Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis

Abstract

Highly efficient genetic transformation technology is beneficial for plant gene functional research and molecular improvement breeding. However, the most commonly used Agrobacterium tumefaciens-mediated genetic transformation technology is time-consuming and recalcitrant for some woody plants such as citrus, hampering the high-throughput functional analysis of citrus genes. Thus, we dedicated to develop a rapid, simple, and highly efficient hairy root transformation system induced by Agrobacterium rhizogenes to analyze citrus gene function. In this report, a rapid, universal, and highly efficient hairy root transformation system in citrus seeds was described. Only 15 days were required for the entire workflow and the system was applicable for various citrus genotypes, with a maximum transformation frequency of 96.1%. After optimization, the transformation frequency of Citrus sinensis, which shows the lowest transformation frequency of 52.3% among four citrus genotypes initially, was increased to 71.4% successfully. To test the applicability of the hairy roots transformation system for gene functional analysis of citrus genes, we evaluated the subcellular localization, gene overexpression and gene editing in transformed hairy roots. Compared with the traditional transient transformation system performed in tobacco leaves, the transgenic citrus hairy roots displayed a more clear and specific subcellular fluorescence localization. Transcript levels of genes were significantly increased in overexpressing transgenic citrus hairy roots as compared with wild-type (WT). Additionally, hairy root transformation system in citrus seeds was successful in obtaining transformants with knocked out targets, indicating that the Agrobacterium rhizogenes-mediated transformation enables the CRISPR/Cas9-mediated gene editing. In summary, we established a highly efficient genetic transformation technology with non-tissue-culture in citrus that can be used for functional analysis such as protein subcellular localization, gene overexpression and gene editing. Since the material used for genetic transformation are roots protruding out of citrus seeds, the process of planting seedlings prior to transformation of conventional tissue culture or non-tissue-culture was eliminated, and the experimental time was greatly reduced. We anticipate that this genetic transformation technology will be a valuable tool for routine research of citrus genes in the future.

Keywords: Agrobacterium rhizogenes; citrus; gene editing; genetic transformation; subcellular localization.

Figure 1

Workflow of the hairy root transformation for citrus by using seeds as explants.

Figure 2

A rhizogenes-mediated hairy root transformation in citrus seeds. (A) Schematic diagram of the germination process of seeds from four citrus genotypes, namely C sinensis, P. trifoliata, C limon and C grandis. (B) Representative images of WT and transgenic hairy roots (TG) at 15 or 30 days post transformation. The scale bar indicates 1 cm.

Figure 3

Transformation frequency of A. rhizogenes-mediated hairy root transformation in C. sinensis seeds with different AS concentration and Agrobacterium concentration selections. Different letters on top of the bars indicate significant differences among groups based on a Tukey’s test (p<0.05).

Figure 4

Protein subcellular localization with C sinensis hairy roots and tobacco leaves. GFP expression and subcellular localization with C sinensis hairy roots [left, (A)] and tobacco leaves [right, (B)] severally. Three citrus endogenous proteins, CsWRKY7, CitSWEET6, and CitF3’H, each fused with GFP protein were employed as the nucleus marker, plasma membrane marker and ER marker, respectively. Fluorescence was detected by a confocal laser scanning microscope. The scale bars of C sinensis hairy roots and tobacco leaves represent 20 µm or 50 µm, respectively.

Figure 5

Positive identification and expression analysis of A rhizogenes-mediated overexpressing hairy roots. (A) Green fluorescence signal visualized in transgenic overexpressing roots (OE) of C sinensis. (B) Genomic PCR identification of the regenerated hairy roots using NPTII-specific primers (upper panel), GFP-specific primers (middle panel) and CaMV 35S/CsWRKY17 primers (lower panel). +: plasmid (used as a positive control); -: ddH₂O; WT: wild-type, 1-20: transgenic lines of hairy roots. (C) Expression levels of CsWRKY17 in WT and ten randomly selected transgenic hairy root lines. The scale bar indicates 1 cm.

Figure 6

A rhizogenes-mediated gene editing in citrus seeds. (A) Gene structure and selected gRNA targets of CsPP2-1. Orange texts indicate gRNAs and blue texts indicate PAMs. (B) Green fluorescence signal visualized in transgenic citrus roots. (C) Genomic PCR identification of the regenerated hairy roots using Cas9-specific primers (upper panel) and GFP-specific primers (lower panel). +: plasmid (used as a positive control); -: ddH₂O; WT: wild-type, 1-8/1-7: transgenic lines of hairy roots. (D) Alignment of the gene-edited sequence of transgenic hairy roots with the gRNA-targeted loci. Orange nucleotides indicate gRNAs and blue nucleotides indicate PAMs. The scale bar indicates 1 cm.