Microstructural and histochemical modifications in leaves at successive stages of in vitro development of the terrestrial orchid Spathoglottis plicata Blume

Substantial progress has been made in the micropropagation of orchid species using biotechnological interventions, but the commercial viability of the technique has been hindered by increased mortality of plantlets out of culture vessels. This is attributed to the development of in vitro-induced structural and physiological abnormalities in the plantlets. This research investigated the chronological series of foliar microstructural and histochemical responses at the microscopic level during various stages of plantlet development in Spathoglottis plicata, an important terrestrial orchid. Plants that were grown under in vitro heterotrophic conditions displayed underdeveloped foliar anatomical and histochemical traits such as a thin cuticle, unorganized epidermal cells, and fewer mesophyll tissue density and vascular elements with reduced deposition of lignins, cutin, pectin, tannins, and polyphenolic compounds. Ex vitro hardening of the plantlets in a mixotrophic environment improved the development of the cuticle and histochemical traits, created a uniform epidermal layer, increased mesophyll and vascular elements. The field-developed plants had well-developed dermal, ground, and vascular tissue systems along with increased deposition of primary and secondary metabolites. These gradual changes positively impacted the microstructural and physiological status of in vitro-propagated plantlets of S. plicata to withstand biotic and abiotic stresses in field conditions. This study suggests that subsequent hardening and an acclimatization period would enhance the structural and histochemical parameters of in vitro-propagated S. plicata plants resulting in the production of healthy regenerants with 100% survival success.