An Improved Procedure for Agrobacterium-Mediated Transformation of ‘Carrizo’ Citrange

Abstract

Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of Agrobacterium cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock ‘Carrizo’ citrange [Citrus sinensis (L.) Osb. × Poncirius trifoliata (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating Agrobacterium with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.

CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane

“…Citrus species can be transformed through different techniques ( Conti et al., 2021 ) ( Figure 3 ), although Agrobacterium -mediated gene transfer is the most commonly used transformation method ( Boscariol et al., 2006 ; Dutt and Grosser, 2009 ; Caserta et al., 2014 ; Souza-Neto et al., 2022 ). Different explants can be used to transform citrus species, mostly obtained from juvenile tissues, such as epicotyls and embryogenic cells, besides protoplasts ( Omar et al., 2007 ; Dutt et al., 2020 ; Li et al., 2022 ; Mahmoud et al., 2022 ; Su et al., 2023 ). However, mature tissues are also possible sources of explants for citrus genetic engineering ( Almeida et al., 2003 ; Peng et al., 2019 ).…”

“…However, mature tissues are also possible sources of explants for citrus genetic engineering ( Almeida et al., 2003 ; Peng et al., 2019 ). Li et al. (2022) have described the transformation of epicotyls in Carrizo citrange, whose explants were pre-treated with cytokinin (6-benzylaminopurine) and auxins (2,4-dichlorophenoxyacetic acid and 1-naphthaleneacetic acid), thus increasing the transformation efficiency from 11.5% to 31.8%.…”

Inhibition of Agrobacterium tumefaciens Growth and Biofilm Formation by Tannic Acid

“…Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium that thrives in the rhizosphere and readily forms biofilms and thus colonizes plant surfaces [ 3 ]. A. tumefaciens exerts its pathogenicity by transferring T-DNA from its tumor-inducing plasmid to host plant cells [ 4 , 5 , 6 ]. Studies have established that various strains of the genus A. tumefaciens are responsible for the neoplastic condition of plants referred to as crown gall [ 7 ].…”