An efficient and easy-to-use protocol for induction of haploids in cucumber through parthenogenic embryo development.

BACKGROUND: Cucumber (Cucumis sativus L.) is a model crop to study cell biology, including the development of haploids and doubled haploids in vegetable crops. In plant breeding, haploid and doubled haploids are valuable tools for developing pure homozygous inbred lines and accelerating genetic progress by reducing the time required for breeding cycles. Besides, the haploids are also valuable in genomic studies. We are reporting the induction of haploids in cucumber involving gynoecious and parthenocarpic genotypes for the first time. This study aimed to assess the efficient induction of haploids through pollination with gamma-irradiated pollen in cucumber. The effect of gamma irradiation dose on pollen viability and germination, fruit setting percentage, seed development, and haploid embryo development in cucumber hybrid genotypes were studied in detail. The goal was to utilize this information to produce haploid plants for genomics and transformation works in this model vegetable crop. RESULTS: Pollination was done on six cucumber genotypes using varying doses of gamma rays (100, 200, 300, 400, and 500 Gy). Genotypes, doses of irradiation, and embryo developmental stage influenced the successful generation of in-vitro haploid plants. The optimal timeframe for embryo rescue was found to be 25 to 30 days after pollination. Haploid embryos were effectively induced using irradiated pollen at 400 to 500 Gy doses. Parthenogenetic plantlets were analyzed, and their ploidy level was confirmed through stomatal physiology, cytology (mitosis), and flow cytometry methods. CONCLUSION: Through parthenogenic embryo development, it is possible to induce a large number of haploids in cucumber. This technique's power lies in its ability to streamline the breeding process, enhance genetic gain, and produce superior cultivars that contribute to sustainable agriculture and food security.

Identification of new stable resistant sources and assessing agro-morphological performance of sponge gourd germplasm against Tomato Leaf curl New Delhi Virus incidence.

Tomato leaf curl New Delhi virus (TolCNDV) causes yellow mosaic disease, which poses a significant biotic constraint for sponge gourd cultivation, potentially resulting in crop loss of up to 100%. In the present investigation, 50 diverse genotypes were screened for 3 years under natural epiphytotic conditions. A subset of 20 genotypes was further evaluated across four different environments. The combined analysis of variance revealed a significant genotype × environment interaction. Eight genotypes consistently exhibited high and stable resistance in the preliminary screening and multi-environment testing. Furthermore, genotype plus genotype × environment interaction biplot analysis identified DSG-29 (G-3), DSG-7 (G-2), DSG-6 (G-1), and DSGVRL-18 (G-6) as the desirable genotypes, which have stable resistance and better yield potential even under diseased conditions. The genotype by yield × trait biplot analysis and multi-trait genotype-ideotype distance index analysis further validated the potential of these genotypes for combining higher yield and other desirable traits with higher resistance levels. Additionally, resistant genotypes exhibited higher activities of defense-related enzymes as compared to susceptible genotypes. Thus, genotypes identified in our study will serve as a valuable genetic resource for carrying out future resistance breeding programs in sponge gourd against ToLCNDV.

Molecular mapping of genomic regions and identification of possible candidate genes associated with gynoecious sex expression in bitter gourd.

