MICROPROPAGATION Direct shoot organogenesis from in vitro-derived mature leaf explants of pistachio E. Tilkat & A. Onay Received: 22 June 2007 / Accepted: 5 September 2008 / Published online: 21 November 2008 / Editor: J. Grosser # The Society for In Vitro Biology 2008 Abstract An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl -1 IAA and 2 mgl -1 BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl -1 BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl -1 of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species. Keywords Leaf explant . P. vera L. . Plant regeneration . Organogenesis Introduction Pistachio (Pistacia vera L.), a member of the Anacardia- ceae, is a virtually cosmopolitan family of the Spindalis/ Rulates comprising 70 genera and 600 species (Wannan and Quinn 1991). Zohary (1952) recognizes 11 species within the genus Pistacia; other authors recognize as many as 15 (Whitehouse 1957). P. vera is the only commercially important species in the genus Pistacia. The tree requires long, hot summers (mean temperatures ≥30°C for 98– 110 d) and moderate winters with at least 1,000 h ≤ 7°C (Ayfer 1990). The pistachio is a dioecious tree species cultivated in Iran, California, Turkey and other warm- temperate to subtropical parts of the world for its edible nuts. Most pistachio cultivation is based on clonally propagated scion cultivars grafted onto seedling rootstocks of the same species or of other Pistacia species. The few cultivars of P. vera L., probably less than 100, described worldwide are thought to be derived from only a few primitive varieties (Maggs 1973) resulting in a high degree of genetic vulnerability. The California pistachio industry is based on the nut producing Kerman and the pollen producing Peters cultivars. Unlike California, in Iran and Turkey (the first and third largest pistachio producing countries), there are many different male and female cultivars in the orchards. The main cultivars grown in Iran are Ohadi, Kalleghochi, Ahmad Aghai, Badami Zarand, Rezaii, and Pust Piazi (Esmail-pour 2001) and in Turkey: Uzun, Halebi, Siirt, Beyazben, Sultani, Değirmi, and Keten Gömleği (Ak and Açar 2001). There are research results that show variability in shell splitting among edible pistachio cultivars (Crane 1984). One of the main areas of interest in current pistachio breeding programs is the percentage of split shells (Hormaza and Wünsch 2007). Split shells make pistachio nuts more attractive to consum- In Vitro Cell.Dev.Biol.-Plant (2009) 45:92–98 DOI 10.1007/s11627-008-9168-4 E. Tilkat (*) : A. Onay Faculty of Science and Literature, Department of Biology, University of Dicle, 21280 Diyarbakir, Turkey e-mail: etilkat@dicle.edu.tr