Pal Minakshi et al / Int. J. Res. Ayurveda Pharm. 5(1), Jan - Feb 2014 95 Research Article www.ijrap.net PLANT REGENERATION STUDIES IN SAFED MUSLI (CHLOROPHYTUM SP.) Pal Minakshi 1 , Sethi Neeraj 2 *, Kaura Sushila 3 , Parle Milind 3 1 Centre for Plant Biotech, Hisar, Haryana, India 2 Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India 3 Pharmacology Division, Dept. Pharm. Sciences, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India Received on: 17/12/13 Revised on: 23/01/14 Accepted on: 20/02/14 *Corresponding author Neeraj Sethi, PhD Scholar (CSIR SRF), Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India Email: 20neerajsethi@gmail.com DOI: 10.7897/2277-4343.05119 ABSTRACT An efficient protocol was developed for the rapid in vitro multiplication of an endangered medicinal herb, Chlorophytum borivilianum (safed musli); by using Basal/crown plate explants. The effects of phytohormones [6-benzylaminopurine (BAP), kinetin (KN), indole-3-acetic acid (IAA), indole-3- butyric acid (IBA) or α-naphthalene acetic acid (NAA)] on in vitro regeneration were investigated. The highest number of shoots (21.67), maximum shoot length (13.14 cm) and the highest response of shoot induction (85 %) were recorded on MS medium supplemented with 3.0 mg/l BAP and 2.0 mg/l KIN. Rooting was best achieved on half-strength MS medium augmented without plant growth regulators IBA and sucrose (3 %, w/v). The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing sterile sand and coco peat (1:1) and grown in a greenhouse with 85 % survival rate. The regenerated plants did not show any detectable morphological variation. The developed method can be successfully employed for large-scale multiplication and conservation of C. borivilianum. Keywords: Chlorophytum borivilianum, Micro propagation, Basal/Crown explants, Kinetin, BAP, IAA. INTRODUCTION Plant tissue culture is the established technique of in vitro maintenance and growth of plant cells, tissues or organs under aseptic conditions on artificial nutrient medium in a suitable container under the control environmental conditions. The technique of plant cell culture occupies the key role in the second green revolution in which gene modification and biotechnology are being used to improve crop yield and quality. The application of tissue culture techniques for the biosynthesis of secondary metabolites, particularly in the plants of pharmaceutical significance, holds an interesting alternative for controlled product of plant constituents. In the recent years, it has become increasingly difficult to maintain constant supply of many of the important medicinal plants due to several factors, such as, their ruthless exploitation, lack of conservation of environment, increasing cost of labour and economical and technical problems associated with the cultivation of medicinal plants. On the other hand, techniques of plant tissue culture can provide ever lasting solution to some of the burgeoning problems in the area of plant biotechnology. Medicinal plant Chlorophytum borivilianum (Safed Musli) is endogenous medicinal plant to India and considered as a 'white gold' or 'divya aushad' in Indian systems of medicine 1 . It is distributed in eastern part of India (Assam, Eastern Ghats, Eastern Himalayas, Bihar and Andhra Pradesh 2 . 13 species are reported to occur in India of which 8 are endemic 3 . It belongs to family Liliaceae and is valued for dried fasciculated storage roots 4 . It is an endangered species 5 . C. borivilianum is a small herbaceous plant. The major contents of safed musli are carbohydrate (42 %), proteins (8-9 %), root fibers (3-4 %) and saponins (2.17 %) 5 . It is an aphrodisiac drug used to cure general debility. Recently, C.borivilianum has gained a well established domestic (Indian) and international market for being the herbal alternative of "Viagra" without any side effects. It is a remedy for diabetes, for rheumatism and joint pains 6 . It is antimicrobial, anti-inflammatory and antitumor agent 7 . The roots of plants are currently sold in market at a price of Rs. 600-700/kg. The overuse of C. borivilianum species made it rapidly decreasing. This herb is traditionally grown through tuberous root that have become scarce in nature. Moreover seed germination is only 14-16 %. Poor seed setting, germination and slow growth in conventional vegetative propagation are major constraints in the large-scale cultivation of this commercially important medicinal plant 8 . High frequency multiplication through plant tissue culture technique would minimize the damage to remnant populations. In this context, an efficient micropropagation protocol has been developed for rapid multiplication and conservation of C. borivilianum. MATERIALS AND METHODS Plant material Basal/Crown plate and peduncle of C. borivilianum were collected from mature plants grown in medicinal and aromatic plant section of the department of plant breeding CCS Haryana Agriculture University. The explants were washed in running tap water for at least 25 minutes and then soaked in a 5 % (v/v) detergent, labolene (Qualigens, India) for 10 minutes. Surface sterilization of the explant was done with KMnO 4 for 10 minutes followed by treatment with 0.1 % HgCl 2 (w/v) for 8-10 minutes and rinsed thoroughly (4 times) with sterile distilled water. All