Determination of appropriate embryo rescue stage and ploidy level in citrus crossbreeds

This study was conducted to obtain new rootstock genotypes through hybridization at the Western Mediterranean Agricultural Research Institute between 2015 and 2019. For this purpose, Common Sour Orange (Citrus aurantium L.) (CSO) and Common Mandarin (Citrus reticulata Blanco) (CM) were crossed with Troyer Citrange (Citrus sinensis L. × Poncirus trifoliata L.) (TC) while King Mandarin (Citrus nobilis L.) (KM) was crossed with Carrizo Citrange (Citrus sinensis L. × Poncirus trifoliata L.) (CC). Embryos obtained as a result of hybridization were taken 110, 120 and 130 days after pollination (DAP) and were germinated on MT (1.5 mg/l GA3, 50 g/l sucrose, 25 mg/l adenine sulfate, 500 mg/l malt extract) culture media. Embryo germination rate was found that higher on the 120th DAP within hybridization combinations (CSO x TC; 95.15%, CM x TC; 96.25%, KM x CC; 95.23%). Therefore, it was concluded that this embryo development stage was suitable for embryo culture studies. Germinated embryos were subcultured on MS culture media and then good results were found that 110th DAP within hybridization combinations (CSO x TC; 17.40%, CM x TC; 11.11%, KM x CC; 6.17%) for trifoliate rate. Survival rates of obtained hybrid plants were determined for different stages (between 72.13% and 90.28% for subcultured, between 57.14% and 85.87% for transplanted to mixture and between 40.17% and 64.71% for transferred to greenhouse). The highest survival rates of obtained 335 genotypes from hybridization combinations was found on the 110th DAP within all combinations. In this study, plant length and stem diameter at plants obtained hybrid plants were measured during one year after transferred to greenhouse and consequently, it was determined that genotypes generally showed linear development. It was defined leaf properties similar to the mother parent in most plants except as morphological differently and trifoliates genotypes.A result of ploidy analysis by flow cytometry, genotypes were found diploid except one from 199 hybrids. The nuclear DNA content of diploid genotypes was found between 0.78 pg/2C and 0.93 pg/2C. The nuclear DNA content of genotype derived from CM x TC combination on 120th DAP was determined to be 1.16 pg/2C and this genotype was determinated as triploid. Key Words: Citrus, Poncirus, Rootstock, Embryo rescue, Polyploidy