Early-flowering sweet orange mutant \'x11\' as a model for functional genomic studies of Citrus

Background: There had been many reports on genetic transformation of Citrus for functional genomic studies but
few included genes associated with flower or fruit traits. A major reason for this might derive from the extensive
juvenile stage of Citrus plants when regenerated from juvenile explants (epicotyls, cotyledon or calli), which delays
the observation of the resulting phenotype. Alternatives include the use of explants from adult tissues, which
sometimes may be recalcitrant to regeneration or transformation, or of early-flowering genotypes. However, there is
no report about the use of early-flowering sweet orange mutants for functional genomic studies.
Results: Here, we propose a sweet orange spontaneous early-flowering mutant, named ‘x11’, as a platform for Citrus
functional genomic studies, particularly for genes associated with flower or fruit traits. We report a procedure for
efficient regeneration and transformation using epicotyl segment explants of ‘x11’ and Agrobacterium tumefaciens as
a proof-of-concept. The average transformation efficiency was 18.6%, but reached 29.6% in the best protocol tested.
Among 270 positive shoots, five were in vitro micrografted and acclimatized, followed by evaluation of transgene
expression by quantitative amplification of reversed transcripts (RT-qPCR) and determination of the number of
copies inserted. Four of these plants, containing from one to four copies of the transgene, exhibited the first flowers
within three months after ex vitro establishment, and the other, two months later, regardless of the period of the year.
Flowers of transgenic plants displayed fertile pollen and gynoecium, with self-pollination inducing fruit development
with seeds. Histochemical staining for β-glucuronidase activity using stem segments, flowers and fruits from 5 to
7 month-old acclimatized transgenic plants confirmed the constitutive transge