An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants

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.

Keywords: Agrobacterium; callus; hybrid poplar; plant transformation; regeneration.

Figure 1

Plant regeneration from 84K poplar calli. (A) Leaves of three-week-old plants used for inducing callus formation. (B) Calli induced from leaves after 6 weeks on callus induction medium 1 (CIM1). (C) Induced calli for shoot induction on shoot induction medium 1 (SIM1). (D) Shoots formed from calli after 5 weeks of growth on SIM1. (E) Shoots rooting on RM. (F) The regenerated plants obtained after 2 weeks of growth on rooting medium (RM).

Figure 2

Effect of hygromycin B on shoot induction from 84K calli. (A–F) The calli were placed on SIM1 supplemented with 200 mg L⁻¹ timentin and 0 (A), 0.5 (B), 1.0 (C), 1.5 (D), 2.0 (E) or 2.5 (F) mg L⁻¹ hygromycin B. After 5 weeks, the induction of shoots was observed. Three replicates were performed; each replicate contained 30 explants.

Figure 3

Factors that affect the transformation frequency evaluated from shoots with positive GUS staining. (A,B) GUS staining in non-transgenic plants (A) and transgenic plants (B). (C–H) The Agrobacterium concentration, infection duration, cocultivation duration, acetosyringone concentration, Ca²⁺ concentration and precultivation duration were analyzed. Three replicates were performed, and each replicate contained 100 callus explants. The results are presented as the means and standard errors from three independent experiments. Within each variable, values with different letters indicate statistically significant differences at the p < 0.05 level.

Figure 4

Regeneration of transgenic 84K plants using the optimized Agrobacterium-mediated transformation system based on calli. (A) Infected calli were cocultivated for 6 days. (B) Infected calli were cultivated on shoot induction medium 1 (SIM1) containing timentin and hygromycin B for 5 weeks. (C) Putative transgenic shoots were transferred and cultured on rooting medium (RM) supplemented with timentin and hygromycin B for 2 weeks.

Figure 5

The growth biomass of callus and callus subculture generation affects the transformation frequency of shoots. (A) Quantification of the biomass of growing calli. The calli were subcultured on CIM1 for 2 weeks, and the fresh weight (FW) was measured. (B) Effect of the callus subculture generation on the transformation efficiency of GUS-positive hygromycin-resistant shoots. S1–S6, Number of subcultured calli. Three replicates were performed; each replicate contained 30 callus explants. The results are presented as the means and standard errors. Within each variable, values with different letters indicate statistically significant differences at the p < 0.05 level.

Figure 6

Molecular analyses of the transgenic plants expressing the GUS gene. (A,B) PCR amplification of the GUS (653 bp) and aadA (265 bp) genes in transgenic lines (22 lines are shown) using genomic DNA as templates. M, 2000 bp DNA marker; N, non-transgenic 84K plants used as a negative control; P, 35S::GUS binary vector used as a positive control; 1–22, the independent transgenic lines displayed bands for GUS but not for the aadA gene.

Figure 7

GUS expression in transgenic plants. (A) RT-PCR analysis of GUS transcript levels in non-transgenic plants (N) and transgenic lines (1–22), as shown in Figure 6. Actin expression was used as an internal control. (B) GUS assay in leaves of non-transgenic (N) and transgenic lines (11 lines are shown). (C,D) Histochemical staining for GUS activity in regenerated non-transgenic (C) and transgenic (D) plants. GUS staining was observed in 3-week-old transgenic plants but not in the non-transgenic plants.

Figure 8

Stepwise protocol for transforming 84K plants using calli as the explant. CIM, callus induction medium; CM, cocultivation medium; SIM, shoot induction medium; RM, rooting medium.