Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus

Abstract

Precocious trifoliate orange (Poncirus trifoliata [L.] Raf), an extremely early flowering mutant of P. trifoliata, is an attractive model for functional genomics research in Citrus. A procedure for efficient regeneration and transformation of this genotype was developed by using green fluorescent protein (GFP) gene as visual marker and etiolated stem segments as explants. In vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic shoots and escapes. Transformation efficiency was 20.7% and the transformants were identified by polymerase chain reaction (PCR) and Southern blot analysis. Moreover, the transgenic lines expressed variable amounts of the GFP gene as revealed by real-time PCR analysis. Fifteen transgenic plants flowered 18 months after transfer to the greenhouse and six of them set fruits. GFP expression was also observed in the transgenic flowers and fruits. To test the utility of this system for functional genomics studies, an Arabidopsis thaliana MAC12.2 gene with the potential to produce seedless fruits was introduced into this genotype, and the traits of the transgenic fruits were characterized. The successful transformation of this perennial woody genotype with extremely short juvenility will allow us to test the function of cloned genes in citrus, the improvement of which is hindered by a long juvenility period.

Regeneration and characterization of transgenic kumquat plants containing the Arabidopsis APETALA1 gene

“…Compared with other Citrus species, this transformation efficiency was higher than that of grapefruit (3.6%) (Yang et al 2000), equivalent to that of Mexican lime (3.5-4.6%) (Peña et al 1997, Domínguez et al 2000, and was lower than that of sweet orange (7.6-15.0%) and precocious trifoliate orange (20.7%) (Peña et al 1995, Mendes et al 2002Tan et al 2009). In kumquat transformation, one possible obstacle leading to low transformation efficiency, is the high frequent regeneration of escapes, and another potential obstacle was the high frequency of chimeras as revealed by GUS analyses.…”

“…In our study, that an elevated concentration (100 mg/l) of Km during development of resistant kumquat shoots effectively inhibited the elongation of non-transformed shoots was also observed. Herein, it was suggested that genotype rather than other factors accounted for low efficiency since much higher transformation efficiency was achieved with another citrus genotypes like precocious trifoliate orange, under the same transformation and regeneration conditions (Tan et al 2009). In transformation of 'Meiwa' kumquat, seedling age of explants significantly affected transformation efficiency.…”

“…Since Citrus trees have a long juvenile phase that delays reproductive development by between 6 and 20 years, depending on the species (Peña et al 2001), to expedite transgenic breeding process, several strategies were applied in citrus transformation: (1) over-expression of flowering meristem identity genes in juvenile tissues leading to a shorter generation time (Peña et al 2001;Endo et al 2005;Cervera et al 2009); (2) direct transformation of mature tissues (Almeida et al 2003;Cervera et al 1998Cervera et al , 2008; and (3) exploiting and using short juvenile genotypes for transformation (Tan et al 2009). The third strategy is specially convenient and helpful for citrus functional Yan-Xin Duan and Jing Fan contributed equally to this research.…”

Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy

“…One of the advantages of using reporter marker genes, like Escherichia coli uidA or Aequorea victoria gfp gene, is to discriminate transgenic events by positive reporter expression in recovery lines (Saika and Toki 2009;Tan et al 2009). Herein, we report a visual selection system for transformed cells using a GFP marker after Agrobacteriummediated transformation in citrus.…”

T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange

“…Four replicates of each reaction were performed. Output data generated by the instrument onboard software Sequence Detector Version1.3.1 (PE Applied Biosystems) were transferred to a custom designed Microsoft Excel macro for analysis (Tan et al 2009). …”