Abstract: A protocol for mass propagation through axillary bud proliferation was established for Rauwolfia serpentina L. Benth. (Apocynaceae). MS medium supplemented with 1.5 mg L-1 BA and 0.2 mg L-1 NAA elicited the maximum number of shoots (4 multiple shoots) from nodal explants. These adventitious shoots were best rooted on half strength MS medium supplemented with 1.0 mg L-1 each of IBA and IAA. The in vitro raised plants were acclimatized in glass house and successfully transplanted to field condition with almost 95% survival.
INTRODUCTION
Rauwolfia serpentina L. Benth. is a medicinally important perennial herb belongs to the family Apocynaceae. The plant is indigenous to Bangladesh, India and other tropical region of the world (Roy et al., 1994). R. serpentina contains some 50 indole alkaloids and most of the total alkaloid content present mainly in root bark (Klyushnichenko et al., 1995). Among all the alkaloids reserpine, yohimbine, serpentine, deserpidine, ajmalicine, ajmaline, etc. are used to treat hypertension (Von Poser et al., 1990) and breast cancer (Stanford et al., 1986). Roots are used in traditional medicine as a valuable remedy for many complex diseases e.g., high blood pressure, insomnia, anxiety, excitement, schizophrenia, insanity, epilepsy, hypochondria and other disorders of the central nervous system (Bhatara et al., 1997; Kirtikar and Basu, 1993; Dastur, 1988). The root extract of this plant is also used to hasten the expulsion of the fetus, to treat painful affection of bowels, diarrhoea (Tona et al., 1999), dysentery, cholera and colic (Ghani, 1998).
For centuries, root of R. serpentina has been used in the traditional Unani and Ayurvedic medicine (Andrew and Chevallier, 1996). But during the middle of 20th century the importance of its major alkaloid reserpine has attracted much attention in the field of allopathic medicine as a remedy for hypertension (Von Poser et al., 1990; Vakil, 1949), insomnia and schizophrenia (Bhatara and Gupta, 1997; Bleuler and Stoll, 1955).
Mass scale collection of this plant from natural habitat by the pharmaceutical industries as well as local ayurvedic and unani practitioners is leading to a depletion of this plant resource. Propagation by means of seeds to replenish the exhausting supply might prove ultimately even unwise, since of its poor seed viability and very low germination percentages (25-50%) that may be ascribed largely to the presence of cinamic acid derivatives in the seeds (Mitra, 1976). However, alkaloid content might get reduced in successive progenies through adverse gene recombination (Anonymous, 1950).
In vitro propagation studies of different plant species have shown that the technique may be a solution for rapid propagation of such selected useful plant species and subsequent exploitation (Bonga and Durjan, 1987). It also has been found that explant of an alkaloid producing plant, cultured in vitro retain the capacity to synthesis alkaloids to that in the intact plant (Sarker et al., 1996). On the other hand in vitro micropropagation has a number of advantages over sexual propagation (Abbott, 1978). In sexual method superior genotypes may lost through recombination but micropropagation can preserve superior gene combinations practically unaltered. In vitro propagation of R. serpentine has been reported by many researchers as Ahmad et al. (2002), Sarker et al. (1996), Roy et al. (1994) and Mathur et al. (1993). The special purpose of current study was to identify most suitable media supporting competence necessary for large-scale propagation scheme to replenish the exhausting supply and to conserve the threatened species.
MATERIALS AND METHODS
The experiment was carried out in the Plant Tissue Culture Laboratory, Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhanmondi, Dhaka, Bangladesh in the year 2007. Nodal segments containing buds of 3-4 years old plants were collected from the medicinal plant garden of BCSIR.
For surface sterilization, explants were cut into small pieces and cleaned thoroughly under a continuous stream of running tap water for 30 min. After that the segments were washed with detergent and kept under running tap water for 15 min. Then treated with 1% Savlon and washed in running tap water for 30 min. The explants were then taken under laminar airflow cabinet and surface sterilized with a 0.1% HgCl2 for 5 min followed by their washing three times with autoclaved double distill water. Explants of approximately 1 to 2 cm in length were cut and inoculated aseptically onto MS (Murashige and Skoog, 1962) media supplemented with different concentrations and combinations of auxin and cytokinine. The pH of the medium were adjusted to 5.8±0.05 before adding agar and the media were autoclaved at 1.1 kg cm-1 for 20 min at 121°C. Cultures were incubated at 25±1°C with a photoperiod of 16 h at 3000 lux light intensity of cool white fluorescent light.
