Plant Cell, Tissue and Organ Culture 34: 307-309, 1993. 1993 Kluwer Academic Publishers.

. Printed in the Netherlands. Research note

In vitro propagation of Gentiana kurroo- an indigenous threatened plant

of medicinal importance
N e e l a m S h a r m a , K.P.S. C h a n d e l & Anderson Paul

National Plant Tissue Culture Repository, N B P G R , N e w Delhi - 110012, India

Received 10 November 1992; accepted in revised form 6 May 1993

Key words: axillary shoot sprouting, clonal propagation

Abstract

Shoot multiplication of Gentiana kurroo Royle, a threatened medicinal plant species, was achieved in vitro using shoot tips and nodal segments as explants. Fifteen-fold shoot multiplication occurred every 6 weeks on Murashige and Skoog's medium (MS) containing 8.9 ~M benzyladenine and 1.1 ~M 1naphthaleneacetic acid. Rooting was accomplished successfully in excised shoots grown on MS basal medium containing 6% sucrose.
Abbreviations: B A - 6-benzyladenine, I A A - Indole-3-acetic acid, I B A - Indole-3-butyric acid, M S -

Murashige and Skoog's medium, NAA - 1-naphthaleneacetic acid

Gentiana kurroo Royle (Indian Gentian), commonly known as 'Karu' or 'Kutki', occurs as a perennial herb in the Himalayan region of India at an altitude of 1500-3300m. The dried rhizomes and roots of this plant species are used as a substitute for the true gentian. In India, it has been used medicinally as a bitter to stimulate gastric secretions, to cure debility and in cases of fevers and urinary complaints. It is also useful in syphilis and leucoderma (Kirtikar & Basu 1975). Fresh roots and rhizomes are the source of the glycosides-gentiopicrine, gentiamarin and gentiin and the alkaloid gentianin (Anonymous 1956). The plant is not cultivated in India. Thus, the pharmaceutical industry is largely dependent upon material procured from naturally occurring stands, which are being depleted rapidly, raising concern about possible extinction of the species (Gupta 1988). Hence, a need for employing tissue culture techniques for the rapid clonal propagation and in vitro conservation is evident. To our knowledge, there are no reports on the micropropagation of G. kurroo. The present

communication is the first report on clonal propagation of G. kurroo through shoot proliferation. Plants of Gentiana kurroo obtained from AICRP M&AP Centre located at Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, were maintained under controlled temperature (15C), humidity (70-75%) and light (10 h dark and 14 h light) conditions in a growth chamber. Stem segments were washed in 'Teepol' detergent for 15 min at slow speed on a magnetic stirrer and later washed thoroughly under running tap water for 2 h. Subsequently these were surface disinfested with 0.1% HgC1 e (BDH) for 10min and rinsed several times with sterile distilled water before implanting vertically on nutrient medium. The basal medium (BM) comprised of the mineral salts and organic nutrients of Murashige & Skoog (1962), 3% sucrose and 0.8% Bacteriological agar (Hi Media). Different concentrations of BA singly and in

