An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf ...
Plant Cell, Tissue and Organ Culture 75: 125–129, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Rapid mass propagation of Tylophora indica Merrill via leaf callus culture Mohd. Faisal & Mohammad Anis* Plant Tissue Culture Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202 002, India ( * requests for offprints; Fax: ⫹91 -571 -400 -528; E-mail: anism1@ rediffmail.com) Received 4 June 2002; accepted in revised form 14 March 2003
Key words: callus culture, endangered, mass propagation, Tylophora indica
Abstract An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 M 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 M Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 M IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot development in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant. Abbreviations: BA – 6-benzyladenine; 2,4-D – 2,4-dichlorophenoxy acetic acid; IAA – indole-3-acetic acid; IBA – indole-3-butyric acid; Kinetin – 6-furfurylaminopurine; MS – Murashige and Skoog medium; NAA – ␣ -naphthalene acetic acid; 2,4,5-T – 2,4,5-trichlorophenoxy acetic acid
Introduction Tylophora indica (Burm.f.) Merrill (Asclepiadaceae) is an endangered medicinal plant commonly known as Antamul, distributed in Assam, West Bengal and Peninsular India, ascending to an altitude of 900 m (Anonymous, 1976).The pharmacological activity of this plant is mainly due to the presence of alkaloid tylophorine and tylophorenine. The leaves and roots of the plant have long been used for the treatment of asthma, bronchitis, whooping cough, dysentery and diarrhoea. They are also effective in rheumatic and gouty pains and hydrophobia. Due to large scale and indiscriminate collection of wild plants from forest and insufficient attempts to either allow its replenishment or its cultivation, T. indica is rapidly disappearing and is now listed as one of the plant species in India vulnerable to extinction. Therefore, it is necessary to devise a method for the
development of a potentially large-scale multiplication protocol for commercial production of this endangered species. In vitro propagation methods offers powerful tools for germplasm maintenance and multiplication. Tissue culture studies on T. indica have largely been restricted to monitor the alkaloid synthesis in callus and protoplast culture and regenerated plants (Rao et al., 1970; Rao and Narayanaswamy, 1972; Benzamine et al., 1979; Mhatre et al., 1984). Micropropagation of T. indica was reported using axillary buds, which yielded a very low number of shoots (Sharma and Chandel, 1992). These methods can not be useful for the large scale production and cultivation of elite clones. It has been shown that shoot organogenesis via callus culture can be used as an effective method for multiplication of medicinal plants (Sarasan et al., 1994; Lusia and Rojas, 1996; Ahroni et al., 1997; Reddy et al., 2001). In the present
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Figure 1. Induction of callus, shoot regeneration and complete plant establishment from leaf segments of Tylophora indica. (a) Callus induction from leaf segments on MS⫹2,4,5-T (10 M). (b) Shoot buds differentiation from organogenic callus on MS⫹Kinetin (5.0 M). (c) Multiplication and elongation of shoots on MS⫹Kinetin (5.0 M). (d) Rooting of in vitro regenerated shoot on 1 / 2 MS⫹IBA (0.5 M). (e) A plant acclimatized to greenhouse conditions.
127 study, we have investigated callus mediated shoot organogenesis as an alternative method to achieve a higher rate of shoot multiplication.
