gene_transfer

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Gene Transfer Techniques in Plants

by Nicole Jovanovic

Have you ever wanted to make apples that repel worms? Well, you might be able to with today’s transgenic technology. Today’s transgenic technology involves gene transfer in plants, as well as bacteria and animals.

First of all, to do genetic engineering, you need a genetic vector. A genetic vector is something used to transport genes into the new host. There are a few things a vector needs to be a good vector. First, a vector needs the DNA sequence that makes sure it gets copied when new cells are made. It also needs to have a way for new DNA to be inserted into it. Then, it needs a reporter gene so scientists can tell whether or not the set of genes has made it into a cell or not. Finally, it needs to be smaller than the new host’s chromosome to get it through a plant cell’s cell wall. This is where bacteria come into the picture. The reasons they are used are that they are easy

to grow, they are easy to manipulate, their molecular biology is known, they have lots of reporter genes, and they have plasmids (Purves 322). The plasmid is a mini-chromosome for the bacterium, as opposed to the main chromosome, which is larger, though both are rings (Purves

267, 322). The plasmid has all four qualities mentioned above. The plasmid makes a good vector (Purves 322).

You are probably wondering how new DNA is inserted

into a plasmid. Well, they are first cut into strips using chemicals called restriction enzymes (Lurquin 44). These chemicals cut DNA at special base pair sequences called recognition sites. Eco RI is a common restriction enzyme (Lurquin 47). When the DNA is cut, tabs called “sticky ends” are left hanging, and new DNA, also with sticky ends, is added and sealed with ligase (Lurquin 47, Purves 322). The result is called recombinant DNA. If you put recombinant DNA into a cell, you will have transfected that cell.

Once you have the new DNA you want in a plasmid, you will need to find a way to get the plasmid back into a bacterium. One way to do this would be to use the Diplococcus pneumoniae bacterium, which naturally absorbs DNA (Lurquin 42). Another way would be to use the calcium chloride heat shock treatment. To do this, heat an Escherichia coli bacterium to 41°Celsius in a certain calcium chloride treatment. Then it also will absorb DNA.

Now that we have covered the main ways to transfect bacteria, we should probably start covering the ways to transfect plants. One way to do this would be to use the

Agrobacterium tumefaciens bacterium. This bacterium is used because it naturally transfects plant cells with its DNA. To use this to your advantage, take the part of the bacterium’s plasmid that will transfect the cell, and put

in up to 10,000 new base pairs of DNA (Lurquin 85, Purves 322). Then, you cut some pieces of leaf, and incubate them with the transgenic A. tumefaciens bacteria for a few hours. Then you can put the transgenic leaf pieces in growth regenerator chemicals to form transgenic plants (Lurquin 85).

Though the plasmid method of vectoring works well, there is a limit of 10,000 on how many base pairs can be inserted. Viruses, though, have a couple of advantages over plasmids in bacteria. One is that in some, you can add up

to 20,000 base pairs of DNA. Another is that all viruses infect cells naturally. Now, viruses are pretty good vectors, but vectoring technology gets even better, with artificial plasmids as vectors. These artificial chromosomes can hold 50,000 to 1.5 million base pairs, and can transfect most cells (Purves 322, 323).

There are also ways to transfect cells without using vectors. One is the protoplast method. This method involves dissolving the cell wall of the plant cell, and leaving the rest of the cell in the membrane, as the protoplast. Then,

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