Micro-morpho-anatomical changes in leaf structure of plantlets during in vitro propagation (micropropagation) of Gardenia jasminoides J. Ellis

The present study aimed to determine the foliar micro-morpho-anatomical features of in vitro cultured Gardenia jasminoides J. Ellis (Rubiaceae) to compare the effect of exogenous supplementation of growth regulators (cytokinins; 6-benzylaminopurine and Kinetin), in order to attenuate heterotrophic nutrition (in vitro) induced structural disorders in the proliferating shoots. Murashige and Skoog’s (MS) medium supplemented with 2.0 mg L⁻¹ 6-benzylaminopurine (BAP) in combination with 0.15 mg L⁻¹ indole-3-acetic acid (IAA) was detected optimal for axillary bud induction. Nutrient medium containing 0.5 mg L⁻¹ BAP and 0.25 mg L⁻¹ IAA was found appropriate combination for proliferation of multiple shoots, and yielded 37.2 shoots (per explant) with 7.45 cm average length after 2nd subculture (8 weeks). Supplementation of NAA with cytokinins resulted in callus formation. The proliferation of shoots on kinetin (Kn) and IAA combination resulted in the formation of fragile shoots and the leaves with increased structural impairments like underdeveloped photosynthetic, vascular, and ground tissue systems, non-functional stomata, and reduced vein density. Comparatively, BAP and IAA treatment favoured healthy shoot proliferation and development of stable tissue systems with reduced structural abnormalities. Half strength MS medium augmented with 3.0 mg L⁻¹ indole-3-butytric acid (IBA) was the optimal medium for root induction (32.0 roots per shoot with 3.5 cm in length). The regenerated plantlets showed 97% survival success during acclimatization and exhibited normal growth characteristics and morphology. The light microscopic evaluation of foliages provided technical support to in vitro regeneration techniques to understand the structural adaptational mechanism of in vitro raised plantlets, thereby substantially contributed in the reduction of the rate of mortality of regenerated plants of G. jasminoides.