Weeding the Gems

Arabidopsis thaliana is a ‘model plant,’ which essentially means that researchers work on it because it is easy to work on.

You can read more about the history of A. thaliana as a model plant in this post.

It was initially a good candidate for a model plant because it has a short six-week life cycle; each plant produces very many seeds; it is easy to grow; and, importantly, A. tumefaciens is able to infect it (the reason that is important will become clear as you read on). Today, A. thaliana is a good research subject due to the treasure trove of resources, from lab protocols to bioinformatics databases, built for it. This guide is an introduction to the basics of A. thaliana research.

Plant research often involves changing the expression of a gene or number of genes. Scientists can approach molecular biology like a lucky dip, using mutagens to alter a plants’ DNA, growing whatever beneficial plant line is generated, and throwing away anything useless. Mutagens like X-rays or ethylmethane sulfonate can be used for this kind of random mutagenesis. The resulting plant is different from the original but is not transgenic – no foreign DNA has been inserted into the plant. Plant breeders have used this technique for over 80 years.

A transgenic organism has a bit of DNA from another organism in its genome. This could be a whole gene, for example putting the gene for human insulin into yeast. Foreign DNA can be used in a number of ways to suppress a gene’s expression. Gene expression can also be increased or even turned on under specific circumstances by using foreign promoters.

Making a transgenic plant for research

1. Put the DNA into a plasmid: The piece of DNA that you want to insert into the plant, which might be a whole or partial gene, promotor, or other piece of DNA, must first be ligated into a plasmid, which can be bought from suppliers. There are many ways of doing this, but they all involve cutting the plasmid and insert so that their ends compliment each other and can be ligated together.

2. Plasmid into E. coli: The plasmid is then inserted into an E. coli host, as this is the easiest way of bulking up the plasmid. E. coli has a reproductive cycle of about 20 minutes, so just leaving the culture overnight gives you more than enough plasmids to work with.

3. Testing for successful transformation: Transforming cells, whether they be E. coli or another organism, is not a perfect process so the cells must be tested. For this reason, the plasmid containing the DNA to be inserted into the plant also contains an antibiotic resistance gene. The transformed E. coli cells are spread on agar plates containing the antibiotic, so untransformed cells die. Colonies are picked from the plates and grown in liquid culture for the next stage of the protocol.

4. Extracting and testing the plasmid: The plasmid is extracted from the cells (this is called a plasmid prep) and the presence of the insert may be confirmed by PCR or restriction digests – the sequence of both the plasmid and the insert are known, so the researcher can see if the DNA fragments are the right size when they are run on an agarose gel.

5. Plasmid into Agrobacterium tumefaciens: Agrobacterium strains used in labs contain a Ti helper plasmid, which contains the genes necessary for the Agrobacterium to insert the second ‘binary’ plasmid, which contains the DNA that will go into the plant genome, into the plant cells. Agrobacterium are more difficult to grow than E. coli, which is why the plasmid is only inserted into Agrobacterium at this point. The transformed Agrobacterium are selected as E. coli was, and the liquid culture is used to transform the plant.

6. Transforming Arabidopsis: Agrobacterium tumefaciens has all the equipment to insert the gene into Arabidopsis – the cells just need to be brought together. This is done by dipping Arabidopsis flowers into the transformed A. tumefaciens culture so that the ovules are exposed to the bacteria. The whole plant is then bagged so that the atmosphere around the flowers is humid, encouraging A. tumefaciens growth. When the seeds are made and ripened, they are harvested. A. tumefaciens cannot be used to transform all plant species, which is one of the reasons A. thaliana is a good plant to work on.

7. Testing the transformed plants: In the same way as the bacteria were tested, the plant transformation is now tested by sowing the seeds on antibiotic plates, as both the antibiotic resistant gene and the required DNA should have been inserted into the genome. It is useful to know if the insert has gone into one or both copies of the gene. It is better to work with homozygous plants and be sure that the effect of transgenesis will be expressed and continued to the next generation. This is done by PCR, and usually the DNA fragment is sequenced to be quite sure the insert is present and correct. DNA extraction can be done with a commercial kit, or with common laboratory solvents. DNA extracted using a kit is usually of high quality, but it is difficult to extract large amounts.

There is a good step-by-step guide to transforming Arabidopsis here as well: http://www.unc.edu/depts/our/hhmi/hhmi-ft_learning_modules/plantmodule/transgenicplants.htm

Image credits: Arabidopsis thaliana by Emmanuel Boutet via Wikimedia. Empty Erenmeyer Flask and Beaker Outline by Pray_For_Eliza; Red Petri Dish by nazanin; Thaliana Flower by gringer, all via Clker.com. Plasmid and ligation by me.