2.7. Experimental design and data analysis
The aforementioned experiments were arranged as a completely randomized design with 10 replications. Each culture vessel was considered an experimental unit, and there were three explants per culture vessel. Collected data were analyzed using SPSS 13.0 for Windows (SPSS, Chicago, IL). When significant differences (P < 0.05) occurred, means were separated using Fisher’s Protected Least Significant Differences (LSD) at P < 0.05 level
2.8. Transplantation and acclimatization
After washing off the rooting medium with tap water, plantlets were transplanted to black plastic pots (10 cm in diameter) containing a substrate comprised of 20% clay soil, 40% carbonized rice hull, 20% perlite, and 20% coarse sand based on volume. They were grown in a shaded greenhouse under a maximum photosynthetic active photon flux density of 200 μmol m−2 s−1, temperature range of 20–28 °C, and a relative humidity from 70% to 100%. Plants were watered through intermittent mist at 10 s per 30 min. After two weeks of growth in the shaded greenhouse, a nutrient solution with N at 100 mg L−1prepared from a 30N-10P2O5-10K2O fertilizer was applied weekly. Survival rates of plantlets were recorded. Morphological characteristics including leaf color and size as well as internode length and plant height were evaluated for the potential occurrence of somaclonal variation. Three months later, plants were transplanted for a field trial