Control of media browning during micropropagation and assessment of biochemical and clonal fidelity of in vitro-derived and mother plants in Thottea siliquosa (Lamk.) Ding Hou., an important ethnomedicinal shrub
Background
Thottea siliquosa(Lamk.) Ding Hou., an important medicinal shrub, is widely used in both ayurvedic and indigenous systems of medicine. Root being the most useful part, the plant is constantly uprooted and thus puts pressure on the natural population. Until date, nomicropropagationstudy is available in this plant. The objective of the study is to develop an efficient in vitro propagation protocol and assessment of clonal fidelity ofT. siliquosa.
Results
Media browning was a serious issue during micropropagation, and the addition of 40.0 mg/Lascorbic acidreduced the media browning. For direct shoot regeneration, the optimum response (92% frequency with 20.9 shoots per explant) was obtained when 7-day-oldcotyledonswere cultured on WPM supplemented with 1.0 mg/Lthidiazuronand 0.25 mg/L α-naphthalene acetic acid. The cultures were transferred to WPM augmented with 0.4 mg/L thidiazuron for shoot elongation and growth. On this medium, 100% of cultures responded with a mean number of 27.6 shoots. For callus induction, MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L N6-benzylaminopurin was used. Shootorganogenesiswas initiated on the same medium, and calli with minute shoots were transferred to MS medium fortified with 0.5 mg/L N6-benzylaminopurin and 0.25 mg/L α-naphthalene acetic acid for highest shoot regeneration (100% cultures responded with a mean number of 26.5 shoots per explant). Maximum rooting frequency (82%) and number (20.8) were obtained on half-strength MS medium with 1.0 mg/L indole-3-butyric acid. The rooted plants were acclimatized and transferred to the field. TheHPTLCandSCoTanalysis revealed thephytochemicaland clonal similarity between the in vitro propagated plants and mother plant.
Conclusions
In this study, it is confirmed that cotyledon is an excellentexplantfor direct and indirect shoot organogenesis inT. siliquosa. For direct shoot induction WPM and indirect organogenesis, MS medium was found to give better response. The true-to-type nature of in vitro-derived plants were confirmed byphytochemicaland SCoT analysis. The protocol described here could be used for the large-scale propagation of elite clones ofT. siliquosa.