Camptothecin (CPT) analogues and derivatives are a novel class of effective anticancer agents that exert their action against DNA topoisomerase (topo) I. CPTs are powerful for several diseases such as lung, colon, ovarian, and uterine cancer, and also against herpes, AIDS, and malaria. The worldwide market size of camptothecin derivatives reached 1.5 billion US dollars in 2002. Due to the cytotoxicity of camptothecin itself, the CPT derivatives, irinotecan and topotecan used for the treatment of various cancers, are synthesized from natural camptothecin extracted from plants. Ophiorrhiza  prostrata D. Don is a herbaceous perennial medicinal plant, producing camptothecin mainly in their roots. Because of slow propagation rate and mass harvest of roots for CPT, the plants became rare. The present study proposes rapid propagation of this valuable medicinal plant using leaf and internode segments.

Young leaves and internode segments O. prostrata washed separately under running tap water followed by 5% (v/v) solution of Extran (a neutral liquid detergent, Merck India Ltd., Mumbai) for 5 min were surface sterilized using 0.1% (w/v) mercuric chloride (7-9 min and 10-12 min respectively for leaves and internodes). These tissues after repeated washes in sterile water cut into appropriate sizes (leaf segments of 10 mm² and internode of 7-15 mm) were cultured on MS (Murashige and Skoog, 1962) medium supplemented with different levels of plant growth regulators (Table 1). For indirect organogenesis and in vitro rooting MS media supplemented with different PGRs were used (Table 2 and Table 3). The media adjusted to pH 5.8 were solidified with 0.8% (w/v) agar, and sterilized at a pressure of 1.06 kg cm^-2 at 121ºC for 20 min in an autoclave. All cultures were incubated at 25 ± 2ºC with 16 hrs light (at an irradiance of 25 µmol m^-2 s^-1)/8 h dark cycle under white fluorescent tubes. Origin of shoot formation was studied by taking random sections at different shoot development stages. Plantlets (derived through in vitro rooting) as well as well-grown shoots (without roots) from the shoot multiplication medium were transferred to small pots containing soil and sand (1:1), and subsequently transferred to field conditions after acclimatization. All the PGR treatments were repeated twice for confirmation with twenty replicates each. The data were analyzed using Duncan’s multiple range test.

Leaf and internode segments cultured on MS basal medium produced directly a mean of 2.9 and 4.0 shoots respectively. Addition of PGRs increased the shoot formation and the number of shoots depended on the types and concentrations of PGRs, BA in particular (Table 1). MS medium with 8.87 µM BA and 2.46 µM IBA developed the highest number of shoots; 76.0 and 90.8 shoots from leaf and internode segments respectively (Table 1). All other levels were inferior for the induction of shoots (Table 1). The shoots on leaf segments were mostly developed adaxially. Of the different regions in the leaf, the segments from basal region of leaf were superior in the induction of shoots. Culture of stem and leaf (basal) segments as well as shoot clumps excised from the primary cultures on MS medium with 8.87 µM BA and 2.46 µM IBA produced shoots, which were difficult to count. The shoots were developed from sub-epidermal cells especially from the region above the vascular bundles of the leaf segments. The calluses developed from leaf and internode segments on MS media with different levels of NAA either alone or in combination with BA or Kn upon subculture developed higher number of shoots (a mean of 20.1) on MS medium with 8.87 µM BA and 2.46 µM IBA (Table 2).

Direct organogenesis is regarded as the most reliable method for clonal propagation because of uniformity among the progeny as has been reported in many plants. Synergy of BA and auxins observed in the present study has been documented in several plants. The difference in the number of shoots formed in leaf and internode segments may be due to the differences in the regeneration potential by the difference in physiological state, age and cellular differentiation among the constituent cells. The high potential of the basal end to the tip may be due to the difference in the maturity between basal and tip of the leaf; basipetal manner of leaf maturity substantiates the same. Callus mediated shoot morphogenesis has been accomplished in several medicinal plants and the high frequency shoot regeneration is useful for the improvement of this valuable medicinal plant through the induction of somaclonal variation.

Shoots transferred to half-strength MS medium with 10.74 µM NAA and 2.32 µM Kn was superior for the induction of roots with a mean of 48.2 roots per shoot (Table 3). The roots developed on all media became reddish brown through golden yellow from white. The high numbers of roots produced under optimal PGR regimes opens the possibility of producing camptothecin as the root is the main source for the chemicals. Plantlets derived after in vitro rooting and directly transferred rootless shoots showed 100% and 50% survival respectively in field conditions. Direct rooting in field looks promising considering the reduction in cost by avoiding the in vitro rooting and use of chemicals and the reduction in labour and time of plantlet establishment from laboratory to land.

The protocol described in this study enables production of more than 75 plants within two months using single internode explant, which in turn will reduce the pressure on the natural population of this plant. Besides, this highly efficient regeneration protocol serves as a tool for improvement of the plant through genetic transformation methods.

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