Biodiversity and Conservation (2006) 15:2325–2338 DOI 10.1007/s10531-005-0770-z
Springer 2006 -1 Propagation and conservation of Picrorhiza kurrooa Royle ex Benth.: an endangered Himalayan medicinal herb of high commercial value BHUWAN CHANDRA1, L.M.S. PALNI1,2,* and S.K. NANDI1 1 G. B. Pant Institute of Himalayan Environment & Development, Kosi-Katarmal, Almora-263 643, Uttaranchal, India; 2State Biotechnology Programme, Government of Uttaranchal, Biotech Bhavan, P. O. Haldi, Pantnagar, US Nagar-263 146, Uttaranchal, India; *Author for correspondence (e-mail: lmspalni@rediffmail.com) Received 19 April 2004; accepted in revised form 7 January 2005 Key words: Conservation, Conventional propagation, In vitro multiplication, Picrorhiza kurrooa Abstract. Picrorhiza kurrooa Royle ex Benth., a high value medicinal herb of alpine Himalaya and a source of hepatoprotective picrosides, is listed as ‘endangered’ due to heavy collection from its natural habitat. The present report deals with successful propagation of this species using both conventional and in vitro techniques. Vegetative propagation was achieved by rooting runner cuttings with indole-3-butyric acid (IBA) or a-naphtheleneacetic acid (NAA) treatment before planting. Nearly 87% rooting success was achieved by treatment of cuttings with 50.0 lM IBA. Seeds were given a presoaking treatment with gibberellic acid (GA3), 6-benzylaminopurine (BAP) or a combination of both to influence germination. More than 11-fold improvement in germination was recorded in seeds treated with 250.0 lM GA3. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segment. Multiple shoots were formed following culture for 3 weeks on Murashige and Skoog (MS; 1962. Physiologia Plantarum 15: 473–497) medium containing 1.0 lM BAP. Cent percent rooting success, without basal callus formation, was observed when individual microshoots were placed in MS medium supplemented with IBA. The plantlets raised using conventional as well as tissue culture methods were hardened and successfully established in the experimental field located at 2450 m elevation. In addition, strategies have been discussed to encourage cultivation and in situ conservation of this highly valued medicinal herb so as to reduce pressure on its natural populations. Introduction Picrorhiza kurrooa Royle ex Benth. (family: Scrophulariaceae; local/trade name: Kutki), an important medicinal herb endemic to alpine Himalaya (Thakur et al. 1989), is distributed between 2800–4800 m altitude. The extract of runners and roots of this plant has been used since long in several ‘Ayurvedic’ preparations, prescribed in hepatic disorders. The plant is also used in the traditional as well as modern systems of medicine as stomachic, purgative, and antiperiodic agent, as a brain tonic and in dyspepsia and fever (Hussain 1984; Kirtikar and Basu 1984). It is known to contain picroside I, II, III and kutkoside as major bioactive compounds (Rastogi et al. 1949; Kitagawa et al. 1969; Weinges et al. 1972; Jia et al. 1999). 2326 The plant perennates through underground runners and the period of active growth is confined only to a few summer months. In nature, the plant regenerates both by vegetative means (underground runners) as well as through seeds. The seed setting and seedling survival has been reported to be generally poor in alpine plants (Pandey et al. 2000). In common with other Himalayan medicinal herbs of high altitude regions, P. kurrooa is also being indiscriminately harvested, often well before seed setting, for subsequent trading of raw material (Farooquee and Saxena 1996; Rai et al. 2000). At times the entire plants are collected by unskilled persons. In addition, lack of organized cultivation has led to considerable depletion of its natural population resulting in its listing as vulnerable species in Red Data Book (Nayar and Sastry 1990) and subsequently under the ‘endangered’ species category, as per IUCN criteria. The use of conventional (vegetative propagation, seed germination) and in vitro techniques for rapid and mass propagation offers possibilities for ‘recovery’ of endangered species, thus reducing the risk of extinction (Nadeem et al. 2000). In view