How genetic modification is done: 1. Agrobacterium

Genetic modification (GM, genetic engineering) for eukaryotes matters because we humans are eukaryotes and we use eukaryotes. As you know, eukaryotes have nucleated cells.
Among the eukaryotes we eat angiosperms (flowering, fruiting plants), herbivores which eat them, and predators which eat herbivores. We use angiosperms for textiles and buildings. We fight eukaryote pests, some of which attack angiosperms.
The most efficient, therefore the most widely-used method for making GM angiosperms uses modified strains of Agrobacterium tumefaciens. Since bacteria, including this one, are prokaryotes (having no cell nucleus) the Agrobacterium method involves prokaryote GM.
Agrobacterial GM has been used for dicots such as cotton (Gossypium hirsutum) and soya (Glycine max). Dicots are dicotyledenous angiosperms, whose seeds’ starchy endosperm is in two halves.
Agrobacterial GM has been used also for monocots such as rice (Oryza sativa) and maize (corn, Zea mays). Monocots are monocotyledonous angiosperms, whose seeds’ endosperm is single.
As an example of agrobacterial GM, here I describe cotton which has been modified to protect itself from the Old World bollworm (Helicoverpa armigera) by making an insecticide in its tissues.
For now I’ll stop short of discussing controversies around GM. For now.

Pests attacking crops
Some invertebrate pests attack crops. For example, moth larvae called bollworms are pests on cotton (Figure 1).
Bollworms attack leaves and stems but their name derives from their invasion of cotton bolls. Bolls are cotton fruits which open into burs when ready to harvest.

Figure 1. Lepidopteran pest on a dicot.
Photograph shows an Old World bollworm caterpillar on a cotton leaf.
Source:  David McClenaghan via the Commonwealth Scientific and Industrial Research Organization (CSIRO).

A bacterium inserting DNA
GM by the Agrobacterium method exploits a pathogenic bacterium, A. tumefaciens. Wild-type Agrobacterium is a pathogen of dicots, causing uncontrolled growth called a crown gall (Figure 2).



Figure 2. Crown gall caused by wild-type Agrobacterium infection of a dicot.
Photograph shows a kalanchoë (Kalanchoe blossfeldiana) twig after infection by Agrobacterium. A rounded gall is visible.
Source: Photograph by Bhai, used under a Creative Commons licence.

Crown galls, and agrobacterial GM, can occur because Agrobacterium transforms plant cells. That is, this bacterium inserts DNA.
Transfection occurs when T-DNA (transfer DNA) invades the host cell’s nucleus and becomes incorporated into the host genome. T-DNA is carried by the agrobacterial Ti (tumour-inducing) plasmid: a small circular DNA molecule performing transfection.
Ti is not required for agrobacterial survival but it is required for the bacterium’s use in GM. Ti carries vir (virulence) genes which are required for transfection, but which are not transferred to the host cell.

Agrobacterial GM
For agrobacterial GM, Ti is transformed. Genetic engineers replace genes on the plasmid, encoding amino acids called opines which lead to gall formation, by genes of their choice.
Engineers isolate (purify) desirable DNA and use it to make a transgenic construct. In such a construct, the chosen gene is accompanied by promotors (sequences which drive gene expression) and by marker genes which will allow transfected cell lines to be identified.
Ti remains within Agrobacterium while the plasmid is transformed by inserting the transgenic construct. The most efficient method for this transformation of Ti is electroporation, in which bacterial cells in a liquid medium receive an electric shock.
Transformed Agrobacterium is mixed with plant cells to allow transfection (Figure 3). This step is performed in a liquid medium.


Figure 3. Agrobacterial transfection.
Diagram shows an A. tumefaciens cell (A) containing that bacterium’s chromosome (B) and Ti plasmid (C). On the plasmid T-DNA (a), vir (b), the origin of plasmid replication (c) and the gene for opine catabolism (d) are indicated. Locations of T-DNA are shown in sequence 1-6. Diagram also shows a plant cell (D) containing a mitochondrion (E), chloroplast (F) and nucleus (G). One mitochondrion and one choroplast each represents several of those organelles which are found in a plant cell.
Source: Chandres via Wikimedia Commons.

After transfection, cells are spread onto solid selective agar containing the antibiotic to which the marker gene confers resistance. Therefore, cells in which transfection was successful are the only cells which grow into colonies.
Cell colonies are picked off the agar and grown into plants. These transgenic plants are tested for transgene expression in the lab, in the greenhouse and in the field.

Example of agrobacterial GM: Bt cotton

Some agrobacterial GM exploits the soil bacterium Bacillus thuringeiensis (Bt) which makes insecticides in its cells. GM exploiting Bt builds on the use of Bt since the early 20th century as a crop treatment.
Insecticides from Bt are the Cry (crystal toxin) proteins encoded by cry genes. Cry proteins are toxic to specific invertebrates, many of which are pests.
When agrobacterial GM has been used to insert cry into crop plants, these plants make Cry in their tissues. Therefore they are insecticidal:  the Bt crops.
For example, cotton resistant to bollworms (Figure 1) has been made by inserting cry genes into the cotton genome. Such Bt cotton has become important in agriculture (Figure 4).

