Induction of Shikonin production in hairy root cultures of Arnebia hispidissima via Agrobacterium rhizogenes-mediated genetic transformation

Abstract

Data presented herein provides a rapid and efficient method for Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima for hairy root cultures as well as for enhancing Shikonin production. Etiolated explants viz. shoot tip, nodal, leaf and internodal segments were co-cultivated with Agrobacterium rhizogenes for induction of hairy root. Among the various explants employed, leaf explant showed maximum 70.7% response followed by shoot tip 48.3%, nodal segment 38.7% and internodal segment 9.3%. Integration of Ri plasmid rolB gene in the transformed hairy root cultures was confirmed by PCR analysis using forward (FrolB) and reverse (RrolB) primers of rolB gene resulting in the amplification of 0 ~ 0.8 kb fragments. Medium compositions have been optimized for in vitro induction of Shikonin in hairy root cultures of Arnebia hispidissima. Hairy roots on hormonefree MS medium showed red spots in the older part of the tissues which turned white after a second subculture. Whereas hairy roots cultured on RC medium showed faster growth and produced large amount of Shikonin. The Shikonin content in transformed hairy root culture was estimated by recording absorbance at 620 nm and quantified against authentic sample of Shikonin. Shikonin content was estimated to be 0.85 mg g -1 fresh weight of tissue at the end of the 50 days of culture. The results presented herein will help to design strategies for bridging the gap between ever increasing demand and supply of raw products necessary for obtaining Shikonin for cosmetic, dyeing, food, medicinal, and pharmaceutical industries.

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

“…In several studies of gene transformation leaf explant was more suitable than other explants as we found in our case also (Wang et al 2006;Sivanesan and Ryong Jeong 2009;Chaudhury and Pal 2010). Also, explants capable of auxin synthesis (intact shoots with shoot tips and young leaves) are expected to show higher transformation rates (Cardarelli et al 1987).…”

“…Being a medicinal plant, T. terrestris is available only during a short period of growing season. Thus, the techniques of plant tissue culture and biotechnological intervention have augmented the production and availability of such plants throughout the year for meeting the growing industrial demand (Chaudhury and Pal 2010).…”

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

“…Not only cell cultures but also hairy root cultures have been developed for shikonin production in plants of the Arnebia genus. Arnebia hispidissima was successfully transformed by Agrobacterium rhizogenes strain A4 (Chaudhury and Pal 2010 ) to produce the hairy root phenotype. Shikonin content in hairy roots growing in RC medium was estimated to be 0.85 mg g −1 FW at the end of the 50-d culture period.…”

“…in vitro culture research has been widely dedicated to improving shikonin production by application of various strategies, among them changing medium composition, addition of different elicitors, and various in situ extraction methods (Fu and Lu 1999 ; Ge et al . 2006 ; Chaudhury and Pal 2010 ; Kumar et al . 2011 ; Malik et al .…”

First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation

“…The hairy roots of different plants are being harnessed for the in vitro production of valuable phytochemicals of medicinal and commercial importance and for wide range of uses such as pharmaceuticals, pesticides, cosmetics, food additives etc. The hairy root culture (HRC)-based in vitro productions were reported for bioactive molecules such as shikonins, azadirachtin, camptothecins, harpagide, ginsenosides, paclitaxel, hyoscyamine and scopolamine [1][2][3][4][5]. Hairy root formation is induced by Agrobacterium rhizogenes and it often grows comparatively as fast as or faster than plant cell cultures [6,7].…”