|Plant Biotechnology Basics|
A simple explanation of gene transfer in plants.
Generating a whole plant from cells. Most gene transfer work in plants relies on the ability to produce whole plants from either a single cell or a group of cells. For successful production of transgenic plants, the idea is to target DNA into those cells and then recover a whole, genetically engineered plant. Plant regeneration from a single cell occurs naturally through the fertilization of the egg to form the zygote or embryo, which gives rise to the seed. In the laboratory, plant embryo formation and development (embryogenesis) can be induced in many tissues from many types of plants. Laboratory produced artificial embryos are a very suitable target for gene transfer in plants. Plant regeneration from groups of cells also occurs naturally when plants clone themselves by sending off runners, splitting or when certain plants fall down and form roots on various points of the stem. Plant regeneration from groups of cells can also be induced in the laboratory from a variety of different tissues. Shoot formation from groups of cells is called shoot morphogenesis. Shoot-forming cells are most easily targeted for gene transfer before the cells have morphed into shoot tissue. The conditions that are used to induce the formation of artificial embryos or shoots from various plant tissues need to be optimized for each plant and tissue type. Once those conditions are defined, DNA delivery is the next step.
DNA delivery. There are two main methods for gene delivery in plants. Agrobacterium and the particle gun.
Agrobacterium is a naturally-occurring bacterium, which has the unique ability to transfer parts of its own DNA into plant cells. In the wild, transfer of a portion of the bacterial DNA (called T-DNA, for "transferred DNA") causes rapid plant cell division leading to the formation of a tumor. Compounds produced in the plant tumor are used to feed other bacteria in the soil. In the laboratory, the tumor forming genes are removed and other genes are substituted. None of the laboratory strains have the wild genes anymore. For Agrobacterium to transfer part of its DNA into plants, the bacterium is usually inoculated with living, wounded plant tissue. After culturing the bacteria with the plant tissues, antibiotics are supplied, eliminating the bacterium from the plant tissue.
The particle gun is a physical method for DNA delivery. For this method, DNA is coated onto small (<1 Ám), dense (gold or tungsten) particles, which are accelerated towards the target plant tissues. The original particle bombardment device was a modified 22 caliber rifle, using 22 blanks to generate the force needed to propel the particles. Most current bombardment devices use a release of compressed helium gas to accelerate the DNA-coated particles. After the particles pass through the plant cell wall, they enter the cytoplasm and preferably the nucleus, where the DNA comes off of the particles and integrates into the chromosome.
Selection. After DNA delivery, the plant cells are placed on an additional medium, which contains a "selective agent". The selective agent is typically an antibiotic or herbicide, which would normally kill all cells. If the introduced DNA also contains a gene for resistance to the herbicide or antibiotic, only those cells with the resistance gene will survive. After sufficient selection, resistant cells will form tissues, which may be capable of generating whole plants. Whole plants are then generated from the single cell or group of cells.
Analysis. Once tissue or transgenic plants are recovered, they are usually analyzed in great detail. In some cases, if everything is done properly, the foreign DNA is integrated and the gene functions as it was designed. In many cases, the introduced DNA does not function or functions at low levels. To test if the foreign DNA is present, PCR (polymerase chain reaction) is used, which give a plus or minus result (foreign DNA is present or absent). To determine if the DNA has integrated as an intact unit and estimate how many copies of the DNA have been introduced, Southern hybridization analysis is used. To determine if the gene is functioning, Northern analysis is performed. Depending on what genes have been introduced, other analysis are also necessary. Analyses are performed on the original plants and progeny, to determine stability of the introduced gene. For most plants, DNA introduction methods are a fraction of the effort of the analysis. Except for soybean, where consistent recovery of transgenics is still challenging!