Ceropegia noorjahaniae Ans. (Family: Asclepiadaceous) is one of the critically endangered, endemic plants growing in the grasslands of Western Ghats of India. Its tubers are edible and flowers have considerable ornamental value. Scarcity of pollinators and poor seed set are hurdles in the natural propagation of this plant, leading to a continuous depletion of its natural population. A rapid in vitro multiplication protocol has been developed to conserve this plant using axillary buds as propagules. Various media combinations have been tried for the induction of multiple shoots, their subsequent rooting and recovery of complete plants. Optimum response in terms of multiple shoot formation was achieved on MS medium supplemented with BA (2mg l^-1). In four weeks 10-12 shoots developed per axillary bud. Shoots were rooted on half strength MS + IBA (2mg l^-1) + NAA (1mg l^-1) + activated charcoal (200mg l^-1). Acclimatization of in vitro derived plants was accomplished by transfer to soilrite in paper cups and subsequently to soil. The developed protocol for in vitro propagation of this plant will significantly contributes towards its conservation.

 Keywords: conservation, critically endangered, endemic, in vitro multiplication, MS medium

BA, 6-benzyl amino purine; IAA, 3-inoleacetic acid; IBA, 3-indolebutyric acid; Kinetin, N-6 furfuryladenine; MS, murashige and skoog (1962) Medium; NAA, 1-nephthaleneacetic acid; TDZ, thiodiazuron

Due to rapid depletion of forest and forest resources, many plant species are became endangered and are on the verge of extinction. Conservation of biodiversity is now a high priority area and needs special attention as far as environment is concerned. Conservation of plant genetic resources for food, fiber, fuel and pharmaceuticals is considered to be a major element of any strategy to achieve sustainable development of other natural resources.¹ According to the World Bank document, India is one of the 12-mega diversity countries in the world, which collectively accounts for 70% of the world’s biodiversity.² Ceropegia noorjahaniae Ans. is a member of family Asclepiadaceous (Figure 1). It is one of the critically endangered, endemic plants of Maharashtra.³ The plant is well known for its peculiar flowers, which have high ornamental value and its edible tubers. The plant can easily be identified by its 2.7cm long, glabrous corolla. Deforestation Western Ghats, Being endemic, scarcity of pollinators and poor seed set are the major causes in its dwindling numbers. The plant is found in its natural habitat in the Maharashtra, one of the states in India - especially in few districts, and the extent of its occurrence is estimated to be less than 500sq. km. with severely fragmented populations. In 1996, only 19 mature individuals were located at Jarandeshwar hills of Satara district MS India.⁴ A typical habitat of this plant is found along ghat slopes in well-drained rocky-gravelly soil above 1000meter altitude. The present investigations focused on in vitro propagation of Ceropegia noorjahaniae Ans. Through axillary bud multiplication and their acclimatization for restoration of this critically endangered plant species.

Figure 1 Habit of Ceropegia noorjahani Ans.

Nodal segments of young C. noorjahaniae shoots were collected from mature plants grown in the botanical gardens of Shivaji University, Kolhapur, MS, India and used as explants. The explants were washed in running tap water for at least 30min followed by a detergent, labolene (5% v/v) for 10min. followed by repeated washing in double distilled water. Surface sterilization of the explants was done with mercuric chloride (0.1% w/v) for 8min. and rinsed thoroughly (4-5times) with sterile double distilled water. Nodal explants (1cm) were cultured on MS⁵ basal medium (major and min or salts, vitamins, FeEDTA, inositol and 170mg l^-1 monobasic sodium phosphate) and supplemented with growth adjuvants such as BA, Kn, TDZ, NAA, IAA, IBA. Rooting of in vitro derived shoots was accomplished on half strength MS medium supplemented with various concentrations of auxins and activated charcoal (200mg l^-1). Sucrose (3%, w/v) was used as the carbon source and the pH of the media was adjusted to 5.8 prior to the addition of gelrite (Sigma, 0.2%). All the media were autoclaved at 1.08kg cm^-2 for 20min. Cultures were maintained at 25±2⁰C with 16-h light/8-h dark photoperiods (Philips TL 34, 25mmol m^-2s^-1). All the experiments were repeated at least twice and data was collected after four weeks of culture. The effect of different treatment was quantified and the data was analyzed using one-way analysis of variance (ANOVA). Means were compared using Duncan’s Multiple Range Analysis at 5% level of significance.

