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.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana

“…It is desirable to monitor the presence of knock-in regularly even among cells that have successfully passed selection and carry a confirmed knock-in, since it is known that cells that produce foreign proteins at a high level tend to be displaced from a heterogeneous culture [36,42]. To reduce the heterogeneity of the cell culture and obtain monoclonal lines with knock-ins, it is necessary to control the cell population for the presence of knock-in, since the heterogeneity of the culture also often leads to loss of cells carrying the transgene [43][44][45] or knock-in. We faced such a problem in our experiment-some of the cell lines with knock-ins were lost during their selection and creation of monoclonal cell lines based on them.…”

Obtaining 2,3-Dihydrobenzofuran and 3-Epilupeol from Ageratina pichinchensis (Kunth) R.King & Ho.Rob. Cell Cultures Grown in Shake Flasks under Photoperiod and Darkness, and Its Scale-Up to an Airlift Bioreactor for Enhanced Production

“…Cell suspension cultures have demonstrated to be suitable for improving the cell growth, producing a higher yield of biomass and not only higher contents but also great variety of bioactive molecules [ 70 , 71 , 72 , 73 , 74 ], when cell cultures are submitted to photoperiod conditions [ 62 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 ]. Similar results have been reported in cell suspension cultures for the production of antioxidant compounds in Melastoma malabathricum [ 84 ], lignans in Linum usitatissimum [ 85 ], and alkaloids in Hyoscyamus muticus [ 86 ], where higher contents were achieved when cultured under photoperiod conditions.…”

A simplified protocol for Agrobacterium-mediated transformation of cell suspension cultures of the model species Medicago truncatula A17

“…These procedures increase the difficulty to transfer the cells in an individualized way (e.g. Wu et al 1998 ; Moniruzzaman et al 2021 ; Badim et al 2022 ).…”