Studies on Genetic Transformation with Phalaenopsis Callus
碩士 === 國立臺灣大學 === 園藝學研究所 === 88 === Callus of Doritaenopsis ( Dtps. ) Taisuco Ladylip was successfully induced from 26% of the protocorms cultured on 1/2 MS medium ( PC medium ) containing 5 mg/l 2,4-D and 5 mg/l TDZ in the dark condition. For the establishment of Dtps. suspension cells the calli we...
| Main Authors: | , |
|---|---|
| Other Authors: | |
| Format: | Others |
| Language: | zh-TW |
| Published: |
2000
|
| Online Access: | http://ndltd.ncl.edu.tw/handle/34209082559612250922 |
| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 88 === Callus of Doritaenopsis ( Dtps. ) Taisuco Ladylip was successfully induced from 26% of the protocorms cultured on 1/2 MS medium ( PC medium ) containing 5 mg/l 2,4-D and 5 mg/l TDZ in the dark condition. For the establishment of Dtps. suspension cells the calli were cultured in liquid medium supplemented with 2000 mg/l of PVP ( polyvinylpyrolidone ). PVP decreased cell necrosis by 83.3 % compared with the control. Suspension cells of Dtps. increased growth weekly by 50 %. Calli and suspension cells of Dtps. subcultured on G7M medium turned green and then produced PLBs in the light. Plantlets was successfully regenerated from calli under illumination for three months. The Agrobacterium-mediated transformation using calli of Phalaenopsis ( Phal. ) Taisuco Kaaladian and Dtps. was established based on the transient expression assay. Acetosyringone ( 100 µM ) could improve the transformation efficiency of Dtps. calli. Phal. calli co-cultivated with Agrobacterium in liquid medium for 2 days had 72.2% of transformation and the transformation efficiency of co-cultivation in liquid medium was higher than co-cultivation on solid medium. Agrobacterium—mediated transformation combined with sonication for 30 seconds using Phal. calli had 40.7% of transformation. GUS enzyme activity assay for the Agrobacterium-mediated transformation using calli of Dtps. confirmed that putative transformants can be selected with antibiotics for 5 months. These results showed the application of Agrobacterium-medium transformation on the gene transformation of Dtps. calli is practicable.
Histochemical GUS activity was assayed on the leaves of putative Phal. transgenic plants derived from the introduction of pBI121、pBCH121-1 and pBACH121-1 into protocorms by bombardment-assisted Agrobacterium transformation or Agrobacterium co-cultivation. The expression pattern of GUS activity on the leaves could be classified into four categories by their location : ( 1 ) almost whole leaf； ( 2 ) leaf margin with randomized patch or leaf margin with vein； ( 3 ) randomized patch or randomized spot；( 4 ) no GUS activity. This result demonstrated the elementary evidence of transformation.
|
|---|