Optimization of potting mixture for hardening of in vitro raised plants of Tylophora indica to ensure high survival percentage

Transcription

1 , ISSN Vol. 1, No. 2, pp , September 2011 RESEARCH ARTICLE Optimization of potting mixture for hardening of in vitro raised plants of Tylophora indica to ensure high survival percentage Harmanjit KAUR*, Manju ANAND, Dinesh GOYAL Department of Biotechnology & Environmental Sciences, Thapar University, Patiala, Punjab, India *Corresponding Author, Tel: , Fax: Article History: Received 2 nd September 2011, Revised 27 th September 2011, Accepted 28 th September Abstract: Micropropagation is an important technique for rapid and large-scale multiplication of selected plants. The type and concentration of plant growth regulators and physiological status of the explant have profound effect on the efficacy of plant regeneration. However, success of any micropropagation protocol depends upon the percentage survival and successful establishment of plants from in vitro to ex vitro conditions. In the present study, a successful attempt has been made to acclimatize the tissue culture raised plants of Tylophora indica using various potting mixes of soil, vermicompost and biofertilizers (Azotobacter and Pseudomonas) in different combinations. Among all the combinations tried, soil: vermicompost: Azotobacter (N 2 fixer): Pseudomonas (phosphate solubilizers) (1:1:1:1) showed the highest percentage survival (92%) upon transplantation of plants to the field conditions followed by soil: vermicompost (1:1) with 88% survival. Different parameters like plant height from the base of stem in the polybags, number of phytosynthetically active leaves/plant and mortality rate were also monitored periodically. Keywords: Tylophora indica; acclimatization; potting mixture; biofertilizers. Introduction Tylophora indica (Burm. f) an important medicinal plant of family Asclepiadaceae has been used as a folk remedy for the treatment of bronchial asthma, whooping cough, bronchitis, inflammation, allergies and dermatitis (Shivpuri et al. 1968; Gore et al and Exoticnatural 2005). It is a perennial climber indigenous to India growing widely in forests of Central and Peninsular India. The roots and leaves of this plant contain pharmacologically active alkaloids like tylophorine and tylophorinine (Govindachari et al. 1961) and anticancerous tylophrinidine (Mulchandani et al. 1971). Due to the shortage of high quality planting material, commercial cultivation of this important medicinal plant is uncommon and only the wild populations are exploited for secondary metabolite extraction. The plant has low seed viability and germination rate (Thomas and Philip 2005) and the destruction of plant caused by harvesting the roots as a source of drug has threatened the very survival of this plant. It is therefore imperative to adopt alternative methods having high multiplication rates to produce large number of plants of improved quality and shortened rotation. In vitro clonal propagation of Tylophora indica has been reported using different explants like axillary buds (Sharma and Chandel 1992), root (Chaudhari et al. 2004), leaf (Jayanthi and Mandal 2001; Thomas and Philip 2005; Chandrasekhar et al and Sahai et al. 2010) and stem and petiole (Faisal and Anis 2005; Verma et al. 2010). However, there is hardly any report on the optimization of potting mixture to ensure high survival percentage of in vitro raised Tylophora plants under field conditions. In vitro propagation system provides an alternative method for the rapid production of plants but its ultimate success depends upon the successful transfer and establishment of these plants in the field conditions. Plants produced under in vitro conditions under controlled high humidity, diffused light and constant temperature need to be acclimatized because transferring of these plant from in vitro to ex vitro conditions is the most traumatic experience for them. Type of potting mixture used during acclimatization is one of the important factors determining the survival percentage of the plants *Corresponding author: ( ) harrykajjal@gmail.com 2011 Open Access Science Research Publisher

