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Making a transgenic plant

This interactive illustrates a technique used by scientists to generate transgenic plants.

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Transcript

1. Designing and making the gene construct

Richard Espley (Plant & Food Research)

When making a transgenic plant, you build what is known as a construct, and that construct contains the gene that you are interested in, the gene you want to test, and then it needs a promoter, and that promoter effectively drives the gene – it makes the gene make messenger RNA, and that gets turned into protein.

So you need a gene, you need a promoter and you need a terminator, and that decides when the gene has stopped transcribing. Apart from that, we put antibiotic-resistant genes in to make it easier for us to select for those plants that have been properly transformed.

2. Introducing the transgene to plants using Agrobacterium

Richard Espley (Plant & Food Research)

In nature, Agrobacterium is able to integrate its own DNA into a plant. That ability we can exploit in the laboratory to move the genes that we are interested in into the plants that we are transforming, and it does this really efficiently.

To transform a leaf with Agrobacterium, first we have to transform our Agrobacterium with a gene that we are interested in coupled to an antibiotic-resistance gene. Then we take our leaf pieces, we damage them – for example, we might cut them – and we introduce the Agrobacterium to those cut leaf pieces.

We leave them just for about 10 minutes, and that’s enough time for the Agrobacterium to integrate its DNA into those exposed cells on the leaf surface. Then we remove those leaf pieces, we let them recover, and then we start growing them on the antibiotic-containing media. The only things that grow on that medium are the transformed cells that contain the antibiotic-resistance gene and our gene of interest.

Agrobacterium in the wild

Agrobacterium tumefaciens is a soil bacterium that can integrate its own DNA into the DNA of plant cells.

T-DNA is the section of Agrobacterium DNA that is transferred into plant cells. Within Agrobacterium, T-DNA is part of a small circular DNA sequence called the Ti plasmid.

To infect a plant, Agrobacterium attaches to a root and makes a transport channel between its own cytoplasm and that of a root cell. A single-stranded piece of T-DNA is released from the Ti plasmid.

The T-DNA passes through the transport channel along with some Agrobacterium proteins.

The T-DNA moves through the plant-cell cytoplasm and into the nucleus.

Finally, the T-DNA is integrated into the plant’s own DNA. The genes on the T-DNA can now be expressed in the plant cell.

Researchers often use Agrobacterium to make transgenic plants in the lab. They replace the Agrobacterium genes on T-DNA with the gene they are studying, and the Agrobacterium integrates the new gene into the plant cell’s DNA.

Biolistics: an alternative method

Richard Espley (Plant & Food Research)

Some plant species are not susceptible to Agrobacterium DNA integration, and in that case, we could use an alternative method for transforming a plant such as biolistics.

Biolistics is like a small gun that shoots tiny gold particles that are coated in DNA into the plant,  and if you shoot enough of these particles into the leaf, for example, of a plant, some of it will get integrated and you get transformed cells.

Acknowledgements
Calvin College, Michigan
Bio-Rad Laboratories Pty Ltd

3. Growing the transgenic plantlets

Richard Espley (Plant & Food Research)

If Agrobacterium is able to successfully transform parts of a leaf, what it’s doing is transforming individual cells in those leaves. These cells will divide and grow and they become a sort of undifferentiated clump of cells growing.

But plants have this amazing ability to effectively recreate themselves – it’s called totipotency – and they can create a whole plant from just one cell.

So what we get is these undifferentiated cells, they start to differentiate, and they start to resemble early leaves and even early stem, and we can help them do that by introducing various hormones. Cytokinin helps the plant grow, the shoot elongate, and auxin will help the roots grow. So we can create a whole plantlet in the lab.

Once we have these little plantlets in the laboratory, we can graft those, for example, with apple, onto an apple rootstock, and from there, they will grow into a tree.

4. Confirming the plants are transgenic

Richard Espley (Plant & Food Research)

When we have transformed a plant, what we need to look at is how successfully that is done. We can analyse gene integration at a very early stage –  even from the plantlet stage, we’d work out where our transgene has been integrated into the genome of, for example, apple.

We now have the whole apple genome so we know pretty much the whole DNA sequence. So we can do a fairly simple procedure called PCR – polymerase chain reaction – to work out where our transgene is in the apple genome.

And we can also work out how many times that transgene has been integrated into the genome – it may be more than once, for example – and we need to know this because it might affect the way the plant performs.

5. What transgenics can tell us

Richard Espley (Plant & Food Research)

The real advantage of using this transformation of plants is so that we can study exactly what individual genes do in a plant. We can take 1 gene and show exactly what it does. We can analyse the tree and how it grows and how efficiently it grows and how tall and how big it grows, so we can predict what would happen, for example, in an orchard situation.

But really importantly, we can look at how it flowers and how much fruit it will produce and ultimately what that fruit will be like, which is what we are really interested in.

So we are using this technology to predict what our breeding programme will eventually produce. Now a breeding programme takes a long time. We can do this transformation relatively quickly and with that knowledge we can look at all those aspects such as how the apple will store, even how it will taste, and we can then predict what the bred apple will be like.

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