Protocol for in vitro regeneration and marker glycoside assessment in Swertia chirata Buch-Ham

Abstract

We have developed a rapid in vitro propagation system, via axillary shoot formation from nodal explants of Swertia chirata Buch Ham. Culture medium supplemented with 2 mg/L. BAP is best for direct shoot regeneration initially formed adventitious buds from axils of the nodal explants after 30 days. The reduced BAP concentration 0.5 mg/L proliferate shoots effectively. Kept the number of hyperhydrated shoots to minimal and induced on an average 22-38 shoots per flask (4.3 cm average length). The regenerated shoots (5- to 6-m long) formed roots very well in Murashige and Skoog (MS) medium devoid of any growth regulator and followed by acclimatization of plants in pre-sterilized sand containing 1% Trichoderma viride and Azatobactor chrococcum as bioinoculants. The regenerated plants don’t show any genomic alterations. This protocol also outlines procedure of assessment of marker iridoid glycosides (amarogentin and amaroswerin) from callus, roots, multiple shoots, regenerated plants, and mother plant. High propagation frequency, reproducibility of procedure, molecular, and phenotypic and chemical stability ensures the efficiency of the developed protocol.