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Transformation of Liquidambar formosana L. via Agrobacterium tumefaciens using a mannose selection system and recovery of salt tolerant lines

Qiao, Guirong, Zhou, Jing, Jiang, Jing, Sun, Yuehua, Pan, Luanyin, Song, Honggai, Jiang, Jingmin, Zhuo, Renying, Wang, Xiaojuan, Sun, Zongxiu
Plant cell, tissue, and organ culture 2010 v.102 no.2 pp. 163-170
Liquidambar formosana, genetic transformation, salt tolerance, Agrobacterium tumefaciens, culture media, mannose, sucrose, antiporters, transgenes, gene expression regulation, protein content, in vitro regeneration, micropropagation, mannose-6-phosphate isomerase, enzyme activity
A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Liquidambar formosana L. This system makes use of the pmi gene, which encodes phosphomannose isomerase. This protein converts mannose-6-phosphate to fructose-6-phosphate. Leaf explants from an axenic plant of L. formosana L. were infected with Agrobacterium. The gene AtNHXI was transformed into L. formosana L. using the transformation procedure developed in this study. We found that supplementing the media with 1.5% (w/v) mannose and 1% sucrose provided the necessary conditions for the selection of transformed plants from non-transformed plants. In this study, 134 positive transgenic plants were obtained. Genetic transformation was confirmed by PCR and Southern blot analysis, while RT-PCR confirmed expression of the foreign gene (AtNHX1) in the regenerated plants. Chlorophenol red assays confirmed the activity of transgenes in regenerated plants. In order to ascertain the tolerance of transformants for salt, further experimentation was done. Results showed that the soluble protein content in three transgenic lines (T-12, T-22, T-43) was higher than that in the control plant, while the MDA content was lower. Our results show that transgenic L. formosana L. plants can be obtained using either antibiotic resistance genes that are not expressed in the microorganisms or an antibiotic-free positive selection system.