A successful procedure was established for in vitro plant regeneration from callus derived from stem and leaf explants of Centella asiatica on semisolid modified ...
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Plant Growth Regulation 24: 13–16, 1998. c 1998 Kluwer Academic Publishers. Printed in the Netherlands.
Successful plant regeneration from callus cultures of Centella asiatica (Linn.) Urban A. Patra1 2 , B. Rai2 , G.R. Rout1 & P. Das1 ;
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Plant Biotechnology Division, Plant Tissue Culture Laboratory, Regional Plant Resource Centre, Bhubaneswar-751015, India; 2 Biochemistry Laboratory, Pt. Ravishankar University, Raipur, Madhya Pradesh, India (*author for correspondence) Received 13 June 1997; accepted in revised form 22 September 1997
Key words: growth regulators, In vitro, medicinal plant, shoot-bud
Abstract A successful procedure was established for in vitro plant regeneration from callus derived from stem and leaf explants of Centella asiatica on semisolid modified Murashige and Skoog’s [7] medium supplemented with 2.0 mg L 1 kinetin and 4.0 mg L 1 a-naphthaleneacetic acid. The rate of shoot-bud regeneration was the highest (42.8 and 54.3 shoots/culture in stem and leaf derived callus respectively) after 4 weeks of subculture on 4.0 mg L 1 6-benzyladenine, 2.0 mg L 1 Kn, 0.25 mg L 1 a-naphthaleneacetic acid and 20 mg L 1 adenine sulfate. Differentiated shoots rooted within 11 days in 1/2 strength MS basal salts supplemented with 0.5 mg L 1 indole3-acetic acid and 2% (w/v) sucrose. About 85% of rooted plantlets were acclimatized and transferred to the greenhouse. Abbreviations: BA = 6-benzyladenine; Kn = kinetin; Ads = Adenine sulfate; NAA = a-naphthaleneacetic acid; IBA = indole-3-butyric-acid; 2,4-D = 2,4-dichlorophenoxyacetic acid; MS medium = Murashige and Skoog’s [7] medium.
1. Introduction The genus Centella (Umbelliferae) comprised of about 33 species of herbs is widely found in tropical and sub-tropical regions. Centella asiatica, an important medicinal plant is distributed from the Himalayas to Ceylon up to an altitude of 1800 m above MSL [3]. It is used in the treatment of various skin diseases, ulceration, chronic rheumatism, leprosy, malaria, fever, epilepsy and enlargement of glands. It also acts as an alternative tonic when given internally and stimulant when applied externally [1]. Pharmaceutical companies largely depend upon material procured from naturally occurring stands which are being depleted rapidly raising concern about possible extinction of the species and providing justification for the development of in vitro propagation techniques for this crop. In this study, we report an efficient protocol for plant regeneration
from callus derived from stem and leaf explants of Centella asiatica (Linn) Urban. 2. Materials and methods 2.1 Plant material Shoots (5–6 cm) were collected from greenhouse grown plants of Centella asiatica and brought to the laboratory with cut ends dipped in in distilled water. Leaves were separated from the stems; both the leaf and stem were washed in 2% (v/v) (Teepol; Qualigen, India) detergent solution, and surface sterilized in a 0.1% (w/v) aqueous mercuric chloride for 20 min. After rinsing 4–5 times with sterile distilled water, juvenile leaves and stem internodes were cut into smaller segments (0.3 cm 0.4 cm and 0.5 cm respectively) were used as the explants.
