Developing practical protocols for micropropagation of 6 important pistachio rootstocks

In this project different stages of micropropagation including surface sterilization, culture establishment, basal culture medium and concentration of growth regulators for shoot proliferation and then for rooting, suitable strategies to overcome encountered propagation problems and finally the efficient methods for transferring plantlets to in vivo conditions and acclimatization were investigated in the Department of Tissue Culture and Gene Transformation of Agricultural Biotechnology Research Institute of Iran (ABRII). Plant materials used were both in vitro germinated seeds and actively-growing healthy current-year twigs from adult trees (15 – 20 years old) of different pistachio rootstocks including Pistacia vera cv. Badami Zarand, P. atlantica, P. mutica, P. khinjuk, P. integrima and UCB1, all supplied by Iranian Pistachio Research Institute (Rafsanjan, Kerman Province). Experiments carried out using twigs in all of the mentioned rootstocks showed little or no success in culture establishment due to severe internal contamination and phenolic exudation from the explants. Only a very few explants slowly responded but these died later because of internal bacterial contamination. Frequent subculturing every 2-3 days within the first two weeks after initial culture did reduce the phenolic exudation problem but most of the explants with no apparent contamination did not respond to shoot induction treatments and remained inactive for long time (4 months), after which were omitted from the cultures. Severe heading and pruning of the donor trees in the winter to using their young shoots in the spring season had also little success. Meanwhile, cultures were established quite successfully using in vitro seedlings of Badami Zarand cultivar and P. mutica and P. khinjuk species. Several experiments were conducted in different stages of their micropropagation particularly for Badami Zarand as one of the most important and popular pistachio rootstocks. A protocol was developed based on the obtained results after studying various aspects of shoot proliferation including the response of different clones to shoot proliferation medium, effects of different basal culture media, vitamin compositions, BAP concentrations, culture vessels and number of explants cultured in each jar, incubation temperatures and subculturing times, sucrose concentrations and replacing table-sugar with sucrose, increasing Fe-Na2EDTA concentrationand in rooting stage types and modifications in basal culture media, vitamins, growth regulators, incubation conditions, length of micro shoots and cutting place, sucrose and agar concentrationsand soil compositions and acclimation methods during hardening stage. Briefly, DKW basal culture medium supplemented with 2.0 mg. L-1 BAP, 10 mg.L-1 thiamin-HCl, 1 mg.L-1 nicotinic acid, 1 mg.L-1 pyridoxine-HCl, 100 mg.L-1 myo-inositol, 73.4 mg.L-1 Fe-Na-EDTA, 30 g.L-1 sucrose and 7 g.L-1 agar found to be the most suitable medium for shoot growth and multiplication. Incubation under a 16-hour photoperiod at 27±1 °C gave more shoot growth and proliferation but was associated with higher shoot tip necrosis. An incubation temperature of 24±1 °C gave less necrosis.Experiments were also conducted on micropropagation of P. mutica and P. khinjuk species using explants from in vitro- germinated seeds. For root induction, modified MS medium (half concentration of macro elements) supplemented with 2.0 mg.L-1 IBA plus 0.5 mg.L-1 NAA and 1 mg.L-1 thiamin-HCl, 1 mg.L-1 nicotinic acid, 1 mg.L-1pyridoxine-HCl, 100 mg.L-1 myo-inositol and 30 g.L-1 sucrose, solidified with 8 g.L-1 agar found to be the most effective medium composition. Using micro-shoots of about 20-30 mm in length and an incubation temperature of 26±1 °C gave better results. A preliminary 6 to 7-day dark incubation and subsequently a further two weeks under 16 h-photoperiod at 26±1 °C was necessary for effective root induction. For root development, micro-shoots with root primordia had to be transferred to a hormone-free culture medium for about 20 days before transferring to in vivo conditions for acclimatization