In-Vitro Plantlet Regeneration and Agrobacterium-Mediated Gene Transformation of Carica Papaya L. Cv. Eksotika
Panjaitan, Syaiful Bahri (2006) In-Vitro Plantlet Regeneration and Agrobacterium-Mediated Gene Transformation of Carica Papaya L. Cv. Eksotika. Masters thesis, Universiti Putra Malaysia.
The study was conducted at the In Vitro Laboratory, Institut Pertanian Persekitaran Terkawal (IPPT), Faculty of Agriculture and Molecular and Cell Biology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor. The main objectives of the study were to develop in-vitro plantlet regeneration systems and to carry out preliminary studies of Agrobacterium-mediated gene transformation of Carica papaya L. cv. Eksotika. The study consisted of in-vitro plantlet regeneration through organogenesis, and somatic embryogenesis using different types of explant and different concentrations and combinations of plant growth regulators whereby the most appropriate system could be applied in the genetic transformation of the crop. The experiments were arranged in a completely randomized design (CRD). The study on organogenesis included shoot induction and rooting. On shoot induction, modified MS (Murashige and Skoog, 1962) medium supplemented with 500 mgL-1 (w/v) casein hydrolysate, 30 gL-1 (w/v) sucrose and 0.2% (w/v) phytagel was used as the basic medium, and different concentrations of BAP (0, 0.1, 1.0, and 2.0 mgL-1 (w/v)) in combination with 0, 0.05, 0.1 and 0.2 mgL-1 (w/v) NAA were applied as treatments. A modified MS medium containing 1 mgL-1 (w/v) BAP combined with 0.05 mgL-1 (w/v) NAA was the most suitable treatment combination to induce shoot proliferation from shoot tip, leaf and petiole explants of field-grown hermaphrodite plant. The mean number of shoots produced was 73 on shoot tip explant, 47 on leaf explant and 10 on petiole explant. The treatment also successfully enhanced the percentage of explant producing shoots which were 60.24% on petiole explants, 70.07% on leaf explants and 100% on shoot tip explants. In the rooting study, shoots obtained above were pretreated in different concentrations of IBA (0, 0.5, 1.0, and 2.0 mgL-1 (w/v)) for one week followed by culture on medium with or without vermiculite. Pretreatment in 1 mgL-1 (w/v) IBA for one week stimulated root initiation on shoots cultured on MS medium with vermiculite supplement. Pretreatment in 1 mgL-1 (w/v) IBA followed by culture on medium with vermiculite was most suitable for root induction on shoots derived from shoot tip, leaf and petiole. Shoots derived from field-grown shoot tip and petiole explants produced 90% rooting while 87.50% rooting occurred on shoots derived from leaf explant. However, the treatments and methodology used for rooting still need to be modified and improved in order to produce roots of good characteristics and root quality. Plantlet regeneration through somatic embryogenesis was successfully achieved on leaf and petiole explants derived from proliferated shoots of hermaphrodite field grown shoot tips as well as from embryogenic cell suspension culture. Modified MS medium with the macro nutrients reduced to half-strength, full-strength of other elements, 500 mgL-1 (w/v) glutamine, 30 gL-1 (w/v) sucrose, 0.2% (w/v) phytagel, and 2,4-D at 1.0, 2.5, 5.0 and 7.5 mgL-1 (w/v) in combination with 0, 0.001, 0.005 and 0.010 mgL-1 (w/v) BAP as treatments was employed for the induction of somatic embryos on leaf and petiole explants. For the establishment of cell suspension culture, two treatments which were liquid full strength MS and liquid half-strength MS medium containing 2 mgL-1 (w/v) 2,4-D were assessed. Treatment containing 5 mgL-1 (w/v) 2,4-D was the most suitable for induction of somatic embryos from leaf explant with the highest percentage of explants that responded to form somatic embryos at 55% and mean number of somatic embryos produced at 76.56. Meanwhile, treatment containing 2.5 mgL-1 (w/v) 2,4-D was most suitable for production of somatic embryos on petiole explant with the highest percentage of explants that responded to form somatic embryos at 20.50% and mean number of somatic embryos produced at 11.79. High frequency of somatic embryo formation was achieved through cell suspension culture method. Liquid full-strength MS medium supplemented with 2 mgL-1 (w/v) 2,4-D significantly produced the highest percentage of somatic embryo formation in cell suspension derived from embryogenic calli of leaf or petiole explants compared to treatment consisting of liquid half-strength MS medium containing 2 mgL-1 (w/v) 2,4-D. 58.33 ± 5.86 cotyledonary somatic embryos were produced per mL of suspension culture derived from embryogenic calli of leaf explant in liquid full-strength MS medium supplemented with 2 mgL-1 (w/v) 2,4-D whereas only 35.67 ± 1.53 cotyledonary somatic embryos were obtained per mL of suspension culture established from embryogenic calli of petiole explant in the same treatment (liquid full-strength MS supplemented with 2 mgL-1 (w/v) 2,4-D). Different concentrations of NAA (0, 0.01, 0.02 and 0.03 mgL-1 (w/v)) in combination with 0, 0.1, 0.2 and 0.3 mgL-1 (w/v) BAP were used as treatments for germination of somatic embryos derived from the leaf and petiole explants and from the cell suspension cultures. For somatic embryos obtained from leaf explant, treatment containing 0.02 mgL-1 (w/v) NAA combined with 0.1 mgL-1 (w/v) BAP gave significantly the highest percentage of somatic embryos germination (92.50%) and also the highest percentage of normal plantlet formation (83%). Whereas for somatic embryos obtained from petiole explant, treatment containing 0.02 mgL-1 (w/v) NAA combined with 0.2 mgL-1 (w/v) BAP produced significantly the highest percentage of somatic embryos germination (93.25%) and also the highest percentage of normal plantlet formation (83%). Meanwhile, treatment containing 0.02 mgL-1 (w/v) NAA combined with 0.1 mgL-1 (w/v) BAP was the most suitable for germination of somatic embryos derived from cell suspension culture, whereby 92% of the somatic embryos germinated into seedlings (plantlets). In the development of genetic transformation protocol via Agrobacterium-mediated gene transfer, the minimal inhibitory concentration (MIC) of hygromycin on target materials such as leaf, petiole and embryogenic calli was evaluated. The different concentrations of hygromycin tested were 2, 4, 6, 8, 10, 12 and 15 mgL-1 (w/v). Other parameters for genetic transformation that were evaluated, were the different bacterial concentrations (1 X 105, and 1 X 107 cfu) combined with co-cultivation periods of 5, 15 and 30 minutes. Hygromycin at 12 mgL-1 (w/v) was chosen as the minimal inhibitory concentration (MIC) that caused the death of leaf explant and embryogenic calli derived from leaf and petiole explants, while 15 mgL-1 (w/v) hygromycin was chosen as the minimal inhibitory concentration for petiole explant. Although plantlet regeneration via organogenesis and somatic embryogenesis and through cell suspension culture were successfully established in this study and applied in the genetic transformation of papaya, no transgenic plant was obtained. This means that further work on the establishment of an efficient genetic transformation protocol via Agrobacterium-mediated gene transfer for papaya cv. Eksotika need to be pursued.
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