and inability to exchange genetic material, new genetic modifications can be effectively induced through mutation in garlic. Hitherto, two garlic clones exposed to different mutagens viz., EMS, sodium azide and gamma irradiation at various doses and durations gave morphological mutants in MV2 generation, to be utilized in further crop improvement programmes. To overcome the challenge posed by its sterile behaviour, various growth regulators were tried for induction of flowering. Though, these treatments altered the morphology of garlic genotypes, affected aspects like germination as well as early and high scape length but were unable to trigger the process of flowering in garlic. However, the variability available in germplasm provided substantial opportunity to develop new clones with table and processing properties.
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Development of monoecious lines in muskmelon (Cucumis melo L.) through marker assisted selection
(Punjab Agricultural University, 2024) Nidhi; Sharma, Sat Pal
In heterosis breeding, monoecious sex expression offers significant advantages over andromonoecy in muskmelon, as it eliminates emasculation and produces fruits with desirable traits, such as earliness, high TSS, thick flesh, small scar size, and large fruit size. Thus, development of stable monoecious lines possessing desirable horticultural traits is critical for hybrid breeding in this crop. This investigation was aimed to identify molecular markers linked to monoecious trait and to transfer this trait to a popular melon cultivar through marker assisted selection. For this purpose, stability of monoecism was assessed under varying environments and identified 18 stable monoecious using the andromonoecious index. A set of identified lines was used to develop KASP markers for the sex-determining gene, CmACS-7, which revealed two mutations in exons 1 and 2, with one SNP at 170th, followed by insertion of ‘GA’ at 222nd nucleotide position. Upon validation, KASP-CT-2 accurately separated homozygous andromonoecious and monoecious parents and was also successfully validated on F2 population segregating for monoecious trait. Subsequently, marker-assisted backcross breeding was carried out to transfer the dominant gene 'A' controlling the monoecious trait into the Punjab Sunheri, a popular muskmelon cultivar. Heterozygous plants in BC1F1 and BC2F1 and homozygous plants in BC2F2 with high recurrent parent genome recovery and monoecious trait were advanced using the cleaved amplified polymorphic sequence (CAPS) marker. In the BC2F2 population, homozygous monoecious plants were identified using the CAPS marker. These plants were self pollinated to generate a BC2F3 progenies, of which 11 out of 13 lines had phenotypic similarities with the recurrent parents. These 11 lines were self pollinated to generate 34 BC2F4 progenies. Eight BC2F4 lines, viz. Mono-6R2, Mono-IOB1, Mono-2OB3, Mono-7R3, Mono-2OV2, Mono-1OB2 were selected based on TSS, TSS/TA ratio, β-carotene, low titratable acidity, pH, flesh thickness, fruit shape index, and yield. The information and developed associated molecular marker will be useful in developing new stable monoecious lines. Furthermore, the generated monoecious lines can potentially be utilized as a female parent for F1 hybrid development in muskmelon.
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Production and Characterization of Haploids in Brinjal (Solanum melongena L.)
(Punjab Agricultural University, 2022) Jasmeen Kaur; Sidhu, Mohinder Kaur
A study was conducted for production and characterization of haploids in brinjal. Induction of haploids in brinjal was attempted through two methods androgenesis and interspecific hybridization. For microspore androgenesis among donor hybrids, PSB-7-2 X P-219 had the highest (14.6 in 63 days) number of calli per ml of microspore suspension, followed by SR-5 x SR-9322 (4.4in 69 days) and SC-15-2 x MR-319 (2.1 in 73 days). Overall, NLN medium supplemented with 2 mg/l 2, 4-D + 20g/l Sucrose induced the maximum number of calli per ml of suspension (13.3 calli in 62 days). In this study, four days temperature pretreatment (35̊ C) in distilled water induced the uppermost number of calli per ml of suspension (11.2 calli in 66 days). The