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

Abstract

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 transgene expression in these organs.

Conclusion: The '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.

Figure 1

Tissues of the ‘x11’ sweet orange plant #1 transformed with pCAMBIA2301 expressing uidA gene. A) cross-section of stem segment; B) floral pedicel; C) flower after anthesis; D ) middle section of the fruit; E) transgenic ‘x11’ sweet orange plant event #1 at the flowering stage, three months after acclimatization; F) transgenic ‘x11’ sweet orange plant with mature fruits, approximately 14 months after acclimatization.

Figure 2

Relative expression of the nptII gene. Relative expression of the nptII gene in control and in transgenic plants #1, #2, #3, #4 and #5, transformed with pCAMBIA2301, in relation to the gene encoding Eukaryotic Translation Initiation Factor 5A (IF5A) used as a reference gene.