of the importance of this species, a holistic approach for the propagation of P. kurrooa, using both conventional as well as in vitro methods, and strategies for its conservation have been described in this paper. Material and methods Plant material Spikes (5–10 cm long, bearing capsules) and runners of Picrorhiza kurrooa Royle ex Benth. were collected from Pindari (306¢ to 3915¢ N and 7055¢ to 805¢ E; 3400 m amsl, District Bageshwar, Uttaranchal) of Kumaun Himalaya in October 1998. The spikes were brought to the laboratory, seeds were separated from the capsules, sorted, air-dried (room temperature, 3 days) and used immediately for germination trials. Runners were also brought to the laboratory, wrapped in moist papers, in plastic bags and used immediately for vegetative and in vitro propagation studies. Seed germination Seeds were pretreated (24 h, dark) in beakers containing 25 ml aqueous solutions of various plant growth regulators (PGRs; all from Sigma Chemical Co., St. Louis, USA), namely gibberellic acid (GA3; 25.0 and 250.0 lM), 6-benzylaminopurine (BAP; 25.0 and 250.0 lM) and abscisic acid (ABA; 25.0 and 250.0 lM). The combined effect of GA3 and BAP (25.0 lM each) was also examined. Stock solutions were prepared in 50% ethanol (v/v) and diluted with water before treatment, so that the final concentration of ethanol in various test solutions (including control) never exceeded 2.0% (v/v). Following various treatments, the seeds were washed (· 2) with water and placed in petridishes 2327 over two layers of moistened filter paper; these were then kept in a seed germinator (Narang Scientific Works, New Delhi) at 10 or 20 C (dark). Each treatment consisted of 20 seeds (in triplicate) and germination was monitored weekly upto 10 weeks. Vegetative propagation Whole runners (including apical parts to the extreme basal portions) were cut into small segments of 3.5–5.0 cm length (with 2–3 nodes) and treated with various PGRs (12 h, dark) by dipping the basal portion (2.0 cm) of cuttings in treatment solutions kept in separate glass beakers. The PGR treatments included indole-3-butyric acid (IBA; 50.0 lM), BAP (10.0 lM), IBA + BAP (50.0 + 10.0 lM), a-naphthaleneacetic acid (NAA; 50.0 lM), NAA + BAP (50.0 + 10.0 lM). The effect of Bavistin, a systemic fungicide (0.01% a.i.; BASF India Ltd., Mumbai, India) was also examined. All treatment solutions were prepared as mentioned above. Following treatment, the runner segments were planted (one per cup) vertically in trays (20 cups per tray; cup diameter 5 cm, depth 10 cm, each cup contained approximately 185 g of a mixture of soil and sand, 2:1), and kept in a mist chamber (25C, 90% RH) of a greenhouse under natural light (15% of ambient). Each treatment consisted of seven cuttings (in triplicate). Data were recorded after 90 days. Initiation of in vitro cultures and plant regeneration Runners were cut into small segments (2.5–5.0 cm) with 2–3 nodes per segment. These were washed with Tween-80 (1%, 10 min) and successively surface disinfected with mercuric chloride (HgCl2; 0.05%, 5 min), and sodium hypochlorite (NaHClO3; 2%, 5 min) solutions. Following surface disinfection the nodal explants were thoroughly washed (· 4) with sterilized double distilled water, and then placed vertically on Murashige and Skoog’s (1962) basal medium containing 0.8% (w/v) agar and sucrose (2%, w/v), in tubes (150 · 25 mm) containing 15 ml medium. The pH of the medium was adjusted to 5.8 before autoclaving (1.05 kg/cm2, 121 C; 20 min). Cultures were maintained at 25 ±1C in 16/8 h light/dark cycle on racks fitted with cool fluorescent tubes (Philips 40 W; 42.0 and 60.0 lmol/m2/s irradiance inside and outside the culture flasks, respectively). Subculturing was carried out at 4–6 weeks interval. Statistical analyses Least significant difference (LSD) and standard error (SE) were calculated following the method of Snedecor and Cochran (1967). 