Sep 17, 2007

Research Live

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SiFo project: Transgenic apple varieties - approaches to preventing outcrossing

“We activate a natural defence mechanism and switch off specifically targeted genes.”

It is the beginning of September once again, the official start of the apple harvest in Germany. In Saxony too, Germany’s third largest fruit-growing region with around 3000 hectares, the branches of the apple trees are bowed under the weight of ripe fruit. At the Institute of Fruit Breeding at the Federal Centre for Breeding Research on Cultivated Plants in Dresden-Pillnitz, scientists are producing disease-resistant apple varieties which need spraying less often. Genetic engineering could also speed up the breeding process, but critics remain sceptical. They are concerned that genetically modified DNA will outcross to other plants. A method currently being tested within the area of biological safety research may provide a solution.

Dr Henryk Flachowsky of the Institute of Fruit Breeding at the Federal Centre for Breeding Research on Cultivated Plants (BAZ) in Dresden-Pillnitz is in charge of the SiFo project

On land belonging to BAZ, genetically modified apple plants are growing in a special safety cage, which reproduces field-like conditions.

Transgenic apple trees in the safety cage

Henryk Flachowsky assesses the success of his work: a red-leafed wild species was grafted onto a genetically modified rootstock which blocks the formation of the red pigment. Initially the leaves were indeed green but now they are gradually turning red again.

In apple growing as in apple breeding, scions of the desired variety are grafted onto a rootstock. This is the only way the variety can survive.

Small apple leaves in a bacterial solution: The genetic transformation of the apple plants is done with the aid of a bacterium (Agrobacterium tumefaciens). The genes to be transferred are inserted in the bacterium and the plant material is infected with it.

Leaves infected with Agrobacterium after three days. They are then rinsed with an antibiotic solution to remove A. tumefaciens…

… cut into strips and placed on the selection medium. This nutritive medium contains an antibiotic on which only those cells in which transformation has been successful can survive. This is what the leaf sections on the selection medium look like after four weeks.

Calli or tiny plantlets after eight weeks

Transgenic shoot cluster

In Dresden-Pillnitz resistant apples begin life in a preserving jar. Twelve jars stand before Katrin Winkler on the laboratory counter, while the laboratory technician transplants plants, a task she performs at least every three weeks. She carefully removes an apple shoot just a few centimetres long from the jar with a pair of tweezers, divides it up and places each of the rootless shoots back on fresh, clear culture medium. The young shoots propagated in this way are being used as the starting material for an exciting experiment: The scientists want to manipulate the characteristics of conventional apple plants by grafting them onto a genetically modified rootstock. The advantage is that the apples and pollen will remain GM-free and the risk of outcrossing will be eliminated.

Whether this approach will work in apple-breeding practice as well as in theory is being put to the test on land belonging to the well-established research institute: In a sealed, insect-proof field cage the young genetically modified apple plants from the laboratory are being tested in simulated field conditions. The scientists have grafted some of them onto a genetically modified, red-leafed wild apple species. This was done about a month ago, and now the plants are waist high. Henryk Flachowsky lets the leaves run through his fingers as he explains: “The visual impact is not as strong as we would have hoped, but initial molecular-biological analyses confirm our assumption.” Part of the crown is in fact green, although the grafted variety has red leaves. The reason for this is that the scientists have blocked the activity of a gene involved in the production of the red pigment anthocyan, but only in the rootstock. So how has the signal been transmitted to the conventional upper part of the plant?

RNA interference: activating the plant’s defences

Henryk Flachowsky takes out a sheet of paper and a pen to demonstrate a complex but useful mechanism which won two American scientists the Nobel prize in 2006: RNA interference. “This is what the gene construct looks like which we have inserted into the rootstock cells,” he explains as he sketches the DNA segment in question. Like all DNA sequences, this is expressed in the cell nucleus, converted to single-stranded messenger RNA (mRNA) and transported to the protein factories in the cell as a ‘protein blueprint’. “But our gene construct is designed to produce complementary mRNA sequences, which bind together like a zip to form a double strand,” the scientist goes on to explain and draws a structure resembling a hairpin on the paper, the ‘hairpin loop’. “Double-stranded RNA is not normally found in plant cells – if one does appear in the cell plasma, it is an alarm signal which alerts the cell to the presence of a hostile virus. In this way we can activate a natural defence mechanism and switch off specifically targeted genes – in this case a gene which is involved in the production of the red pigment in the apple plants.”

The main attraction of this approach is that the snippets of RNA affect not just the genetically modified rootstock, but are also transported across the graft union to the non-GM parts of the plant along the nutrient pathways (phloem). This has already been demonstrated in herbaceous plants such as tobacco. Scientists at the Institute of Fruit Breeding now want to find out whether this systemic acquired silencing also works in apples. Blocking pigment synthesis is a means of providing an optical test system, which makes the successful transportation of RNA visible. “At first the effect was very clear, but it has gradually faded over time. We had hoped that the leaves would remain entirely green, but that’s science for you,” explains Flachowsky, who in the next stage is planning to verify the reduced gene activity using molecular-biological and biochemical methods as well, and to test the system on different varieties and rootstocks.

Apple breeding: a lengthy process with an uncertain outcome

In addition to increasing resistance, this method could also be used to force early flowering. “If we manage to manipulate the grafted-on genetically unmodified apple plants so they flower after just one year, that would represent a massive saving in terms of time and money for classic fruit breeding,” Flachowsky points out. In conventional apple breeding, researchers must wait four to five years when the apple trees first bear fruit, before they can see how successful a cross has been. Breeding a new variety can take up to seventy years, if wild apples with small fruit but pronounced resistance are used as parents. “A good fruit grower depends on his predecessors and works for his successors,” comments Flachowsky. The apple diseases firebrand, apple scab and apple powdery mildew, which are difficult to control, are, in his view, a further argument in favour of genetic engineering: “Most consumers have no idea how many times an apple has normally been sprayed with pesticides before it ends up on the shelf.” Even in organic fruit farming, apple diseases are often controlled with large quantities of pesticides containing copper and sulphur. Resistant apple varieties can reduce pesticide use, but classic breeding methods have their limits. New varieties produced by conventional breeding methods are difficult to establish on the market. “Consumers want one apple to look much like another and to still be as fresh as the day it was bought a week later, with no bumps or bruises – not every variety can provide that.”

Genetic engineering as a useful tool

The fact that genetic methods still encounter scepticism is, in Flachowsky’s view an educational problem: “Many consumers would like to be better informed, I have noticed this time and time again. I am often asked to explain what it actually involves.” Many growers, on the other hand, are clearly in favour. They have problems and need solutions. “If we actually manage to transfer species-specific genes from variety A to variety B using a suitably elegant approach, without genetically modifying the product, i.e. the apple, then I think public acceptance will be very high,” says Flachowsky with conviction.