Agrobacterium-mediated plant transformation is typically conducted by inoculating plant tissues with an Agrobacterium suspension containing approximately 108-109 bacteria mL-1, followed by a 2-3-d co-culture period. Use of longer co-culture periods could potentially increase transformation efficiencies by allowing more time for Agrobacterium to interact with plant cells, but bacterial overgrowth is likely to occur, leading to severe tissue browning and reduced transformation and regeneration. Low bacterial inoculum levels were therefore evaluated as a means to reduce the negative outcomes associated with long co-culture. The use of low inoculum bacterial suspensions (approximately 6 × 102 bacteria mL-1) followed by long co-culture (15 d) led to the production of an average of three transformed sunflower shoots per explant while the use of high inoculum (approximately 6 × 108 bacteria mL-1) followed by short co-culture (3 d) led to no transformed shoots. Low inoculum and long co-culture acted synergistically, and both were required for the improvement of sunflower transformation. Gene expression analysis via qRT-PCR showed that genes related to plant defense response were generally expressed at lower levels in the explants treated with low inoculum than those treated with high inoculum during 15 d of co-culture, suggesting that low inoculum reduced the induction of plant defense responses. The use of low inoculum with long co-culture (LI/LC) led to large increases in sunflower transformation efficiency. This method has great potential for improving transformation efficiencies and expanding the types of target tissues amenable for transformation of different plant species.

中文翻译：

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较低的根癌农杆菌接种水平和较长的共培养期会导致植物防御反应降低并增加向日葵（Helianthus annuus L.）的转基因芽产量。


农杆菌介导的植物转化通常通过用含有大约108-109细菌mL-1的农杆菌悬浮液接种植物组织，然后进行2-3天的共培养期来进行。使用更长的共培养时间可能会通过让农杆菌有更多的时间与植物细胞相互作用来提高转化效率，但可能会发生细菌过度生长，导致严重的组织褐变并减少转化和再生。因此，低细菌接种水平被评估为减少与长期共培养相关的负面结果的一种手段。使用低接种量细菌悬浮液（约 6 × 102 细菌 mL-1），然后进行长时间共培养（15 天），导致每个外植体平均产生 3 个转化的向日葵芽，而使用高接种量（约 6 × 108 细菌 mL-1) 随后进行短暂共培养 (3 d)，导致没有转化芽。低接种量和长时间共培养具有协同作用，两者都是向日葵转化改良所必需的。通过 qRT-PCR 进行的基因表达分析显示，在共培养 15 d 期间，与植物防御反应相关的基因在低接种量处理的外植体中的表达水平普遍低于高接种量处理的外植体，这表明低接种量降低了植物防御反应的诱导。植物防御反应。使用低接种量和长时间共培养（LI/LC）导致向日葵转化效率大幅提高。该方法在提高转化效率和扩大适合不同植物物种转化的目标组织类型方面具有巨大潜力。