Abstract: The influence of media composition on callus induction and subsequent regeneration of Rauwolfia serpentina L. Benth has been studied. High frequency (96.43%) callus induction was obtained when nodal segments from in vitro raised shoots were cultured on MS medium supplemented with 0.5 mg L-1 BA and 2.0 mg L-1 NAA. The callus differentiated into adventitious shoots when it was subcultured on MS medium supplemented with 2.0 mg L-1 BA with 0.2 mg L-1 NAA. Regenerated shoots were best rooted on half-strength MS medium with 1.0 mg L-1 each of IBA and IAA.
INTRODUCTION
An endangered woody shrub Rauwolfia serpentina L. Benth belongs to the Apocynaceae family holds the tremendous potentialities for massive propagation and isolation of indole alkaloids through in vitro culture. Alkaloids of this plant have a great medicinal importance to treat cardiovascular diseases (Anitha and Kumari, 2006), hypertension (Von Poser et al., 1990), arrhythmia (Kirillova et al., 2001), various psychiatric diseases (Bhatara et al., 1997; Kirtikar and Basu, 1993), breast cancer (Stanford et al., 1986), human promyelocytic leukemia (Itoh et al., 2005) like complicated diseases.
Explant of an alkaloid producing plant, cultured in vitro, has been found to retain the capacity to synthesis alkaloids identical to that in the intact plant (Yoshimatsu and Shimomura, 1991). Sometimes, high yield of secondary metabolites is observed in tissue grown as callus masses produced during differentiation (Benavides and Caso, 1993; Maheshwari et al., 2007). Beside this callus culture facilitates optimization of alkaloids production (Yamamoto and Yamada, 1986; Premjet et al., 2002; Anitha and Kumari, 2006) and subsequent isolation (Kirillova et al., 2001).
As propagation by means of seeds is very much difficult due to low germination percentage (Salma et al., 2008) and still now traditional Unani and Ayurvedic practitioners are dependent on crude extract of roots of this plants; in vitro propagation scheme is utmost necessary to keep pace with its demand.
We here reported the influence of media composition on effective callus induction to facilitate modern approaches of alkaloids production as well as regeneration for the conservation of the threatened genotype.
MATERIALS AND METHODS
The experiment was carried out in the year 2007 in the Plant Tissue Culture Laboratory, Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhanmondi, Dhaka, Bangladesh.
Nodal explants of R. serpentina from actively field grown plants were cleaned thoroughly under a continuous stream of running tap water for 30 min. After that the explants 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. Explants were surface sterilized with 50% (v/v) ethanol (1 min) and followed by 0.1% (w/v) HgCl2 (3 min). Finally the explants were washed thoroughly three times with autoclaved double distilled water.
The nodal explants were then cut into appropriate size (6-8 mm) and cultured on MS (Murashige and Skoog, 1962) medium containing 1.5 mg L-1 BA and 0.2 mg L-1 NAA. Nodal pieces (6-8 mm) taken from 6-week-old in vitro shoots were used to initiate callus culture. In this study we report the influence of plant growth regulators such as BA and NAA for effective callus and shoot induction. Regenerated shoots were rooted on half strength MS medium with IBA and IAA.
Media were adjusted to pH 5.8 before addition of 0.6% agar and autoclaved at 121 °C for 15 min. All cultures were incubated at 25 ± 1 °C with a photoperiod of 16 h at 3000 lux light intensity of cool white fluorescent light.
Weekly visual observation of culture was made. 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
Nodal segments from 3-4 years old field grown plants produced multiple shoots within two weeks of culture. Within seven to fifteen days of culture callus formed at the cut surface of nodal pieces of in vitro raised shoots, when grown on MS medium supplemented with 0.5 and 1.0 mg L-1 BA in combination with 0.2-2.5 mg L-1 NAA. Maximum (96%) callus induction was observed on MS medium fortified with 0.5 mg L-1 BA and 2.0 mg L-1 NAA after two successive subcultures. In this combination yellowish green calli were developed (Fig. 1a, b). Percentages of callus induction, degree of callusing and type of callus have been shown in Table 1.
