Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple

• GL-3, a genotype with high regeneration capacity and sensitive to Agrobacterium, was screened out from the seedling population of apple, and this strategy could be applied in other perennial woody plants.
• GL-3 was compared with its parent Royal Gala on the regeneration capacity, and the data showed that the number of buds per explant of GL-3 was 2.6 fold higher than that of Royal Gala.
• The data of the transient expression of GUS in leaf segments and the percentage of explants with kanamycin-resistant buds showed that GL-3 was sensitive to Agrobacterium.

Agrobacterium-mediated transformation has been the preferred system for transforming apples. Although transgenic lines have been obtained form a number of genotypes, the transformation efficiency was not high. Apple is highly heterozygous genetically, so its seedling populations segregate heavily. We speculate that a genotype with high regeneration capacity and sensitive to Agrobacterium could be screened from the seedling population of apple. In order to verify this hypothesis, we developed in vitro seedling clones of apple (Malus × domestica) and compared their regeneration capacity and susceptibility to Agrobacterium tumefaciens in this study. Royal Gala, a genotype had been widely used in apple transformation, was selected as the source of seeds. First, 10 clones with higher multiplication capacity and normal leaf shape and smooth leaf surface, named GL-1 to GL-10, were screened out from 100 in vitro seedling clones. The capacity of adventitious bud regeneration from leaf segments were compared among the ten clones, and the result showed that GL-3 exhibited extremely higher adventitious regeneration capacity. GL-3 was also compared with its parent on the regeneration capacity, and the data showed that the number of buds per explant of GL-3 was 2.6 fold higher than that of Royal Gala. The susceptibility of GL-3 to Agrobacterium was compared with Royal Gala by monitoring the transient expression of GUS in leaf segments. For GL-3, the percentage of explants with large GUS-positive patches was 46.7 ± 1.1%, which was obviously higher than that (25.1 ± 7.2%) of Royal Gala. After the leaf segments of GL-3 were transformed with A. tumefaciens strain EHA105 carrying the AtmiR156a antisense gene, the percentage of explants with kanamycin-resistant buds reached 27.5%. In conclusion, a genotype with high regeneration capacity and sensitive to Agrobacterium was screened out from the seedling population of apple, and this strategy could be applied in other perennial woody plants.