Developing protocols for mass propagation of important pistachio rootstocks and commercial cultivars using tissue culture techniques

• Main Authors: Vatan Pur Azghandi, A`li, Habashi, A`li Akbar, Taj Abadi Pur, A`li, Mojtahedi, Narges, Zadeh Parizi, Reza, Rostami, A`li Mardan, Rafi`ei, A`bbas, Shakib, A`li Mohammad
• Format: Publication
• Language: Persian
• Published: Karaj (Iran) Agricultural Biotechnology Research Institute of Iran 2008
• Subjects: CULTIVO DE TEJIDOS; CULTURE DE TISSU; DESARROLLO EMBRIONARIO; DEVELOPPEMENT EMBRYONNAIRE; EMBRYONIC DEVELOPMENT; IRAN ISLAMIC REPUBLIC; IRAN REPUBLICA ISLAMICA; IRAN REPUBLIQUE ISLAMIQUE; Kerman ( Province ); MATERIALES DE PROPAGACION; MATERIAU DE MULTIPLICATION; MICROPROPAGACION; MICROPROPAGATION; MULTIPLICATION DES PLANTES; PISTACHE; Pistachio Reseach Institute; PISTACHIOS; PISTACHO; PISTACIA VERA; PLANT PROPAGATION; PORTAINJERTOS; PORTE GREFFE; PROPAGACION DE PLANTAS; PROPAGATION MATERIALS; Rafsanjan ( Iran ); ROOTSTOCKS; TISSUE CULTURE

The research "Developing protocols for mass propagation of important pistachio rootstocks and commercial cultivars using tissue culture techniques" was conducted in 5 different projects including: 1. 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 Transfer of Agricultural Biotechnology Research Institute of Iran (ABRII). Plant materials used were nodal explants taken from actively-growing healthy current-year twigs from adult trees (15 – 20 years old) of 6 different pistachio rootstocks including Pistacia vera cv. Badami Zarand, P. atlantica, P. mutica, P. khinjuk, P. integrima and UCB1, and in vitro germinated seeds of these rootstocks, all supplied by Iranian Pistachio Research Institute (Rafsanjan, Kerman Province). Briefly, DKW basal culture medium supplemented with 2.0 mgL-1 BAP, 10 mgL-1 thiamin-HCl, 1 mgL-1 nicotinic acid, 1 mgL-1 pyridoxine-HCl, 100 mgL-1 myo-inositol, 73.4 mgL-1 Fe-Na-EDTA, 30 grL-1 sucrose and 7 grL-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 mgL-1 IBA plus 0.5 mgL-1 NAA and 1 mgL-1 thiamin-HCl, 1 mgL-1 nicotinic acid, 1 mgL-1 pyridoxine-HCl, 100 mgL-1 myo-inositol and 30 grL-1 sucrose, solidified with 8 gL-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. 2. Developing practical protocols for micropropagation of some important pistachio commercial cultivars and 2 selected male varieties: In this project different stages of micropropagation of 4 pistachio commercial cultivars (Owhadi, Kalleh Ghoochi, Akbary and Ahmad Aghaii) and 2 male varieties (R-31 and R-20) were optimized using nodal explants taken from actively-growing healthy current-year twigs from adult trees of 15 – 20 years old as well as in vitro-germinated seeds, all supplied by Iranian Pistachio Research Institute (Rafsanjan, Kerman Province). Experiments carried out using twigs in all of the mentioned cultivars showed little or no success in culture establishment due to severe internal contamination and phenolic exudation from the explants. 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 did not respond to shoot induction treatments and remained inactive for long time (about 4 months). Severe heading and pruning of the donor trees was carried out in the winter and the young shoots in the spring season were used but this also had little success. However, it was possible to establish cultures from Owhadi and R-31 and shoot proliferation and rooting stages were optimized for them. DKW basal culture medium with doubled concentration of FeNa2EDTA supplemented with B5 vitamins and 2 mg/L BAP was the best medium composition for shoot proliferation of Owhadi and R-31 cultivars. For rooting, a modified MS medium (half strength of its macro and micro elements and normal concentration of FeNa2EDTA) supplemented with 2.5 mg/L NAA and 0.1 mg/L IBA gave the best results. 