A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Area of Science:

• Plant biotechnology
• Molecular biology
• Biofuel crops

Background:

• Grasses are crucial biofuel crops but challenging for genetic transformation.
• Previous methods for switchgrass (Panicum virgatum L.) transformation include Agrobacterium cocultivation and biolistics.
• Enhancing transient gene expression is key to improving stable transformation efficiency.

Purpose of the Study:

• To develop a high-throughput Agrobacterium-mediated transient gene expression system for switchgrass.
• To identify optimal Agrobacterium strains and conditions for switchgrass seedling inoculation.
• To increase the efficiency of gene delivery in planta for switchgrass.

Main Methods:

• In planta inoculation of germinating switchgrass seedlings using Agrobacterium.
• Comparison of four Agrobacterium strains, with AGL1 identified as most infective.
• Evaluation of various wounding pretreatments (sonication, carborundum, centrifugation, vacuum infiltration, heat shock) and chemical additives (L-cysteine, dithiothreitol, acetosyringone, surfactants).

Main Results:

• Agrobacterium strain AGL1 demonstrated superior infectivity in switchgrass seedlings.
• Wounding pretreatments significantly increased transient reporter gene (GUSPlus) expression.
• The addition of L-cysteine and dithiothreitol with acetosyringone boosted GUS expression, while surfactants reduced it.
• Peak GUS enzyme activity was observed 3-5 days post-cocultivation, depending on the plasmid used.

Conclusions:

• Agrobacterium strain AGL1 is more effective for switchgrass seedling transformation than strains C58, GV3101, and EHA105.
• Multiple wounding treatments combined with thiol compounds (L-cysteine, dithiothreitol) and acetosyringone markedly increased transient expression from 6% to 54%.
• Different plasmids (pCambia1305.1 and pCambia1305.2) influenced the temporal expression of the GUS gene.