Plant transformation vector

A custom DNA plasmid sequence can be created and replicated in more than one way, but all methods generally share the following processes.

Plant transformation using plasmids begins with the propagation of the binary vector in E. coli. When the bacterial culture reaches the appropriate density, the binary vector is isolated and purified. Then, a foreign gene can be introduced. The engineered binary vector, including the foreign gene, is re-introduced in E. coli for amplification.

The engineered binary factor is isolated from E. coli and is introduced into Agrobacteria containing a modified (relatively small) Ti plasmid. This engineered Agrobacteria can be used to infect plant cells. The T-DNA containing the foreign gene gets inserted into a plant cell genome. In each infected cell, the T-DNA gets integrated at a different site in the genome.

The entire plant will regenerate from a single transformed cell, which results in an organism with transformation DNA integrated identically across all cells.

A selector gene can be used to distinguish successfully genetically modified cells from unmodified ones. A selector gene is integrated into the plasmid together with the desired target gene and provides the cells with resistance to an antibiotic, such as kanamycin, ampicillin, spectinomycin or tetracycline. The desired cells (along with any other organisms growing within the culture) can be treated with an antibiotic allowing only the modified cells to survive. The antibiotic gene is not usually transferred to the plant cell but instead remains within the bacterial cell.

Plasmids replicate to produce many plasmid molecules in each host bacterial cell. The number of copies of each plasmid in a bacterial cell is determined by the replication origin, the position within the plasmids molecule where DNA replication is initiated. Most binary vectors have a higher number of plasmid copies when they replicate in E. coli, the plasmid copy-number is usually lesser when the plasmid is resident within Agrobacterium tumefaciens. Plasmids can also be replicated using the polymerase chain reaction (PCR).

T-DNA contains two types of genes: the oncogenic genes encoding for enzymes involved in the synthesis of auxins and cytokinins and responsible for tumor formation; and the genes encoding for the synthesis of opines. These compounds, produced by condensation between amino acids and sugars, are synthesized and excreted by the crown gall cells and consumed by A. tumefaciens as carbon and nitrogen sources.

Outside the T-DNA, are located the genes for the opine catabolism, the genes involved in the process of T-DNA transfer from the bacterium to the plant cell, and the genes involved in bacterium-bacterium plasmid conjugative transfer. (Hooykaas and Schilperoort, 1992; Zupan and Zambrysky, 1995). The T-DNA fragment is flanked by 25-bp direct repeats, which act as a cis-element signal for the transfer apparatus. The process of T-DNA transfer is mediated by the cooperative action of proteins encoded by genes determined in the Ti plasmid virulence region (vir genes) and in the bacterial chromosome. The Ti plasmid also contains the genes for opine catabolism produced by the crown gall cells and regions for conjugative transfer and for its own integrity and stability. The 30 kb virulence (vir) region is a regulon organized in six operons that are essential for the T-DNA transfer (virA, virB, virD, and virG) or for the increasing of transfer efficiency (virC and virE) (Hooykaas and Schilperoort, 1992; Zupan and Zambryski, 1995, Jeon et al., 1998). Different chromosomal-determined genetic elements have shown their functional role in the attachment of A. tumefaciens to the plant cell and bacterial colonization: the loci chvA and chvB, involved in the synthesis and excretion of the b -1,2 glucan (Cangelosi et al., 1989) the chvE required for the sugar enhancement of vir genes induction and bacterial chemotaxis (Ankenbauer et al., 1990, Cangelosi et al., 1990, 1991) the cell locus, responsible for the synthesis of cellulose fibrils (Matthysse 1983); the pscA (exoC) locus playing its role in the synthesis of both cyclic glucan and acid succinoglycan (Cangelosi et at. 1987, 1991) and the att locus, which is involved in the cell surface proteins (Matthysse, 1987).

• Technical Focus:a guide to Agrobacterium binary Ti vectors Trends in Plant Science 5(10): 446-451 2000