308 combination with NAA, IAA or IBA were added for initiation of buds and elongation of microshoots. For rooting the medium was supplemented with NAA, IAA, IBA, activated charcoal or 6% sucrose. All the adjuvants were added to molten agar and the pH of media were adjusted to 5.8 before autoclaving at 106 kPa (121C) for 15 min in culture tubes (150 x 25 mm, rimless) closed with polypropylene caps. The cultures were maintained at 25 + 2C under a 16-h photoperiod provided by cool white fluorescent tubes (38-40 i~mol m -2 s-l). Each treatment was replicated 12 times and all experiments were repeated at least twice. Regenerated shoots were subcultured after 68 weeks. At each subculture, the individual shoots were separated and stem segments with a node were transferred to fresh medium. The total number of culturable segments obtained from each cutting at the end of each multiplication cycle was used to calculate the shoot multiplication rate. Nodal segments from plants maintained under controlled conditions were used for the initiation of cultures. MS medium supplemented with 2.222 ixM BA and 2.3-23 ~M kinetin alone or in combination either with NAA (0.54-5.4 txM) or IAA (0.57-5.7~M) were tested in the first experiment. Only in the medium containing 8.9 ~M BA and 2.7 ixM NAA did 50% of the axillary buds sprout within 10-15 days of inoculation. None of the other combinations induced axillary shoot sprouting. The shoots regenerated from nodal cuttings were subcultured after 6 weeks onto fresh medium of the same composition to establish an initial stock of shoots. Subsequently, nodal explants from these in vitro-grown shoots were used to assess optimal growth regulator requirements for plantlet development. In the second set of experiments, nodal explants from in vitro-grown shoots were cultured on MS medium supplemented with various concentrations and combinations of BA, kinetin, IAA, IBA and NAA. Data obtained after 6 weeks of culture revealed that 8.9 ~M BA and 1.1 txM NAA gave an optimal response with regard to overall plantlet development, as assessed in terms of number of shoots and shoot growth (Table 1). Shoot emergence was observed within 10 days of culture and 75% of the explants produced multiple shoots (up to 10 shoots) on this medium. The average number of shoots per culture was 3.5. The shoots attained an average length of 4.2cm with 3-4 nodes, thereby providing about 15 propagules for the next cycle of shoot multiplication. Most of the other combinations resulted only in singleshoots. BA was found to be better than kinetin for the production of multiple shoots. Replacing NAA with IAA or IBA at about the same concentrations in the presence of 8.9 ~M BA was not effective in improving multiple shoot formation (data not shown). Therefore, the medium containing 8.9~xM BA and 1.11xM N A A was used routinely for shoot multiplication. This medium has supported the persistence of the regeneration capacity of the system for more than 2 years so far. For rooting, individual shoots with 3-4 nodes were implanted onto rooting media consisting of MS supplemented with IAA, NAA or IBA or activated charcoal (0.5%) or MS basal, 1/2 strength basal and MS basal with 6% sucrose. Of the various media tested, the best rooting re-

Table 1. Effect of various growth regulator treatments on shoot multiplication in Gentiana kurroo. Treatments IxM B A 4.4 B A 8.9 B A 8 . 9 + N A A 1.1 B A 8.9 + N A A 2.7 B A 8.9 + N A A 5.4 Kinetin 9.2 + N A A 1.1 Kinetin 9.2 + N A A 2.7 Basal m e d i u m : MS, Growth period: 6 weeks. Cultures with multiple shoots ( % ) 50.0 66.6 75.0 25.0 16.6 33.3 16.6 No. of shoots per explant S.E. 2.6 0.6 1.5 0.4 3.5 0.7 1.1 + 0.3 1.1 0.3 1.5 +- 0.4 1.3 0.3

309 sponse in terms of days of initiation and number of roots was observed in MS basal with 6% sucrose. In this medium 90-100% shoots produced an average of 8 thick, white roots within 2 weeks. Rooting also occurred in basal medium ( 6 0 - 7 0 % ) and medium containing 1.1-5.4 txM N A A (50-80%). The roots produced in NAA-suplemented medium were feathery and remained on the surface with only a few penetrating the medium. Rooting did not occur in the media containing either activated charcoal or I A A or IBA. Induction of multiple shoots through axillary branching is now recognised as a useful technique for propagation and in vitro conservation of threatened plants, especially those in which roots or rhizomes contain the active compound (Constabel 1990; Sharma & Chandel 1992). The present work demonstrates a simple procedure for rapid clonal propagation of G. kurroo through axillary branching. Attempts are underway to devise methods for in vitro conservation of this threatened plant species. for the encouragements and Dr. Rajendra Gupta, Project Co-ordinator, All India Coordinated Research Project on Medicinal and Aromatic Plants, for help in procuring material. We also thank Mr, D.K. Nerwal for technical assistance. Financial support provided by the Department of Biotechnology, Ministry of Science and Technology, New Delhi is gratefully acknowledged.

References
Anonymous (1956) In: The Wealth of India, Raw Materials. Vol IV, Publication and Information Directorate, CSIR, New Delhi, India Constabel F (1990) Medicinal plant biotechnology. Planta Med. 56:421-425 Gupta R (1988) Genetic resources of medicinal plants. Indian J. Plant Genet. Resources 1:98-102 Kirtikar KR & Basu BD (1975) Indian Medicinal Plants. Vol Ill Jayyed Press, New Delhi, India Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497 Sharma N & Chandel KPS (1992) Low temperature storage of Rauvolfia serpentina Benth. ex Kurz- An endangered, endemic medicinal plant. Plant Cell Rep. 11:200-203

Acknowledgements
We express our thanks to Dr. R.S. Rana, Director, National Bureau of Plant Genetic Resources