Bitter gourd is an important vegetable crop grown throughout the tropics mainly because of its high nutritional value. Sex expression and identification of gynoecious trait in cucurbitaceous vegetable crops has facilitated the hybrid breeding programme in a great way to improve productivity. In bitter gourd, gynoecious sex expression is poorly reported and detailed molecular pathways involve yet to be studied. The present experiment was conducted to study the inheritance, identify the genomic regions associated with gynoecious sex expression and to reveal possible candidate genes through QTL-seq. Segregation for the gynoecious and monoecious sex forms in the F2 progenies indicated single recessive gene controlling gynoecious sex expression in the genotype, PVGy-201. Gynoecious parent, PVGy-201, Monoecious parent, Pusa Do Mausami (PDM), and two contrasting bulks were constituted for deep-sequencing. A total of 10.56, 23.11, 15.07, and 19.38 Gb of clean reads from PVGy-201, PDM, gynoecious bulk and monoecious bulks were generated. Based on the ΔSNP index, 1.31 Mb regions on the chromosome 1 was identified to be associated with gynoecious sex expression in bitter gourd. In the QTL region 293,467 PVGy-201 unique variants, including SNPs and indels, were identified. In the identified QTL region, a total of 1019 homozygous variants were identified between PVGy1 and PDM genomes and 71 among them were non-synonymous variants (SNPS and INDELs), out of which 11 variants (7 INDELs, 4 SNPs) were classified as high impact variants with frame shift/stop gain effect. In total twelve genes associated with male and female gametophyte development were identified in the QTL-region. Ethylene-responsive transcription factor 12, Auxin response factor 6, Copper-transporting ATPase RAN1, CBL-interacting serine/threonine-protein kinase 23, ABC transporter C family member 2, DEAD-box ATP-dependent RNA helicase 1 isoform X2, Polygalacturonase QRT3-like isoform X2, Protein CHROMATIN REMODELING 4 were identified with possible role in gynoecious sex expression. Promoter region variation in 8 among the 12 genes indicated their role in determining gynoecious sex expression in bitter gourd genotype, DBGy-1. The findings in the study provides insight about sex expression in bitter gourd and will facilitate fine mapping and more precise identification of candidate genes through their functional validation.

Elucidating the role of key physio-biochemical traits and molecular network conferring heat stress tolerance in cucumber.

Cucumber is an important vegetable crop grown worldwide and highly sensitive to prevailing temperature condition. The physiological, biochemical and molecular basis of high temperature stress tolerance is poorly understood in this model vegetable crop. In the present study, a set of genotypes with contrasting response under two different temperature stress (35/30°C and 40/35°C) were evaluated for important physiological and biochemical traits. Besides, expression of the important heat shock proteins (HSPs), aquaporins (AQPs), photosynthesis related genes was conducted in two selected contrasting genotypes at different stress conditions. It was established that tolerant genotypes were able to maintain high chlorophyll retention, stable membrane stability index, higher retention of water content, stability in net photosynthesis, high stomatal conductance and transpiration in combination with less canopy temperatures under high temperature stress conditions compared to susceptible genotypes and were considered as the key physiological traits associated with heat tolerance in cucumber. Accumulation of biochemicals like proline, protein and antioxidants like SOD, catalase and peroxidase was the underlying biochemical mechanisms for high temperature tolerance. Upregulation of photosynthesis related genes, signal transduction genes and heat responsive genes (HSPs) in tolerant genotypes indicate the molecular network associated with heat tolerance in cucumber. Among the HSPs, higher accumulation of HSP70 and HSP90 were recorded in the tolerant genotype, WBC-13 under heat stress condition indicating their critical role. Besides, Rubisco S, Rubisco L and CsTIP1b were upregulated in the tolerant genotypes under heat stress condition. Therefore, the HSPs in combination with photosynthetic and aquaporin genes were the underlying important molecular network associated with heat stress tolerance in cucumber. The findings of the present study also indicated negative feedback of G-protein alpha unit and oxygen evolving complex in relation to heat stress tolerance in cucumber. These results indicate that the thermotolerant cucumber genotypes enhanced physio-biochemical and molecular adaptation under high-temperature stress condition. This study provides foundation to design climate smart genotypes in cucumber through integration of favorable physio-biochemical traits and understanding the detailed molecular network associated with heat stress tolerance in cucumber.

Simple cryopreservation protocol for Luffa pollen: enhancing breeding efficiency.

This study aimed to develop a long-term pollen storage protocol for Luffa species (L. acutangula, L. cylindrica, L. echinata, and L. graveolens) and assess its potential for crop improvement. The optimal medium for in vitro pollen germination varied by species, with Brewbaker and Kwack (BK) medium with 10% sucrose suitable for L. acutangula, L. cylindrica, and L. echinata, and BK medium with 3% sucrose ideal for L. graveolens. Overestimation in staining tests compared to in vitro pollen germination was observed. The best results for cryopreservation were achieved with desiccation periods of 20, 30, and 40 min, maintaining moisture content between 14.04% and 18.55%. Pollen viability was negatively correlated with storage temperature (25, 4, and -20°C) and duration. Cryopreserved pollen at -196°C exhibited the highest viability over a prolonged period (2 months) and was comparable to fresh pollen in terms of germination, ovule fertilization, and fruit and seed set. This study presents a simple and reproducible pollen cryopreservation protocol applicable across Luffa species, facilitating long-term storage and its use in crop improvement efforts.

