2.3 From infected cells to transgenic plants
Unlike the 'natural' infection process, where only the cells at the site of the crown gall are affected by the inserted T-DNA, scientists wanted to introduce new genes into all the cells of the plant. Fortunately, most plant cells are totipotent, which means that any cell from any part of the plant is capable of dividing into cells that can form any or all of the plant's tissues. This means that, using appropriate growth hormones and other tissue culture techniques, a single infected plant cell can be induced to divide and form an entire, new, fertile plant.
In order to produce genetically modified plants, A. tumefaciens carrying the artificial Ti plasmid and helper vector are incubated with plant fragments (explants) for 48 hours. The explants could be leaf discs or cotyledon (seed leaf) slices or root segments. They have a cut surface and the wounded cells produced by the cut are the sites of DNA transfer from A. tumefaciens. The explants are then placed on culture plates containing nutrient medium, kanamycin and a growth regulator to stimulate the division of the cells to produce new plants.
What is the purpose of the kanamycin?
Kanamycin is used to selectively kill plant cells that have not been transformed.
Remember that genetically modified plant cells contain the kanamycin resistance gene, which we introduced as a plant selectable marker gene (Section 2.2 and Figure 5a). This means that modified plant cells can produce an enzyme that breaks down the otherwise toxic kanamycin. Untransformed cells cannot produce the enzyme and are killed.
New tissue develops at the site of wounding on the explants. New shoots that develop from this tissue are separated from the explant and induced to root. The plantlets that develop can be tested for the appropriate phenotype. If the results are positive, the plantlets are allowed to develop into mature plants.
The modified plants initially produced are crossed with established high-yielding varieties, using conventional plant breeding methods. The offspring are repeatedly crossed with the established varieties until a true-breeding transgenic line is produced.
Box 1: The gene gun - biolistics
Transformation via A. tumefaciens has been successfully practiced for many years and is now the main route used to create transgenic dicots. However, until relatively recently, this gene transfer method was thought to be of little use with monocots. This was a problem, because so many of the world's staple crops, like rice and wheat, are monocots. Alternative methods for transformation of plant cells have been developed, and microprojectile bombardment, sometimes referred to as biolistics (ballistics using biological components) is probably the most important. In this technique, a particle gun (or gene gun) literally shoots genes into plant cells. The DNA to be delivered is attached to tiny gold or tungsten balls (1-2 μm in diameter). These are put onto a disk which is placed inside the gene gun. A blast of high pressure gas propels the disk forwards at roughly the same speed as a bullet leaving a rifle. A screen stops the disk and the tiny gold or tungsten balls are launched towards the target cells. The balls penetrate the cell membrane and release the DNA-carrying particles. In a minority of cases, the DNA particles will then be successfully integrated into the host cell's DNA.