Application of RSM- CCD methodology and image J. for modeling and optimization of orchid protocorm encapsulation

Synthetic seed technology is an excellent method for large-scale production of unique hybrids, valuable and unstable genotypes, and genetically modified plants that unable to produce plants that require symbiosis of mycorrhiza fungi for seed germination. This technology is now considered as an efficient way to propagate and store several important commercial orchids. The evaluation of physical properties of synthetic seeds has been recognized as one of the critical aspects for seed quality enhancement, which resulted in higher germination and viability at storage duration. The main aim of this study was to optimize the encapsulation conditions to reach the physical properties of Phalaenopsis orchid synthetic seeds using the response surface methodology. A device was designed to control the dripping of alginate for a given temperature in order to wrap the protocorm. The central composite design has been used to investigate the effect of encapsulation variables on the physical properties of orchid synthetic seed such as volume, sphericity Index (SI) and Concentricity Index (CI). Four independent variables were considered to optimize the physical properties of orchid synthetic seed including two sodium alginate solution temperature (4 and 25 °C), three calcium chloride solution temperature (25, 30, and 35 °C), three alginate droplet height (10, 13, and 16 mm), and three stirring rate of calcium chloride solution (zero (no stirring), 1, and 2 rpm). To determine the synthetic seeds, digital images were taken, and response variables were extracted using the image J processing platform. The ANOVA results showed significant effects of linear and interaction terms of models on all studied factors (P < 0.001). The results indicate that stirring rate has most significant effect on the synthetic seed sphericity Index (p < 0.0001). The results showed that the optimum values of volume, sphericity Index and concentricity Index CI were 1285.27 mm³, 0.927 %, and of 2.436 mm, respectively, with a desirability of 0.756. Optimum conditions for orchid protocorm encapsulation were obtained with stirring rate of 0.83 rpm, droplet height of 10 mm, high calcium chloride solution temperature of 35 °C, and sodium alginate solution temperature of 25 °C. In general, results showed that the use of image J and the response surface methodology is a useful tool to obtain the optimal physical properties of synthetic seeds. The modeling and optimizing of physical characteristics of synthetic seed can be developed for encapsulation of any explants of plant species.