14 2.2 Culture medium and culture conditions Leaf (0.3 cm 4.0 cm) and stem (0.5 cm) explants were placed on semi-solid basal MS [7] medium supplemented with different concentrations and combinations of BA (0.0–5.0 mg L 1 ), Kn (0.0–5.0 mg L 1 ), NAA (0.0–5.0 mg L 1 ) and 2,4-D (0.0–5.0 mg L 1 ) for callusing. The pH of the media was adjusted to 5.8 using 0.1N HCI or 0.1N NaOH before autoclaving. Routinely, 25 mL of liquid medium with 0.8% (w/v) agar (Qualigen, India) was dispensed into culture tubes (25 150 mm) and plugged with non-absorbent cotton wrapped in one layer of cheese-cloth. The cultures were sterilized at 121 C and 104 kPa for 15 min and maintained by regular subculturing at 4-week intervals on fresh medium with the same compositions. To induce shoot buds, the 8 weeks old proliferating friable callus (200 20 mg) derived from both stem and leaf explants were transferred to various regeneration media containing BA (0.0–5.0 mg L 1 ), Kn (0.0– 5.0 mg L 1 ), NAA (0.0–5.0 mg L 1 ), 2,4-D (0.0–5.0 mg L 1 ) and Ads (5.0–30.0 mg L 1 ). The different regeneration media were: R1-MS + 3% sucrose R2-MS + BA 2.0 mg L 1 + Kn 1.0 mg L 1 + 3% sucrose R3-MS + BA 2.0 mg L 1 + Kn 2.0 mg L 1 + 3% sucrose R4-MS + BA 3.0 mg L 1 + Kn 2.0 mg L 1 + NAA 0.25 mg L 1 + Ads 20 mg L 1 + 3% sucrose R5-MS + BA 4.0 mg L 1 + Kn 2.0 mg L 1 + NAA 0.25 mg L 1 + Ads 20 mg L 1 + 3% sucrose R6-MS + BA 5.0 mg L 1 + Kn 2.0 mg L 1 + NAA 0.25 mg L 1 + Ads 20 mg L 1 + 3% sucrose R7-MS + BA 4.0 mg L 1 + Kn 1.5 mg L 1 + NAA 0.5 mg L 1 + 3% sucrose All the cultures were incubated under 16 h photoperiod in cool, white fluorescent light (55 mol m 2 s 1 ) at 25 2 C. 2.3 Induction of rooting and acclimatization Excised shoots (1–2 cm) regenerated from the callus were cultured on rooting medium containing halfstrength basal MS semi-solid medium without vitamins and myo-inositol either alone or in combination with different concentrations of IBA or NAA (0.1, 0.25, 0.5 and 1.0 mg L 1 ) and 2% (w/v) sucrose for root initiation. One excised shoot was cultured in each tube (25 150 mm) having 15 ml of culture media.
All the cultures were incubated at 25 2 C under 16 h photoperiod with cool white fluorescent lamps (Philips, India). Rooted micropropagules were thoroughly washed to remove the adhering gel and planted in 2.5 cm earthen pots containing a mixture of soil, sand and well rooted cow dung manure in the ratio of 1:1:1 (w/v) and kept in a greenhouse maintaining at temperature 25–30 C and 60–70% RH for acclimatization. 2.4 Observations of cultures and presentation of results Each treatment had 20 replications and each experiment was repeated three times. The data pertaining to mean percentage of cultures regenerated, mean number of shoot buds/culture were statistically analysed by the Post-Hoc Multiple Comparison test at the level of P 0.05 [5]. 3. Results and discussion 3.1 Effect of growth regulators on callus induction Leaf and stem explants enlarged and developed callus at the cut surfaces in all media within 10–12d of inoculation and this subsequently covered the entire surface of the explants. There was no sign of callus formation when explants were cultured in media without auxin or cytokinin. Globular and greenish calli developed on the surface of the explants 3–4 weeks after culture initiation on MS basal medium supplemented with varying concentrations of BA or Kn with NAA (Table 1). The medium containing 2,4-D (3.0 mg L 1 ) and BA or Kn promoted rapid callus growth from both leaf and stem explants which subsequently turned brown within 4weeks of culture. Most of the media that induced rapid callus growth included NAA in combination with Kn; both leaf and stem explants produced the best callus growth in the medium containing 2.0 mg L 1 Kn and 3.0–4.0 mg L 1 NAA. The appearance of the calli varied with both the explant type and the medium but the responses were similar. The callus derived from leaf explants was invariably greenerand more granular than the stem-derived callus on the medium containing 2 mg L 1 Kn with 4.0 mg L 1 NAA. Higher concentration of NAA induced compact calli which was not desirable. On media containing BA in combination with NAA did not induce any significant callus growth. Of the two explant types used, callus was formed on the
15 Table 1. Effect of various media on shoot bud regeneration in calli derived from stem and leaf explants of Centella asiatica after 4 weeks of subculture. A – Mean percentage of cultures regenerated; B – Average number of shoot buds/culture MS medium + 3% sucrose + additives
R1 R2 R3 R4 R5 R6 R7
Stem A*
Source of callus Leaf B* A*
B*
0 13.6a 18.7b 54.3d 62.8f 42.8c 58.4e
0 8.3a 13.6b 38.5e 42.8f 18.7c 30.6d
0 14.2a 18.4b,c 16.8b 54.3e 24.8c 42.6d
0 16.8a 24.5b 61.4d 73.4e 43.6c 61.3d
* Means having the same letter in a column were not significantly different by Post-Hoc Multiple Comparison test P 0.05 level.
