The effects of different application to induce haploid embryo and plant formation on eggplant (Solanum melongena L.) microspore culture

In the study, two F1 ("A117" and "Amadeo") eggplant cultivars were studied on the possibilities of obtaining haploid and DH plants by microspore culture. For this purpose, microspores were firstly isolated from the anthers of the appropriate microspore developmental stage (mostly vacuole microspores and young twin pollen) and subjected to pretreatment application at 35°C for 3 days in dark conditions. Afterwards microspores were incubated with 2 % sucrose, 0.5 mg/l NAA and 0.5 mg/l BAP, pH 5.9, and incubated in the dark at 25°C for one month. At the end of one month in order to determine the responds of cultivars to microspore culture, percentage of alive microspores at 1st day of microspore isolation, percentage of alive microspores at the end of 3rd day after pretreatment, number of total calli/petri, 1 mm at the end of culture (1st day) number of callus/petri and number of embryos obtained were evaluated. Experimental results revealed that "Amadeo" was prominent in a comparision to "A117" variety in terms of callus and embryo formation rates. Later on in the study, the effects of arabinogalactan proteins (AGPs), abscisic acid (ABA) and different hormone concentrations were investigated in order to improve the microspore culture method. To determine the AGPs effect on microspore culture, different of doses (10 mg/l, 1 mg/l and 0.1 mg/l) were evaluated and AGP application was found to have no stimulating effect on microspore culture in the cultivars used. ABA was applied to microspores at different doses and durations throughout the pre-treatment period to determine ABA effects. First of all, after stress pre-treatment, the effects of ABA apllied at different doses and durations was compared with control group in terms of microspore viability. It was determined that all applied ABA concentrations and durations in both cultivars enhanced the microspore viability percentage. The highest percentage of microspores viability percentage in the "A117" was obtained by application of 0.1 mg/l ABA for 24 hours. In the "Amadeo" cultivar, the increase in the viability percentage was recorded when 1 mg/l ABA was applied for 24 hours. Based on these findings, microspores were cultured in accordance with standard protocol with applications of 0.1 mg/l ABA for in "A117" and 1 mg/l ABA for in "Amadeo" cultivar in the first 24 hours of 3 days pre-treatment at 35°C. At the end of one month, no callus development occurred in the "A117" in both control and ABA application. When the total callus and callus numbers greater than 1 mm were evaluated in the "Amadeo" cultivar, no statistically significant difference was found between the control group and ABA applications. In addition to AGPs and ABA applications, effects of different doses of 2,4-D and kinetin added to media were investigated. For this purpose, 16 different media combinations containing different concentrations of 2,4-D and kinetin were prepared. At the end of the study, while the most effective medium in terms of total number of calli and number of calli bigger than 1 mm was media 12 (0.5 mg / l 2,4-D +1 mg / l KIN), control medium gave the best result in terms of total number of calli and number of calli bigger than 1 mm for Amadeo. When the microspore culture technique used in the study was evaluated, the plantlets were successfully obtained from the calli and embryos obtained from microspores. A total of 18 plants from 36 calli for "A117" cultivar and 25 plants from 70 calli were obtained for "Amadeo" cultivar. The flow cytometry analyzes of the plants developed from the calli obtained from microspore culture revealed that the ploidy levels of obtained plants were diploid and triploid. The DH plant percentage of plants obtained from calli was determined as 68 % in the "A117" cultivar and 60 % in the "Amadeo" cultivar. On the other hand, only haploid plants were obtained from the embryos obtained from microspore culture. The plants obtained from the microspore culture were transferred to field conditions for selfing. After selfing obtained seeds were planted in the field conditions and the fruits were harvested when the fruits were mature. The obtained seeds were re-seeded to obtain double haploid homozygous lines.