Articles
Studies on apple genetic transformation by using fluorescence analysis of green fluorescence protein
Article number
1261_22
Pages
137 – 142
Language
English
Abstract
By using fluorescence analysis of GFP (green fluorescence protein) we studied the target gene transformation and expression in transgenic apple plants.
Although cutting damage is used in most apple transformation operations, our study found that by using crush damage the regenerated calluses or plantlets on leaves almost all are homogeneous unlike chimera plantlets, only the plantlets regeneration rates are lower than when using cutting damage.
No difference was found in transgenic plantlet regeneration rates when different types of plasmid vectors with the same antibiotic selection marker gene were used.
The difference in plantlet regeneration rates was clear even on the same plasmid vector with different antibiotic selection marker genes.
Fluorescence analysis showed that by using high antibiotic selection pressure (50 mg L-1) all the regenerated transgenic calluses had strong green fluorescence.
While low selection pressure (10 mg L-1), besides the escape and chimera calluses, resulted in much more regenerated calluses where different degrees of fluorescence were observed.
We think that plantlets from these calluses could be used in the expression study of apple transgenic plants.
Gene silencing was tested on apple transgenic plantlets after one year continued observation by using fluorescence analysis.
In the newly obtained transgenic plantlets, strong green fluorescence was observed.
Meanwhile after one year of subculture, fluorescence of shoot tips and young leaves was lost on some transgenic plantlets, and calluses with strong green fluorescence could be induced on the leaves of those plantlets.
Although cutting damage is used in most apple transformation operations, our study found that by using crush damage the regenerated calluses or plantlets on leaves almost all are homogeneous unlike chimera plantlets, only the plantlets regeneration rates are lower than when using cutting damage.
No difference was found in transgenic plantlet regeneration rates when different types of plasmid vectors with the same antibiotic selection marker gene were used.
The difference in plantlet regeneration rates was clear even on the same plasmid vector with different antibiotic selection marker genes.
Fluorescence analysis showed that by using high antibiotic selection pressure (50 mg L-1) all the regenerated transgenic calluses had strong green fluorescence.
While low selection pressure (10 mg L-1), besides the escape and chimera calluses, resulted in much more regenerated calluses where different degrees of fluorescence were observed.
We think that plantlets from these calluses could be used in the expression study of apple transgenic plants.
Gene silencing was tested on apple transgenic plantlets after one year continued observation by using fluorescence analysis.
In the newly obtained transgenic plantlets, strong green fluorescence was observed.
Meanwhile after one year of subculture, fluorescence of shoot tips and young leaves was lost on some transgenic plantlets, and calluses with strong green fluorescence could be induced on the leaves of those plantlets.
Publication
Authors
Yongjie Wu, Yusheng Li, Long Chen, Hehe Cheng, Yanhua Zhao, Yaqin Wu, Shengjian Zhao
Keywords
apple, genetic transformation, GFP, gene silence
Groups involved
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