3. Developing practical protocols for callus and cell suspension cultures and plant regeneration via somatic embryogenesis or organogenesis for important pistachio rootstocks and commercial cultivars: In order to develop an efficient plant regeneration protocol for pistachio, several experiments were conducted using both mature and immature kernels of Kallehghouchi, Owhadi, Fandoghi-48, Ahmadaghaii and Badamizarand cultivars at three different stages after pollination (4-6, 8-10, 10-12 weeks). Immature fruits were pre-sterilized with washing liquid detergent for 10 min followed by a rinse with distilled water. These presterilized fruits were then exposed to a 2.5% (v/v) solution of sodium hypochlorite for 15 min and rinsed 3 times with sterile distilled water. The surface sterilized fruits were cracked and immature kernels were removed and used as explants. Effects of culture media (liquid or solid MS, full or half strength of macro and micro elements), different combinations of growth regulators (BAP, Kinetin, Zeatin, IAA, 2,4-D, NAA) in various concentrations and incubation conditions (light or dark) were investigated on plant regeneration. Direct shoot regeneration (DSR) obtained only from immature cotyledon explants of Kallehghouchi cultivar, cultured on half strength solid MS medium supplemented with 2.0 mg.L-1 BAP and 2.0 mg.L-1 Zeatin and 1.0 mg.L-1 IAA or the same culture medium containing 4.0 mg.L-1 BAP and 2.0 mg.L-1 Zeatin and 1.0 mg.L-1 IAA. Regenerated shoots were differentiated directly from the surface of immature cotyledons and produced numerous leaf-like organs. These organs did not elongate enough to produce normal shoots and gradually turned to brown colour and became inactive.4. Developing a practical protocol for pistachio micrografting in vitro and in vivo In this project, pistachio (Pistacia vera L.) micrografting technique was studied both in vitro and in vivo. For in vitro micrografting, rootstocks and scions (Badami- Zarand, Owhadi, Akbari, Ahmadaghaii and R-31 selected male variety) were propagated using micropropagation technique. Young seedlings (1-2-month-old) of Pistacia vera L. cv. Badami-Zarand were used as rootstock for in vivo micrografting experiments. To prepare the scions of micrografts, auxiliary nodes at least with one leaf from micropropagated-shoots were used and two wedge-shaped oblique cuts were made in their basal parts. Microscions were grafted on rootstocks which were decapitated to remove all leaves and a vertical slit was made on the stump. The scion base, cut in a V-shape, was fitted to the slit. In vitro success rate was evaluated through percentages of regenerated shoots, callus production on grafted area and leaf numbers one and two months after grafting. Results showed that vertical grafting was suitable for in vivo and in vitro grafting. The best culture medium for growing rootstocks after micrografting was MS supplemented with 2 mgL-1 NAA, in which callus production decreased. Zeatin was the best plant growth regulator for in vitro micrografting and increased micrografting success rate. The age of scion had no significant effect on success rate. Parafilm tape was used to increase connection of rootstock and scion for in vivo micrografting. Male and female cultivars showed no differences in micrografting success rate and both of them can be used as rootstocks in micrografting. 5. Comparison of growth and morphologic characteristics of in vitro micropropagated with in vivo seed-raised pistachio plants, under field condition: Field evaluation of growth and development of micropropagated pistachio plants would be very useful before large scale application of this technique. This project was conducted to study the growth and morphological characteristics of micropropagated plants in compare with conventionally pistachio seedlings of Badami Zarand cultivar (an important rootstock) under field conditions. The pistachio plants of both types were produced during 2002 and 2003 simultaneously and transplanted into the field in four different regions of Kerman province (Rafsanjan, KowsarRiz, Sirjan and Kerman) under similar conditions. After establishment, monitoring and recording of vegetative growth parameters such as height and diameter were carried out regularly based on complete randomized design with five replications. The results obtained showed that micropropagated pistachio plants had always greater heights and diameters compared with seed-raised plants but the differences for the diameter were not statistically significant. No particular morphological differences or distinguishable variations were observed between two types of plants. Seedlings and tissue culture plants grew normally with better vegetative growth characteristics for micropropagated plants.