In Vitro Shoot Proliferation of Pyrus communis cv. ʽShekariʼ in Different Medium Nutrition by Design Expert Software

Micropropagation is important for both multiplication and preservation of a wide range of nursery plants, including many fruit crops. A number of studies exist on optimization of growth in in vitro condition for one or two cultivars, but often these results cannot be used for the other genotypes because individual cultivars may differ greatly in their requirements. Therefore, genotype-specific medium are usually empirically developed for many plants including pear. Pear cultivars and species are often recalcitrant to tissue culture manipulations and Murashige and Skoog (23) (MS) basal nutrient medium at full or half strength or with slight modifications is the most media were used. The QL, DKW, and WPM media are also used and they differ mostly in types or amounts of calcium and nitrogen in compared with MS. Developing growth media for specific and unique cultivars is complex and time-consuming. Currently, improved experimental design and using statistical softwares allow much more efficient approaches to be utilized for the improvement of micropropagation media and conditions. Improving of growth medium for in vitro propagation of plants depends on type and quantities of mineral nutrients and plant growth regulators as important ones. The existence of statistical softwares to manage effective factors is very needed to access an optimized growth medium for in vitro propagation of plants. Design Expert is used as auxiliary software to identify essential factors in in vitro culture. Since the in vitro proliferation parameters of Pyrus communis cv. ʽShekariʼ need to optimize for growth better, we were designed and performed a multifactor surface response experiment by Design Expert software to following two goals. First, to find optimized amount of some elements in medium and second, to show the response surface method can be useful for improving in vitro culture.

One experiment was designed by Design Expert software and was performed to improve in vitro proliferated shoots of Pyrus communis cv. ʽShekariʼ. Shoots were grown in a modified MS medium (supplement with 1 mg l-1 of N6-benzyladenine) were used for this experiment. The experiment was included 20 model points randomly based on three nutritional factors: NH4NO3 (0.5-1.5×), Fe (0.5-1.5×) and micro nutrients (1-2×) in different concentration of their MS amounts. Media enriched with sucrose (30 g l-1) and agar (8 g l-1) after pH adjustment at 5.7. Cultures were grown at 25°C under a 16-h photoperiod with 70–90 μM m-2 s-1 irradiance provided by a combination of cool- and warm-white fluorescent bulbs and were transferred to new medium every 3 weeks. Several responses were recorded after two months: Proliferated shoot number, proliferated shoot length (cm), total leaf number, leaf chlorophyll a (mg g-1), leaf carotenoids (mg g-1), and vegetative growth (cm). Responses for each point were the mean of 5 replicates. Experimental design, model evaluation, and analysis were done by Design-Expert® 8 (2010) software and the highest-order polynomial model that was significant for each response was used for ANOVA.

Factors statistically were significant for responses according to ANOVA in linear, 2FI and quadratic models. Reduced NH4NO3 (×0.5) and enhanced Fe (×1.5) induced the higher number of proliferated shoots up to 4.43 folds of control according a quadratic model. NH4NO3 and Fe×Micro had negative liner relationships with shoot length, while leaf number negatively was affected by micros. Fe and NH4NO3 were effective factors on leaf chlorophyll a and carotenoids contents. Increasing Fe (×1.5) and decreasing NH4NO3 (×0.5) led to 2 folds higher production of chlorophyll a and carotenoids. Vegetative growth of Pyrus communis cv. ʽShekariʼ  in a quadratic-order method (negatively controlled by NH4NO3 and micros) increased by high values of proliferated shoot number and shoot length induced by reduced NH4NO3 (×0.5). Optimized amount of three studied factors based on two important responses, maximum amount of proliferated shoot number and length, were 0.9, 1 and 0.5× for Fe, micro and NH4NO3 in MS medium, respectively.

Design Expert software and response surface method were used successfully for in vitro optimizing of Pyrus communis cv. ʽShekariʼ regenerated shoots. Fe, Micro and NH4NO3, were the effective factors for shoot regeneration responses in linear, 2FI and quadratic models. The multifactor investigation in surface response design will enable us to predict an optimal medium for several effective factors and estimate suitable responses. Outputs of these types of experiments provide a suitable background to increase optimization accuracy for future experiments.