Multiple shoots were initiated from the nodal explants on all media combinations in four weeks (Table 1). The cytokinin, BA (2mg l^-1) was most potent for axillary bud initiation and proliferation resulting in 10.04±0.87 shoots per axillary bud (Figure 2). However, the shoots were found to be delicate. Similar observations have been recorded in Ceropegia jainii and C. bulbosa, where BAP was found to be most effective (Patil, 1998). In contrast to this, multiple shoots produced on MS+BA (2.0mg l^-1) + IBA (0.5mg l^-1) produced shoots (7.54±0.83 shoots per axillary bud) that were healthy and exhibited vigorous growth. A similar observation of a synergistic effect (on shoot multiplication) of BAP in combination with an auxin has been reported in many members of Asclepiadaceous such as Seshagiria sahyadrica,⁶ Frerea indica Desai et al.,¹ Ceropegia candelabrum,⁷ Holostemma ada-kodien,⁸ Hemidesmus indicus⁹ and Holostemma annulare Sudha et al.¹⁰ In subsequent cultures too enhanced shoot multiplication was observed in our studies and this is in accordance with previous reports in other Asclepiadaceous members such as Ceropegia candelabrum Beena et al.,⁷ Holostemma ada-kodien,⁸ Hemidesmus indicus⁹ and Gymnema sylvestre.¹¹ Beena et al.⁷ have reported leaf and shoot tip abscission in micro propagated shoots of Ceropegia candelabrum. In our studies too when MS medium devoid of monobasic sodium phosphate shoot tip abscission occurred. It is speculated that accumulation of ethylene in the culture vessel could have been the cause of necrosis and abscission of the leaves and shoot tips as reported in Gymnema sylvestre and Hemidesmus indicus.¹¹ A synergistic effect of monobasic sodium phosphate, NH⁺₄ ions and cytokinin has been reported in grape for reduction of hyperhydricity in cultures.¹²

Figure 2 Multiple shoot induction on MS basal medium supplemented with BA (2 mg l^-1).

Half strength MS (liquid) medium with activated charcoal (200mg l^-1), IBA (2mg l^-1) and NAA (1mg l^-1) was found most effective in the induction of healthy roots (80% rooting; 2.63±0.38 roots per shoot). This medium also supported robust shoot growth. (Table 2), (Figure 3). The roots on this medium were found to be thicker which has an added advantage during planting. It has been shown that combinations of two or more auxins are more effective in induction of roots in Syzygium cumini Yadav et al.¹³ and Seshagiria sahyadrica Kedage et al.⁶ Effectiveness of IBA in root induction has been reported in Frerea indica Desai et al.,¹ Ceropegia candelabrum Beena et al.,⁷ Holostemma ada-kodien,⁸ Hemidesmus indicus,⁹ Gymnema sylvestre¹¹ and Tylophora indica.¹⁴ The beneficial effect of activated charcoal on rooting was well documented in Hemidesmus indicus, Mishra et al.¹⁵ and Seshagiria sahyadrica Kedage et al.⁶ and is similar to our finding in this study. Shoots, with well-developed roots were transferred to small plastic pots containing sterile soil rite for acclimatization (Figure 4). Plants were transferred subsequently to field conditions where they grew well and exhibited morphological characters similar to parent plants. The field-grown plants developed tubers and flowered normally.^16,17 Shoot proliferation via axillary bud culture of selected individuals has great potential for micropropagation. Our studies in Ceropegia noorjahaniae are significant in the conservation of this critically endangered, endemic plant species.

Figure 3 In vitro rooting on half strength MS basal medium supplemented with IBA (2 mg l-1) and NAA (2 mg l^-1).

Figure 4 Hardened plant after one month.

Authors are thankful to Head, Department of Botany and University authorities of Shivaji University, Kolhapur MS India for providing laboratory facility to carry out this research work.

The author declares no conflict of interest.

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