INTRODUCTION:

Scoparia dulcis has been traditionally used for treating stomach ailments, hypertension, diabetes, inflammation, bronchitis, hemorrhoids, hepatosis, an analgesic and antipyretic agent (Riel et al., 2002, Ratnasooriya et al., 2005). Extracts of the plant contains antidiabetic activity (Pari et al., 2004), anticancer activity (Nishino et al., 1993), antimalarial activity (Riel et al., 2002), antiviral activity (Hayashi et al., 1988), neurotrophic activity (Li el al., 2004) and anti-inflammatory activity (Ahmed et al., 2001). Conventional vegetative propagation on commercial scale has limitations (Thakur et al., 1998). Therefore, micropropagation offers a reliable method for mass production of plants in a shorter time without seasonal constraints. The development of polyploids can be a useful and valuable tool to improve this trait in breeding programs (Notzuka et al., 2000). Polyploidization was used successfully to increase the size of flowers, intensify the colours of leaves and flowers, modify plant shape and restore fertility in ornamental species (Horn, 2002). So far, there are only few reports on in vitro colchicine treatment for this plant in order to improve its cultivation (Escandon et al., 2005). Therefore this present investigation offers an effective alternative method of propagation for this important multipurpose medicinal plant.

MATERIALS AND METHODS:

In vitro multiplication

Healthy and profusely growing shoots of S. dulcis were collected from Madras Christian College campus, Chennai and were identified using regional floras (Gamble, 1967; Henry et al., 1987) in Centre for Floristic Research, Department of Botany, Madras Christian College, Chennai, India. The explants were washed with soap (soap powder) in running tap water for 1 hour. This is necessary to remove the exudates (phenolics, tannins and mucillages) present within the tissues. The explants were washed with Tween 20 (2%, w/v) until traces of soap was removed. Later these explants were surface sterilized with 0.1% mercuric chloride (w/v) for 8 minutes and washed thrice using sterilized distilled water. Under aseptic conditions, explants were inoculated on basal MS (Murashige and Skoog, 1962) medium containing 3% (w/v) sucrose, supplemented with 0.5 mg/l KIN and 2.0 mg/l IAA (Karthikeyan et al., 2009). The pH was adjusted to 5.7 prior to the addition of 0.8% agar and autoclaved at 121°C (1.06 kg/cm²) for 15 min. Cultures were then incubated at 26 ± 2°C with a 16 hours photoperiod by cool white fluorescent tubes and 70-75% relative humidity (Mukherjee et al., 1991). The plantlets were placed at 70% to 80 % humidity, 25 ± 2 °C under a 12-hours photoperiod for acclimatization. After the plants get acclimatized, the plants were transferred to pot with farmyard mixture: sand (1: 1 v/v) and placed in green house (Karthikeyan et al., 2009). After three weeks of development, these hardened plants were transferred to the field and the survival rate was recorded.

Twenty cultures were used per treatment and each experiment was repeated at least three times. Percentage of success was scored four weeks after culture.

In vitro plant polyploidization

Nodal segments from in vitro plant of S. dulcis were submerged in 1% DMSO solution containing the following doses of colchicine (v/v): 0.0; 0.0001; 0.005; 0.001 and 0.01 for 48 hrs. As control treatments, a group of nodal segments was untreated, and other segment groups were submerged in water or in 1% DMSO (water solution) (Escandon et al., 2005). The culture medium was MS supplemented with with 0.5 mg/l KIN and 2.0 mg/l IAA. The culture conditions and the acclimatizing protocol were those mentioned in the above section. Once established under greenhouse conditions, the recovered treated plants were phenotypically analyzed. Number and length of shoots and roots were measured. Data collected were statistically analyzed and results presented in the tables.

RESULTS:

Simultaneous regeneration of shoots and roots and in vitro flowering were achieved from the nodal explants on MS medium supplemented with KN and IAA. Best response was noticed with 0.5 mg/l KN and 2.0 mg/l IAA (Karthikeyan et al., 2009).

The regenerated plants were phenotypically normal after the colchicines treatment. Table 1 shows the means of shoot per explants obtained from the different colchicine treatments. Except for the treatments 0.1% colchicines, the others treatments ranging very good results and no significance differences were found between them. The exception was S. dulcis that showed less regeneration, followed by drying of the plantlet and ending with explants death under the assayed culture conditions. Best results were observed with 0.001 % colchicines where 6.5 cm length of shoots, 2.34 cm length of roots and average range of 5-6 flowers were recorded.

DISCUSSION:

Chromosome duplication is caused by abnormalities during mitosis and it may occur spontaneously in most plants. Although, these disruptions during cell division can be artificially induced with colchicine and thus, in vitro polyploidization was proposed back in the 1960’s as an alternative tool to obtain polyploid plants (Murashige and Nakano, 1966). This methodology was extensively used during the last 30 years in many species such as banana (Baziran and Ariffin, 2002), grapes (Notsuka et al. 2000), blueberry (Lyrene and Perry, 1982), potato (Hermsen et al., 1981), and sugarcane (Heinz and Mee, 1970). Under in vitro controlled conditions polyploidization was applied in several ornamental crops, such as Alocasia (Thao et al. 2003), Rhododendron (Eeckhaut et al., 2002), Cyclamen (Takamura and Miyajima, 1996) and S. monteviediensis (Escandon et al., 2005).

S. dulcis rooted easily using the proposed protocol, even with the previous BAP treatments (Escandon et al., 2005). This, together with the fact that no problems were found either in the acclimatization step, or in the multiplication rate, indicates that this material is suitable for commercial multiplication (Escandon et al., 2005).

Direct shoot multiplication is preferred for generating true-to-type plants than callus regeneration. This study also supports the rapid multiplication of this useful medicinal plant by in vitro conditions. This report provides a simple protocol for the micropropagation of S. dulcis and for developing new verities using colchicines treatments. Tissue culture combined with the polyploidization treatment showed to be a very interesting alternative to obtain the needed variability in Scoparia genus to start a breeding program.

ACKNOWLEDGEMENT:

Authors are thankful to the Management of Biozone Research Technologies, Chennai, India for providing the infrastructure for the present study.

Table 1: Effects of different concentrations colchicine in MS medium supplemented with 0.5 mg/l KN and 2.0 mg/l IAA for simultaneous shoot and root regeneration and in vitro flowering from node explants of Scoparia dulcis.

Concentration of colchicine	% of explant showing response	No. of shoots	Average length of shoots (cm)	No. of roots	Average length of roots(cm)	No. of flowers
0.0	97.2	1.8	5.88	4.4	2.08	4.8
0.0001	97.0	2.0	5.95	5.1	2.12	4.8
0.005	96.5	1.9	5.98	5.2	2.18	5.1
0.001	96.7	2.0	6.5	5.8	2.34	5.4
0.01	81.2	1.1	4.8	4.4	1.6	2.3

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Received on 20.02.2012 Modified on 12.03.2012

Research J. Pharm. and Tech. 5(5): May2012; Page 632-634