2328 Results Effect of PGRs on seed germination Presoaking treatment with different PGRs and subsequent incubation at 10C resulted in only minor change in germination values (over control) with the exception of 250.0 lM GA3 (Figure 1a). Treatment with GA3 (250.0 lM) caused a marked improvement (78.3% compared to 1.7% in control) in germination in the 2nd week itself; thereafter the values increased marginally and reached a plateau (83.3% compared to 15.0% in control) within the next 8 weeks. This treatment also reduced the time taken for germination as more than 80% germination occurred within the first 3 weeks (Figure 1a and Plate 1A). Incubation at 20C generally improved germination significantly (over control values) in almost all PGR treatments, except in case of ABA (Figure 1b). Maximum increase was observed following treatment with 250.0 lM GA3, and even after 1 week 33.3% germination (compared to 0.0% in control) was recorded; this increased to 90.0 and 93.3% after 2nd and 4th weeks, respectively, and then remained constant (Figure 1b). The pattern of germination with PGR treatments (Figure 1b) was similar as in Figure 1a, i.e., there was a marked increase in germination within the first few weeks and subsequently the values remained constant (Figure 1a). Both GA3 (25 and 250 lM) and BAP (25 lM), either alone or GA3+BAP (25 lM each) were found to advance the time of germination. Although the lower concentration of ABA (25 lM) resulted in marginal but significant improvement in germination (LSD at p=0.05=17.5, at 4th week), it was found to be inhibitory at higher (250 lM) concentration (Figure 1b). Rooting of runner cuttings Table 1 summarizes the effect of various PGRs on rooting. Among various treatments, 50.0 lM IBA resulted in maximum rooting (86.7% compared to 53.3% in control) and highest root number (4.0 roots/cutting compared to 1.7 roots/cutting in control). Cuttings treated with 50.0 lM NAA, and the combined treatments of NAA + BAP (50.0 + 10.0 lM) and IBA + BAP (50.0+10.0 lM) also enhanced root formation (80.0% and above) as well as stimulated number of roots (Plate 1B). Marginal, yet significant stimulation (66.7%) in rooting was noticed when the cuttings were treated with 10.0 lM BAP; however, bavistin did not show any stimulation (Table 1). Although root initiation in cuttings treated with IBA and NAA was observed within 7–9 days of planting, the final rooting percent values in various treatments were recorded after 90 days of planting. 2329 Control GA3 250 µM BAP 250 µM ABA 25 µM GA3 25 µM BAP 25 µM GA3+ BAP (25 µM) ABA 250 µM (a) 100 90 % Germination 80 70 60 50 40 30 20 10 0 1 2 3 4 5 10 1 2 3 4 5 10 (b) 100 90 % Germination 80 70 60 50 40 30 20 10 0 Time (Weeks) Figure 1. Time course of P. kurrooa seed germination following PGR treatment. (a) Germination at 10C, experiment conducted in triplicate with 20 seeds/petridish. Germination values remain essentially unchanged during weeks 4–10; (b) Germination at 20C, experiment conducted in triplicate with 20 seeds/petridish. Germination values remain essentially unchanged during weeks 4–10. In vitro shoot multiplication The nodal segments sprouted, giving rise to axillary shoots after 2 weeks of inoculation on MS basal medium (Plate 1C). Only 60% explants were found to be contamination-free after 4 weeks. These were further cultured on the same 2330 Table 1. Effect of various plant growth regulators and chemicals on rooting of runner cuttings of P. kurrooa. Treatments % Rooting Avg. no. of roots/cutting Avg. no. of sprouts/cutting Avg. no. of leaves/cutting Avg. length of roots (cm) Avg. length of longest root (cm) Control IBA (50 lM) BAP (10 lM) NAA (50 lM) IBA (50 lM)+BAP (10 lM) NAA (50 lM)+BAP (10 lM) Bavistin (0.01%, a. i.) LSD (p=0.05) 53.3±2.7 86.7±2.7 66.7±2.7 80.0±4.7 80.0±0.0 83.3±2.7 53.3±5.4 12.3 1.7±0.15 4.0±0.23 1.9±0.14 2.5±0.21 2.5±0.04 3.1±0.15 1.4±0.12 0.59 2.1±0.02 2.1±0.14 1.8±0.09 1.7±0.15 1.8±0.09 1.5±0.05 1.5±0.12 0.38 13.4±1.2 11.9±0.97 12.4±1.5 11.7±0.84 11.3±0.76 9.7±0.29 7.9±0.76 3.57 6.6±0.46 7.7±0.38 6.1±0.43 7.9±0.33 8.3±0.86 7.3±0.50 5.2±0.87 2.12 10.7±0.67 11.4±1.07 11.0±0.53 11.8±0.99 11.3±0.75 12.5±0.52 9.1±0.48 2.7 Data were recorded after 90 days of planting; n=7 (in triplicate); Mean±SE; a. i.=active ingredient. 