ferentiated shoots were recorded after 4 weeks of inoculation. Root formation
Materials and methods Plant material and explant source The first fully expanded and second leaf over 1 cm long from apical buds were collected from plants grown at the Botany Department, Aligarh Muslim University, washed with tap water for at least 30 min followed by soaking in 5% (v / v) teepol for 5 min. The leaves were disinfected by immersing in 0.1% (w / v) HgCl 2 for 3 min. After four rinses in sterile distilled water, leaves were trimmed into pieces of about 1 cm 2 and then inoculated onto culture medium. Media and culture conditions Murashige and Skoog (1962) medium containing 3% (w / v) sucrose was used in all experiments. The pH of media was adjusted to 5.8 prior to the addition of 0.8% (w / v) agar and autoclaving at 1.06 kg cm ⫺2 for 20 min. All cultures were maintained at 25⫾2 ⬚C with 16-h light / 8-h dark photoperiods (light intensity 50 mol m 2 s ⫺1 ) Callus induction Various concentration of 2,4-D (0.5–10 M) and 2,4,5-T (0.5–10 M) were tested for their effect on callus formation from leaf explant. MS medium lacking growth regulators served as control. Leaf segments were inoculated on slanted media and data on callus was recorded as the percent of the leaf explant forming callus. Ten replicates were used per treatment and the experiment was repeated thrice. Shoot differentiation The light yellow friable callus was transferred onto MS medium supplemented with either Kinetin or BA at four concentration level 0.5, 2.5, 5 and 10 M. To evaluate the effect of cytokinin / auxin, the callus was also cultured on MS medium supplemented with Kinetin (5 M), BA (5 M) with NAA (0.5–2.5 M). All the cultures were transferred to fresh medium every 2–3 week. Data on percentage of calluses forming shoots, number and length of dif-
In vitro differentiated shoots measuring 3–4 cm in length were excised from leaf derived callus and cultured on MS basal medium supplemented with IBA (0.5–5 M). The shootlets were also cultured on auxin-free full and half strength MS medium. Data were recorded on percentage of rooting, mean number and length of the roots, 4 weeks after transferring onto rooting media. Acclimatization The regenerated plantlets with well developed shoots and roots were transferred to pots containing sterile vermiculite under diffuse light (16 / 8-h photoperiod) conditions. Potted plants were covered with transparent polythene membrane to ensure high humidity, and watered every 3 days with half strength MS salt solution for 2 weeks. Polythene membranes were opened after 2 weeks in order to acclimatize plants to field conditions. Plants were transferred to pots containing garden soil and watered with tap water. Data analysis All the experiments were repeated thrice. The effect of different treatments was quantified and the data was analyzed using one-way analysis of variance (ANOVA), and means were compared using the Tukey test at the 0.05 level of significance.
Results and discussion Leaf segments were cultured on MS medium supplemented with 2,4-D or 2,4,5-T. Within 4 weeks of inoculation, callus was proliferated from cut edge of the explants. Morphology and growth of the callus varied with different level of 2,4-D and 2,4,5-T. On lower concentration of 2,4-D (0.5 M), 73.5% cultures formed callus. When 2,4-D concentration was increased up to 10 M, almost all the cultures showed callusing. The leaf explant produced highly proliferating light yellow friable callus in the medium containing 2,4,5-T (Figure 1a). On increasing the concentration of 2,4,5-T from 0.5 to 10 M, a gradual
128 Table 1. Effect of auxin on callus induction from leaf explants in MS medium after 4 weeks of culture Auxin
Concentration ( M)
2,4-D
0.5 2.5 5.0 10
73.5 85.0 92.5 96.0
2,4,5-T
0.5 2.5 5.0 10
87.0 90.0 95.0 100
increase in percentage of cultures forming callus was noted (85.6–100%) (Table 1). The light yellow friable callus was sub-cultured on MS medium supplemented with either Kinetin or BA alone and adventitious shoot buds differentiated from the surface of the callus within 4 weeks of culture (Figure 1b). Formation of leaves and shoot elongation occurred within 6 weeks of culture (Figure 1c). Among various concentration of Kinetin tested, the highest shoot regeneration frequency (85%) and highest number of shoots (64.8⫾0.74) were recorded at 5 M concentration (Table 2). Increasing the concentration of Kinetin from 5 to 10 M, resulted in a decrease in the rate of shoot regeneration ability. It is in contrast to the earlier reports (Sharma et al., 1991; Reddy et al., 2001) on Coleus forskholii where the
