Developing micropropagation protocol for two native dwarfing apple rootstocks (Morabbaei and Zinati).

The influence of dwarfing rootstocks on controling shoots, increasing number of trees per hectare and consequently yield increase is well undrestood. Famous apple vegetative rootstocks such as Maling and Maling Merton regardless of having clonal benefits have some weakness points such as succeptibility to crown rot. Native dwarfing cultivars are adapted more to Iranian fauna and flora, heavy soil texture and high calcareous soils. Slow growing and dwarfinf native cultivars named as Zinati and Morabbaei have high levels of tolerance to different common stresses in apple plantation areas such as drought, heavy texture and calcic soils. If these native cultivars have suitable graft comptibility with commercial cultivars, and in future research show good fruit-set, low abscission, low alternate bearing and high yield could be introduced as commercial native dwarfing rootstocks. This research was carried out to study and optimize different stages of micropropagation for these two cultivars. Plant materials were provided from healthy and actively growing current-year shoots of twenty-year-old trees during spring to winter from apple cultivars collection of Kamalshahr Horticultural Research Station, Karaj, Iran. Explants consisting of 1 or 2 buds from softwood, semi hardwood and hardwood parts of the shoots were prepared and after surface sterilization cultured on MS medium containing 0.2-0.5 mg/L BAP for establishment. Different treatments such as washing under tap water, 70% ethanol, various concentrations of sodium hypochlorite and exposure times with continous shaking and also Mercuric chloride were applied for surface sterilization. Effects of different sampling dates during growing season were investigated on sterilization and growth parameters. Several treatments of different growth regulators, BAP, 2iP, zeatin, GA3 and kinetin, alone or in combination with IBA and IAA were tested on shoot growth and proliferation. Also, effects of basal culture media, WPM, QL, MS and DKW were investigated in establishment and proliferation stages. Experiments in each stage were set up as simple completly randomized design or factorial based, at least with 5 replications, each replicate consisting 5 explants. Recorded data on number of buds activated, bud breake percentages, shoot length, shoot and leaf number per explant and visual growth quality index were statistically analysed. Micro shoots produced from proliferation stage were used for optimizing rooting and the effects of different treatments of growth regulators, basal culture media and pHysical conditions were investigated and finally plantlets were transferred to soil and acclimatized. Results showed that using 70% ethanol for 45 sec, 2.5% nano silver for 10 min, 25% howsehold bleach with 1-2 drops of Tween-20 for 8-10 min and 4-5 times washing with sterile distilled water was effective for surface sterilization. Sampling date and type of explant affected successful rates considerably and hardwood samples taken in the spring produced the highest shoot number per explant, shoot and leaf length compared with samples from automn and winter seasons. Zinati cultivar was more responsive and had more in vitro establishment rate compared to Morabbaei. In establishment, number of shoots per explant was not affected by basal culture media but number of leaves per explant, shoot length and visual growth quality index were higher on MS medium compared to other 3 media. Reducing the strength of macro and micro elements to half had no positive effect on recorded parameters and even had negative effect on number of leaves per explant. In proliferation stage, slight differences were observed among BAP concentrations tested and 2.0 mg/L BAP caused better growth quality compared with 0.5 and 1.0 mg/L. Number of shoots per explant for Morabbaei cultivar increased with increasing BAP concentration and the highest was obtained in 2.0 mg/L but for Zinati cultivar this trend was not observed and 0.5 mg/L gave the highest shoot number. The longest shoots for Morabbaei were produced in 1.0 mg/L BAP and for Zinati cultivar in 2.0 mg/L. Comparing 3 basal culture media; MS, QL and QL, the greatest number of leaves per explant, the longest shoots and the best visual growth quality index was obtained in WPM and Zinati cultivar producd longer shoots. When growth and proliferation of both cultivars were compared in QL and MS media supplemented with 1.0 mg/L, 1.0 mg/L GA3 and 0.01 mg/L IBA, it was observed that Zinati responded better in MS and Morabbaei in QL medium. Using sucrose as carbon source in culture medium compared with sorbitol caused better shoot quality and sorbitol application had no positive effect on shoot growth and proliferation. In rooting stage, different concentrations of NAA were studied and 90% rooting was achieved in 0.25 mg/L NAA added to 1/2QL. About 100% rooting rate was obtained for both cultivars (100% and 96% for Morabbaei and Zinati respectively) in 1/2QL supplemented with 1.0 mg/L IBA+0.5 mg/L NAA. Plantlets were transferred to a peat: perlite soil mixture (1:1) for acclimation in several times. Considering the slow growing and dwarfing habits of these cultivars, extra care, better controled envirnomental conditions (greenhouse), regular irrigation and fertilizer was neccesary for the first 8 weeks after transfer for an effective and suucessful acclimation. In good conditions, an average of 85% survaival rates was achieved but some times the rate was even less than 50% and as a pilot, several numbers of plantlets ware produced and acclimatized successfully to in vivo conditions. Over all, based on the results obtained, an optimized protocol can be developed for micropropagation of these two native dwarfing apple rootstocks. Keywords: Apple (Malus domestica Borkh.), micropropagation, clonal rootstock, dwarfing