Abstract
BackgroundFunctional genomic studies using genetics approaches of conifers are hampered by the complex and enormous genome, long vegetative growth period, and exertion in genetic transformation. Thus, the research carried out on gene function in Pinus tabuliformis is typically performed by heterologous expression based on the model plant Arabidopsis. However, due to the evolutionary and vast diversification from non-flowering (gymnosperms) to flowering (angiosperms) plants, several key differences may alter the underlying genetic concerns and the analysis of variants. Therefore, it is essential to develop an efficient genetic transformation and gene function identification protocol for P. tabuliformis.ResultsIn the present study we established a highly efficient transgene Agrobacterium-mediated transient expression system for P. tabuliformis. Using a β-glucuronidase gene (GUS) as a reporter gene expression, the highest transformation efficiency (70.1%) was obtained by co-cultivation with Agrobacterium strain GV3101 at an optical density at 600 nm of 0.8, with 150 μM acetosyringone for 30 min followed by 3 days in the dark at 23 ± 1 °C. This protocol would be applied to other conifers; GUS staining was observed 24 h post-infection.ConclusionsWe report a simple, fast, and resilient system for transient Agrobacterium-mediated transformation high-level expression of target genes in P. tabuliformis, which will also improve transformation efficiency in other conifer species.
Highlights
Functional genomic studies using genetics approaches of conifers are hampered by the complex and enormous genome, long vegetative growth period, and exertion in genetic transformation
Functional genomic research in P. tabuliformis is typically performed by heterologous expression based on the Arabidopsis as a model plant
Transient expression methods including particle bombardment, protoplast transformation using polyethylene glycol, electroporation, and Agrobacterium-mediated transformation have been used in conifer plants [19]
Summary
Functional genomic studies using genetics approaches of conifers are hampered by the complex and enormous genome, long vegetative growth period, and exertion in genetic transformation. The research carried out on gene function in Pinus tabuliformis is typically performed by heterologous expression based on the model plant Arabidopsis. Transient expression methods including particle bombardment, protoplast transformation using polyethylene glycol, electroporation, and Agrobacterium-mediated transformation have been used in conifer plants [19]. Compared to commonly used particle bombardment and protoplast transformation, Agrobacterium-mediated transformation is used more frequently in many plant species. This method is one such versatile simple, rapid and transient expression of target genes can be detected within a few hours [8, 16, 27]. The transformation system for P. tabuliformis in combination with high-throughput sequencing technologies are capable to improve the transformation efficiency for other conifer species
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