Abstract: A protocol has been developed for in vitro shoot proliferation from callus cultures of Aconitum heterophyllum Wall. Callus initiation occurs from nodal segments on MS media fortified with NAA (0.5 mg L-1) and BAP (0.25 mg L-1). Callus was transferred on Msmediasupplemented with BAP (0.25 mg L-1) for shoot proliferation. The best response for shoot proliferation was obtained on MS media+ NAA (0.25 mg L-1) + BAP (0.5 mg L-1). The well-developed micro shoots were transferred to root induction media containing MS basalmedia + IAA (1.0 mg L-1). The rooted plantlets were finally transferred to green house for hardening and field transfer.
INTRODUCTION
Aconitum heterophyllum Wall. commonly known as ‘Atis’ or ‘Patis’ belongs to the family Ranunculaceae and is reputed for its medicinal and pharmaceutical values since long. It is a perennial herb, distributed over temperate parts of western Himalaya, extending from Kashmir to Kumaon. Its chief habitat is in the alpine and sub-alpine areas at elevations of 3000-3500 m, like Gulmarg, Kilanmarg, Sonamarg (Uniyal et al., 2002). The tuberous roots of genus Aconitum contain alkaloids: benzoylmesaconine, mesaconitine, aconitine, hypaconitine, heteratisine, heterophyllisine, heterophyllisine, heterophylline, heterophyllidine, atidine, isotisine, hetidine, hetisinone and benzolylheteratisine (Zhaohong et al., 2005; Pelltier et al., 1968) plant contain Alkaloids heteratisine, heterophyllisine, heterophylline, hetrophyllidine, atidine, Isoatisine hetidine, hetsinone and benzoylheteratisine (Zhaohong et al., 2005). The roots, which have been used mostly as poison than as drug, are now reported to possess significant antipyretic and analgesic properties and a high therapeutic index The Aconitum alkaloids mesaconitine and 3 acetylaconitine have been shown to possess anti inflammatory activity (Ameri et al., 1998). The plant is used for the treatment of diseases of the nervous system, digestivesystem, rheumatism and fever. Aconitum has biological and pharmacological activities such as anti-fungal, anti-bacterial, insecticidal and Brime shrimp cytotoxic activities (Anwar et al., 2003). The plant possesses potent immunostimulant property (Atal et al., 1986).The root extract exhibits anti-viral activity against Spinach MosaicVirus (SMV) (Patwardhan et al., 1990). It is also used for curing hysteria, throat infection, dyspepsia, abdominal pain and diabetes. In the indigenous system of medicine this plant is considered as valuable febrifuge, nervine tonic, especially in combating debility after malaria and in hemoplegia. In view of the continued popularity of Aconitum heterophyllum in indigenous as well as modern system of medicine coupled with unscientific and indiscriminate extraction from wild sources has reduced this high value plant species towards rarity and is already in red list (Dar et al., 2001). Organized cultivation of Aconitum is therefore necessary to ensure the quality and continuous supply of drug. Moreover, the tubers derived from seeds result in large genetic variability, and are frequently infected with fungal diseases, mainly Verticelium sp. (Pirone et al., 1978). Seedlings are plagued by their variation in quality and quantity, natural uniformity is not maintained throughout. In the past many programmes have been initiated for the in situ propagation of this endangered medicinal plant. However they are limited by several factors. The potential for regeneration of medicinal plants out of their natural habitat is poor. The germination of seeds and establishment of seedlings is also poor (Khan and Khanum,1998). The number of propagules produced by the natural methods is limited and insufficient for large-scale planting in the wild or under field conditions. Using in vitro and associated biotechnological interventions, the removal of such otherwise problems could be possible.
Tissue culture techniques have been used for endangered plants to generate large numbers of propagules, which can be reintroduced in their native habitat. It can enable the mass propagation of this herb from a minimum of plant material so that large quantities of biomass required for extraction of active constituents can be made available throughout the year, without causing further endangerment of the species. This study on Aconitum heterophyllum was taken up with a view to develop techniques for its in vitro multiplication.
MATERIALS AND METHODS
Fresh viable seeds of Aconitum heterophyllum were procured from the Gene bank of Regional Research Laboratory (CSIR), Sanatnagar, Srinagar, Kashmir in the year 2004-2005. Voucher specimen was deposited in the repository of RRL Srinagar, (Voucher No.RRL/AC/Srinagar-2004). Seeds were washed with detergent and 2-4 drops of Tween-20 (Himedia) under running tap water, followed by final rinsing with double-distilled water. These seeds were soaked in double-distilled water for 2-3 days at 4°C in a refrigerator. Surface sterilization of soaked seeds was achieved by using 0.1% (w/v) mercuric chloride for 3-4 min, followed by three times rinsing with autoclaved double distilled water to remove all traces of sterilant. The sterilized seeds were then kept in the dark at 20±2°C on moist absorbent cotton in the petri plates. The germinated Seedlings were inoculated on MS (Murashige and Skoog, 1962) basal media fortified with 3% sucrose. pH of the media was adjusted at 5.8 by using 0.1N NaOH and 0.1N Hcl before gelling the medium with 0.8% agar-agar type (Himedia). The media was finally dispensed into culture tubes, which were plugged and autoclaved for 20 min. at 15 lb pressure and 121°C temperature. These cultures were maintained at 25±2°C with 55-65% relative humidity and exposed to 16 hours photoperiod provided by cool fluorescent tubes (3000 lux).
