Plant genetic engineering or the introduction of foreign genes into plants has revolutionized the study of plant biology and provided a new avenue for the production of agriculturally superior crop plants. Crops resistant to herbicides, pathogens and insect pests have been produced and the possibilities at present are endless, limited only by our knowledge of how plants function (Gasser and Fraley, 1989). The Agrobacterium tumefaciens system of DNA introduction (transformation) is a vector system that can be used for plant genetic engineering. A. tumefaciens, a pathogen of dicotyleonous plants, is a natural genetic engineer. A. tumefaciens, normally infects wounded plant cells and causes tumors or galls to form. During the infection process a region of A. tumefaciens DNA called the T-DNA (transfer DNA) is transferred into the host plant cells where it becomes integrated into the chromosomes. This mobilization of the T-DNA requires the trans-acting products of the virulence (vir) genes. Both the vir genes and the T-DNA are normally borne on a large plasmid called the tumor-inducing or Ti plasmid. The T-DNA is flanked by 25 nucleotide direct repeat sequences that are involved in excision. Between these T-DNA border sequences lie genes which encode enzymes required for the biosynthesis of auxin and cytokinin plant hormones, as well as genes for the biosynthesis and utilization of amino acid/sugar derivatives called opines. After excision, the T-DNA is transferred into the host plant cells where it becomes integrated at random into the chromosomes. Integration and expression of the T-DNA borne genes results in a hormone imbalance within the plant cells and leads to uncontrolled growth and the production of a crown gall or tumor. The transformed plant cells also produce opines and the enzymes needed for the bacteria to utilize these compounds as a food source (Hooykass and Schilperoort, 1992).
Fortunately it has been possible to produce disarmed strains of A. tumefaciens, which can still transfer their T-DNA to plant cells, but no longer induce tumors. These disarmed strains make ideal vectors for plant tranformation. Foreign genes can be inserted between T-DNA border sequences and introduced into such disarmed strains. The engineered Agrobacteria are then incubated with plant cells or wounded plant tissues. After a short time the T-DNA becomes stably integrated into some of the plant cells. These transformed cells, because they are totipotent, can then be regenerated to yield an entire plant that harbors the "new" gene in all of its cells.
In this experiment we will be using a binary vector system to transform tobacco. In such a system a T-DNA, which lacks the phytohormone genes but contains the genes to be transferred, is carried on one plasmid (pBI121 in our case) while all of the other required functions (e.g. mobilization genes) are carried on a helper plasmid. You will use an engineered disarmed A. tumefaciens strain (LBA4404/pBI121) to introduce two new genes into cells of the tobacco plant, Nicotiana tabacum. The bacterial strain we will use as our “engineer”, LBA4404/pBI121 is a nonvirulent strain which carries a T-DNA on the small plasmid, pBI121. The T-DNA of pBI121 contains two foreign genes that can be expressed in plants. One, the NPTII gene, encodes neomycin phosphotransferase, an enzyme that breaks down the antibiotic kanamycin. Cells transformed with this gene are resistant to the antibiotic kanamycin. The NPTII gene will serve as our selectable marker. Only the transformed plant cells will grow on plates that contain kanamycin. The other gene encodes an E. coli enzyme, β-glucuronidase (GUS). The GUS gene is under the control of the strong constitutive 35S promoter of the cauliflower mosaic virus.
The T-DNA region of pBI121
At the end of the experiment you will use two methods to measure GUS activity in your transformed plants; one a traditional enzyme assay, and the other, a histochemical stain. The presence of GUS activity will be positive evidence that the plants have been transformed since plant cells do not normally contain this activity. You will also test the putative transformants and untransformed controls for the presence of the introduced GUS gene using the polymerase chain reaction (PCR).