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Explant collection is important for P. vera because shoot tips or nodal buds that have regeneration potential are available only for a limited period of the year for establishing in vitro cultures (Table 2.3.2). Pistachio leaves emerge in late April to mid-May although current year shoot tips and nodal buds are available until September, senescing in October with bud dormancy from October to March depending on weather conditions (Onay et al., 2012). Dormant shoot tips and nodal buds collected between October and February are less responsive than explants collected between March and September that are derived from the previous year’s dormant nodal and apical buds (Tilkat et al., 2013).

Table 2.3.2. Micropropagation of P. vera L.

Explants are collected from three sources: (i) fresh sprouts from the trunk; (ii) apical tips and nodal orthotropic buds distal to the root–shoot junctions (Fig. 2.3.1a); and (iii) plagiotropic apical tips and nodal buds distal to the root–shoot junction. Forcing flushing buds from 20- to 50-year-old trees provide good explants (Fig. 2.3.1b; Onay, 2000b; Tilkat et al., 2008; Onay et al., 2012). Explants are disinfested with 5–20% sodium hypochlorite (NaOCl) followed by a rinse 3–5 times with sterilized distilled water (Tilkat et al., 2013). Fungal and bacterial contaminants are major problems (Bustamante-Garcia, 1984; Barghchi and Alderson, 1985; Abousalim, 1990; Onay, 1996; Ozden-Tokatli et al., 2005; Tilkat et al., 2014). Decontamination of explants from mature pistachio trees is best achieved when the explants are obtained from meristem tips, actively growing shoot tips and forced growing shoot cultures (Onay, 1996; Tilkat et al., 2008).

Fig. 2.3.1. Regeneration of pistachio (P. vera). Explants used for in vitro culture initiation: (a) current year apical and nodal buds and (b) forced flushing buds. (c) Shoots regenerated from apical and nodal buds. (d) Forced apical-shoot tips of ‘Atlı’ on initiation medium after 4 weeks (Tilkat et al., 2009a, 2013). (e) Shoot proliferation from in vitro-derived leaf explants of pistachio ‘Siirt’ (Tilkat and Onay, 2009). (f) Rooted microshoots of ‘Atlı’ on modified MS medium supplemented with 9.8 μM IBA. (g) Acclimatized plantlets (Tilkat and Onay, 2009). (h) One-year-old micropropagated pistachio plant in the field.

Browning, especially during explant establishment, inhibits establishment of in vitro cultures (Ozden-Tokatli et al., 2005; Tilkat, 2006; Tilkat et al., 2008). Use of antioxidants to prevent browning has received some attention (Onay, 2000b). Polyvinylpyrrolidone (PVP) alleviates browning (Ozden-Tokatli et al., 2004), and inclusion of up to 48.0 μM AgNO₃ in the culture medium also improves shoot growth and the regeneration, while reducing basal callus formation in regenerated shoots (Ozden-Tokatli et al., 2005). Ascorbic acid, citric acid and PVP had no affect on inhibition of browning of mature shoot tip cultures (Tilkat et al., 2013). Apical necrosis occurs if explants are not subcultured to fresh culture medium 3 weeks after initiation. Exudation of phenolics has been reported to cause apical necrosis of many tree species (Bellarosa, 1988; Standardi and Romani, 1990). Browning can be avoided with weekly subcultures and removal of dark tissues until the cultures are established. Tilkat et al. (2013) showed that shoot survival of 100% is possible if sterilized shoots are washed 2× for 1 h in sterile distilled water at 150 rpm. Culture initiation from adult shoot tips should involve: (i) vigorous shoots removed during May from mature trees; (ii) surface disinfestation in 10% (v/v) NaOCl for 30 min; (iii) rinsing 3× in sterile distilled water at 150 rpm for 1 h; (iv) culturing on MS medium (Murashige and Skoog, 1962) with Gamborg vitamins (Gamborg et al., 1968), 3% (w/v) sucrose, 8.8 μM 6-benzylaminopurine (BA) and 0.55% (w/v) agar. MS medium containing 4.4 μM BA is used for maintenance and shoot proliferation.

Tilkat (2006) and Tilkat et al. (2008) developed a method that allows faster initiation and establishment of adult material. Apical shoot tips were excised from mature trees. Softwood shoot tips were cut into 1- to 2-cm-long pieces. Following disinfestation, explants were rinsed in twice sterile distilled water for 1 h to remove disinfectant and to control exudation of phenolics. Explants were further dissected to 4–6 mm and placed on initiation medium yielding 100% sterile and nonbrown explants.

Shoots developed from apical and nodal buds and from the forced apical shoot tips of ‘Atlı’ after 4 weeks (Fig. 2.3.1c and d; Tilkat et al., 2013). These shoots were cut into segments containing a shoot or nodal tip and were subcultured on fresh medium (Fig. 2.3.1e). Rooting of shoots was induced on semisolid half-strength MS medium supplemented with 12.25 μM indolebutyric acid (IBA) in darkness for 7 days followed by transfer to auxin-free medium after root emergence (Barghchi, 1982). Rooting can also be induced with a 5 min dip of microshoots in 49 μM IBA (Abousalim, 1990). Plantlets were readily established in peat-based compost (Table 2.3.2; Fig. 2.3.1f and g). Figure 2.3.1h shows a micropropagated pistachio plantlet in the field.