embryogenesis (%) of microspore derived calli was the most effective in MS + 4 mg/l Zeatin + 0.2 mg/l IAA+ 20 g/l of sucrose + 8g/l agar (R1) as compared to MS + 2 mg/l Zeatin + 0.2 mg/l IAA+ 20 g/l of sucrose + 8g/l agar (R2) for all the donor hybrids. In total, 260 plants were rooted on half-strength MS medium and only 65 (25.0 %) could be established as complete plants after hardening. In the confirmation, the molecular analysis with three SSR markers established complete homozygosity (microspore origin) in all the plants developed from two donors. Chromosome counting (n=12) at diakinensis or metaphase stage of meiosis in pollen mother cells categorized 65 regenerants into 5 haploids, 30 doubled-haploids, 29 mixoploids, and one triploid. Many chromosomal abnormalities in PMCs also affcetd the reproductive features of heteroploids. In anter androgenesis of donor hybrids, SC-15-2 x MR-319 and SR-5 x SR-9322 were at par with each other for percentage of calli formation from expanded anthers with values 73.4 % and 71.3 % respectively. Donor hybrid SR-5 x SR-9322 also showed direct embryo regeneration from expanded anthers with value 2.8 %. Among different media combinations, the maximum calli formation from expanded anthers with in minimum days was observed in MS + 2 mg/l IAA + 0.5 mg/l Kinetin (65.8 % and 28.5 days). Direct embryo regeneration from expanded anthers was observed only in media composition MS + 2 mg/l IAA + 0.5 mg/l Kinetin (1.6 %). However, the highest calli regeneration was observed in donor hybrid SR-5 x SR-9322 (15.5%) with in minimum days (21.7 days). Among media combinations, MS + 0.2 mg/l IAA + 4 mg/l Zeatin (19.5 %) had the maximum calli regeneration in the minimum number of days (21.4 days). Plants obtained through anther androgenesis via direct embryos regeneration from expanded anthers in SR-5 x SR-9322 at media combination MS + 2 mg/l IAA + 0.5 mg/l Kinetin also confirmed their genetic origin. Morphological observations and molecular marker EEMS 37 confirmed the possibility of the haploid nature of these plants. In wide hybridization, no chromosome elimination was observed in plants. In conclusion, microspore culture of F1 donor was the best approach to develop homozygous lines of genetic origin and considerable morphological and genetic variability.
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Improving longevity of carrot (Daucus carota L.) seeds using ultra dry storage condition
(Punjab Agricultural University, Ludhiana, 2022) Sushil Kumar; Rajinder Singh
The studies on ―Improving longevity of Carrot (Daucus carota L.) seeds using ultra dry storage conditions‖ were conducted at the Seed Physiology Laboratory, Office of Director (Seeds), Punjab Agricultural University, Ludhiana during 2020 and 2021. In the present investigation carrot seeds cv PC-161 were dried using different drying methods, then packed in aluminium foil packs and stored under ambient storage environment. Result showed that, silica gel drying achieved the lowest seed moisture content as compared to oven dry that had highest germination percentage and seed vigour up to 30 months of the storage period under ambient conditions. Study investigating optimum seed moisture content, packaging material and storage conditions revealed that, carrot seeds ultra-dried to 3% MC when packed in aluminium foil packs and stored in cold store, enhanced the seed longevity and recorded for lowest gain in moisture content, higher germination percentage and seed vigour. Whereas, the activity of antioxidant enzymes, α-amylase enzyme, lipid peroxidation and seed storage reserves were found least affected in ultra-dried seeds. Accelerated ageing test predicted that artificially aged carrot seeds (PC-161 and Punjab Black Beauty) for 4-5 days were equivalent to naturally aged seeds of 16 months under cold store and 12 months under ambient storage conditions, in terms germination percentage, germination rate, seedling length and seed vigour index. In carrot seeds stored for 16 months, seed proteins (30 - 70 kDa) and RNA integrity degraded with storage time and the lowest degradation of RNA and protein profile was observed in seeds dried with silica gel and stored in aluminium foil with 3% moisture content under cold storage conditions.