High-quality Momordica balsamina genome elucidates its potential use in improving stress resilience and therapeutic properties of bitter gourd.

Introduction: Momordica balsamina is the closest wild species that can be crossed with an important fruit vegetable crop, Momordica charantia, has immense medicinal value, and placed under II subclass of primary gene pool of bitter gourd. M. balsamina is tolerant to major biotic and abiotic stresses. Genome characterization of Momordica balsamina as a wild relative of bitter gourd will contribute to the knowledge of the gene pool available for improvement in bitter gourd. There is potential to transfer gene/s related to biotic resistance and medicinal importance from M. balsamina to M. charantia to produce high-quality, better yielding and stress tolerant bitter gourd genotypes. Methods: The present study provides the first and high-quality chromosome-level genome assembly of M. balsamina with size 384.90 Mb and N50 30.96 Mb using sequence data from 10x Genomics, Nanopore, and Hi-C platforms. Results: A total of 6,32,098 transposons elements; 2,15,379 simple sequence repeats; 5,67,483 transcription factor binding sites; 3,376 noncoding RNA genes; and 41,652 protein-coding genes were identified, and 4,347 disease resistance, 67 heat stress-related, 05 carotenoid-related, 15 salt stress-related, 229 cucurbitacin-related, 19 terpenes-related, 37 antioxidant activity, and 06 sex determination-related genes were characterized. Conclusion: Genome sequencing of M. balsamina will facilitate interspecific introgression of desirable traits. This information is cataloged in the form of webgenomic resource available at http://webtom.cabgrid.res.in/mbger/. Our finding of comparative genome analysis will be useful to get insights into the patterns and processes associated with genome evolution and to uncover functional regions of cucurbit genomes.

Identification of a major QTL, Parth6.1 associated with parthenocarpic fruit development in slicing cucumber genotype, Pusa Parthenocarpic Cucumber-6.

Parthenocarpy is an extremely important trait that revolutionized the worldwide cultivation of cucumber under protected conditions. Pusa Parthenocarpic Cucumber-6 (PPC-6) is one of the important commercially cultivated varieties under protected conditions in India. Understanding the genetics of parthenocarpy, molecular mapping and the development of molecular markers closely associated with the trait will facilitate the introgression of parthenocarpic traits into non-conventional germplasm and elite varieties. The F1, F2 and back-crosses progenies with a non-parthenocarpic genotype, Pusa Uday indicated a single incomplete dominant gene controlling parthenocarpy in PPC-6. QTL-seq comprising of the early parthenocarpy and non-parthenocarpic bulks along with the parental lines identified two major genomic regions, one each in chromosome 3 and chromosome 6 spanning over a region of 2.7 Mb and 7.8 Mb, respectively. Conventional mapping using F2:3 population also identified two QTLs, Parth6.1 and Parth6.2 in chromosome 6 which indicated the presence of a major effect QTL in chromosome 6 determining parthenocarpy in PPC-6. The flanking markers, SSR01148 and SSR 01012 for Parth6.1 locus and SSR10476 and SSR 19174 for Parth6.2 locus were identified and can be used for introgression of parthenocarpy through the marker-assisted back-crossing programme. Functional annotation of the QTL-region identified two major genes, Csa_6G396640 and Csa_6G405890 designated as probable indole-3-pyruvate monooxygenase YUCCA11 and Auxin response factor 16, respectively associated with auxin biosynthesis as potential candidate genes. Csa_6G396640 showed only one insertion at position 2179 in the non-parthenocarpic parent. In the case of Csa_6G405890, more variations were observed between the two parents in the form of SNPs and InDels. The study provides insight about genomic regions, closely associated markers and possible candidate genes associated with parthenocarpy in PPC-6 which will be instrumental for functional genomics study and better understanding of parthenocarpy in cucumber.