MATERIALS AND METHODS

Leaf base segments were excised aseptically from in vitro grown cultures, which were originally established from the rhizome buds of field grown mature plants. The sheathing basal parts of excised leaves were chopped into pieces (1-1.5 mm) and segments were placed on the agar-gelled medium. The leaf base explants were ultimately cultured on MS medium^[12] supplemented with various concentrations of 2, 4-D alone and in combination with BA for callus induction. Seven weeks old callus was thus subcultured on MS medium containing different combinations of BA with NAA or IBA for somatic embryogenesis.

The embryogenic calluses were subcultured at monthly intervals on same medium for somatic embryos formation and development of complete plantlets with shoots and roots.

The pH of the culture media was adjusted to 5.7±0.1 before addition of agar and sterilized by autoclaving for 20 min at 1.1 kg cm^-2 pressure at 121°C.

For solidifying the medium, 7-8 g L^-1 agar or 2.0 g L^-1 gelrite was used. The tubes or flasks containing explants were incubated on culture racks in the growth chamber. The cultures were maintained at 25±2°C under the cool white fluorescent lights for 16 h photoperiod with a photon flux density of about 70 μ mol m^-2s^-1.

RESULTS AND DISCUSSION

Induction of callus: Excised in vitro leaf base segments were cultured on MS medium with various levels of 2, 4-D alone and in combination with BA for induction of callus. After six weeks of culture incubation callus mass increased to a transferable size (Fig. 1A). Morphogenic potentialities of the explant found to differ depending on the growth regulator supplements (Table 1).

Among different combinations of the auxin and cytokinin tested, 2, 4-D + BA was found highly effective for induction of callus (results of the other combinations are not shown in the Table 1). It was mostly soft, faster growing, light creamy-white and friable. The highest frequency of callus induction (85%) was recorded at the end of 7 weeks on medium containing 1.5 mg L^-1 2, 4-D with 1.0 mg L^-1 BA. Similar combination of auxin with cytokinin for callus induction has been reported in the past by Malamug^[6] on ginger, Vincent et al.^[11] on Kaempferia galanga, Muthukumar et al.^[13] on Datura metel. Callus induction from leaf base explant has also been achieved in cardamom using 2, 4-D+ Kn in combination^[8].

Embryogenesis from the subcultured callus: The callus tissues developed from leaf base explants were subcultured on MS medium with various concentrations of BA with NAA and BA with IBA for somatic embryogenesis (Table 2).

Table 1:	Effects of different concentrations of 2, 4-D alone and in combination with BA for callus induction from leaf base explant. There were 20 explants in each treatment and data (±SE) were collected after 7 weeks of culture
(-) Indicate no response; (*) slight callusing; (**) considerable callusing and (***) profuse callusing; Cr = Creamy; CrW = Creamy-white; W = White

Table 2:	Effects of different concentrations and combinations of BA with NAA or IBA for somatic emtayogenesis on MS medium from leaf base derived callus. There were 20 cultures in each treatment and data (x±SE) were collected after 6 week of culture

After three weeks of subculturing creamy white sectors with well-developed embryoids were seen that later turned into green shoots (Fig. 1B). Although root initiation started from these calli after two weeks of subculturing but complete plants were seen in the differentiating callus after three weeks (Fig. 1C) and a single leaf to multiple shoots were observed after five weeks of culture (Fig. ID).

Fig. 1: A-F:	Plantlets regenatation from the leaf base explant through somatic embryogenesis of Kaempferia galcmga L.

The maximum percentage of embryogenic callus that formed embryos was 75% at the BA+NAA concentration of 2.0 mg L^-1+ 0.1 mg L^-1. The highest number of somatic embryos per culture was 35.4±2.19, which was also recorded in the same concentration of BA+NAA after ten weeks of culture (Fig. 1E). Complete plant regeneration by embryogenesis was observed on different explants cultures of garlic^[14-15], ginger^[5], Muntmgia calabura^[16]. Using BA+NAA combinations adventitious shoot regeneration from leaf base explant has been observed on cardamom^[8]. Qrganogenesis and plant formation in presence of BA or Kn with NAA was also reported from various explants via callus culture of ginger^[6], Kaempferia galanga^[10] and sugarcane^[17] .

Plantlets were at first established in small plastic pots with a view to easy handling and maintenance during acclimatization and transplantation. About 95% of the plantlets established under ex vitro condition when they were initially transferred on coco-peat as potting mix. On the other hand, 90% survival rate of the plantlets was observed when they were transferred on ice cream pots containing garden soil, compost and sand (2:2:1). It was also observed 85% of the plantlets could be established under ex vitro conditions when they were transferred on earthen pots containing soil and organic manure (Fig. 1F).