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leaf explant early and this callus proliferate very fast. Stem explants produced callus at a relatively slow rate. 3.2 Differentiation of shoots from callus After 8 weeks on callus-induction medium (MS + 2.0 mg L 1 Kn + 4.0 mg L 1 NAA), the calli were subcultured onto media containing different concentrations of BA, Kn and NAA for shoot regeneration. Green nodular structures which developed into dark green shoots were evident over the entire surface when calli were cultured in medium supplemented with BA (4.0 mg L 1 ), Kn (2 mg L 1 ), NAA (0.25 mg L 1 ) and adenine sulphate (20 mg L 1 ) after 4 weeks and 6 weeks of culture. Shoot-bud regeneration was better when calli were derived from leaves (73.4%) as compared to stems (62.8%). Low frequency regeneration of shoot buds also took place at low concentration of BA (1.0–2.0 mg L 1 ) (data not shown). Similar results have been reported for root explants of Digitalis obscura [9] and petiole explants of Valeriana wallichi [6]. The frequency of regenerated shoots/culture varied from 8.3–42.8% in stem and 14.2–54.3% in leaf derived calli respectively (Table 1). The differential response could be due to the varying concentrations of the growth regulators used in the medium and the explant types [2, 3, 6, 10]. The rate of regeneration of shoot buds per culture was stable upto fifth to sixth subculture which subsequently declined (data not shown). Calli derived from both leaf and stem explants were successfully maintained over eight months.
Table 2. Effect of IBA and NAA on rooting of excised shoots of Centella asiatica on 1/2 MS + 2% sucrose MS + Growth regulators (mg L NAA IBA
0 0.1 0.25 0.50 1.0 0 0 0 0
0 0 0 0 0 0.1 0.25 0.5 1.0
1)
% of shoots rooted s.e.*
Average number of roots/shoot s.e.*
Days to rooting
0 1.18 1.82 2.76 2.38 1.06 3.73 4.34 1.26
0 14 14–15 15a 17a 14 13 11 16a
0 20.4 41.8 62.6 52.8 24.6 76.4 92.2 47.4
0.4 0.7 0.6 0.8 0.4 0.3 0.6 0.8
0.5 0.6 0.4 0.3 0.2 0.4 0.2 0.4
* Data represent mean of 20 replicates/treatment in three repeated experiments. a – basal callusing at the cut end.
3.3 Root development in regenerated plants Leafy shoots regenerated from both stem and leaf callus were separated and transferred to the rooting medium to form complete plantlets. MS medium without growth regulators did not promote root induction; roots were formed on excised shoots grown in MS media containing either IBA (0.1–1.0 mg L 1 ) or NAA (0.1–1.0 mg L 1 ) with 2% (w/v) sucrose (Table 2). A high percentage of shoots (92%) rooted in the medium containing half strength MS basal salts with 0.5 mg L 1 IBA + 2% (w/v) sucrose. Root initials formed with 11d and developed into a good root system in 15d (Figure 1e). Root initiation was achieved at a higher concentration of NAA (1.0 mg L 1 ) but with intervening callus at the cut end of the shoots. The roots were thin and varied in number from 1.06 to 4.34 per shoot (Table 2). Root development was, however, slow at lower concentration of NAA (data not shown). 3.4 Acclimatization of rooted plants Rooted plantlets were transferred into pots containing soil:sand:well rooted cowdung manure in the ratio of 1:1:1. The plants were supplied with one-fourth strength MS inorganic solution two times, once a week before transfer to the greenhouse. The percentage of survival of the plantlets were 55–65%. The plants grew normally. In conclusion, an efficient protocol was developed for successful in vitro shoot bud regeneration for an
16 important medicinal plant Centella asiatica. These in vitro technique may help in the conservation of the species and possibly lead to the synthesis and extraction of active compounds from callus sources.
Acknowledgement The authors wish to acknowledge the help of the Department of Forest and Environment, Govt. of Orissa for providing necessary facilities.
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