A rapid in vitro propagation of Holarrhena antidysenterica has been developed. Seedling cotyledonary nodes on Murashige and Skoog medium (MS) containing 2 mg dm -3 N 6 -benzyladenine (BA) produced highest number of multiple shoots. The shoot numbers were increased further upon subculture on MS medium supplemented with 0.5 mg dm -3 BA. By repeated subculture of derived shoots, a high multiplication rate was established. The excised shoots were rooted on MS basal medium without growth regulators. The in vitro formed shoots were also rooted ex vitro by dipping them in 2 mg dm -3 of indole-3-butyric acid (IBA) solution for 2 min before transferring them onto the hardening medium. Successful hardening and further establishment (survival 90 %) of micropropagated plants under natural conditions was observed.Additional key words: benzyladenine, ex vitro rooting, indole-3-butyric acid, micropropagation.
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Holarrhena antidysenterica Wall. belonging to the familyApocynaceae is medicinally important tree species. The regeneration through seeds is not sufficient due to poor seed viability and dependence on season (Ravindra et al. 2005). Clonal propagation like rooting of cuttings and grafting has not been successful (Ahmed et al. 2001). Although a few attempts were made on in vitro propagation of H. antidysenterica (Ahmed et al. 2001, Raha and Roy 2001, 2003, Ravindra et al. 2005, Mallikarjuna and Rajendrudu 2007, studies on in vitro propagation using aseptic seedling explants are lacking. Earlier studies (Mallikarjuna and Rajendrudu 2007) have shown that mature nodal bud culture was effective for the production of genetically stable plants but the technique is rather difficult due to frequent contamination, presence of latex and highly differentiated tissue. However, aseptic seedling tissues are free from secondary metabolites and microbial contamination and hence widely used for micropropagation. In addition to this, in vitro culture using seedling results in the production of genetically variant plants which may contain desired traits. Nevertheless, the progeny of single (seed-derived) seedling should be genetically homogenous if multiplied via micropropagation in vitro. In this paper, based on detailed study, we report rapid in vitro propagation of H. antidysenterica using seedling explants, namely cotyledonary nodes.Five hundred seeds of Holarrhena antidysenterica Wall. were collected from 15 to 20-year-old trees from Tirumala Hills, Andhra Pradesh, India and were initially washed with 5 % Teepol (Chemisynth, New Delhi, India) for 15 min followed by washing in running tap water and finally 4 -5 times with distilled water. The seeds were surface sterilized by dipping in 0.1 % HgCl 2 solution for 5 min and in 70 % alcohol for 15 s. Each surface sterilization was followed by 5 -6 rinses in sterile double distilled water. The seedlings were grown in test tubes containing 10 -15 cm 3 of water-agar medium (0.8 % agar and 2 % sucrose), Murashige and Skoog (1962; MS) liquid medium and MS half strength liquid medium, separat...
