MADS-box genes similar to Arabidopsis thaliana SHORT VEGETATIVE PHASE (SVP) have been implicated in regulation of flowering in annual species and winter dormancy in perennial species. It was demonstrated that viable seeds were produced and that the uidA gene was inherited. To verify that the transgenes could be transferred to offspring, crosses were conducted between three transgenic lines of ‘Victoria Fanny’ and two wild type pollen donors. Integration of the transgenes in the plant genome was confirmed using PCR and Southern blot hybridisation. The highest number of potentially transformed shoots was regenerated after co-cultivation of ‘Victoria Fanny’ leaf explants with LBA 4404. In ‘Victoria Fanny’ shoots were formed faster and without a callus phase after co-cultivation with LBA 4404 or C58. Regeneration of transgenic plants after co-cultivation with A281 was independent of cultivar, and all explants produced callus followed by indirect shoot formation. Three bacterial strains (LBA 4404, A281 and C58) all carrying the binary vector, p35S-GUS-INT, and harbouring the uidA gene coding for β-glucuronidase (GUS) were used. A transformation system for the two cultivars Victoria Fanny and Victoria Jane was developed by co-cultivation of leaf explants with Agrobacterium tumefaciens. Addition of dichlorophenoxyacetic acid (2,4-D) promoted callus formation, but inhibited shoot elongation. Addition of 0.1 mg l−1 indole-3-acetic acid or naphthaleneacetic acid increased the total number of shoots per explant, but not the number of shoots longer than 1 cm. A Murashige and Skoog medium with 1.0 mg l−1 6-benzyladenine induced adventitious shoot formation in 15 out of 19 cultivars. All rights reserved.Ī protocol for adventitious shoot formation in Symphyotrichum novi-belgii was developed after investigating the effects of cultivar and hormone combinations. CRISPR/Cas9‐ mediated mutagenesis of AcCEN4 and AcCEN may be a valuable means to engineer Actinidia amenable for accelerated breeding, indoor farming and cultivation as an annual crop. The number of affected genes and alleles and severity of detected mutations correlated with the precocity and change in plant stature, suggesting that a bi‐allelic mutation of either AcCEN4 or AcCEN may be sufficient for early flowering, whilst mutations affecting both genes further contributed to precocity and enhanced the compact growth habit. Mutation of these genes transformed a climbing woody perennial, which develops axillary inflorescences after many years of juvenility, into a compact plant with rapid terminal flower and fruit development. In this study, CRISPR/Cas9‐ mediated manipulation enabled functional analysis of kiwifruit CEN‐like genes AcCEN4 and AcCEN. Previously, multiple kiwifruit CENTRORADIALIS (CEN)‐like genes have been identified as potential repressors of flowering. Kiwifruit (Actinidia chinensis) is a recently domesticated fruit crop with a short history of breeding and tremendous potential for improvement. More than 700 transgenic plants in four Actinidia species have been produced during the last three years.Īnnualization of woody perennials has the potential to revolutionize the breeding and production of fruit crops and rapidly improve horticultural species. Agrobacterium-mediated transformation using EHA105 is currently being used for high-throughput functional genomic studies in the genus Actinidia. GUS activity was detected in all calli and the transgenic nature of regenerated plants was confirmed by PCR and Southern blot analysis. The highest efficiency was obtained with strain EHA105. However, less than 20% of calli from the virulent A281 strain produced normal shoots and roots. Over 70% of calli derived from the three avirulent strains regenerated shoots and all these shoots initiated roots. Each strain harboured the pART27-10 binary vector which encodes an nptII gene for kanamycin resistance and the uidA gene for histochemical GUS staining. Leaf strips from in vitro grown kiwifruit plants were transformed using three avirulent Agrobacterium tumefaciens strains (GV3101, EHA105 and LBA4404) and one virulent strain (A281). The choice of Agrobacterium strain is one of the key parameters required for development of an efficient Agrobacterium-mediated transformation.
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