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Enhancing productivity and industrial sustainability of snack seeded pumpkin through interspecific hybridization
(Punjab Agricultural University, Ludhiana, 2022) Garcha, Karmvir Singh
seeded pumpkin through interspecific hybridization’ was planned to combine hull-less seed, bush growth habit and virus resistance in a single Cucurbita genotype. Interspecific hybridizations among ten C. pepo (hull-less) and two C. moschata (hulled) rendered HLP-36 (C. pepo) and PVR1343 (C. moschata) as the best cross compatible genotypes. Molecular characterization of the virus symptoms revealed the predominance of two begomovirus species viz., Squash Leaf Curl China Virus and Tomato Leaf Curl New Delhi Virus. The intraspecific F2 populations of C. moschata displayed monogenic dominant and digenic recessive inheritance for bush growth habit and begomovirus resistance, respectively, whereas for hull-less seed monogenic recessive expression was observed in C. pepo. However, these best fit ratios were not found with the interspecific F2 populations due to genomic instability among C. pepo and C. moschata. Multiple crosses involving HLP-36, HLP-639 and PVR-1343 led to development of three genotypes having all the targeted traits. To map hull-less character, a 2.06 Mb genomic region on chromosome 12 of C. pepo was used for developing 21 KASP assays. Genotyping of F2 population derived from HP111 × HLP36 cross with eight polymorphic KASP assays lead to limit the hull-less seed locus (Cphl-1) to 40.3cM partial genetic linkage map. The most closely linked marker viz., Cp_3430407 (10 cM) accurately predicted 93.33% hull-less genotypes in the diversity panel. The functional analysis of candidate genomic region decoded 182 genes, out of which 19 shared homology with A. thaliana genes related to cell wall, cellulose or lignin biosynthesis. Two of these genes viz., ‘NAC’ and ‘MYB’ act as master regulators of lignin biosynthesis and were differentially expressed in hulled and hull-less seeds at different time intervals. The transcriptional differences between hull-less and hulled seeds at 15 and 20 days after pollinations displayed 3124 differentially expressed genes. Out of these 482 (15.43%) were related to cell wall biosynthesis, which indicated the seed coat initiation between 15 to 20 DAP. Further, functional characterization of the gene(s) involved in the lignin/seed coat development will improve the understanding of hull development in pumpkin. The newly developed hull-less lines with bush growth habit and begomovirus resistance will revolutionize the hull-less seed industry, while the KASP markers will be highly useful in marker assisted selections from the segregating populations.
Open Access
Inheritance and molecular mapping of gene(s) associated with yellow vein mosaic virus disease (YVMVD) resistance in pumpkin (Cucurbita moschata Duch. ex Poir)
(Punjab Agricultural University, Ludhiana, 2022) Verma, Neha; Dhatt, Ajmer Singh
Worldwide begomoviruses causative diseases are major reason of quantitative and qualitative losses in pumpkin. The three types of viral symptoms were molecularly characterized as SqLCCV, ToLCNDV and their mixed infection (MI-Sq/To). Artificial screening, histopathological study and PCR confirmed PVR-1343 as a novel resistant source against both begomoviruses. To understand the inheritance and mapping of begomovirus resistant gene(s), segregating populations between PVR-1343 and five susceptible lines (P-135, P-6711, Punjab Samrat, MBN-6242 and P-278) were developed and artificially inoculated with Bemisia tabaci Asia II strain against both viruses and their mixed infection in enclosed structures for three consecutive years (2018-2020). Disease scoring at 75DAS rendered all F1 progenies as susceptible indicating recessive nature of resistance with no maternal or cytoplasmic effects. Chi-square analysis showed best fit of 9(S):3(MS):3(MR):1(R) and 1(S):1(MS):1(MR):1(R) ratio in F2 and backcross with resistant parent respectively. Phenotypic segregation ratio of F2:3 confirmed the F2 genotypic ratio of 1:2:2:4:1:2:1:2:1. This substantiated that multiple begomovirus resistance in PVR-1343 is controlled by a digenic recessive gene. Moreover, F2:3 progenies resistant to SqLCCV were also resistant to ToLCNDV and MI-Sq/To and vice versa, suggesting that common or tightly linked gene(s) are involved in resistance to both viruses. To map these genes, WGRS coupled with QTLseq of two extreme bulks (resistant and susceptible) of cross PVR-1343×P-135 along with parents were employed and two genomic regions viz., qMI-Sq/To7.1 on chromosome 7 (3.18 to 4.70Mb) and qMI-Sq/To17.1 on chromosome 17 (7.30 to 8.17Mb) were identified. Nine polymorphic SNPs identified within the highly significant qMI-Sq/To7.1 region were converted into KASP marker. The KASP genotyping of 177 F2 individuals narrowed down the qMI-Sq/To7.1 interval to a 103Kb region flanked by Cmo3914729 and Cmo4018182 KASP markers. One of the flanking SNP (Cmo4018182 KASP marker) imparting resistance against MI-Sq/To, accurately predicted the disease reaction of 91 per cent of diverse Cucurbita genotypes. This SNP showed nonsynonym substitution in the coding region of putative candidate syntaxin-121 protein and might be responsible for diminishing or abolishing the viral infections. This study has thus, seeded the path for marker assisted selection for efficient introgression of resistance into elite C. moschata lines and providing a viewpoint for gene cloning.