All cultures were initiated in 150x25 mm2 glass tube containing
15-20 mL of medium. The cultures were regularly subcultured on fresh medium
at 4 weeks intervals in glass tubes or 100 mL flasks. Observations were
recorded every 5 days following inoculation and subculturing. For inducing
adventitious shoots and their development, nodal segments containing buds
were cultured on MS medium supplement with 0.5-2.0 mg L-1 BAP
with NAA (0.2, 0.5) mg L-1. Auxiliary shoots produced within
four weeks of culture were isolated and regularly subcultured on fresh
medium at four-week intervals. In rooting experiments proliferated shoot
of approximately 2-4 cm length were rescued aseptically from culture vessels
and transferred to freshly prepared half strength MS macro and micro-nutrients
contained in glass tube. IBA and IAA (0.1-1.5) mg L-1 were
used as supplementary with the media for root induction. Culture tubes
containing rooted plantlets were kept in a room of normal temperature
(30±2°C) and normal daylight for 7 days. Plantlets were then
taken out from the culture tubes and washed carefully under running tap
water for complete removal of media. Then the plantlets were transplanted
to small plastic pots containing garden soil and compost in a ratio of
2:1. The pots were immediately covered with polythene bag to prevent desiccation.
After the hardening period the plantlets were transferred to field condition
(Fig. 1).
| Fig. 1: | Mass Propagation of Rauwolfia serpentina from shoot node derived culture: (a) Formation of Multiple Shoots in test tube on MS media supplemented with BA 1.5 mg L-1 + 0.5 mg L-1. (b) Shoot multiplication in flask on media same as Fig. (a). (c) Development of adventitious roots on half strength MS medium supplemented with IAA 1.0 mg L-1 + IBA 1.0 mg L-1. (d) Transplantation of plantlets into plastic pots |
Observations were recorded every week following inoculation and subculturing. All experiments were repeated twice with at least 18 cultures per treatment and data were taken after 4-6 weeks of culture.
RESULTS AND DISCUSSION
Shoot induction was observed on all the used concentrations and combinations of BA and NAA. MS supplemented with very low level of BA with NAA exhibited low percentage of shoot formation and the response for average shoot length was also very poor. Multiple shoots emerged from the nodal explants within two weeks of incubation. Among different concentrations of growth hormone tested, 1.5 mg L-1 BA and 0.2 mg L-1 NAA induced 90% shoot formation and elicited the maximum number of shoots (4 multiple shoots) from nodal explants (Table 1). These shoots attained a height of 4-6 cm within four weeks of additional culture. There were significant differences in shoot formation frequencies, number of shoots per culture and length of shoots per culture.
Rooting experiments were conducted in half-strength MS supplemented with IAA and IBA. Root induction was found to be more prominent in the medium containing 1 mg L-1 each of IAA and IBA resulted in 80% root initiation (Table 2). Roots elongated up to 4-5 cm within 15 days of incubation period.
The rooted plantlets were successfully transferred to hardening and well established in field condition (Fig. 1). The survival rate was 95% and plant showed normal growth with similar phenotype of mother plants.
Multiple shoot formation from nodal segment was reported using higher concentration of growth hormone (Verma et al., 2002; Selvakumar et al., 2001; Sudha and Seeni, 1996). The present study exemplifies a positive modification of shoot induction and multiplication efficacy on MS basal media supplemented with very low concentrations of auxin with cytokinin.
Although Kn is reported to promote shoot bud initiation in nodal explants
of many plant species, BA is the most efficient cytokinin for the axillary
bud initiation and subsequent proliferation (Hamdy and Hattori, 2006;
He et al., 2005; Baskaran and Jayabalan, 2005; Gupta et al.,
2001). Addition of auxin at low concentration significantly enhanced the
growth of culture and elongation of proliferated shoots (Hu and Wang,
1983). Manipulation of cytokinin (BA) and auxin (NAA) concentration was
found to be very important for effective mass scale propagation. Excision
and culture of
| Table 1: | Effect of different concentrations and combinations of BA and NAA on shoot proliferation |
| Table 2: | Effect of different concentrations of IBA and IAA in half strength MS medium on root formation in regenerated shoots the nodal segments from in vitro derived shoots facilitated the development of increased number of shoots. |
The effectiveness of half-strength MS basal medium supplemented with auxins on root induction has been reported in many medicinal plants (Ahamed et al., 2005; Mederos-Molina, 2004; Huda et al., 2003). Root induction was reported on half strength MS supplemented with IAA and IBA alone (Ahamed et al., 2005; Vesperinas, 1998) with an afficacy around 73%. But this study shows a higher root induction efficacy of 80% on half strength MS supplemented with IAA and IBA combination. The root lengths were varied in all media concentrations.
The ultimate success of in vitro propagation lies in successful establishment of plants in the soil. 80-85% survival of plantlets were recorded in the experiments of previous workers (Sudha and Seeni, 1996; Roy et al., 1994). The high survival rate in the present study indicates that this procedure could be easily adapted for large-scale propagation. As R. serpentina holds the tremendous potentialities for massive propagation towards the commercialization of many invaluable indole alkaloids this present study will be fruitful for this novel application and will help in the conservation study of such threatened species.