2 under ex-vitro conditions. In vitro raised plantlets of Garcinia indica showed 76% survival rate when hardened on cocopeat as a potting mixture (Chabukswar and Deodhar 2005). Press mud cake mixed with soil was used as the optimal medium for producing sturdy plants during the secondary hardening process of banana plantlets (Vasane and Kothari 2006). An eff i- cient one-step hardening technique for tissue culture raised orchid seedlings was reported on chips of charcoal, bricks and decayed wood as an alternate substratum (Deb and Imchen 2010). Vermicompost was shown to be the most suitable planting substrate for hardening which ensured high frequency survival (96%) of regenerated plants of Tylophora indica prior to their field transfer (Rani and Rana 2010). In the present paper, we describe an efficient hardening procedure for in vitro raised plantlets of Tylophora indica ensuring high survival percentage on different potting mixtures including biofertilizers, which was not reported earlier. Material and methods Plant material and Explant sterilization Leaves were collected from 3 years old healthy field grown plants of Tylophora indica maintained at Thapar University Campus, Patiala. Leaves were washed under running tap water for 30 minutes followed by their immersion in teepol solution 0.1% (v/v) for 5 minutes and subsequent washings with tap water. The explants were then treated with bevistin ( 0.1% w/v) for minutes followed by repeated washings with water. Surface sterilization was carried out in laminar flow hood by treating leaves with 0.1% (w/v) aqueous solution of mercuric chloride for 3-4 minutes followed by 3-4 washings in sterile distilled water to remove all traces of mercuric chloride. Culture media and Growth conditions Explants were cultured on MS (Murashige and Skoog s 1962) medium augmented with various growth regulators, 2% sucrose and 0.8 1% agar with ph adjusted to 5.8 before the addition of agar. Culture vessels containing media 84 were autoclaved at 121 o C for 15 lbs/inch 2 for 15 minutes. All the inoculated cultures were incubated in growth room in controlled conditions at a temperature of 25 ± 2 o C, 16 hr light/8 hr dark photoperiod and continuous illumination provided by cool white fluorescent tubes at 50µmolm -2 s -1. Twenty replicates were used for each treatment and subculturing was done at an interval of 4-5 weeks. Plant regeneration in vitro Leaf explants were excised (4-5 mm in size) and planted on Basal Murashige and Skoog s medium, supplemented with different concentrations of 6- benzyladenine ( μM) either alone or in combination with adenine sulphate for de novo adventitious shoot bud formation. Regenerated shoots were made to root on half strength basal MS medium. Plantlets thus formed were then acclimatized on moist cotton in the jars and were kept under growth room conditions followed by their transfer to polybags containing different potting mixtures. Results and Discussion Leaf explants were cultured on variously supplemented MS medium for de novo adventitious shoot formation directly from the explant. MS medium supplemented with 6- benzyladenine (9.84µM) either alone or in comb ination with adenine sulphate (0.5mg/l) pr oduced the greatest number of shoot buds per explant. Nodular meristemoids differentiated from the cut ends and from the abaxial surface of the leaf lamina after 8-10 days of culturing and within 3 weeks the entire surface was covered with theses meristemoids (Figure 1a). Eventually these meristemoids developed into green leafy shoots in 85% cultures (Figure 1b). Repeated su bculturing accelerated the formation of shoots in large number (50-60/flask) without any decline in proliferation. Regenerated shoots were excised and subjected to rooting on half strength basal MS medium where root initiation occurred after 10 days of culturing and 4-6 healthy roots were formed in 90% of the cultures (Figure 1c). The roots formed were long and branched.

