유전자총을 이용한 팔레놉시스 형질전환 효율향상에 삼투압 조절제 및 발사횟수차이가 미치는 영향
Effects of osmoticum treatments and shooting chances on the improvement of particle gun-mediated transformation in Phalaenopsis
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초록
This study was carried out to develop an efficienttransformation protocol via particle bombardment withPLBs (protocorm-like bodies) in Phalaenopsis. To achievethis aim, osmoticum treatment and an increasing shootingchances in particle bombardment process were applied forthis study. In addition, pCAMBIA3301: ORE7 vector whichcontains a herbicide-resistance bar gene as a selectablemarker and ORE7 gene as a gene of interests were employed. With regard to the increasing chances of shooting in particlebombardment, double shooting was the best results with 1.5 ~2.5 times higher than those of a single or triple shootingtreatment in the productioon of PPT (D-L-phosphinothricin)-resistant PLBs. However, regeneration rate of shoots indouble shooting was not high as a single shooting. Further,double shooting showed 35 ~ 40% higher than that of asingle shooting in the frequency of browning. Regardingeffects of different osmotic treatments, combination of 0.2M sorbitol with 0.2 M mannitol showed the best results intransformation efficiency, regeneration of transformants andreduction of browning. Putative transgenic Phalaenopsisplants were analyzed by PCR analysis and confirmed thepresence of bar and ORE 7 gene. Also, real-time PCR wasconducted by using 21 transgenic plants and showed only 4plants had one copy of transgene; whereas, the other 17plants had more than 2 copies of transgene. Transgenicphalaenopsis plants produced in this study were transferredto pots and flowered normally without morphologicalvariations in flower and leaf.
Abstract
This study was carried out to develop an efficienttransformation protocol via particle bombardment withPLBs (protocorm-like bodies) in Phalaenopsis. To achievethis aim, osmoticum treatment and an increasing shootingchances in particle bombardment process were applied forthis study. In addition, pCAMBIA3301: ORE7 vector whichcontains a herbicide-resistance bar gene as a selectablemarker and ORE7 gene as a gene of interests were employed. With regard to the increasing chances of shooting in particlebombardment, double shooting was the best results with 1.5 ~2.5 times higher than those of a single or triple shootingtreatment in the productioon of PPT (D-L-phosphinothricin)-resistant PLBs. However, regeneration rate of shoots indouble shooting was not high as a single shooting. Further,double shooting showed 35 ~ 40% higher than that of asingle shooting in the frequency of browning. Regardingeffects of different osmotic treatments, combination of 0.2M sorbitol with 0.2 M mannitol showed the best results intransformation efficiency, regeneration of transformants andreduction of browning. Putative transgenic Phalaenopsisplants were analyzed by PCR analysis and confirmed thepresence of bar and ORE 7 gene. Also, real-time PCR wasconducted by using 21 transgenic plants and showed only 4plants had one copy of transgene; whereas, the other 17plants had more than 2 copies of transgene. Transgenicphalaenopsis plants produced in this study were transferredto pots and flowered normally without morphologicalvariations in flower and leaf.
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