RESULTS AND DISCUSSION
Seeds of Aconitum heterophyllum, cultured on moist absorbent cotton, resulted in full-fledged seedling formation after 4 weeks of their culture (Fig. 1). Nodal explants were excised aseptically from these seedlings and cultured on different phytohormonal regimes, the effect of which is depicted in Table 1. Compact callus was formed on MS medium supplemented with various concentrations of 2,4-D (0.1- 0.4 mg L-1), with 40% response. With NAA (0.1 mg L-1), single shoot, followed by root formation was obtained from the nodal bud. However, comparatively higher concentrations of NAA (0.6 mg L-1) resulted in non-differentiating callus formation with 70% response.
Various BAP concentrations (0.10-0.50 mg L-1) stimulated direct axillary shoot-initiation, proliferation and elongation. The maximum number of shoots survived and grow best on BAP (0.25 mg L-1) with 80% response (Fig. 2). However, higher concentration of BAP (0.5 and 1.0 mg L-1) reduced the number of axillary shoots per explant. The combined interaction of BAP (0.1 mg L-1) + 2, 4-D (0.1 mg L-1) also resulted in shoot proliferation but with lower percentage of shoot formation.
| Fig. 1: | Seed germination |
| Fig. 2: | Shoot proliferation |
| Table 1: | Morphogenetic response of nodal explants of Aconitum heterophyllum to various phytohormonal regimes |
| *Data recorded after 4 weeks of culture | |
| Table 2: | Rooting response in Aconitum heterophyllum after inoculation on MS media supplemented with different concentrations of auxins (observation recorded after 35-45 days). |
| *The data is based on 5 replicate cultures, while the experiment was repeated thrice mg L-1 = milligram per liter | |
| Fig. 3: | I: Friable callus; II: Green callus |
| Fig. 4: | In direct shoot proliferation |
| Fig. 5: | Rooting |
| Fig. 6: | Plant in field |
Profuse callus was formed on MS medium supplemented by BAP (0.25 mg L-1) + 2,4-D (0.5 mg L-1) with 80% response (Fig. 3). The combined interaction of BAP (0.5 and 1.0 mg L-1) + NAA (0.25 and 1.0 mg L-1) resulted in compact callus formation at the basal ends, which was followed by multiple shoot regeneration and elongation on the same media with 90% response (Fig. 4). Direct root initiation and elongation was observed after sub- culturing of isolated shoots into MS basal media within 4 weeks of their culture period (Table 2). The rooting response of shoots was also initiated on MS basal media fortified with IAA (1.0 mg L-1) after 4 weeks of culturing (Fig. 5). Complete plantlets (6-10 cm) were recovered after 6-8 weeks on rooting media. The healthy plantlets were deflasked and transferred into pots containing sand, soil and vermiculite mixture (1:1:1) (Fig. 6).
In the present study, the maximum multiple direct adventitious shoot regeneration and elongation was observed by culturing nodal segments of A. heterophyllum on MS media augmented with BAP (0.25 mg L-1).The results are very much in conformity with other previous studies (Giri et al., 1993). Our results are also supported by the observations in liquid culture of A. napellus (Watad et al., 1995). Callus regeneration was observed at the basal cut ends of each explant, which is in agreement with the anther culture of A. carmichacli (Hatano et al., 1987).
Combined effect of BAP (1.0 mg L-1) + NAA (1.0 mg L-1) on nodal segments promoted multiple indirect adventitious shoot regeneration and elongation. However, the combined interaction of BAP (1.0 mg L-1) + 2, 4-D (1.0 mg L-1) on nodal segments showed low percentage of response. Such findings are strongly supported by those of Giri et al. (1993) who reported the callus formation on MS medium containing 2, 4-D, Kinetin, Coconut water and maintained on 1.0 mg L-1 level of NAA. Rooting of the elongated shoots was initiated on MS basal medium fortified by IAA (1.0 mg L-1) 4 weeks after culturing. However, root formation results recorded by Giri and Watad in A. heterophyllum and A. napellus was obtained best on IBA (1.0 mg L-1) and NAA (1.0 mg L-1), respectively.
The detailed review of the earlier studies reveal that there is only scanty published data on organogenesis of this plant species. However, there are few published reports regarding liquid culture of A. napellus, A. balfourii and A. carmichalli. (Watad et al., 1995; Hatano et al., 1987; Pandey et al., 2004 and Hatano et al., 1988).
Results of the study reveal that the protocol developed for them micro-propagation of A. heterophyllum has the potential to be reproduced and utilized for large-scale multiplication viz a viz conservation of this medicinal herb, an indigenous endangered medicinal plant. Friable callus formation was obtained on NAA (0.6 mg L-1) (Fig. 3), but callus maintenance was difficult due to excessive leaching of apparent phenolic compounds. The problem was more acute on hormonal combinations of BAP (0.5 mg L-1) and with 2, 4-D (0.25 mg L-1).
Inclusion of anti-phenolic substances, such as ascorbic acid (10 mg L-1) polyvinylpyrolodine (pvp) 0.5% and activated charcoal (2%) could not overcome this problem. Hence callus induced on 2, 4-D supplemented medium was transferred to NAA (1.0 mg L-1) containing medium. The cultures were sub-cultured after one week’s time, so as to prevent the leaching of exudates. The study indicates that A. heterophyllum populations in the North-western Himalaya are genetically diverse. At present, rate of its propagation is far less as compared to its exploitation. These species, or at least a significant proportion of its genetic diversity may be lost in near future, if appropriate measures are not taken for its conservation.
ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Dr. G.N. Qazi Director, Regional Research Laboratory (CSIR). Jammu for research facilities.