Optimization of steps in the technology of obtaining doubled haploids of summer squash (<i>Cucurbita pepo</i> L.) in the culture of unpollinated ovules <i>in vitro</i>

Abstract

Relevance. To create an effective technology for obtaining doubled haploids (DH-technology) of zucchini in unpollinatedseedpod culture in vitro it is necessary to select the optimal values of many factors, the degree of influence of each of which on gynogenesis can vary significantly. The aim of the study was to optimize the individual stages of the technology.Methods. Liquid and agarized (7 g/L) IMC medium with different sucrose concentrations (20 to 80 g/L) and different plant growth regulators (2 mg/L 2,4 D; 0.2 mg/L TDZ ; 0.8 mg/L 2,4 D and 1.2 mg/L NUC) were used for induction of embryogenesis.Results. Optimal for the studied zucchini genotypes was pre-isolated from the evening, plucked in the morning opened bud. Sterilization of zucchini ovaries by short-term burning after treatment with 96% alcohol, allows significant reduction of the time required for this step without loss of embryogenic potential. IMC nutrient medium with sucrose (20 to 40 g/l) can be used for induction of gynogenesis in the unpollinatedzucchini ovary culture. The use of nutrient media with 2 mg/l 2,4 D for most genotypes was more effective and resulted in higher number of embryoids compared to nutrient media containing 0.2 mg/l TDC and media with 0.8 mg/l 2,4 D and 1.2 mg/l NAA. Embryoid formation was observed after 5 weeks of cultivation.Conclusion. We were able to complete the full cycle of technology for obtaining doubled haploids in unpollinatedseedpod culture in vitro for 30 zucchini genotypes and obtain DHplants, which are valuable source material for both breeders and genetic research. Optimization of the individual steps of the technology made it possible to achieve the maximum result for individual genotypes – 55 embryoids per 100 cultivated ovules.