| Summary: | 博士 === 國立中興大學 === 園藝學系所 === 94 === The objectives of this study were to: 1. develop an in vitro tissue regeneration system for Pak-choi (Brassica campestris L. ssp. chinensis L. Makino), 2. explore the possibility for improving the in vitro tissue regeneration derived from mature or aging explants of Pak-choi, 3. establish the Agrobacterium transformation system of Pak-choi, 4. establish the gene-gun transformation system of Pak-choi, and 5. explore the possibility for improvement of Pak-choi with stresses resistance via Agrobacterium-mediated and gene-gun transformation.
The sources of explants of Pak-choi and the components of culture medium were studied to develop a tissue culture system for Pak-choi. The results indicated that the MS basal medium (Murashige and Skoog, 1962) supplemented with 2% sucrose, 1 mg/L BA, 0.5 mg/L NAA, 0.25mg/L GA3, 3mg/L AgNO3 and 5mg/L putrescine was the best components of culture medium for the explants regeneration of ''Tainung No. 1'' and ''Tainung No. 2'' Pak-choi, and 66.7% to 70% of regeneration rate could be achieved. Higher regeneration rates were obtained from the explants derived from the cotyledons than those of from hypocotyls. In general, calli were first regenerated from the explants of Pak-choi, and then adventitious shoots and roots were regenerated on the regeneration medium subsequently, ie. via “organogenesis pathway”. However, embryoids rather than calli were regenerated on the regeneration medium supplemented with polyamines (e.g. putrescine or spermidine), ie. via “somatic embryogenesis pathway”.
The effects of types, ages, and positions of explants of ''Tainung No. 1'' and ''Tainung No. 2'' Pak-choi on in vitro tissue regeneration were performed. Effects of plant growth regulators, amino acids, polyamines, casein hydrolysate, and silver nitrate on shoot regeneration and root formation derived from the 15-days-old and 21-days-old explants of Pak-choi was also conducted. The highest rates of shoot regeneration and root formation were found in the 3-days-old explants. As the ages increased, the rates of shoot regeneration and root formation decreased. However, the high differentiation capacity was also found in the 27-days-old explants. Significantly increased the rates of shoot regeneration and root formation was found in the 15-days-old explants cultivated in the regeneration medium of Pak-choi (MS basal medium containing 2% sucrose, 1 mg/L BA, 0.5 mg/L NAA, 0.25mg/L GA3, and 3mg/L AgNO3) supplemented with 50 ml/l coconut juice. Improving the in vitro tissue regeneration of 21-days-old explants could be achieved by addition of 0.1 mg/L ABA into the regeneration medium of Pak-choi. Nevertheless, the efficient regeneration medium for 27-days-old explants was the same culture medium as the 3-days-old and 9-days-old explants.Attempts had been made to co-transfer the superoxide dismutase (sod62), and catalase (cat78) genes of Chinese cabbage into the ''Tainung No.1'' and ''Tainung No.2'' Pak-choi. Cotyledon and hypocotyl explants of Pak-choi were co-infected with two Agrobacterium carrying a distinct disarmed T-DNA containing rbcS-sod62 (pKrScn) and rbcS-cat78 (pKrCcn) genes, respectively. Results indicate that regenerated rates of Pak-choi were 16%. After 20 mg/L of kanamycin selection, the survival rate was 1.6%, and co-transformation rate of two genes was 0.075%. The results of PCR and Southern bolt hybridization indicated that the rbcS-sod62 and rbcS-cat78 genes had been transferred into Pak-choi, and inserted into their genomes. Two co-transformed plants of ''Tainung No.2'' Pak-choi were obtained. Increases in the transformation efficiency were obtained in the co-culture medium supplemented with the emery or 200µM acetosyringone (AS). Cotyledon and hypocotyl explants of Pak-choi were co-infected with two Agrobacterium carrying a distinct disarmed T-DNA containing rbcS-TP-sod62 (pKrTScn) and rbcS-TP-cat78 (pKrTCcn) genes, respectively. Results indicate that regenerated rates of Pak-choi were 18%. After20 mg/L of kanamycin selection, the survival rate was 2.2%, and co-transformation rate of two genes was 0.202%. The results of PCR and Southern bolt hybridization indicated that the rbcS-TPsod62 and rbcS-TPcat78 genes had been transferred into Pak-choi, and inserted into their genomes. Two and four co-transformed plants of ''Tainung No.1'' and ''Tainung No.2'' Pak-choi were obtained, respectively. T0 plants of rbcS-TP-sod62 and rbcS-TP-cat78 genes transformed Pak-choi were allowed to self-pollinated, and seeds were collected at maturity. T0 seeds were germinated on MS medium containing 20 mg/L of kanamycin for two weeks. Survival T1 seedlings were subjected to PCR and Southern bolt hybridization analysis. The results of genetic analysis indicated that the transformed rbcS-TP-sod62 and rbcS-TP-cat78 genes had been stably inherited in T1 progeny plants. Significant higher SOD and CAT activities were detected in the T1 transgenic progeny of Pak-choi than those of control plants.
Following settings were suggested when Pak-choi explants was bombarded with Biolistic PDS-1000/He (Bio-Rad) particle gun apparatus: gold particle size less than 1.0 μm in diameter, 4mm spacer ring in thickness, 4mm spacer bar in diameter, 4.5cm for distance of bombardment, 3cm for distance from microcarrier launch assembly to the recipient explants. High transformation efficiency was achieved from the 6-days old Pak-choi explants bombarded with 650psi~900psi rupture disks. Cotyledon and hypocotyl explants of Pak-choi were bombarded with two plasmids containing sod62 (pASCCSOD) and cat78 (pASCCCAT) genes, respectively. After 10 mg/L of spectinomycin selection, the survival rate was 3.86%, and co-transformation rate of two genes was 0.43%. The results of PCR and Southern bolt hybridization indicated that the sod62 and cat78 genes had been transferred into Pak-choi, and inserted into their genomes. Two and twenty-nine co-transformed plants of ''Tainung No.1'' and ''Tainung No.2''Pak-choi were obtained, respectively. T0 plants of sod62 and cat78 genes transformed Pak-choi were allowed to self-pollinated, and seeds were collected at maturity. T0 seeds were germinated on MS medium containing 10 mg/L of spectinomycin for two weeks. Survival T1 seedlings were subjected to PCR and Southern bolt hybridization analysis. The results of genetic analysis indicated that the transformed sod62 and cat78 genes had been stably inherited in T1 progeny plants. Significant higher SOD and CAT activities were detected in the T1 transgenic progeny of Pak-choi than those of control plants.
|
|---|
