Unraveling Factors Affecting Micropropagation of Four Persian Walnut Varieties

Walnuts are considered recalcitrant to tissue culture, with a great genetic determinism on all stages of micropropagation; while other factors, also with great impact, become more complicated with the reproduction of newly realized varieties. In this research, a holistic approach aimed to determine the influence of genotype and the nutritive formulation throughout the whole cycle of micropropagation of four Persian walnut varieties (Juglans regia L.) was presented. During the in vitro establishment it was determined that besides genotype and culture medium, the effect of collection season and the likely interaction amongst factors had a great influence on the successful introduction of all four genotypes. However, all cultures were affected by a deep decay, being necessary the introduction of ethylenediamine di-2-hydroxyphenyl acetate ferric, as iron source, and Phloroglucinol in both Murashige and Skoog (1962) and the corrected Driver and Kuniyuki (1987) formulations. These modifications allowed the stabilization of cultures, maintaining thereafter a steady quality. Either proliferation, rooting and ex vitro survival of four clones were affected by the culture medium, obtaining the best results with the corrected Driver and Kuniyuki (1987) formulation. Finally, in vitro plants produced from all clones were acclimated with high survival rates (75.9–91.1% for the best culture medium), depending of clone and the culture medium used. Microsatellite analysis showed that micropropagated plants maintained the same genetic profiles of their corresponding donor trees. These results might contribute to deepening of the understanding of factors that determine the success of micropropagation of walnuts, and the extents of its influence; whereas, it sets the basis for the commercial micropropagation of all four clones.