<em>In vitro</em> rapid multiplication and determination of triterpenoids in callus cultures of <em>Achyranthes aspera</em> Linn

Abstract

151-159Achyranthes aspera Linn. was studied for its in vitro rapid multiplication, callus initiation, proliferation and triterpenoid determination. The plant was also evaluated for pretreatment, sterilization and culture initiation during the study. Best results for vigorous cultures were obtained for plants pretreated with 0.2% (w/v) carbendazime + 3 drops of Tween 20 for 10 min, and double sterilized using both sodium hypochloride (NaOCl) (4.0% v/v for 2 min) and mercuric chloride (HgCl2) (0.1% w/v for 2 min) solutions. In vitro plantlet regeneration in A. aspera was achieved from axillary explants cultured on Linsmaier & Skoog (LS) fortified with 6-benzyl amino purine (BAP) alone and/or in combination with Zeatin (Zn), Kinetin (Kn), thidiazuron (TDZ), naphthalene acetic acid (NAA). The combination of BAP (3.00 mg/L) and TDZ (0.10 mg/L) was the best for multiple shoot induction and rapid proliferation. The developed shoots were successfully rooted on medium containing NAA (1.5 mg/L). Rooted plantlets were regenerated and were successfully established in soil with a survival rate of 90%. The protocol developed for multiplication is rapid and efficient for in vitro propagation of A. aspera. Further, a combination of 2,4-D (3.00 mg/L) and BAP (0.50 mg/L) fortified in LS basal medium, showed significant response with enhanced growth and proliferation of callus. Callus obtained from the 10 combinations were extracted individually using an ultrasonicator, and the extracts were subjected for reverse phase ultra-fast liquid chromatography (RP-UFLC) analysis. Higher amount of betulinic acid (3-hydroxylup- 20(29)-en-(28)-oic acid) and oleanolic acid (3β-hydroxyolean-12-en-28-oic acid) were accumulated on the medium supplemented with 3.00 mg/L of 2, 4-D and 0.50 mg/L of BAP