Micropropagation of two globba species and the genetic fidelity assessment of Globba brachyanthera K. schum.micropropagated plantlets

Kho, Pei Ee (2010) Micropropagation of two globba species and the genetic fidelity assessment of Globba brachyanthera K. schum.micropropagated plantlets. Masters thesis, Universiti Malaysia Sarawak, (UNIMAS).

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Abstract

Globba brachyanthera K. Schum. and Globba atrosanguinea Teysm. & Binn. are miniature gingers belong to Zingiberaceae family. These endemic gingers have great ornamental value, however propagation of these endangered species through conventional method is beset with many problems. Consequently, the present study was carried out in order to develop a rapid in vitro multiplication method for conservation and provide adequate planting material for large-scale cultivation of these species. An efficient surface sterilization protocol was successfully developed for G. brachyanthera and G. atrosanguinea by surface sterilized bulbils in 20% and 35% of CloroxTm solution for 20 minutes respectively. Synergic effect of PPM and tetracycline had produced 75%-80% of axenic cultures and dramatically diminished the degree of fungal and bacteria contamination) For direct organogenesis pathway, shoot-tip explants derived from in vitro cultured plantlets were induced to form shoots on Gamborg B5 medium augmented with 20% sucrose, 2.8 g/L Gelrite and various concentrations of plant growth regulators such as TDZ, Kinetin and BAP either alone or in combination with NAA. Among the cytokinins tested, BAP at 3.0 mg/L was superior in micropropagation of G. brachyanthera with the highest multiplication rate of 8.6 shoots per shoot-tip explant within eight weeks of inoculation. Besides, shoot-tip explants of G. brachyanthera produced more multiple shoots on Gamborg B5 medium as compared to MS medium. The in vitro micropropagated plantlets were acclimatized successfully with 100% survival rate. The genetic fidelity of plantlets developed via the direct organogenesis was assessed by the random amplified polymorphic DNA (RAPD) analysis. DNA from young leaves of G. brachyanthera and the micropropagated plantlets were extracted successfully by using Promega Wizard® Genomic DNA purification Kit with minor modification. Tweleve RAPD primers (OPA 03, OPA 13, OPB 08, OPB 10, OPB 17, OPC 02, OPC 05, OPD 02, OPD 03, OPD 05, OPD I1 and OPD 18) generated a total of 92 scorable bands ranging from 300 bp to 2000 bp in size. The primers giving rise to monomorphic banding patterns across mother plants and their micropropagated propagules analyzed, confirming the genetic stability of the micropropagation protocol to produced true-to-type plantlets. Indirect organogenesis pathway was achieved in callus cultures established from the leaf-sheath explants of G. brachyanthera on B5 medium amended with 5.0 mg/L 2,4-D within four months of culture. After transfer onto B5 medium fortified with 3.0 mg/L BAP, this callus produced small globular embryos first and later developed into plantlets by further subculturing on fresh medium of the same composition.

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