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Generation of frost tolerant potato (Solanum tuberosum L.) clones through intervarietal hybridization and expression analysis of putative frost tolerance genes
(Punjab Agricultural University, Ludhiana, 2022) Angmo, Dechen; Sharma, Sat Pal
The present study was carried out with an overall aim to generate frost tolerant potato clones and optimization of phenotyping techniques in order to complement the natural field selection criteria. The physiological responses to low temperature stress were explored using four potato genotypes viz. Kufri Anand, J-2/19, MS/7-645 and Kufri Pukhraj under in vitro controlled conditions. Significant variation was observed amongst genotypes for parameters associated with frost tolerance in potato such as cell membrane stability, activity of PSII, photosynthetic efficiency and leaf morpho-anatomical traits. The genotypes J/2-19 and Kufri Anand exhibited higher PN, gs and ΔF/Fm’values and lower electrolyte leakage as compared to the sensitive genotype Kufri Pukhraj upon cold acclimation and during recovery days after freezing exposure. Similarly, J-2/19 genotype also recorded higher adaxial trichome density than the sensitive genotype MS/7-645. In order to identify the fold changes in the expression of three candidate genes involved in cold stress responses in potato viz. dehydrin, hsp70 and SAD a qRT-PCR study was also performed. Overall an increase in gene expression was observed in cold acclimated plants of all the genotypes compared to non-acclimated plants. However, the increase was more pronounced in tolerant genotype (J-2/19) as compared with susceptible genotypes (MS/7-645 and Kufri Pukhraj). In the summer 2018, the segregating progenies were developed through hybridization among contrasting parents viz. J-2/19  MS/7-645 (PAU3), MS/7-645  J-2/19(PAU7) and MS/7-645  CP-3765 (PAU9) at Keylong, HP. In F1C1 and F1C2 generations, the characterization of the generated potato clones was accomplished on the basis of leaf morpho-anatomical traits and electrolyte leakage assay. To determine the association between the key traits and potential frost tolerant potato clones, various statistical approaches, such as cluster analysis, principal component analysis and genotype by trait biplot analysis were performed. Total 11, 10 and 8 clones from PAU3, PAU7 and PAU9 crosses, respectively were identified exhibiting potential frost tolerant characteristics with desirable horticultural traits for their further evaluation. Thus, the findings of this study concluded that in addition to electrolyte leakage the chlorophyll fluorescence and leaf gas exchange parameters can be effectively utilized for screening frost tolerant potato genotypes, but with certain limitations. Furthermore, the leaf morpho-anatomical traits have been confirmed to be associated with frost tolerance through decrease in foliage damage and electrolyte leakage. Therefore, these traits can potentially be used for screening of large segregating populations for freezing tolerance in the early clonal generations in potato.
