Establishment of the Transformation System for Phalaenopsis and Construction of Antisense Gene for 1-aminocyclopropane-carboxylate Synthase

• Main Authors: Huang, Shenq-Yow, 黃聖佑
• Other Authors: Huang Pung-Ling
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/23096766643774106010

碩士 === 國立臺灣大學 === 園藝學系研究所 === 85 === Abstract Antisense gene for 1-aminocyclopropane-1-carboxylate synthase (ACS) dr iven by ethylene-inducible and flower-specific ACS promoter was constructed in pBI121, named pBACC121, which contains reporter gene GUS and selection marker NPTII gene. pBACC121 was introduced into the seedlings of Phalaenopsis True L ady (Lucky Lady x Pinlong Cardinal) "79-19" and P. True Lady x P. True Lady th rough the Agrobacterium-mediated transformation.Seeds sown after 5 to 9 days w ere chosen for transformation. 0.35% of treated seedlings by bombarding Agroba cterium cells (LBA4404/pBACC121) into Phalaenopsis seeds survived under the an tibiotic selection. 0.03% of seedlings exposed under reduced pressure 25 inch- Hg in a sterile vacuum chamber for 25 minutes post 25 minutes of ultrasonic vi bration showed resistant to G418. Cocultured with Agrobacterium cells (LBA4404 /pBACC121) after wounding by carborundum shaking at 250 rpm for 30 minutes, 0. 04% of Phalaenopsis seedlings showed resistance to G418. Transformants obtaine d by these three methods expressed GUS enzyme activity. Addition of potato int o cocultured liquid medium elevated the frequency of infection. The number of Agrobacterium cells for higher transformation efficiency were 1.8×106 cells p er (L.Different selection schemes were established for transformants via polle n tube pathway and Agrobacterium-mediated method. 100 mg/l of G418 and gradual increase of its concentration was applied into G7 medium for selection of one month old seedlings derived from pollen tube pathway transformed capsules. Us ing specific primers to synthesize GUS gene, one out of 31 G418-resistant plan ts displayed the whole 1.8 kb gene fragment through polymerase chain reaction (PCR) and nine of these transformants possessed 0.12 kb gene fragment. After t ransformation treatment, the seeds were cultured on G7 medium containing 200 m g/l cefotaxime for seven days, and then 50 mg/l G418 were added into the mediu m for another 14 days. Finally, the transformed seedlings were maintained on G 7 medium containing 100 mg/l G418 and 200 mg/l cefotaxime. The results of PCR indicated that exogenous DNA was integrated into Phalaenopsis genome using gen omic DNA from transformants as template to synthesize GUS gene fragment.Callus of Phalaenopsis was successfully induced by combining auxin and cytokinin i n medium. Proliferated callus was formed from the protocorms and flower stem n odes cultured on 1/2 MS medium containing 10 mg/l 2,4-D and 5 mg/l TDZ in the dark condition. 0.09% of protocorms transformed via Agrobacterium formed G418- resistant callus. GUS enzyme activity was detected on the outer tissue of thes e callus.