Date of Award

2010

Thesis Type

PhD

Document Type

Thesis

Divisions

Faculty of Science

Department

Institute of Biological Sciences

Institution

Universiti Malaya

Abstract

In vitro regeneration of Gerbera jamesonii Bolus ex. Hook f. was successfully carried out in this study. Petiole and leaf explants from 8-week-old aseptic seedlings were used as source of explants. Different response was observed when different explants were cultured on the culture media. MS (Murashige and Skoog, 1962) supplemented with 2.0 mg/l BAP and 0.5 mg/l NAA was identified as the optimum medium for shoot regeneration, while petiole explant was identified as the most responsive explant in this study. Petiole explants regenerated shoots (94.3 } 2.5 %) and produced 9.3 } 0.6 shoots per explant. MS basal medium was the optimum medium for plantlet root induction. Various concentrations of auxin such as NAA, IAA, IBA and 2,4-D at a range of 0.1-2.0 mg/l and cytokinin such as BAP, Kinetin, Zeatin and 2iP at a range of 0.1-3.0 mg/l were used in order to achieve in vitro regeneration of G. jamesonii. Callus was also induced from G. jamesonii petiole and leaf explants. Optimum callus induction was obtained when petiole explant was cultured on MS medium supplemented with 2.0 mg/l 2,4-D and 1.0 mg/l BAP. Green compact callus were induced. Through screening of secondary metabolites in Gerbera callus, flavonoid, terpenoid and conjugated chain compounds were found in Gerbera callus. Studies on indirect somatic embryogenesis of G. jamesonii were carried out using leaf explant. Induction of embryogenic callus was obtained when petiole explants were cultured on MS media supplemented with 0.01-2.0 mg/l 2,4-D. White friable callus was induced. Embryogenic callus was distinguished from non-embryogenic callus through double staining method. Embryogenic callus obtained were transferred to MS liquid medium fortified with 0.1-2.0 mg/l 2,4-D added with 0.1 or 1.0 mg/l NAA. Somatic embryos produced from embryogenic callus cultured in liquid medium were then transferred to MS medium supplemented with 1.0 mg/l BAP and 0.1 mg/l NAA added with 50 mM L-Proline. A total of 29.8 } 1.2 embryos per explant were obtained. Synthetic seeds of G. jamesonii were successfully produced when micro shoots, globular and cotyledonary phase somatic embryos were encapsulated with encapsulation matrix composed of 3.0% sodium alginate dissolved in Ca-free MS liquid medium supplemented with 2.0 mg/l BAP and 0.5 mg/l NAA. Germination rates obtained from encapsulated micro shoots was 74.5 } 2.6%, 34.8 } 1.2% for encapsulated globular somatic embryos and 54.2 } 1.3% for encapsulated cotyledonary phase somatic embryos. Synthetic seeds from encapsulated micro shoots could be stored at 4 } 1 oC for 180 days while synthetic seeds from encapsulated globular and cotyledonary phase somatic embryos could be stored at 4 } 1 oC up to 90 days only before being germinated. Generally, in vitro regeneration from petiole explants were reduced when explants were exposed to gamma irradiation (10-60 Gy). As the irradiation dose increased, in vitro shoots regeneration was declined and abnormal shoots were formed. In vitro plantlets exposed to gamma irradiation also showed height reduction as the irradiation dose was increased. Effects of gamma irradiation were also observed on callus tissues. Growth of G. jamesonii callus tissues was also reduced with the increase of the irradiation dose. Reduction of chlorophyll content in callus tissues was observed when Gerbera callus were exposed to gamma irradiation. Soluble protein content in callus tissues were also reduced with the increase of irradiation dose. Gerbera jamesonii plantlets obtained from tissue culture system through direct regeneration, somatic embryogenesis, synthetic seeds and also irradiated plantlets were successfully transferred and acclimatized to the new environment. Garden soil and vermiculite were used as sowing media. Growth and development of plantlets were optimum when plantlets obtained from regeneration of petiole explants were acclimatized in garden soil (combination of black soil and red soil at 2:1) with 86.0 } 0.9% survival. Ex-vitro flowering was achieved after 6 months. Gerbera jamesonii plants obtained from in vitro regeneration showed similar morphological characters to the mother plant.

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Additional Information

Thesis (PhD) - Faculty of Science, Universiti Malaya, 2010.

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