2331 Plate 1. (A) Seedlings raised in thermocole trays and kept in the greenhouse; seeds were given presoaking treatment with 250 lM GA3 in the dark for 24 h. (B) Plants raised through rooting of runner cuttings treated with various plant growth regulators; 1: Control; 2: IBA (50.0 lM); 3: NAA (50.0 lM); 4: IBA +BAP (50+10 lM); 5: NAA +BAP (50+10 lM). (C) A well-sprouted nodal explant on MS basal medium 4 weeks after inoculation. The axillary shoots were excised and used for further multiplication. (D) Multiple shoot formation on MS medium supplemented with 1.0 lM BAP and 2% sucrose. (E) IBA (1.0 lM) induced in vitro rooting of microshoots (bottom view of the culture flask). (F) A well-rooted shoot, free from the formation of callus at the base of the shoot, and ready for ex vitro transfer. (G) Hardened in vitro produced plants in earthen pots under greenhouse conditions. The photograph was taken 4 months after ex vitro growth. 2332 medium for two more weeks. The axillary shoots were then separated from the mother explants, i.e., nodal segments, and transferred on to MS medium supplemented with 1.0 lM BAP. Multiple shoot formation started within 3 weeks in 66% of cultured shoots. The average number of shoots per cultured axillary shoot was recorded to be 9.2 after 4 weeks of culture with a maximum of 14 shoots from a single axillary shoot. These were further subcultured after 4 weeks in the form of shoot clumps (Plate 1D). After two subcultures, the shoot clumps (five shoots per clump) were transferred on to MS medium containing higher concentrations of BAP (2.5, 5.0 and 10.0 lM). While there was no significant improvement in shoot production at these concentrations, some callus formation was observed at the base of the microshoots. Hence only 1.0 lM BAP was used routinely for shoot proliferation. Rooting of microshoots and hardening of plantlets Transfer of individual microshoots (3.0–5.0 cm length) to MS basal medium resulted in 66.7% rooting after 11 days of transfer to PGR-free medium (Table 2). The rooting percentage could be improved (up to 100%) by the addition of IBA (1.0 and 2.5 lM) in the MS medium. The time of root initiation was also found to be advanced by 4 days in medium containing 2.5 lM IBA. The mean number of roots per shoot (5.4) was recorded to be highest when 0.5 lM IBA was used and lower concentrations (0.5 and 1.0 lM) of IBA resulted in callus-free rooting (Plates 1E and F). Maximum callusing (30.0%), however, occurred in MS basal medium. The rooted plants were transplanted in thermocole cups containing a mixture of soil and sand (1:1) and hardened under high relative humidity (80.0±5.0%) for 6 months in the greenhouse with approximately 65% survival (Plate 1G). These plants were then transferred to a high altitude field site (2450 m amsl, village Khaljhuni in District Bageshwar) for further growth and monitoring, with 80% survival 3 month after field planting. Table 2. Effect of different concentrations of IBA in the culture medium on rooting of microshoots of P. kurrooa. Treatment (lM) % Rooting No. of roots Avg. length of longest root (cm) % shoots with basal callus Control IBA 0.5 IBA 1.0 IBA 2.5 IBA 5.0 66.7±9.8 80.0±0.0 100.0±0.0 100.0±0.0 40.0±0.0 2.6±0.9 5.4±1.2 3.2±0.9 3.4±0.6 2.2±0.7 6.5 10.0 10.0 8.5 12.5 30.0±4.7 0.0 0.0 20.0±8.2 13.3±2.7 n=10 (in triplicate); Data were recorded 4 weeks after inoculation. 2333 Discussion The germination was poor in freshly harvested seeds of P. kurrooa, both at 10 and 20C. This is possibly due to dormancy, exhibited by many alpine seeds as an adaptation to overcome the harsh climatic conditions prevalent at high altitudes (particularly after seed shedding in October), where naturally controlled germination can be crucial to seedling survival (Kaye 1997; Nadeem et al. 2000; Pandey et al. 2000). Like many other alpine species, seeds of P. kurrooa in nature are subjected to chilling at subzero temperatures during winters which may assist germination in the following spring. Earlier report (Nautiyal 1988) indicates upto 60% germination in P. kurrooa by incubation of seeds at 20C in light. In a soil-based experiment inside a polyhouse upto 58% seed germination has been reported without any chemical treatment when seeds were sown in sandy loam soil covered with moss (Nautiyal et al. 2001). In this study, presoaking treatment with GA3 significantly improved percent