Novel Agrobacterium fabrum str. 1D1416 for Citrus Transformation

Abstract

Citrus is one of the world's most important and widely produced fruit crops, with over a 100 million metric tons harvested from nearly 10 million hectares in 2023. Challenges in crop maintenance, production, and fruit quality necessitate developing new traits through Agrobacterium-mediated genetic transformation. While a few Agrobacterium strains (EHA105, GV3101, LBA4404) are known to transform citrus, many wild strains remain untested. We screened forty-one wild-type Agrobacterium strains isolated from various woody species and identified five capable of DNA transfer into citrus cells. Strain 1D1416 demonstrated the highest transient transformation frequency in Carrizo epicotyl explants (88%), outperforming the control EHA105 (84%) with comparable shoot regeneration rates (32% and 42%, respectively). Notably, 1D1416 exhibited no overgrowth and had the lowest necrosis and mortality rates in transformed tissues. It efficiently transferred the DsRed gene and induced galls in mature tissues of Mexican lime (70%), lemon (48%), Washington navel orange (25%), and clementine (6%). Genome sequencing of 1D1416 allowed for the disarming of the native T-DNA and addition of GAANTRY technology. This novel strain, combined with an optimized transformation procedure, make it a valuable tool for advancing citrus transformation.

Keywords: Agrobacterium rhizogenes; Agrobacterium tumefaciens; GAANTRY system; citrus.

Figure 1

(A) DsRed expression in Carrizo epicotyls after transformation with different wild-type Agrobacterium strains carrying pCTAGV-KCN3. Transformed explants cultured on kanamycin selection medium. (EHA105 positive control.) Independent groups of cells expressing the DsRed gene are defined and counted as transgenic foci. (B) The effect of surfactant (SF) on the transformation of Carrizo epicotyls with different Agrobacterium strains and the average DsRed expression frequency observed. The negative control included explants put through the transformation process without the presence of agrobacterium. No tissue growth or DsRed expression was observed (not shown). Three replicates were performed; each replicate contained at least 30 explants. Statistics were performed on the average of the three technical replicates. Significant difference (*) between SF-treated and non-treated samples (p < 0.05, student’s t-test). See Supplementary Table S2 for data set.

Figure 2

Tissue response after transformation with different Agrobacterium strains carrying pCTAGV-KCN3. (A) EHA105 (mix of proliferating and brown necrotic tissues). (B) Wild-type 1D1416 (healthy and proliferating tissues). (C) Wild-type 1D1104 (bacterial overgrowth, necrotic and dying tissues). (D) The effect of surfactant (SF) on the mortality rate of transformed Carrizo tissue with different Agrobacterium strains. Three replicates were preformed; each replicate contained at least 30 explants. Statistics were performed on the average of the three technical replicates. Significant difference (*) between SF-treated and non-treated samples (p < 0.05, Student’s t-test). See Supplementary Table S2 for data set.

Figure 3

Gall formation and stable DsRed expression in Carrizo epicotyls transformed with wild-type strains carrying pCTAGV-KCN3. (A) Strain 1D1416. (B) Strain 1D159. (C) The effect of surfactant (SF) on the different Agrobacterium strains’ gall formation and shoot regeneration frequency. Gall formation study contained three technical replicates with at least 30 explants per replicate; shoot formation frequency was assessed in a single replicate study. Statistics were performed on the average of the technical replicates. Significant difference (*) between SF-treated and non-treated samples (p < 0.05, Student’s t-test). See Supplementary Table S2 for data set.

Figure 4

Gall tissue formation and stable DsRed expression in internodal segments of mature citrus tissues transformed with wild-type strain 1D1416/pCTAGV-KCN3. (A) Mexican Lime. (B) Navel Orange. (C) Clementine. (D) Lisbon Lemon 8A. Gall tissue formation and stable DsRed expression in mature citrus tissues transformed with wild-type strain 1D1416. (E) Average DsRed expression and gall formation frequency in internodal segments of mature citrus tissues transformed with wild-type strain Agrobacterium strain 1D1416/pCTAGV-KCN3. See Supplementary Table S2 for the data set.

Figure 5

Shoot regeneration from Carrizo epicotyls transformed with 1D1416/pCTAGV-KCN3. (A) Abnormal shoot bud regenerated from tissue without gall formation, no surfactant (SF) added. (B,C) Normal regenerated transgenic shoots from epicotyl tissues treated with SF on SMM (upper images are from a light microscope and bottom images are captured using a DsRed fluorescent filter). (D) Carrizo shoots isolated from the edge of gall tissue. (E) Isolated Carrizo shoots in rooting medium.

Figure 6

PCR analysis of nptII gene (T-DNA) and nptIII gene (backbone) in gall tissue and regenerated shoots from Carrizo explants transformed with Agrobacterium strain 1D1416/pCTAGV-KCN3. (A) NptII gene (T-DNA). (B) NptIII gene (backbone). Lanes 1–10: gall tissue; lanes 11–20: putative kanamycin-resistant regenerated shoots; lane 21: blank; lane 22; negative control (water); lane 23: blank; lane 24: wild-type non-transformed Carrizo negative control; lane 25: blank; lane 26: Agrobacterium strain 1D1416/pCTAGV-KCN3 positive control. See Supplementary Table S1 for primers used.

Figure 7

PCR analysis of gall tissue and regenerated shoots from Carrizo explants transformed with Agrobacterium strain 1D1416/pCTAGV-KCN3. (A) NptII (Inside pCTAGV-KCN3 T-DNA). (B) C protein (Cpro), (Inside 1D1416 T-DNA, LB). (C) D-Lysopine/D-Octopine dehydrogenase (LOd), (Inside 1D1416 T-DNA, RB). (D) Glycerophosphoryl diester phosphodiesterase (Outside 1D1416 T-DNA, LB). (E) Multi species NAD/NADP Octopine/Nopaline dehydrogenase (NNONd), (Outside 1D1416 T-DNA, RB). Lanes 1–10: Galls; lanes 11–20: putative kanamycin-resistant regenerated shoots; lane 21: blank; lane 22: negative control (water); lane 23: blank; lane 24: wild-type non-transformed Carrizo negative control; lane 25: blank; lane 26: 1D1416/pCTAGV-KCN3 positive control. See Supplementary Table S1 for primers used.