How to make a transgenic plant

A transgenic plant is a plant with an insertion of DNA from another organism. This technology can be used as a tool for researchers to gather evidence about hypotheses (Step 3) and insert useful genes as a means of improving crops (Step 4).

Transgenic plant step 1

1. Isolate DNA that codes for the protein you want to express.
This protein could be anything! In this case we want to express a protein that is found in most normal plants, but isn't made in our mutant plant because of an insertion in the DNA (basically, the cells of the plant can't read the instructions and make the protein because the insertion is in the way). Therefore, we will use a piece of DNA from a normal plant that has the correct set of instructions.

Transgenic plant step 2

2. Insert the DNA into a plasmid.
A plasmid is a piece of DNA that can replicate on its own. Bacteria are used to plasmids and will readily accept them even if they contain foreign DNA.

Transgenic plant step 3

3. Insert the plasmid into bacteria. Grow a large amount of bacteria containing this plasmid.
This isn't just any bacteria! The type of bacteria that researchers use for creating transgenic plants is called Agrobacterium tumefaciens (more on this below!)

Transgenic plant step 4

4. Dip the flowering plant into a large amount of bacteria.
It is important to wait until plants are flowering to dip them because in order to grow new plants with this piece of DNA it is necessary to insert the DNA into the cells that make new plants - the sex cells! Specifically, we want the bacteria to insert DNA into egg cells found at the base of flowers. Remember - in this case we are dipping our mutant dying plant since we want to see if the instructions help "fix" the dying phenotype.

Transgenic plant step 5

5. Give bacteria the opportunity to insert the DNA into the plant cells.
Agrobacterium tumefaciens has the natural ability to insert new DNA into a plant's genome. Click here to read more about how researchers have learned to utilize this natural phenomenon!

Agrobacteria have evolved to be able to transfer part of their DNA into plant cells. The DNA that agrobacteria naturally insert causes changes in a plant that result in it forming a tumor at the infection site (the tumor produces nutrients that the bacteria need). After studying this naturally occuring phenomenon, researchers learned to alter the bacterial plasmid so that it can be used as a tool! The DNA that causes tumors is no longer included in the plasmid that researchers use in the lab - this has been replaced by whatever piece of DNA is to be inserted. The bacteria have special proteins that allow them to evade plant defense systems and insert DNA into a plant's genome.


Transgenic plant step 6

6. Select for plants that have the insertion.
The agrobacteria insert this new DNA into a huge number of cells. Some of these cells (the egg cells, specifically) are the cells that form seeds which will grow into new plants. Therefore, it is this next generation of plants that may contain the insertion! Not all of the offspring will have this insertion, however. In order to only continue studying the plants with the insertion, an extra piece of DNA is included in the plasmid used in the second step. This extra DNA encodes for resistance to a specific antibiotic. Basically, a huge number of seeds will be sown out on either soil or agarose that contains a specific antibiotic and only the seeds that have the DNA encoding resistance to this particular antibiotic (and therefore the seeds that also contain the DNA that we are interested in) will grow!

At this point, we have plants with a specific piece of DNA inserted! Usually we will study the offspring (or offspring's offspring) of this plant in order to make sure that the plant is homozygous for the insertion (has two identical copies of it). If the plant's phenotype (appearance) looks more like a normal plant than a dying plant, then this transgenic plant is evidence that supports our hypothesis that a certain piece of DNA (the DNA that we inserted) encodes a protein that is important for plant immunity!

These particular plants and the protein that we have been studying throughout the research story represent one small piece in the puzzle of plant immunity. The more we can figure out about proteins like this one, the more we can add to global knowledge of plant defense systems and the more we can help solve problems in plant defense (particularly in agriculture!) Continue on to read about the huge impact transgenics have had on agriculture!