Cotton bolls

Figure 4. Bt cotton ready to harvest.
Photograph shows healthy cotton burs in a field near Narrabri, New South Wales.
Source: Jane Kahler via CSIRO.

Making the most of Agrobacterium
Some GM crops made by the Agrobacterium method carry multiple inserted genes. These ‘stacked’ genes may include multiple cry genes, providing complementary attacks on the pest, and other useful genes.
Not everybody is impressed by Agrobacterium or by GM in general. As promised at the start of this article, I’ll discuss the controversies in due course.

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Biotechnology in Action

My column Biotechnology in Action will go live soon. I’ll promote each article – on the first day of each calendar month, starting September 2015 – here on WordPress.

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New contact details

Hello after a year’s silence. I don’t expect to write Science on the Land for a while, perhaps nevcr, but soon I’ll be writing on Science 2.0. My new column there will be about applied biotech, working title Biotechnology in Action, under my real name Sam Mason.

If you’re still reading Science on the Land, thank you. Any messages you’ve sent to me here since mid-2014 will have gone to my old Hotmail address but I no longer use that and I’ve lost its password. Oops. Instead I now have another email address, where I’ll see any comment you make to Science on the Land from now on.

See you on Biotechnology in Action if that interests you.

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Neonic makers might pay for research about neonics on the land

Here in Europe, three insect-killing neonicotinoids are under a temporary ban. During the ban our UK Government is welcoming new research. It’s now become known that pesticide manufacturers might fund some of this research. Vested interests!

Last year my fellow blogger manuelinor at Ecology is Not a Dirty Word told us about scientists calling for the land to be rid of neonics. But soon after that science came out, our UK Secretary of State for Environment Food and Rural Affairs (Owen Paterson, at that time) said there wasn’t enough evidence that neonics harm bees. So he assertively refused to vote for or against the ban. One of Mr Paterson’s senior staff, Minister of State David Heath, agreed. Neonics aren’t known to harm bees in the field, said Mr Heath.

I don’t know which of the chemical giants are now offering to pay for science about neonics in the field. But I’m guessing Syngenta and Bayer, who challenged the European ban. Syngenta requested an exemption for autumn-sown oilseed rape (Brassica napus) this year, but soon withdrew that request. Perhaps they think they’ll gain more by paying for evidence.

Perhaps I’m being unfair on Syngenta. They’re proud to support British farmers and they’re behind the international Operation Pollinator.

This is one of the first big issues for Elizabeth Truss, the Secretary of State who took over from Mr Paterson. Will she use her new power to act for transparent research about neonics?

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A neonic that’s bad news for birds

Here in Europe, three insect-killing neonicotinoids are under a temporary ban. One of the banned neonics is called imidacloprid. Six months into the neonic ban, here’s new science about imidacloprid and insect-eating birds in the Netherlands.

The chemical and biotech giant Bayer Cropscience makes imidacloprid. Bayer gave info to the European Food Safety Authority (EFSA) about this particular product when it was licenced in 2009 for use on crops including tomatoes (Solanum lycopersicum), apples (Malus domestica) and sugar beet (Beta vulgaris subspp vulgaris). For follow-up discussions in 2011, Bayer gave info relevant to ‘risk assessment for operators and workers’ and ‘risk to birds and mammals.’ Now, EFSA has been reviewing the evidence about imidacloprid. Here’s the EFSA report published at the start of July 2014. It’s wordy! I’ll pick out some of the words about birds.

‘[R]isk assessments for… use on apple indicated a low risk to birds (including insectivorous birds)… two applications were considered separately… result[ing] in some uncertainty (imidacloprid is known as persistent and mobile in plants)…

‘A low risk was also identified for birds consuming pelleted sugar beet seeds [and for] birds consuming sugar beet seedlings… EFSA could not agree with some of the aspects considered in [some German] assessments, in particular with the approach for the estimation of the residues in insects.’

That was on 2nd July this month. A week later, along came this Dutch report about imidacloprid and insect-eating birds. According to this new science, it’s not good enough to test for acute bird poisoning. Imidacloprid builds up in soil, water and insects. And there aren’t so many insects when this neonic has been used. Many kinds of bird rely on insects as food.

The scientists say, ‘Our results suggest that the impact of neonicotinoids on the natural environment is even more substantial than has recently been reported and is reminiscent of the effects of persistent insecticides in the past*. Future legislation should take into account the potential cascading effects of neonicotinoids on ecosystems.’

I’m grateful to my fellow blogger at The Naturephile for drawing attention to this.

* They don’t name any particular insecticide ‘in the past’ but it doesn’t take a genius to work out that they’re thinking of DDT.

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Hello Ms Truss

Our new UK Secretary of State for Environment, Food and Rural Affairs is Elizabeth Truss. Er… who? She’s been working for improvements to education, very important, yes, but I haven’t seen much to suggest that she knows anything about the land. Where will you lead us, Ms Truss? We’re watching you.

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Goodbye Mr Paterson

Here in Britain, our Secretary of State for Environment Food and Rural Affairs has been Owen Paterson. Not any more! Today, in a Cabinet reshuffle, Mr Paterson’s been sacked.

I wonder who’ll take his place. Whoever that person is, I hope they’ll bring wellies as Mr Paterson didn’t when he visited the flooded areas of Somerset.

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