3 85 Figure 1: a). Formation of nodular meristemoids (arrows) and their subsequent growth into multiple shoots on BA (9.8 µm) and 0.5mg/l of adenine sulphate. b). Formation of shoots in large numbers on the same medium c). Rooting of microshoot on half strength BMS media after 10 days of culturing. The hardening of in vitro raised plantlets is essential prior to transplantation of plants to the soil. Direct transfer of tissue culture raised plants to field conditions is not possible due to high mortality rate as the plants were kept under controlled environmental conditions having heterotrophic mode of nutrition and uncontrolled loss of water. It is therefore necessary to transfer the plants to field through various weaning stages in order to increase the survival percentage. In vitro raised plantlets were rescued carefully from culture tubes and washed under tap water to remove traces of agar and were initially transferred to the culture bottles containing moist cotton covered with perforated plastic covers and were kept for a period of 15 days under growth room conditions. Plantlets kept on moist cotton registered increase in shoot and root length, increase in the number of phytosynthetically active leaves and the better survival percentage in the subsequent weaning process. Plantlets were further transferred to plastic cups containing different potting mixtures in equal ratios i.e. soil: vermicompost (T0), soil: vermicompost: Azotobacter (N 2 fixers): Pseudomonas (phosphate solubilisers) (T1), soil: Azotobacter: Pseudomonas (T2), soil: Azotobacter (T3) soil: Pseudomonas (T4). The soil without any organic matter was taken as control. The pots were covered with perforated plastic bags to maintain internal humidity and aeration and were kept inside the growth room for 15 days. Plantlets were then transferred to plastic bags containing same potting mixture and were kept in growth room for another 2 weeks. Plantlets were monitored, watered periodically. The hardened plantlets in plastic bags were then transferred to the green house for another 2 weeks before transferring them to full sunlight outdoor (Figure 2). By this time, plants have become sturdy, developed an efficient root system, formed new leaves and became phytosynthetically active. Different parameters like plant height, number of phytosynthetically active leaves and percentage survival of the plants were measured at this stage (Table 1).

4 86 Figure 2: Plantlets acclimatized in different potting mixtures after 45 days Table 1: Effect of various potting mixtures on hardening of in vitro raised plantlets of Tylophora indica Potting mixtures Number of plantlets transferred Survival Percentage Shoot height (cm) Number of leaves/plant Soil: vermicompost (T0) 40 88± ± ±0.5 Soil:vermicompost:Azotobacter: Pseudomonas(T1) 40 92± ± ±0.33 Soil:Azotobacter:Pseudomonas(T2) 40 65± ± ±0.7 Soil: Azotobacter(T3) 40 61± ± ±0.7 Soil: Pseudomonas(T4) 40 59±0.5 10± ±0.5 Data in terms of mean±se values after 45 days of culture on potting mixtures Using various hardening and acclimatization stages the survival rate was found to be much higher than as compared with the direct transfer of the plantlets to the field. A similar response was recorded in three different orchid species where transplants exhibited 80-85% survival in natural conditions when plantlets were passed through pre acclimatization phase of nearly 3 months (Deb and Imchen 2010). Different potting substrates have been employed for hardening of in vitro raised plants by various workers like soilrite for Carica papaya (Agnihotri et al. 2004), soaked cotton for Saccharum offinarum (Gill et al. 2004), cocopeat and sand for Garcinia indica (Chabukswar and Deodhar 2005) and charcoal chips, bricks and decayed wood or moss for epiphytic or terrestrial orchid plantlets (Deb and Imchen 2010). In the present study, out of various potting mixtures used for hardening, the highest survival percentage was found in T1 (92%), followed by T0 (88%), T2 (65%), T4 (59%) and T3 (61%) respectively. Howe ver poor growth of plants occurred on control (soil without any organic matter) as compared to other treatments. Use of biofertilizers along with basic potting mixture also exhibited better shoot growth and number of leaves formed per plant and reduced the mortality rate by 4-5 % on transplantation of the plants to the field. The present results are in accordance with the reports in banana where better response of biofertilizer treatment over other treatments was reported (Vasane and Kothari 2006). It is obviously due to the complementarity in the function of two biofertilizers, Azotobacter in N 2 fixation and Pseudomonas in phosphate solubilisation, both contributing to the growth promoting characteristics of biofertilizers. Although the inclusion of biofertilizers in the soil: vermicompost (T1) treatment gave the best results in terms of plant growth and percentage survival (92%), treatment T0 (soil: verm i-

