High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics

Abstract

Fragaria vesca L., a diploid (2n = 2x = 14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6-7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l(-1) hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14-15 weeks to complete its life cycle in different seasons in the greenhouse.

Mutation inTERMINAL FLOWER1Reverses the Photoperiodic Requirement for Flowering in the Wild StrawberryFragaria vesca

“…Strawberry transformations were performed as described earlier (Oosumi et al, 2006). Plants of the T1 or T0 generation were analyzed in Arabidopsis and F. vesca, respectively.…”

TheFragaria vescaHomolog of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 Represses Flowering and Promotes Vegetative Growth

“…Vectors carrying overexpression and RNAi constructs were electroporated into Agrobacterium tumefaciens strain GV3101 and transformed into SD strawberry and H4 as described previously (Oosumi et al, 2006). Several transgenic lines were generated for both constructs in each genetic background.…”

In vitro REGENERATION OF Fragraria PLANTS

“…It has been proven, that strawberry regeneration rate from stipules is higher than from leaf explants [Że-browska and Hortyński 2002]. In case of studies about Fragaria vesca the most common primordial explant were seeds [El Mansouri et al 1996, Haymes and Davis 1998, Alsheikh et al 2002, Morozova 2002, Zhao et al 2004, Oosumi et al 2006, Yildirim and Turker 2014, Zhang et al 2014.…”

“…Species, which belongs to Rosaceae family take third place in terms of economic among species cultivated in moderate climate zone [Oosumi et al 2006]. World production of strawberries is about 4 500 000 tons per year while leading European producers are Spain (about 290 000 ton/year), Germany (about 156 000 ton/year) and Poland (about 150 000 ton/year) [Sowik et al 2015].…”

“…is heterozygous oktoploid (2n = 8x = 56) [Owen i Miller 1996], differently from the woodland strawberry (Fragaria vesca L), which genome size is much less than Arabidopsis thaliana genome [Oosumi et al 2006]. This is because the woodland strawberry is diploid (2n = 2x = 14) [El Mansouri et al 1996].…”