germination (above 80%) and also reduced the time required for germination, irrespective of the germination temperature. The seed treatment with GA3 (250.0 lM) is thus routinely being used for raising seedlings in the greenhouse for subsequent field plantation (Plate 1a). Germination of various herbaceous species, including those of alpine regions, is known to be accelerated by the treatment of seeds with GA3 or BAP, or a combined treatment with both, as in the present investigation (Son et al. 1999; Nadeem et al. 2000; Pandey et al. 2000). GA3 (250 lM) alone or in combination with BAP (250 lM) reportedly doubled (over control values) germination as well as advanced the time taken for germination in Podophyllum hexandrum, another alpine herb (Nadeem et al. 2000). Germination of Aconitum balfourii and A. heterophyllum (both alpine herbs) seeds could be significantly improved by the treatment of 250 lM GA3 or 250 lM BAP (Pandey et al. 2000). Use of BAP (111.0 lM) alone has been recently reported to improve germination of Gentiana scabra seeds by upto 90% (Son et al. 1999). Thus GA3 is able to replace the chilling requirement of alpine seeds and/or break dormancy. It must be emphasized that lower and physiological concentrations of PGRs should be applied to obtain a response (Pandey et al. 2000). Cytokinins are also involved in breaking seed dormancy and stimulation of germination (Walker et al. 1989); it has been suggested that they may not be directly involved in the breaking of dormancy but play a ‘permissive’ role by decreasing the level of germination inhibitors and making the seeds more sensitive to gibberellins (Walker et al. 1989). In this investigation, ABA inhibited seed germination both at 10 and 20C. Literature reports indicate that ABA is involved in regulating the onset of dormancy and maintaining the dormant state (Bewley 1997). Thus a dormant state could be maintained over an extended period by exogenously applied ABA. Clonal propagation using ‘vegetative means’ provides a simple method for multiplication of P. kurrooa. Nearly 87% rooting success could be achieved when runner cuttings were treated with IBA or NAA, either alone or in 2334 combination with a low concentration of BAP (10.0 lM). While laboratory scale multiplication using leafy explants and single node cuttings has been reported for this species, no details were given (Ahuja 2001). In an earlier study more than 90% rooting of apical segments taken from runners has been reported, however, rooting in basal cuttings was poor (39%; Nautiyal et al. 2001). In the present study, whole runners were used to obtain cuttings (in which apical segments contribute only a small portion of around 10% of the total cuttings made from each runner) and the observed average rooting success of nearly 87% is a significant advancement. As this is a listed endangered species, the availability of material from nature for multiplication is limited; hence the entire runners can be used for raising cuttings for large-scale propagation. Auxins are well-known as rooting agents and their application (naturally occurring or synthetic) for large-scale multiplication has been well documented (Hartmann et al. 2002). It may be mentioned that IBA or NAA treatment has been found to be effective for rooting rhizome/tuber segments of Podophyllum hexandrum (Nadeem et al. 2000), Aconitum atrox (Rawat et al. 1992), and stem cuttings of Taxus baccata (Nandi et al. 1996) and Cedrus deodara (Nandi et al. 2002), etc., high altitude Himalayan plants. In this investigation, there was no improvement in percent rooting following bavistin treatment although stimulation has been reported in certain gymnosperms, i.e., Taxus baccata and Cedrus deodara stem cuttings (Nandi et al. 1996, 2002). Such an effect of this fungicide on rooting may be related to the auxin-like property of carbendazim (Nandi et al. 2002). In general cytokinins inhibit root induction in stem cuttings (Schrandolf and Reinert 1959), however, root initiation was observed in pea cuttings when applied at very low concentrations (Eriksen 1974). In this investigation, BAP, a natural cytokinin (Nandi et al. 1989), either alone or in combination with