Determination of resistance to important diseases of some cucumber (Cucumis sativus l.) genotypes by molecular methods and optimization of double haploid technique

The development of pure lines used in hybrid seed production is tedious, costly and time consuming (6-8 years). Tissue culture (doubled haploid technique) seems to be an alternative to speed up the process. Using doubled haploid technique 100% homozygous parental lines can be obtained in a time relatively sort (1-2 years). Among the techniques used in doubled haploid plants production, there are parthenogenesis (pollination with irradiated pollen followed by embryo rescue), androgenesis (anther or microspore culture) and gynogenesis (ovul or ovary culture). Among these techniques, gynogenesis seems to be more efficient but depends on genotypes. Therefore, protocol optimization is required for genotypes.In this study, the purpose was to optimize protocols for doubled haploid plants production in 28 genotype by ovule culture and determine the resistance status of these genotypes to Zucchini Yellow Mosaic Virus (ZYMV), Cucumber Mosaic Virus (CMV) and Papaya Ring Spot Virus (PRSV) using molecular markers previously developed. For doubled haploid optimization, three main protocols (P1, P2 and P2K3) were used. According to P1 protocol, the highest embryo induction rate was 71% with genotype 22, the lowest rate was 3% with genotype 15. The average embryo induction rate was 20%. The highest regeneration (shoot induction) rate was 140% (Average of 1.4 shoot/eksplant) with genotype 18. The lowest rate was 0.5% with genotype 25. The average shoot induction rate was 6%. Despite regeneration, regenerants could not develop in planlets.In the P2 medium, the highest embryo induction rate (75%) was found in genotype 3. The lowest embryo rate which is 5.8% was found in genotype 6. The average embryo induction rate was 20%. In terms of rejenerasyon the highest rate was 4.6% obtained in genotype 18. The lowest regeneration rate was 0.5% with genotype 7. The average regeneration rate was 6%. Although regeneration was observed in some genotypes, plantlets could not be obtained. Optimization may be required for the development of regenerants.P2K3 medium is a protocol developed by modifying P2 protocol. In P2K3 medium the highest embryo induction rate was 104% (an average of 1.04 embryo/explant) obtained with genotype 4. The lowest embryo induction rate was 2.5% (genotype 2) and the average embryo induction rate was 20%. For regeneration rates, the highest one obtained in genotype 14 was 470% (an average of 4.7 shoot/explant). The lowest regeneration rate was 1% (genotype 12). In P2K3 medium, some of regenerants developed to give plantlets. Including 3 diploid plants from genotype 7, 4 haploid plants from genotype 11 and 11diploid plants from genotype 14, 19 plants were obtained in total. In short, including 4 haploid and 15 diploid plants, 19 plants were obtained totally. In doubled haploid optimization study, 19 plants are obtained in total with modified P2K3 protocol. Among these plants, 4 are haploid and 15 are diploid. This developed new protocol is promising for cucumber doubled haploid lines development programs. In the molecular analysis studies the molecular markers developed previously for Zucchini Yellow Mosaic Virus (ZYMV), Cucumber Mosaic Virus (CMV), Papaya Ring Spot Virus (PRSV) resistance were optimized and the resistance status of 28 cucumber genotypes to ZYMV, CMV, PRSV were determined. As markers, CAPS-T86C was used for ZYMV resistance screening and SSR11-177 was used for PRSV resistance screening. For CMV disease screening, two flanking markers were used (SSR9-56 and SSR11-1).