Vol.2 No. 1, 61-66 (2013) Received: June 2012; Accepted: April.2013 Conservation of Piper mullesua Buch-Ham.: An important medicinal plant from Arunachal Pradesh through in vitro culture strategies Purnima Dubey and Padmanabh Dwivedi 1 Laboratory of Plant Tissue Culture, Department of Botany, Rajiv Gandhi University, Itanagar- 791112, Arunachal Pradesh 1 Reader, Department of Crop Physiology, BHU, Varanasi, Uttar Pradesh Abstract Arunachal Pradesh, one of the hot spots of biodiversity harbours a rich diversity of medicinal plant species. Over the centuries, the collection of many important medicinal plant species for commercial purpose and traditional practice has exerted tremendous pressure on their existing population in wild. Many species of medicinal plants growing in natural habitats are becoming scarce followed by poor regeneration. Consequently, their unlimited collection as raw materials may lead to the complete disappearance and extinction of certain species and varieties. Such deterioration is further augmented due to unplanned and ruthless exploitation by pharmacological industries, biopiracy and deforestation. The endangered medicinal plants which are also slow propagating and thus having low regenerative ability require rapid clonal multiplication through tissue culture strategies. Piper mullesua Buch-Ham., an important medicinal plant from Arunachal Pradesh is facing threat owing to over-exploitation by the commercial establishments and local people for their medicinal utility, is taken up for in vitro clonal propagation and multiplication. Micropropagation protocol was developed for this plant and the in vitro regenerated plantlets were successfully transferred to the botanic garden of the University. Key words: Conservation, micropropagation, Piper mullesua. Introduction Arunachal Pradesh is a global biodiversity hotspot harbouring a rich diversity of medicinal plants. There are 120 ethnic communities in this state. These communities still almost fully depend on nature for their livelihood. The unsustainable utilization of natural resources through the practice of Jhuming (Shifting cultivation), hunting, fishing, trapping and also gathering plants and animals for medicines, ornaments, decoration and supplementing food by the aborigines has led to the depletion of the natural resources. Harbouring a rich diversity of medicinal plants, Arunachal Pradesh is facing tremendous pressure on their existing population, which are being collected for commercial purpose and traditional practice. Many species of medicinal plants are becoming scarce due to seed dormancy coupled with poor regeneration. Consequently, their unlimited collection as raw materials may lead to the complete disappearance and extinction of certain species and varieties. Such deterioration is further augmented due to unplanned and ruthless exploitation by pharmacological industries, biopiracy and deforestation. 61
The endangered medicinal plants which are also slow propagating and thus having low regenerative ability require rapid clonal multiplication through tissue culture strategies. Piper mullesua Buch-Ham., an important medicinal plant from Arunachal Pradesh facing threat owing to overexploitation by the commercial establishments and local people for its medicinal utility, was taken up for in vitro clonal propagation and multiplication. Micropropagation protocol was developed for this plant and the in vitro regenerated plantlets were successfully transferred to the Botanic Garden of Rajiv Gandhi University, Itanagar. Piper mullesua Buch-Ham., commonly known as Pahari peepal, belongs to the family Piperaceae. It is highly distributed in subtropical Himalayas from Shimla to Bhutan upto the height of 1500 m in Khasi hills and in Nilgiris. In Arunachal Pradesh, it is found in Dibang Valley (Roing), Lohit (Hayuliang, Wakro), West Siang (Along) and East Siang (Pasighat). The plant is economically important because almost each and every part of the plant is medicinally useful. The stem portion is crushed and applied for toothaches, the fruits are used for curing headaches and stomach aches, it is also used as spice. The roots also possess medicinal value and are used in treatment of asthma, bronchitis, dyspepsia and anorexia. Chemical isolation from the hexane fraction of alcoholic extracts of Piper mullesua shows the presence of Myristin (4-Methoxy-6{2- propynyl} 1, 3-bengodioxole) [5].Due to the excessive utilization of plant by local people of the area for medicinal purpose, especially the bark part, the population of Piper mullesua is declining. The plant is also used by commercial establishment companies of India like Dabur for preparation of certain digestive tablets e.g. Hajmola. In addition, owing to some religious purpose, the seed is consumed by the aborigines which has led to the less availability of seed and hence, decline in the plant population. In spite of the immense utility and limited work on germplasm conservation of this species; the in vitro culture of Piper mullesua Buch-Ham., has highly been called for, thus necessitating the need of micropropagational study. Material and methods Plant material and Culture media: The explants used for culture initiation were collected from the seedlings, obtained from State Forest Research Institue, Itanagar, Arunachal Pradesh and established in the garden of the University. The nodal segment was used as culture explant; small stem twigs were collected and the leaves were removed, the nodal explants were washed in agitated solution of liquid detergent for 15 min and later on washed in running tap water for 1 hour. Surface sterilization in case of Piper mullesua was done with 0.1 % mercuric chloride for 2 min. Explants were thoroughly washed with double distilled water followed by explants trimming into 1.0-1.5 cm long size. This was followed by further treatment with 0.1% ascorbic acid for 8 min, 0.2% ampicillin and 0.5% bavistin together for 5 min. Explants were thoroughly washed with sterile double distilled water after 62
