Citrus Cell Suspension Culture Establishment, Maintenance, Efficient Transformation and Regeneration to Complete Transgenic Plant

Abstract

Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, "Sweet orange"-Egyptian cultivar (Citrus sinensis), "Shatangju" (Citrus reticulata) and "W. Murcott" (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli were also investigated. The result showed that the ovules' age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in maintaining the vigor and regeneration potential of the cells.

Keywords: Citrus; cell suspension; de novo organogenesis; genetic engineering; tissue culture.

Figure 1

Somatic embryo induction.Data were recorded after 6–8 weeks of incubation. The statistical difference (p ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using Statistical Package for the Social Sciences (SPSS-version 23), and results were expressed as mean ± standard error of three independent experiments. (A) Culture media effect, all explants on the individual medium were considered as one group irrespective of genotypes (B) Genotypes effect, all explants of the individual genotype on all three media were considered as one group.

Figure 2

Effect of ovule age on embryonic callus induction. Data were recorded after 6–8 weeks of incubation. All explants of the individual age group (i.e., 4 to 6) from all three cultivars on all three media were considered as one group. The statistical difference (p ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using SPSS (version 23), and results were expressed as mean ± standard error of three independent experiments.

Figure 3

Effects of inoculation volume of suspension cells and subculture intervals on embryo production. The suspension cells were subcultured in H+H medium, and then the embryos were produced on EME-malt medium. The embryos were counted after 10 to 12 weeks of incubation on an EME-malt medium. All embryos produced from all genotypes of an individual subculture interval (i.e., 2 W) of the same inoculation volume (i.e., 1 mL) were considered as one group. The statistical difference (p ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using SPSS (version 23), and results were expressed as mean ± standard error of three independent experiments.

Figure 4

Embryonic callus induction, somatic embryo production, suspension cell culture establishment and plant regeneration of the “Sweet orange” (Citrus sinensis) cultivar. (Aa) Embryonic callus induction from 8 weeks old ovule on EME medium (Ab) and somatic embryo development, (B) Suspension cell culture establishment in an H+H medium, (C,D) Callus formation and embryo germination from suspension cell culture on an EME malt medium (the bars represent 0.5 mm), (E) Axis elongation of germinated embryos on an B+ medium, (F) Plants on RMAN rooting medium for root induction, (G) In vitro shoot grafted rootstock plant and (H) In vitro rooted plant transplanted on the soil. The bars represent 1 cm, except C and D.

Figure 5

Gel electrophoresis of PCR amplified DNA from transgenic plants. 2000 kb DNA ladder (M), transgenic plants samples (1–5), positive control (+), negative control (−). (A) PCR amplicon (1139 bp) from CaMV35S forward and CsDMR6 reverse primers.(B) PCR amplicon (429 bp) from Bar (bacterial bialaphos resistance gene) forward and reverse primers. (C) T-DNA constructs showing corresponding primer sites.

Figure 6

RT–PCR-mediated expression level analysis of CsDMR6 in transgenic plants. Results were expressed as mean ± standard error of three independent experiments.