Micropropagation of Tongkat Ali (Eurycoma Longifolia Jack) Via Somatic Embryogenesis and Direct Plant Regeneration Techniques

Eurycoma longifolia Jack or Tongkat Ali is well known among the local communities mainly for its aphrodisiac properties and its effectiveness as the cytotoxic, anti-malarial, anti-ulcer, anti-tumor promoting and anti-parasitic agent. In view of its potential commercial value as a plantation crop as well as to conserve its germplasm, the somatic embryogenesis and direct plant regeneration of E. longifolia were carried out as these in vitro micropropagation protocols had not been reported. In attempts to establish the somatic embryos of E. longifolia, the potential of cotyledon, zygotic embryo, leaf, petiole, stem and taproot in forming embryogenic callus were examined in the basal Murashige and Skoog (MS) medium supplemented with different auxins at various concentrations. Only cotyledon explants were able to form embryogenic callus in the presence of 1.0 mg/L (w/v) of 2,4-dichlorophenoxyacetic (2,4-D) at 30%. A higher yield (60%) of embryogenic callus was obtained when the Type 4 method dissected cotyledon explants were cultured in basal MS medium containing 0.5 mg/L (w/v) of kinetin and 1.0 mg/L (w/v) of 2,4-D. The highest number of somatic embryos (45 ± 2) was observed in the same medium formulation with the addition of 1.0 g/L (w/v) activated charcoal. Subsequent transfer of these mature somatic embryos in basal MS media supplemented with 1.0 mg/L (w/v) of kinetin produced a 90% of plantlet regeneration. Addition of activated charcoal, casein hydrolysate, abscisic acid, proline and polyethylene glycol (PEG) at various concentrations into the regeneration medium did not stimulate the conversion of E. longifolia somatic embryo into plantlet. The differences between the embryogenic and non-embryogenic callus were also determined based on histological studies. Successful direct plant regeneration was obtained from the root, stem, shoot tip, axillary and adventitious bud explants. Each explant generally requires different combinations of media and plant growth regulators to produce the highest regeneration percentage. In the root explant, the best medium formulation determined was basal Juglans medium (DKW) supplemented with 1.0 mg/L (w/v) of kinetin + 1.0 mg/L (w/v) of zeatin whereas in the stem explants, basal woody plant medium (WPM) enriched with 2.0 mg/L (w/v) of BAP and 2.0 mg/L (w/v) of zeatin was found to be the best medium formulation in increasing the regeneration rate and healthy plantlets formation. Stem and root explants that were 2 cm distant from one another as has been identified as the most suitable position for attaining the maximum percentage of direct plant regeneration. Successful direct plant regeneration from in vitro and in vivo shoot tip explants of E. longifolia was achieved in basal MS medium supplemented with 3.0 and 5.0 mg/L (w/v) of kinetin, respectively. As for the direct plant regeneration from axillary bud explant, explants produced the highest regeneration capability (90%) in basal Nitsch medium (NM) supplemented with 10.0 mg/L (w/v) of zeatin while basal NM supplied with 6.0 mg/L (w/v) of zeatin produced the highest regeneration percentage in adventitious bud explants. Rooting induction of in vitro plantlets of E. longifolia was also achieved and the highest induction rate was attained in basal MS medium supplemented with 0.5 mg/L (w/v) of Indole-3-butyric acid (IBA). Acclimatization of in vitro plantlets regenerated from somatic embryogenesis and direct plant regeneration survived well with no morphological differences from the parent plants after two months of transplantation to the soil.

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