High frequency direct plant regeneration, micropropagation and Shikonin induction in Arnebia hispidissima

Abstract

The data presented herein reports a rapid and efficient method for direct plant regeneration at high frequency without intervening callus formation from shoot tip (93%) and nodal segment (60%) cultured on MS media supplemented with 0.5 mg l -1 KIN, 0.25 mg l -1 BAP, 0.1 mg l -1 IAA and 100 mg l -1 CH. Conversely, leaf and internodal explants were poorly responsive. Adventitious shoot buds arose not only from the cut ends but all along the surface of the explants leading to the formation of clusters with multiple shoots. Multiple shoots upon transfer to MS media supplemented with 2.0 mg l -1 IBA induced efficient rooting (80%). In vitro flowering was observed when tissue culture-raised plantlets were maintained for extended period in culture. Shikonin was induced in roots of regenerated plants which often exudates in the culture medium was quantified spectrophotometerically by recording absorbance at 620 nm and estimated to be 0.50 mg g -1 fresh weight of tissue at the end of the 50 days of culture. The regenerated plants were successfully acclimatized, hardened, and transferred to soil in green house for micropropagation. The protocol developed here will be very useful for the supply of Arnebia hispidissima all year as a raw product necessary for obtaining Shikonin for the cosmetic, dyeing, food, and pharmaceutical industries.

Micropropagation and Quantification of Bioactive Compounds in Mertensia maritima (L.) Gray

“…There are several studies detailing the in vitro propagation of plant species in Boraginaceae including Arnebia hispidissima [15,16], Cordia verbenacea [17], Hackelia venusta [18], Heliotropium indicum [19], Sericostoma pauciflorum [20], and Trichodesma indicum [21,22]. However, to the best of our knowledge, no report has been published on in vitro propagation of Mertensia species and for M. maritima , only the callus induction protocol has been developed using leaf and apical shoot explants [14].…”

Micropropagation of Arnebia hispidissima (Lehm). DC. and production of alkannin from callus and cell suspension culture

“…The alkannin has recently attracted the interest of researchers due to its anti-inflammatory, antimicrobial, antitumor activities and wound-healing properties. In addition, it has wide applications as a colorant in food, cosmetic and textiles industries (Pal and Chaudhury 2010).…”

“…have been made (Praveen and Murthy 2010). Recently, Pal and Chaudhury (2010) reported the regeneration of A. hispidissima and production of shikonin using tissue culture technique. However, for the large-scale application of process, several other methodologies should be exploited for improved production of alkannin.…”

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

“…High frequency direct plant regeneration from various explants such as shoot tip, nodal segment, leaf and internodes as well as callus induction, micropropagation and induction of Shikonin production in Arnebia hispidissima has been reported from our laboratory (Pal and Chaudhury 2010). However, development of hairy root cultures in the present investigation has resulted in a twofold higher Shikonin production of 0.85 mg g -1 of the fresh tissue as compared to callus samples in 50 days of culture period Pal 2004, 2005;Pal 2005). This is the first report of Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima using wild-type strain A4 and conditions have been optimized for induction of hairy root cultures as well as induction of Shikonin production.…”

“…Field-grown plants of Arnebia hispidissima were Shikonin Production in Arnebia by Agrobacterium washed thoroughly under tap water with a drop of mild detergent (Teepol) and surface sterilized with 0.1% mercuric chloride (HgCI2) for 5 -7 minutes and subsequently washed three times with double distilled water to remove traces of HgCl2. Various explants: shoot tip, leaf, nodal and internodal segments were inoculated on MS basal media (Murashige and Skoog 1962) supplemented with BAP 0.25 mg l -1 , Kinetin 0.5 mg l -1 , IAA 0.1 mg l -1 , CH 100.0 mg l -1 , and the shoots obtained were multiplied on the same medium for 3 -4 weeks as described by Pal (2005); Chaudhury and Pal (2004;2005). This led to the formation of clusters of multiple shoots.…”