Clonal micropropagation of hybrid Pistacia L. genotypes and determination of genetic stability with IRAP-ISSR molecular markers

In vitro clonal micropropagation protocols were developed for seedlings derived from axenically germinated seeds of hybrid Pistacia L. genotypes obtained by controlled pollination. IRAP and ISSR molecular markers were used to test genetic stability in long-term micropropagated cultures of hybrid genotypes. 8 hybrid genotypes were produced from the inter- and intra-species of P. khinjuk Stocks, P. vera L., P. terebinthus L. and P. atlantica by controlled pollination between the male and female genotypes of the Pistacia species. Surface sterilization techniques have been developed to the in vitro cultures from the seeds of the hybrid genotypes such as P. khinjuk × P. khinjuk, P. khinjuk × P. terebinthus, P. khinjuk × P. atlantica, P. vera × P. terebinthus, P. vera × P. vera, P. vera × P. atlantica, P.terebinthus × P. terebinthus and P. vera × P. khinjuk. The best shaking concentration for the axenic germination of the seeds of the P. khinjuk × P. khinjuk genotype was obtained in 10% sodium hypochlorite for 15 min, while the best maintaining time for other hybrid genotypes was 10 minutes. The highest germination rate was 86.70% in the P. vera × P. terebinthus genotype among the 8 hybrid genotypes when the hybrid seeds were cultured in MS medium supplemented with 1 mgl-1 BA or without any plant growth regulator to the establishment of in vitro culture. Detailed micropropagation methods were described for the clonal propagation of three rootstock candidate P. vera × P. vera, P. khinjuk × P. atlantica ve P. khinjuk × P. terebinthus mong 8 hybrid genotypes which may be used as rootstock for pistachio, showing better development during in vitro germination studies. For the regeneration of clonal shoots, P. vera × P. vera gave the best results in terms of an average number of 2.45 shoots cultured on MS medium supplemented with 1 mgl-1 BA among different media (MS, WPM, and SH) tested. The effects of MS medium strength (1/4, 1/2, 1/1, 2/1) on shoot proliferation among the clones were investigated. The P. vera × P. vera gave the best result with the mean number of 2.60 shoots and with an average shoot length of 17.75 mm when the explants were cultured on a double strength MS medium supplemented with 1 mgl-1 BA. For rooting of hybrid clones, the 8 mgl-1 IBA treatment gave the best results among different IBA concentrations, and 40% rooting was obtained in the P. khinjuk × P. terebinthus genotype. Three cm long shoots were found to give the best results in rooting P. vera × P. vera, P. khinjuk × P. atlantica ve P. khinjuk × P. terebinthus (40%, 35% and 35%, respectively) when the effects of shoot length on rooting success are evaluated. And the use of fully sterilized peat is appropriate for acclimatization of in vitro rooted hybrid clones. 6 hybrid genotypes that may be rootstocks to pistachio trees (P. khinjuk × P. khinjuk, P. khinjuk × P. terebinthus, P. khinjuk × P. atlantica, P. vera × P. terebinthus, P. vera × P. vera and P. vera × P. atlantica) were studied with 5 ISSR and 7 IRAP primers to identify possible somaclonal variations that may occur during in vitro propagation. Obtained data reveal that micropropagation of these hybrid genotypes showed "reasonable polymorphism" (0.25 ≤ PIC ≤ 0.5). These results showed that the optimized micropropagation techniques can be used safely for the clonal propagation of hybrid Pistacia genotypes. In conclusion, this is the first study on the micropropagation of hybrid Pistacia genotypes and the determination of somaclonal variations of subcultured explants during tissue culture. Keywords: Pistacia, clonal rootstock micropropagation, ISSR, IRAP, somaclonal variation.