every treatment and finally, were inoculated onto the nutrient medium. The basal medium consisted of the mineral salts and organic nutrients of MS medium [3], 3% sucrose and 0.8% agar. Depending upon the experiment, the basal medium was variously supplemented with combinations of different growth regulators such as Kn, BAP, IBA, IAA and NAA at different concentrations (0.5-2.0 mg/l). All of the supplements were added to the molten agar, and the ph of the medium was adjusted to 5.8, before autoclaving it at 121 C at a pressure of 15 psi for 15 min in culture tubes (150 x 25mm) or 100 ml conical flasks. The cultures were maintained at 25± 2⁰ C under 16-h photoperiod with a light intensity of 3000 lux provided through Philips coolwhite fluorescent tubes and with 60-70% relative humidity. Shoot growth was periodically observed. There were 10 explants per treatment and the experiments were repeated thrice. Acclimatization and establishment of plants in soil Four-to-six week-old regenerants with well-developed roots were removed from the culture tubes and washed free of agar. They were then dipped in 0.2% bavistin for 2 min and subsequently transplanted into plastic trays containing sterilized soil and river sand (1:1). The tray containing plantlets were covered with a transparent polythene lid to maintain high humidity, and the microcuttings were moisted twice every day. Following three 3 weeks at 24± 2⁰C under a 16 hr photoperiod, the lid was removed and the plantlet were transferred singly to earthen pots containing sterile sand, soil and humus (1:2:1) at ambient room temperature (28±2⁰C) with indirect sunlight. After 2 months, the well acclimatized plantlets were planted outside in the nature and finally transferred to the Botanic Garden of the University. Results and discussion In Piper mullesua, shoot induction was not found in MS (Murashige and Skoog) basal medium even after four weeks of culture similar to the findings in Gloriosa superba L. [2].After four weeks of culture, of the various plant growth regulators tried, best response in terms of % of shoots induced (60.7± 2.5) was observed in MS medium supplemented with 1.5 mg/l BAP (Fig.1 A). At this concentration an average of 2.4 ± 0.3 shoots/explants was produced. However, in terms of both the parameters studied viz. % of shoot induced/ culture (56.3± 1.5) and no. of shoots produced/explant (4.2± 0.2), the combination of 0.75 mg/l each of Kn + BAP in MS medium was found best (Table 1) (Fig.1 B) [4]. Rooting in Piper mullesua was very difficult owing to the semi woody nature of the plant. After several trials, and combination of auxins, supplementationof ½ MS with 1.0 mg/l IAA showed good number of root formation (2 roots/ shoot) 30 days after culture (data not shown). IAA also reported effective for root induction in 63
Table 1: Effect of growth regulators in MS basal medium on shoot induction and number of shoots per culture established from nodal explant of Piper mullesua after 4 weeks of culture. Growth regulators (mg/l) Percentage of cultures with induced shoots (%) Number of Shoots per explants Control Kn - - 0.5 10.7 ± 1.3 1.8 ± 0.2 1.0 20.9 ± 2.0 1.7± 0.4 1.5 30.1 ± 2.3 2.1 ± 0.2 2.0 25.2 ± 1.7 2.0± 0.4 BAP 0.5 45.3 ± 1.6 1.7± 0.2 1.0 50.6 ± 2.2 1.9± 0.3 1.5 60.7± 2.5 2.4± 0.3 2.0 55.8 ± 1.8 2.1± 0.2 Kn + BAP 0.25+ 0.25 48.6 ± 1.5 2.5± 0.4 0.50 + 0.50 55.3 ± 1.7 3.8±0.3 0.75 + 0.75 56.3 ± 1.5 4.2±0.2 1.00 + 1.00 50.1 ± 1.6 3.4 ± 0.3 64
TECHNOFAME- A Journal of Multidisciplinary Advance Research Fig.1 A, B, C: In vitro shoot induction and multiplication in Piper mullesua. D: The mother plant in nature [1] Tectona grandis.rooted microshoots were acclimatized and transferred to the garden behind the laboratory. 50% survivality observed after 30 days of field transfer.the result of the present study thus provides a promising protocol for the propagation of Piper mullesua through in vitro culture strategies on commercial scale as well for the conservation of their superior genetic strains. References 1. Aini, A.S.N., Goh,B.L. and Ridzuan,R. (2009). The effects of different indole-3-butyric acid (IBA) 65
concentrations, two light regimes of in vitro rooting and acclimatization of in vitro teak (Tectona grandis L.f) plantlets. African Journal of Biotechnology. 8 (22): 6158-6161. 2. Hassan, A.K.M.S. and Roy,S.K. (2005). Micropropagation of Gloriosa superba L. through high frequency shoot proliferation. Plant Tissue Culture 15 (1): 67-74. 3. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiolgia Plantarum, 15: 473-497. 4. Roy, S. K., Islam, M. S. and Hadiuzzaman, S. (1998). Micropropagation of Elaeocarpus robustus Roxb. Plant Cell Rep.17: 810 813. 5. Srivastava, S., Gupta, M.M., Prajapati, V., Tripathi, A.K., and Sushil Kumar (2002). Insecticidal activity of myristicin from P. mullesua. Pharmaceutical Biol. 39, 226-229 66