Vol 10, Issue 7, 2017 Online - 2455-3891 Print - 0974-2441 CURBING ACTINOMYCETES AND THIDIAZURON ENHANCED MICROPROPAGATION IN THE RARE ALPINIA GALANGA - A MEDICINAL ZINGIBER BARADWAJ RG, RAO MV*, SENTHIL KUMAR T Department of Plant Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Email mvrao_456@yahoo.co.in Received: 14 February 2017, Revised and Accepted: 13 April 2017 ABSTRACT Objective: Elimination of endophytic actinomycetes before micropropagation using antibiotic pre-treatment in rhizome bud explants of Alpinia galanga. Formulation of an operative protocol for micropropagation from the disinfected rhizome bud explants in Alpinia galanga. Methods: A treatment of mercury chloride and carbendazim, alone and in combination was used as surface sterilants. A pre-treatment of rifampicin and fusidic acid was used against actinomycete endophyte disinfection of rhizome bud explants. Then, Murashige and Skoog (MS) medium supplemented with various concentrations of cytokinins was used for micropropagation of disinfected explants. Results: A treatment of 0.1% (w/v) mercury chloride and 0.1% (w/v) carbendazim, one after the other for 5 minutes gave the best sterility of 83.3%. A pre-treatment of Rifampicin 100 mg/l and fusidic acid 100 mg/l for 2 hrs gave the best disinfection of 70% against actinomycete endophytes. A combination of thidiazuron (TDZ) 0.45 µM and 6-benzyladenine 13.32 µM in MS medium resulted in 9.4 shoots per explant. MS medium fortified with 10.74 µMof 1-naphthaleneacetic acid gave the best rooting of 20 roots/shoot. Inter simple sequence repeat marker genetic similarity of regenerants with the mother plant was confirmed. Conclusion: This study shows the potency of Rifampicin and Fusidic acid to disinfect explants from actinomycete endophytes and is significant as the first report on curbing actinomycetes endophytes in plant tissue culture of A. galanga. This is also the first report conferring the dissimilar regeneration capabilities of TDZ in comparison to other cytokinins in Zingiberaceae. Keywords: Alpinia galanga, Rifampicin, Fusidic acid, Actinomycetes, Thidiazuron, Inter simple sequence repeat. INTRODUCTION Effective in vitro plant regeneration is greatly essential for application of most modern biotechnological practices in crop enhancement [1]. Alpinia galanga has been propagated via tissue culture using micropropagation, direct and indirect organogenesis with rhizome buds as explants [2-5], but the success of these efforts varies significantly, possibly due to the presence of endophytic actinomycetes in the rhizomes of A. galanga [6-8]. This interference in regeneration capability could be caused by the plant growth promoting the activity of indole-3- pyruvic acid and indole-3-acetic acid (IAA) produced by the endophytic Actinomycetes [9,10]. The presence of endophytic actinomycetes greatly affects commercial tissue culture both qualitatively and quantitatively; moreover, tissue cultured plants harboring these endophytes are not suitable for export due to plant quarantine. A. galanga (L.) Willd. (Zingiberaceae) occurring in the Western Ghats of south India, northeastern parts of India and Thailand is the type species of the genus Alpinia Roxb. It is an important cultivated medicinal crop in India and is recognized in different traditional systems of medicine to treat microbial infections, inflammations, rheumatic pains, chest pain, dyspepsia, fever, diabetes, burning of the liver, kidney disease, tumors, and even HIV [11]. Galangin, a flavone present in A. galanga, has exhibited potent cardioprotective activity against doxorubicin-induced cardiomyopathy in Wistar rats [12]. The plant comprises many such pharmacologically important phytochemicals, namely, 1’S’-1’-acetoychavicol acetate, 1’S’-1’-acetoyeugenol acetate, 1’S’-1’-hydroxychavicol acetate, trans-p-coumaryl alcohol, trans-p-hydroxycinnamyl acetate, trans-p- coumaryl diacetate and nortrachelogenin, caryophyllene, cubenol and eucalyptol [13-18]. The validating genetic integrity of in vitro regenerated plants with regard to the mother plant is indispensable for the maintenance of certain horticultural and agronomic traits [19]. There are odds of getting somaclonal variations among the regenerants in plant tissue culture due to the impact of factors such as donor genotype, explant type, culture medium composition, physical culture conditions, the duration between successive subcultures and particular reagents used for various techniques which are possibly heritable [20]. Thus, there ascends the need to appraise whether the regenerants are genetically identical to the mother plant. Due to the increased therapeutic and pharmaceutical demand, the status of A. galanga in the wild has been significantly affected, and it has gained a threatened to endangered status in the Western Ghats of India [21]. Therefore, to fulfill the demand of pharmaceutical industries and as a preliminary prerequisite for reintroduction or cultivation of the medicinal crop, the establishment of an efficient in vitro regeneration system void of glitches is required. The past reports of in vitro plant regeneration in this important medicinal crop are at best ineffective and unreproducible due to the non-elimination of endophytic actinomycetes pedant in the rhizome of the plant as mentioned above. The short-term use of antibiotics such as rifampicin has long been used in plant tissue culture for disinfecting plants without affecting their growth [22]. As there are no methods for successful disinfection of actinomycete in plant tissue culture yet and since the maintenance of genetic characters of the high-class mother plant in in vitro raised plants is imperative, the present study was devised to use antibiotic pre-treatments as an effective technique to avail actinomycete free explants of A. galanga for micropropagation and to assert the inter-simple sequence repeat (ISSR) genetic fidelity of in vitro produced plantlets. © 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i7.17734 Research Article