Novel and Simple Method for in Vitro Production of Psoralen via Cotyledonary Culture of An Endangered Leguminous Taxon– Psoralea Corylifolia Linn.

First time, a novel and simple protocol has been successfully developed for in vitro production of psoralen through rapid and recurrent callus cultures from one week old cotyledon of Psoralea corylifolia Linn. Amongst the various auxins and cytokinins tried alone or in combinations, 10 μM 2,4-dichlorophenoxyacetic acid (2, 4-D) supplemented to Gamborg et al. (1968) basal medium (B5) was optimum for inducing profuse amount of callus in cent percent cultures of cotyledon. The cotyledon calluses were subcultured continuously at an interval of 35-40 days on 5μM 2, 4-D medium up to two year and good amount of callus proliferation was achieved in cent percent cultures. However, some cultures were maintained under optimum cultural condition on B5+5μM 2, 4-D+5μM BA with 0.5% polyethylene glycol (w/v) up to 3 months. These callus cultures showed the maximum psoralen content of 16.15 mg/g FW. Psoralen has been detected time to time from calluses of different time durations grown on different type of culture media under optimum cultural conditions through HPLC by using calibration curve of standard psoralen. Psoralen estimation was also done for mature seeds samples collected from various localities of central India. Introduction: Psoralea corylifolia (Linn.) is enlisted as rare and endangered leguminous taxon (Jain, 1994). Four species are found in India of which P. corylifolia is medicinally valuable plant (Anonymous, 1989; Bhattacharjee, 1998). It is used in indigenous medicine as laxative, aphrodisiac, anthelmintic, diuretic and diaphoretic and specially recommended for the treatment of vitiligo , leprosy, psoriasis and inflammatory diseases of the skin in ayurvedic as well as allopathic system of medicines(Anonymous,1989). The medicinal value is due to the presence of furanocoumarin especially a psoralen in seeds that cost around US $ 4634/per gram (Sigma-Aldrich Chemical Company, USA). In recent time, it is compulsory to have some alternative source of these seeds that produce the highly important bioactive compound known as Psoralen. Till date, there is no report of in vitro production of Psoralen , therefore, the present study becomes globally novel, simple and reproducible in which cotyledonary callus cultures were used for the continuous production of psoralen at commercial/ industrial scale. Materials and methods: (i) Source of seed materials: Mature and immature seeds of Psoralea corylifolia Linn. has procured from different areas/ populations of central part of India . 1. Pharmacopoeial Laboratory for Indian Medicine, Kamla Nehru Nagar, Ghaziabad, U.P., India. 2. Botanical Garden, University of Jamia Hamdard, Delhi, India. 3. National Bureau of Plant Genetic Resources, Pusa Campus, Delhi, India Accessions No. IC-111249, IC111228, IC-111238 4. National Botanical Research Institute, Lucknow, India. 5. Pratap Nursery, Dehradun, Uttrakhund, India. 6. Kharibawali, Delhi, India. (ii) Explants: Cotyledonary callus cultures was developed using the 7 day old cotyledonary explants taken from above mentioned sources. (iii) Sterilization: a) Glasswares, instruments, etc.: Glasswares, instruments (wrapped in aluminium foil) and nutrient media in glass vessels were sterilized by autoclaving them at 1.06 Kg cm-2 pressure for 15-20 min at 1210C. b) Seeds: Mature and immature seeds were washed under running tap water for 10 min in a beaker. They were then immersed in 1% (v/v) germicide-detergent Cetrimide solution (Nicholas Piramal India Ltd., Bombay) and stirred vigorously on magnetic stirrer for 10 min. These were rewashed under running tap water to remove any traces of Cetrimide. After pouring out excess water, the seeds were treated with 0.1% Bavistin and 0.1% Amoxicillin solution for 10-15 min. Later, washing with tap water, seeds were soaked in 15% concentrated sulfuric acid (H2SO4) for 20 min or in hot water at 500C for 3 hours in order to soften the hard seed coat. After decanting the acid or hot water, seeds were rinsed with autoclaved distilled water 4 or 5 times. Subsequently, the seeds were treated with freshly prepared 0.1% (w/v) HgCl2 solution (Merck, India) for 10-15 min, keeping the vessel on the shaker with constant stirring. Finally, the seeds were given 4 or 5 washings with sterilized distilled water under laminar flow cabinet and inoculate onto medium. (iv) Culture media: Cotyledon were implanted on different basal media, viz. Knop’s (Knop, 1865), MS (Murashige and Skoog, 1962,), B5 (Gamborg et al., 1968), and NN (Nitsch, 1969) to assess their germination potential. Stock solution of these media were prepared using analytical grade (AR) salts (Qualigens, Merck, Hi-media, SRL and BDH) and stored in reagent bottles in the refrigerator. The basal media were used alone or in combination with various plant growth regulators such as (a) cytokinins, viz. BA, 2iP, Kn and /or (b) auxins, viz. NAA and 2,4-D procured from Sigma-Aldrich Chemical Company, USA. In addition, adjuvants like adenine sulphate, picloram, polyethylene glycol and coconut water were also supplemented to the medium. The thermolabile growth regulators, and antibiotic (ampicillin) were filter sterilized using 0.22 mm millipore filter and then added to the autoclaved and cooled (45-480C) medium under a laminar flow cabinet As a source of carbon, sucrose (DCM, Daurala) at 3% concentration (w/v) were used in all experiments unless mentioned specifically. The media were gelled with 0.8% agar (Qualigens, Bombay) for morphogenesis. The pH of the