An improved thin cell layer culture system for efficient clonal propagation and in vitro withanolide production in a medicinal plant Withania coagulans Dunal

In vitro micropropagation based on transverse thin cell layer (tTCL), which utilizes a very small number of cells or tissues, has now emerged as a powerful tool in plant tissue differentiation and regeneration. In this study, intense, fast rate shoot multiplication has been achieved utilizing thin cell layer explants for endangered pharmaceutical plant Withania coagulans Dunal (family Solanaceae) having high hypoglycemic potential. To develop an efficient protocol for rapid in vitro propagation of W. coagulans stem node, shoot apical meristem (SAM), and tTCL were used as explants and inoculated in different strength of MS medium. The tTCL explant showed high frequency of shoot regeneration and was also affected by the concentration of plant growth regulators. The full strength solid MS medium was optimal for shoot regeneration (31.94 +/- 1.39%) and the highest percent of shoots (93.05 +/- 2.77) were observed in MS medium fortified with 2.0 mgl(-1) BAP and 0.5 mg(l-1) NAA. In contrast to tTCL, SAM cultured on the same medium showed significantly lower multiplication rate with only 1.16 +/- 0.08 average shoots per responsive explants. The best rooting of the regenerated shoots was achieved in half strength liquid MS medium supplemented with 2mgl(-1) IBA. Plants were successfully transferred to the field after acclimatization with a survival rate of 90.6%. Monomorphic nature of ISSR and RAPD markers in this study confirmed the genetic fidelity of the in vitro raised tTCL clones. Quantification of withanolides content through high performance liquid chromatography (HPLC) showed 1.4 fold increases in withaferin A content and 1.6 fold withanolide A content in acclimatized field grown plants compared to field grown (wild type) plants. In acclimatized field grown plants the over-expression of SQS gene further attested the improved withanolide production in tTCL micropropagated plants having valuable anti-diabetic potential. The current study reveals the fast rate, cost effective micropropagation and conservation of W. coagulans through tTCL technique for its use as anti-diabetic agent at commercial level.