Abstract
BackgroundThere 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.ResultsHere, 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 transgene expression in these organs.ConclusionThe ‘x11’ sweet orange is suitable for functional genomics studies with a satisfactory transformation rate, and it can be considered a good model for functional genomic studies in commercial sweet oranges, for traits related to flower and fruit.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-511) contains supplementary material, which is available to authorized users.
Highlights
There had been many reports on genetic transformation of Citrus for functional genomic studies but few included genes associated with flower or fruit traits
Genetic transformation of ‘x11’ sweet orange Several experiments were previously conducted to optimize the conditions of genetic transformation of ‘x11’ epicotyl explants, including the determination of 6-benzylaminopurine (BAP) concentration on the regeneration media; kanamycin concentration for selection of transgenic events; and inoculation and co-cultivation conditions (Additional file 1 Table S1)
The best transformation and regeneration conditions for epicotyl segments of ‘x11’ sweet orange tested, resulted in regeneration efficiency of shoots ranging between 1.0 and 3.8 shoots per explant in experiments without co-cultivation with A. tumefaciens, or 0.2 to 0.5 shoot per explant, when explants were co-cultivated in bacterial solution
Summary
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. There is no report about the use of early-flowering sweet orange mutants for functional genomic studies. A major reason for the limited number of reports might derive from the extensive juvenile stage of Citrus plants when regenerated from juvenile explants, which delays the observation of the resulting phenotype
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