5 compost) was also closer to it. Therefore, if we consider cost factor, T0 treatment is most cost effective and almost in par with the treatment T1 showing 88% survival of regenerated plants in the field. After 45 days of hardening, the plants were subsequently transplanted to earthen pots containing only soil: vermicompost and 87 were transferred to open field conditions (Figure 3). The acclimatized plants showed welldeveloped shoot and root systems with large number of secondary and tertiary branches and all the plants are thriving very well in field conditions. Figure 3: Transfer of plants to earthen pots in open outdoor. References Agnihotri, S., Singh, S.K., Jain, M., Sharma, M., Sharma, A.K., Chaturvedi, H.C In vitro cloning of female and male Carcica papaya through tips of shoots and inflorescence. Indian Journal of Biotechnology, 3: Chabukswar, M.M., Deodhar, A. Manjushri Rooting and hardening of in vitro plantlets of Garcinia indica Chois. Indian Journal of Biotechnology, 4: Chandrasekhar, T., Mohammad, H.T., Gopal, G.R., Srinivasa, Rao, J.V Somatic Embryogenesis of Tylophora indica (Burm.f.) Merril, an Important Medicinal Plant. International Journal of Applied Science and Engineering, 4 (1): Chaudhari, K.N., Ghosh, B., Jha, S The root: a potential new source of competent cells for high frequency regeneration in Tylophora indica. Plant Cell reports, 22: Deb, C.R., Imchen, T An efficient in vitro hardening technique of tissue culture raised plants. Biotechnology, 9(1):

6 Faisal, M., Anis, M In vitro regeneration and plant establishment of Tylophora indica: Petiole callus culture. In vitro Cellular and Development Biology- Plant, 41: Gill, N.K., Gill, R., Goshal, S.S Factors enhancing somatic embryogenesis and plant regeneration in sugarcane ( Saccharum offinarum L.). Indian Journal of Biotechnology, 3: Gore, K.V., Rao, K., Guruswamy, M.N Physiological studies with Tylophora asthmatica in bronchial asthma. Indian Journal of Medical Research, 71: Govindachari, T.R., Lakshmikantham, M.V., Rajadurai, S Chemical exmaintion of Tylophora asthmatica- V. Structure of tylophorinine. Tetrahedron, 14: Jayanthi, M., Mandal, P.K Plant regeneration through somatic embryogenesis and RAPD analysis of regenerated plants in Tylophora indica (Burm. F. Merrill). In Vitro Cellular and Developmental Biology-Plant, 37: Mulchandani, N.B., Iyer, S.S., Badheka, L.P Structure of tylophorinidine. A new potential antitumor alkaloid from Tylophora indica. Chem India, 19: Murashige, T., Skoog, F A revised medium, for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15: Exoticnatural m. 88 Rani, S., Rana, J.S In vitro Propagation of Tylophora indica-influence of Explanting Season, Growth Regulator Synergy, Culture Passage and Planting Substrate. Journal of American Science, 6(12): Sahai, A., Shahzhad, A., Anis, M High frequency plant production via shoot organogenesis and somatic embryogenesis from callus in Tylophora indica. Turkish Journal of Botany, 34: Sharma, N., Chandel, K.P.S Effect of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. F.) Merrill. Plant cell Tiss. Organ Culture, 29: Shivpuri, D.N., Menon, M.P., Prakash, D Preliminary studies in Tylophora indica in the treatment of asthma and allergic rhinitis. Journal of Association of Physicians India, 16 (1): Thomas, D., Philip, B Thidiazuroninduced high-frequency shoot organogenesis from leaf-derived callus of a medicinal climber, Tylophora indica. In vitro Cellular and Development Biology Plant, 41: Vasane, R.S., Kothari, R.M Optimization of hardening process of banana plantlets (Musa paradisiaca L. var grand nain). Indian Journal of Biotechnology, 5: Verma, R.N., Jamal, S.M., Sharma, M.M., Rao, D.V., Amla, B Regulation of organogenesis using leaf, internode and petiole explants in Tylopohra indica (Burm f) Merill. International Journal of Pharmaceutical Science Review and Research, 5 (1).