Transformation Of Cell Suspension Cultures Research Articles

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

This manuscript describes a unique protocol for the rapid transformation of Medicago truncatula A17 cell suspension cultures mediated by Agrobacterium tumefaciens. Medicago cells were collected on day 7 of the growth curve, which corresponded to the beginning of the exponential phase. They were then co-cultured with Agrobacterium for 3 days before being spread onto a petri dish with appropriate antibiotic selection. The Receptor Binding Domain of the Spike protein of SARS-CoV-2 was used as a model to develop this protocol. The presence of the transgene was assessed using PCR, and the integrity of the product was evaluated by SDS-PAGE and Western-blotting.

Embryo-specific expression of a visual reporter gene as a selection system for citrus transformation.

The embryo-specific Dc3 gene promoter driving the VvMybA1 anthocyanin regulatory gene was used to develop a visual selection system for the genetic transformation of citrus. Agrobacterium-mediated transformation of cell suspension cultures resulted in the production of purple transgenic somatic embryos that could be easily separated from the green non-transgenic embryos. The somatic embryos produced phenotypically normal plants devoid of any visual purple coloration. These results were also confirmed using protoplast transformation. There was minimal gene expression in unstressed one-year-old transgenic lines. Cold and drought stress did not have any effect on gene expression, while exogenous ABA and NaCl application resulted in a minor change in gene expression in several transgenic lines. When gas exchange was measured in intact leaves, the transgenic lines were similar to controls under the same environment. Our results provide conclusive evidence for the utilization of a plant-derived, embryo-specific visual reporter system for the genetic transformation of citrus. Such a system could aid in the development of an all-plant, consumer-friendly GM citrus tree.

Increasing anthraquinone production by overexpression of 1-deoxy-d-xylulose-5-phosphate synthase in transgenic cell suspension cultures of Morinda citrifolia

A Morinda citrifolia cell line was obtained by overexpresion of 1-deoxy-D: -xylulose 5-phosphate synthase (DXS) from Catharanthus roseus, a key enzyme of the metabolic pathway of anthraquinones (AQs). This cell line increased AQs production by about 24% compared to the control cell line. This transgenic cell line which carries dxs cDNA isolated from Catharanthus roseus, was achieved by direct transformation of cell suspension cultures of M. citrifolia using a hypervirulent Agrobacterium tumefaciens strain. The effects of the overexpression of the dxs gene also resulted in increased levels of dxs mRNA transcripts and DXS activity compared to the control cell line. In addition, total phenolics and phenylalanine ammonia-lyase activity were evaluated and were significantly higher in the transgenic line than in controls.

Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare)

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures, which had been initiated from immature embryo-derived (IED) and microspore-derived (MSD) callus, were co-cultivated with various Agrobacterium tumefaciens strains. The T-DNA vectors contained visually-detectable marker genes (C1/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also drug resistance genes (hph or bar) to facilitate selection of stably transformed cell lines. A set of normal binary vectors in a super-virulent Agrobacterium strain [EHA101(pBECKS)] and also a super-binary vector [LBA4404(pTOK233)] were used in this study. Cells of the suspension cultures which received T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolated which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted in a loss of the capacity to regenerate plants from the transgenic callus lines. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated with DNA which was distinct from that of the original Agrobacterium plasmid. Furthermore, independent transgenic lines showed diverse patterns of hybridising bands. These data suggest that the T-DNA fragment was stably maintained through integration into the genomes of the barley cell lines.