Abstract
A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.
Highlights
We developed a simple, rapid and high-throughput Agrobacterium-mediated transformation system for hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’) using callus as the explant
The 3rd to 5th fully expanded leaves of 3-week-old 84K plantlets were cut with a scalpel; the incised leaf explants were cultured on four groups of callus induction medium (CIM1-4) containing 20 g L−1 sucrose and 0.5 g L−1 4-morpholineethanesulfonic acid (MES)
A large number of transformed shoots are induced from calli; high throughput, simplicity and convenience are achieved
Summary
Forests cover 30% of the Earth’s surface and provide natural and societal services, such as climate regulation, carbon storage and timber supply [1]. Forest trees have many specific characteristics that distinguish them from herbaceous plants, such as secondary growth, perennial growth and seasonal adaptation [2]. The underlying mechanisms of these characteristics are poorly studied. Traditional genetic methods, including crossing and mutagenesis, to disseminate these biological processes are difficult because of the long lifespan of tree species [3]. Forward genetics is required to study tree biology using model tree species such as Populus [4]. Poplars are deciduous trees of the Salicaceae family. More than 52 species are distributed in temperate regions of the Northern
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