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In vitro propagation of Decalepis hamiltonii Wight & Arn., an endangered shrub, through axillary bud cultures.

One of the essential requirements for the successful application of plant propagation technology to agriculture is the capacity to regenerate elite plantlets. During the past decade, the demand for these plants has witnessed a steep rise. To meet the ever-growing commercial requirements, the realization of in vitro multiplication of a large number of clonal plants with the improved characteristics has been gaining significance. Decalepis hamiltonii Wight & Arn. (Swallow root), a monogeneric climbing shrub native of the Deccan Peninsula and forest areas of Western Ghats finds use as a culinary spice due to its highpriced aromatic roots. The roots of D. hamiltonii are used as a flavouring principle, appetizer, blood purifier and preservative. Of late, the highly aromatic roots have been subjected to overexploitation by destructive harvesting that has endangered the survival of this plant in its wild habitat. Moreover, the absence of any organized cultivation of this plant (M. Sanjappa, pers. commun., Botanical Survey of India, Calcutta), calls for immediate conservation measures. George et al. were able to regenerate plantlets of D. hamiltonii W&A from leaf callus. In the earlier reports by George et al., it was observed that the aromatic roots of D. hamiltonii proved to be a potent bioinsecticide on storage pests at lethal and sub-lethal levels (Indian Patent No. 1301/Del/98). The supercritical extracts of these roots proved to be potent antimicrobial agents. Induction of rooting is an important step in plant propagation. The classical root induction method uses a shock of high auxin concentration; however, the roots are stunted and malformed. It has been shown that interaction of auxins with thiol compounds also stimulates rooting in apple shoot cultures. Ma et al. demonstrated that the use of ethylene inhibitors such as AgNO3 and CoCl2 might promote root formation in shoot cultures of apple. We demonstrated the promotive effect of AgNO3 on shoot morphogenesis and in vitro flowering in both normal and Agrobacterium rhizogenes-induced genetically altered shoot cultures of Cichorium intybus. There are no reports on the micropropagation studies of D. hamiltonii. The present communication reports the clonal propagation of D. hamiltonii and use of AgNO3 as supplement for in vitro rooting in D. hamiltonii axillary bud cultures. Healthy plants of D. hamiltonii W&A were collected from Gumballi forest ranges located between 11 to 13∞N and 77 to 78∞SE in BR hills, Mysore district, India. Axillary buds of D. hamiltonii were washed under running tap water to remove soil and other superficial contamination. Single bud explants (1 cm each) (upper portion) were washed with Tween-20 (5% v/v) for 5 min followed by thorough washing under running tap water for 15 min. The explants were surface sterilized with 0.15% (w/v) mercuric chloride for 3 to 5 min and later rinsed 4 or 5 times with sterile distilled water. For all the experiments, MS, with 100 mg/l of myo-inositol was used. The pH was adjusted to 5.8 ± 0.2 using 1N HCl/1N NaOH before adding 0.8% (w/v) of agar (Himedia, Mumbai). The medium was subsequently autoclaved under 15 psi at a temperature of 121°C for 15 min. Explants were cultured in conical flasks (150 ml) covered with non-absorbent cotton plugs and kept in controlled conditions of temperature (25 ± 2°C) and light (45 μ mol m s) for 16 h day using fluorescent lights (Philips India Ltd) and 60 to 70% relative humidity. Various growth regulators, viz. 1naphthaleneacetic acid (NAA) (0.05– 2.5 mg/l) and 6-benzylaminopurine (BAP) (0.05–2.5 mg/l) were tried individually or in combination to obtain the most suitable growth hormone level for the proliferation of shoots in established explants. Experiments were performed with a minimum of five replicates and repeated twice. Observations were recorded after an interval of four weeks. The basal rooting medium (RM) contained MS salts with 0.5 mg/l of indoleacetic acid (IAA) supplemented with the similar sucrose and agar concentration as given for shoot multiplication medium. Ethylene action inhibitor AgNO3 (SRL India Ltd) was filter sterilized using 0.22 μm filters (Sartorius Ltd). AgNO3 was incorporated into the culture medium at a concentration range of 10 to 50 μM, respectively, per 40 ml of culture medium. Shoots measuring 3 to 4 cm from 4 week cultures were taken and the top 2 cm was cut and transferred to the RM. The first set of treatments had exogenous supplementation of AgNO3 (10–50 μM) to the RM. The experiment was repeated twice with five replicates each. The duration of root emergence was recorded for each treatment, to calculate specific growth rate expressed as mm growth/week. Rooting efficiency was calculated as the percentage of shoots producing roots after 4 weeks of culture in all the treat-