% Response
higher concentration enhanced the shoot multiplication. The callus when sub-cultured onto the MS medium containing different concentrations of BA revealed that highest shoot regeneration frequency (70%) and number (44.5⫾2.41) per callus clump were obtained at a concentration of 5 M BA (Table 2). However, the regenerants were stunted and had short internodes where as shoots regenerated from Kinetin supplemented medium showed better growth and elongation. The addition of NAA with optimal concentration of Kinetin or BA significantly reduced the frequency of shoots formation (Table 3), which is in contrary to the earlier reports where NAA promotes the proliferation and elongation of shoots in Petasites hybridus (Wldi
Table 2. Effect of cytokinin on shoot regeneration from leaf derived callus of Tylophora indica in MS medium after 6 weeks of culture Treatments
% Regeneration
Mean no. of shoots / callus
Mean shoot length (cm)
Kinetin (0.5 M) Kinetin (2.5 M) Kinetin (5.0 M) Kinetin (10 M) BA (0.5 M) BA (2.5 M) BA (5.0 M) BA (10 M)
17 65 85 55 14 35 70 30
25.0⫾0.70 e 37.4⫾1.32 c 64.8⫾0.74 a 20.8⫾1.05 de 18.0⫾1.41 e 24.0⫾1.21 d 44.5⫾2.41 b 17.4⫾0.83 e
2.46⫾0.05 d 3.58⫾0.06 bc 4.50⫾0.12 a 3.28⫾0.09 c 1.74⫾0.14 e 3.32⫾0.20 c 3.92⫾0.21 b 2.62⫾0.06 d
Values represent means⫾standard error of 10 replicates per treatment in three repeated experiments. Means followed by the same letter not significantly different by the Tukey test at 0.05% probability level. Table 3. Effect of NAA with optimal concentration of Kinetin / BA on shoot regeneration from leaf derived callus of Tylophora indica in MS medium after 6 weeks of culture Treatments
% Regeneration
Mean no. of shoots / callus
Mean shoot length (cm)
Kinetin (5 M)⫹NAA (0.5 M) Kinetin (5 M)⫹NAA (2.5 M) BA (5 M)⫹NAA (0.5 M) BA (5 M)⫹NAA (2.5 M)
50 25 45 10
26.0⫾1.41 a 10.6⫾1.20 c 16.0⫾3.50 b 6.40⫾1.35 c
3.40⫾0.14 a 2.48⫾0.11 b 2.82⫾0.13 ab 2.14⫾0.21 c
Values represent means⫾standard error of 10 replicates per treatment in three repeated experiments. Means followed by the same letter not significantly different by the Tukey test at 0.05% probability level.
129 Table 4. Effect of MS-strength and IBA concentration on root induction from in vitro raised shoots of Tylophora indica after 4 weeks of culture Treatments
% Rooting
Mean no. of roots / shoot
Mean root length (cm)
MS 1 / 2 MS 1 / 2 MS⫹IBA (0.5 M) 1 / 2 MS⫹IBA (2.5 M) 1 / 2 MS⫹IBA (5.0 M)
50 75 90 70 30
1.80⫾0.25 c 2.76⫾0.43 b a 4.30⫾0.47 2.06⫾0.51 c 1.80⫾0.26 c
2.34⫾0.16 c 3.46⫾0.37 a a 3.85⫾0.17 3.04⫾0.16 b 2.57⫾0.50 c
Values represent means⫾standard error of 10 replicates per treatment in three repeated experiments. Means followed by the same letter not significantly different by the Tukey test at 0.05% probability level.
et al., 1998), Eucalyptus grandis (Luis et al., 1999), Hybanthus enneaspermus (Prakash et al., 1999). The regenerated shoots from leaf derived callus were transferred to different rooting media and 10 days after inoculation root formation was induced from basal cut portion of the shoot (Figure 1d). The shoots were transferred onto auxin-free MS medium which produced 1 or 2 thin roots. Reduction of MSsalt solution to half strength enhanced root formation in shootlets. The incidence of root formation in auxin free medium may be due to the presence of higher quantity of endogenous auxin in in vitro shootlets. 1 / 2 MS medium supplemented with 0.5–5 M IBA induced higher frequency of rooting (Table 4). The maximum frequency of root formation was achieved on 1 / 2 MS medium supplemented with 0.5 M IBA. Success of IBA for efficient root induction is also reported in Swaisona formosa (Jusaitis, 1997) and Cunila galoides (Fracro and Echeverrigaray, 2001). The regenerated plantlets with well developed shoots and roots were transferred to pots containing sterile vermiculite for hardening at diffuse light (16 / 8-h photoperiod) conditions. The potted plantlets were covered with polythene membrane to ensure high humidity and watered every 3 days with half strength MS-salt solution free of sucrose. After 1 month they were transferred to green house where all plants grew normally (Figure 1e). To conclude, the present communication presents a rapid method for large scale mass propagation of Tylophora indica through a dedifferentiated callus state. The protocol described is an efficient and could be used as a means of propagation and multiplication of Tylophora indica – a potential medicinal plant for commercial exploitation.
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