An improved procedure for Agrobacterium-mediated transformation of citrus

Authors: LI, YANGJUN - University Of Connecticut; TANG, DAN - University Of Connecticut; Liu, Zongrang; CHEN, JIANJUN - University Of Florida; CHENG, BAOPING - Guangdong Academy Of Agricultural Sciences; LI, YI - University Of Connecticut

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2022
Publication Date: 5/30/2022
Citation: Li, Y., Tang, D., Liu, Z., Chen, J., Cheng, B., Li, Y. 2022. An improved procedure for Agrobacterium-mediated transformation of citrus. Plants. https://doi.org/10.3390/plants11111457.
DOI: https://doi.org/10.3390/plants11111457

Interpretive Summary: The application of gene editing technology for improvement of agronomically important traits in peach, apple and citrus requires development of a highly efficient transformation system. To develop a highly efficient transformation system in citrus, a variety of parameters were tested and optimized. The optimized method can enhance over 400% of transformation frequency compared to the old method, which will be to facilitate gene editing in citrus and other fruit trees.

Technical 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-incubation to enhance transformation efficiency using a commercially used rootstock 'Carrizo' Citrange as a model plant. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone (AS) liquid medium for 6 hours at 25 ' 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 hours at 25' prior to Agrobacterium infection. In the co-cultivation medium, we added 30 µM paclobutrazol (PBZ) and 10 µM lipoic acid (LA). Each of these three treatments significantly increased the efficiencies of transformation. 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 cultivated citrus varieties.