BA and NAA was the best hormones for callus induction as reported by
a number of researchers (Polanco et al., 1988; Biswas et al.,
2007). According to Dixon and Gonzales (1994), inclusion of an auxin and
cytokinin will be necessary for callus growth and somewhat higher auxin
concentration may be required for callus initiation. There were significant
differences in callus induction frequencies and degree of callusing. Frequency
of callusing increased with increasing concentration of auxin. However,
when the auxin concentration surpassed 2.0 mg L-1 decreased
level of callus induction was observed.
| Fig. 1: | Callus induction and regeneration of Rauwolfia serpentina L: (a) and (b) Induction of callus on MS medium supplemented with 0.5 mg L-1 BA + 2.0 mg L-1 NAA, (c) Shoot morphogenesis from callus on MS medium supplemented with 2.0 mg L-1 BA + 0.2 mg L-1 NAA, (d) Regenerated shoots in test tube on media same as Fig. (c), (e) Regenerated shoots in culture flask on media same as Fig. (c) and (f) Development of adventitious roots on half strength MS medium supplemented with 1.0 mg L-1 IAA + 1.0 mg L-1 IBA |
| Table 1: | Effect of different concentrations and combinations of BA and NAA on callus induction |
| * + = Similar size of explant, ++ = Twice size of explant, +++ = Thrice size of explant | |
| Table 2: | Shoot organogenesis from callus cultures of R. serpentina |
| Table 3: | Effect of different concentrations of IBA and IAA in half strength MS medium on root formation in regenerated shoots |
Percentage of response for different growth regulator combination on shoot bud differentiation from callus was observed. The highest shoot morphogenesis (Fig. 1c) and regeneration (Fig. 1d, e) was obtained when callus were subcultured on MS medium supplemented with 2.0 mg L-1 BA and 0.2 mg L-1 NAA (Table 2). Though Kn with IAA and other media composition were used for regeneration purposes, BA and NAA were reported by many researchers (Sinha and Roy, 2002; Huda et al., 2003). Shoot regeneration via a callus phase was the way to induce somaclonal variation and thus pave the way for improvement of the species (Thorpe et al., 1991) and such indirect organogenesis have been described in many medicinal plant species including Asparagus cooperi (Ghosh and Sen, 1989), Plumbago zeylanica (Das and Rout, 2002), Holostea ada-kodein (Martin, 2002), Rotula aquatica (Martin, 2003), Gloriosa superba (Sivakumar et al., 2003), Phellodendron amurense (Azad et al., 2005).
Rooting experiments were conducted in half-strength MS medium supplemented with IAA and IBA. Root induction and elongation (Fig. 1f) was found to be more prominent in the medium containing 1.0 mg L-1 each of IAA and IBA resulted in 80% root initiation (Table 3). Roots elongated up to 4-5 cm within 15 days of incubation period.
The effectiveness of half-strength MS basal medium supplemented with auxin on root induction has been reported in many medicinal plants (Huda et al., 2003; Mederos-Molina, 2004; Ahmed et al., 2005). Root induction of R. serpentina was reported on half-strength MS medium supplemented with IAA or IBA alone (Ahmed et al., 2005) with an efficacy around 73%. But this study shown a higher root induction efficacy of 80% on half-strength MS medium supplemented with IAA and IBA combination. The root lengths were varied in all media concentrations.
R. serpentina is a medicinally important endangered herb. Propagation through tissue culture of this plant has been described earlier but most of them have focused on mass propagation by means of stimulating axillary shoot growth (Mathur et al., 1993; Roy et al., 1994). For the production of alkaloid identical to the mother plant (Yoshimatsu and Shimomura, 1991), high yield of secondary metabolites (Benavides and Caso, 1993; Maheshwari et al., 2007) and optimization of alkaloid production (Yamamoto and Yamada, 1986; Anitha and Kumari, 2006; Premjet et al., 2002) and subsequent isolation (Kirillova et al., 2001) effective callus mass production is necessary. Though some studies have been reported induction of callus and regeneration of plants from callus culture of R. serpentina (Sarker et al., 1996) but callus induction efficacy and the number of regenerated plantlets were not so high. So this study will be fruitful to make up the demand of this plant for commercial utilization and production of alkaloids as well as will help in the conservation of such threatened genotype.