Open Access
Introgression of yellow vein mosaic disease resistance in okra
(Punjab Agricultural University, Ludhiana, 2021) Gagandeep Singh; Pathak, Mamta
The present investigation was undertaken with the objectives to screen okra wild relatives, study inheritance of yellow vein mosaic disease, identification of marker(s) linked to YVMD resistance and to evaluate introgressed lines for economic traits. Two year consecutive artificial YVMD screening of sixteen entries of seven wild species and two varieties of cultivated okra (Punjab Padmini and Pusa Sawani) revealed Abelmoschus angulosus accessions IC-470751 and IC-203833 and Hibiscus acutetus accessions SBT-12557 and SBT-12592 highly resistant to YVMD. A. moschatus accession IC-140986, A. caillie and A. manihot (PAUAcc-1) exhibited moderately resistant reaction at 90 DAI. Whereas, A. moschatus accession IC-141056, A. manihot accession EC-329394, A. tuberculatus and cultivated okra variety Punjab Padmini manifested susceptible and cultivated okra variety Pusa Sawani highly susceptible reaction to YVMD up to 90 DAI. Deng Begomovirus specific universal primer was used to confirm the YVMD resistance. The YVMD resistant plants of A. angulosus (IC-203833) and A. manihot PAUAcc-1 were used as pollen parent for the development of F1, BC1F1, BC1F2 and BCIF3 population using Punjab Padmini as the female parent. Two recessive genes were found to govern resistance to YVMD as revealed by the appearance of susceptible and resistant plants in the ratio of 9:7 in the BC1F2 generation. Out of 200 SSR markers, 31.5% were found polymorphic between the two parents Punjab Padmini and A. manihot (PAUAcc-1). Four primer pairs viz. Okra 032, Okra 049, Okra 129 and Okra 270 differentiated the two contrasting YVMD resistant and susceptible bulks in bulk segregant analysis. A set of 137 BC1F3 progenies of Punjab Padmini x A. manihot (PAUAcc-1) were evaluated along with five checks (Punjab Padmini, Pusa Sawani, PORL, POL-44 and Punjab Suhawani) for yield and its contributing traits. Fifty seven progenies were found to be free from YVMD up to 90 DAI. Seventy four progenies having marketable yield in the range of 112.5 to 189.6 g/plant were observed to be significantly superior to standard check Punjab Suhawani (111.9 g/plant). Progeny PAUMIOL-56 was at top for yield (189.62 g/plant) followed by PAUMIOL-36, PAUMIOL-138, PAUMIOL-105 and PAUMIOL-142 and had no YVMD incidence upto 90 DAI. Similarly, 88 BC1F3 progenies of Punjab Padmini x A. angulosus (IC203833) were accessed along with seven checks (Punjab Padmini, Punjab Suhawani, PORL, POL44, POL-78 and Pusa Sawani) for economic traits. Six progenies recorded marketable yield in range of 108.2 to 130.2 g/plant significantly higher than standard check Punjab Suhawani (97.9 g/plant). Forty one progenies were free from YVMD up to 90 DAI. Progeny PAUAIOL-324 yielded maximum (130.2 g/plant) followed by progeny PAUAIOL-251, PAUAIOL-301, PAUAIOL-256 and PAUAIOL-283 and had no YVMD incidence up to 90 DAI.
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Molecular mapping and interspecific hybridization for hull-less seed trait in pumpkin for value addition in food processing industry
(Punjab Agricultural University, Ludhiana-, 2020) Barinder Kaur; Dhatt, Ajmer Singh
The present investigation entitled ‘Molecular mapping and interspecific hybridization for hull-less seed trait in pumpkin for value addition in food processing industry’ was conducted in the Department of Vegetable Science, Punjab Agricultural University, Ludhiana from 2016 to 2020. Segregating populations were developed using hulled and hull-less genotypes of Cucurbita pepo for understanding the genetics and molecular mapping of hull-less seed trait. Chi-square analysis of segregating populations revealed that single recessive gene is responsible for controlling this trait. The sequence based bulked segregant analysis of two extreme bulks (hulled and hull-less) of cross HP111 × HLP36 along with their parental lines identified one QTL on chromosome 12 (Cp4.1LG12) between 1891261 to 3863267 bp covering 1.97 Mb genomic region with 396 number of SNPs for hull-less seed trait. In interspecific hybridization, total 916 crosses between C. pepo (HLP36, HLP44, HLP53, HLP72) and C. moschata (HM1404, HM108, HM1343, HM1022, HM2211, HM6711) were attempted to transfer hull-less seed trait, however success was achieved in 5.57% crosses only. The fruit setting, number of seeds/fruit, seed germination and survival of F1 seedlings were high when C. pepo was used as female parent in cross HLP36 × HM1343 (66.67%, 15.00, 45.24% and 49.48%), followed by HLP36 × HM1022 (22.73%, 23.00, 44.35%, 47.06%) and HLP44 × HM1022 (13.79%, 12.00, 45.85%, 45.46%), respectively. However, in reciprocal crosses (C. moschata × C. pepo) success rate was very poor, which was overcome through in vitro ovule culture after 17 to 19 days of pollination in cross HM1343 × HLP53 and HM6711 × HLP72. The hybridity of putative Cucurbita interspecific hybrids was confirmed through molecular, morphological and micromorphological markers and their results were found to be highly consistent. The phenotyping of F2 populations of interspecific crosses confirmed the transfer of hull-less seed trait. Findings of the present study will facilitate in genetic dissection of candidate gene(s) and to enhance productivity, improve quality and multi-season cultivation of hull-seeded pumpkin in India.

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