IBA or NAA significantly improved rooting of runner segments. Raising crops through vegetative methods is not only useful for eliminating the difficulties associated with seed germination and seedling survival, but it reduces the overall duration of the cultivation cycle. In addition, being a clonal means of propagation, genetic uniformity of the crop is assured. In vitro multiplication of P. kurrooa has been achieved using nodal explant. The application of BAP at lower concentrations (1.0 lM) has proven extremely beneficial for induction of multiple shoots and subsequent shoot multiplication as also reported earlier (Lal et al. 1988; Upadhyay et al. 1989). Higher concentrations of cytokinins like BAP and kinetin have been known to result in lower shoot numbers and, in some cases, also inhibit shoot growth, e.g., in Glycine max (Cheng et al. 1980). In this study, when higher concentrations (2.5–10.0 lM) of BAP were used during subsequent subculture, no improvement in shoot multiplication was observed; however, 5.0–10.0 lM BAP resulted in production of vitrified shoots with callus formation at the base of microshoots. Transfer of vitrified shoots on medium containing lower levels of cytokinins or a mixture of cytokinin and auxin at low concentrations resulted in regeneration of normal shoots from the base of vitrified shoots. 2335 Vitrification of shoots in cytokinin supplemented medium has been reported earlier in P. kurrooa (Upadhyay et al. 1989). Rooting is an important step for developing a complete in vitro propagation protocol. Successful rooting of microshoots in P. kurrooa could be achieved by incorporation of an auxin in the culture medium. While the extent of in vitro rooting has not been stated in an earlier report by Lal et al. (1988), good rooting of microshoots was reported with 1.0 lM NAA in 20 days (Upadhyay et al. 1989). In the present investigation, cent percent in vitro rooting of microshoots has been observed with IBA (1.0 or 2.5 lM). Further, the time required for root initiation was also reduced to 7 days; callus free rooting could also be obtained in some cases. Successful hardening and post-hardening establishment of in vitro raised plants in a natural habitat is an important step towards propagation and conservation of commercially valuable species. In vitro raised rooted plants of P. kurrooa were hardened under greenhouse conditions, and after 6 months these plants were transferred to a high altitude field station with 80% survival. In continuation of the present study in vitro raised plants of P. kurrooa, developed from cotyledonary node explants, have shown more than 90% survival (as against 65% survival in the present study) during ex vitro hardening when the soil was inoculated with some bacterial isolates (e.g., Bacillus subtilis) having antagonistic properties against selected pathogenic fungi (Chandra et al. 2004). This is not surprising since maximum mortality is caused by fungal infection during early weeks of transfer of rooted plants to the soil. The growth of these plants was found to be identical to that of seedling or cutting raised plants with normal runner formation. The 80% survival of tissue culture raised plants following transfer to field, as observed in the present study, can perhaps be improved if the time taken during transport of plantlets from the hardening chamber to the field planting is reduced. Since the field site was situated in a distant and difficult to reach place in the subalpine Himalayan zone and replanting could be done only after 72 h, a major climatic shift, in addition to transport shock, may have further affected the final survival in the field. Conclusions Being included in the negative list of Indian plants export of P. kurrooa, in any form, is not permitted by the Export Authority of India (Purohit 1997). Majority of medicinal plants used by the pharmaceutical companies are presently harvested from their natural habitat. In order to facilitate collection and marketing of medicinal plants, a co-operative has been set up by the State Government that allows for the collection of these herbs through issue of a license. However, illegal trade still continues due to low (and variable) price offered by the co-operative, as compared to the open market price. For example, in the neighbouring state of Uttar Pradesh, dried runners of 2336 P. kurrooa are purchased by the co-operative @ Rs. 22.25/kg (1 US $=approx. Rs. 45.0) as against Rs. 56.0/kg in the open market (Purohit 1997) and in the state of Sikkim the price is Rs. 202.0/kg (Rai et al. 2000). Apart from providing scientific back up for mass propagation of elite material (in close proximity of field sites), a prerequisite for promoting organized cultivation of medicinal plants, it is crucial that the State Forest Departments, non-governmental organizations and Government policies should be in the interest of farmers, and work towards minimizing the intervention of middle men and illegal traders. Habitat protection and proper management of existing populations provide far better options to conserve rare and endangered plant species. Since this species in particular and collection and trade of medicinal plants in general form an important part of the economic activity in the region, it is imperative that improvement in propagation techniques, both conventional and tissue culture based methods, and eventually the domestication of relevant species will serve the purpose of providing alternate cash crops for the farmers. These efforts will substantially ease the pressure on unregulated exploitation from the natural populations by fulfilling the demand of the traders. Based on the findings reported in this communication, farmers may be provided trainings and readymade solutions in easy to use form for raising nurseries using seeds and runner segments. Some efforts should also be placed in identifying elite populations (in terms of active ingredient content) suitable for raising large number of propagules. In spite of the endangered status of P. kurrooa (Samant et al. 1998) there have been very few attempts to restore its natural status and/or to initiate cultivation. In this regard, the propagation protocols reported in this communication will be useful for initiating a programme on systematic cultivation of this high value species. Presently efforts are also in progress to develop farmer friendly package of agrotechnniques at the high altitude field station. Thus, demonstration of propagation techniques and distribution of elite propagules to farmers interested in their commercial cultivation, will pave the way for in situ conservation of endangered species like P. kurrooa. Acknowledgement The financial assistance provided by the Department of Biotechnology and the Ministry of Environment, Govt. of India, New Delhi is gratefully acknowledged. References Ahuja P.S. 2001. Current status of propagation of medicinal plants in Indian Himalaya. In: Samant S.S., Dhar U. and Palni L.M.S. (eds), Himalayan Medicinal Plants: Potential and Prospects. Gyanodaya Prakashan, Nainital, pp. 207–230. 2337 Bewley J.D. 1997. Seed germination and dormancy. Plant Cell 9: 1055–1066. Chandra B., Palni L.M.S. and Nandi S.K. 2004. Micropropagation of Picrorhiza kurrooa Royle ex Benth., an endangered alpine herb, using cotyledonary node and shoot tip explants. Phytomorphology 54(3&4): 303–316. Cheng T.Y., Saka H. and Voqui-Dinh T.H. 1980. Plant regeneration from soybean cotyledonary node segments in culture. Plant Sci. Lett. 19: 91–99. Eriksen E.N. 1974. Root formation in pea cuttings III. The influence of cytokinin at different developmental stages. Physiol. Plant. 30(2): 163–167. Farooquee N.A. and Saxena K.G. 1996. Conservation and utilization of medicinal plants in high hills of the central Himalayas. Environ. Conserv. 23(1): 75–80. Hartmann H.T., Kester D.E., Davies F.T. and Geneve R.L. 2002. Plant Propagation: Principles and Practices. Prentice Hall, New Jersey, 880 pp. Hussain A. 1984. Conservation of genetic resources of medicinal plants in India. In: Jain S.K. (eds), Conservation of Tropical Plant Resources. Botanical Survey of India, Howrah, pp. 110–117. Jia Q., Hong M.F. and Minter D. 1999. Pikuroside: a novel iridoid from Picrorhiza kurroa. J. Nat. Prod. 62: 901–903. Kaye T.N. 1997. Seed dormancy in high elevation plants. In: Kaye T.N., Liston A., Love R.M., Luoma D.L., Meinke R.J. and Wilson M.V. (eds), Conservation and Management of Native Plants and Fungi. Native Plant Society of Oregon, Corvallis, Oregon, pp. 115–120. Kirtikar K.R. and Basu B.D. 1984. Indian Medicinal Plants. Vol. III. Bishen Singh Mahendra Pal Singh, Dehradun, pp. 1824–1826. Kitagawa I., Hino K., Nishimura T., Mukai E., Yosioak I., Inouye H. and Yoshida I. 1969. Picroside I: a bitter principle of Picrorhiza kurrooa. Tetrahedron Lett. 43: 3837–3840. Lal N., Ahuja P.S., Kukreja A.K. and Pandey B. 1988. Clonal propagation of Picrorhiza kurroa Royle ex Benth. by shoot tip culture. Plant Cell Rep. 7: 201–205. Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497. Nadeem M., Palni L.M.S., Purohit A.N., Pandey H. and Nandi S.K. 2000. Propagation and conservation of Podophyllum hexandrum Royle: an important medicinal herb. Biol. Conserv. 92: 121–129. Nandi S.K., Letham D.S., Palni L.M.S., Wong O.C. and Summons R.E. 1989. 6-Benzylaminopurine and its glycosides as naturally occurring cytokinins. Plant Sci. 61: 189–196. Nandi S.K., Palni L.M.S. and Rikhari H.C. 1996. Chemical induction of adventitious root formation in Taxus baccata cuttings. Plant Growth Regul. 19: 117–122. Nandi S.K., Tamta S. and Palni L.M.S. 2002. Adventitious root formation in young shoots of Cedrus deodara L. Biol. Plant. 45(3): 473–476. Nautiyal M.C. 1988. Germination studies on some high altitude medicinal plant species. In: Proceedings of Indigenous Medicinal Plant Symposium. Today and Tomorrows Printers and Publishers, New Delhi, pp. 107–112. Nautiyal B.P., Prakash V., Chauhan R.S. Purohit H. and Nautiyal M.C. 2001. Assessment of germinabiltiy, productivity and cost benefit analysis of Picrorhiza kurrooa cultivated at lower altitudes. Curr. Sci. 81(5): 579–585. Nayar M.P. and Sastry A.R.K. 1990. Red Data Book of Indian Plants, Vol. I. Botanical Survey of India, Howrah. Pandey H., Nandi S.K., Nadeem M. and Palni L.M.S. 2000. Chemical stimulation of seed germination in Aconitum heterophyllum Wall. and A. balfourii Stapf.: important Himalayan species of medicinal value. Seed Sci. Technol. 28: 39–48. Purohit A.N. 1997. Medicinal Plants – need for upgrading technology for trading the tradition. In: Nautiyal A.R., Nautiyal M.C. and Purohit A.N. (eds), Harvesting Herbs-2000: Medicinal and Aromatic Plants and Action Plan for Uttarakhand. Bishen Singh Mahendra Pal Singh, Dehradun, India, pp. 49–75. Rai L.K., Prasad P. and Sharma E. 2000. Conservation threats to some important medicinal plants of Sikkim Himalaya. Biol. Conserv. 93(1): 27–33. 2338 Rastogi R.P., Sharma V.N. and Siddiqui S. 1949. Chemical examination of Picrorhiza kurrooa Benth. Indian J. Sci. Ind. Res. 8B: 172. Rawat A.S., Pharswan A.S. and Nautiyal M.C. 1992. Propagation of Aconitum atrox (Bruhl) Muk. (Ranunculaceae), a regionally threatened medicinal herb. Econ. Bot. 46(3): 337–338. Samant S.S., Dhar U. and Palni L.M.S. 1998. Medicinal Plants of Indian Himalaya: Diversity, Distribution and Potential Value. Gyanodaya Prakashan, Nainital, 163 pp. Schrandolf H. and Reinert J. 1959. Interaction of plant growth regulators in regeneration processes. Nature 184: 465–466. Snedecor G.W. and Cochran W.G. 1967. Statistical Methods. Oxford and IBH Publishing, New Delhi, 593 pp. Son B.G., Choi Y.W., Ahn C.K., Cho D. and Kang J. 1999. Effect of maturity, storage and growth regulators treatment on the seed germination and longevity of Gentiana scabra Bunge var. buergeri Max. J. Korean Soc. Horticult. Sci. 40(1): 129–133. Thakur R.S., Puri H.S. and Hussain A. 1989. Major Medicinal Plants of India. CIMAP, Lucknow, pp. 404–407. Upadhyay R., Arumugam N. and Bhojwani S.S. 1989. In vitro propagation of Picrorhiza kurroa Royle ex Benth. – an endangered species of medicinal importance. Phytomorphology 39(2,3): 235–242. Walker M.A., Roberts D.R., Waite J.L. and Dumbroff E.B. 1989. Relationship among cytokinin, ethylene and polyamines during the stratification–germination process in seeds of Acer saccharum. Physiol. Plant 76: 3236–3332. Weinges K., Kloss P. and Henkels W.D. 1972. Natural products from medicinal plants. XVII, picroside II, a new 6- vanilloyl catapol from Picrorhiza kurroa. Liebigs Annalen Chem. 759: 173– 182.