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Transgenic apple varieties: Approaches to preventing outcrossing – possible effects on micro-organisms

(2005 – 2008) Federal Centre for Breeding Research on Cultivated Plants, Institute of Fruit Breeding, Dresden (since 2008 Julius Kühn Institute (JKI), Federal Research Centre for Cultivated Plants)

Topic

The aim of the first part of this project was to clarify what effect genetically modified apple trees with increased resistance to bacteria can have on plant-associated bacteria.

The second part aimed to look at whether the activity of genes in non-transgenic apple plants can be affected when these plants are grafted onto a transgenic apple rootstock (targeted [[L:6|gene silencing]]). If this mechanism works in fruit trees, this would have the advantage that the reproductive part of the plant would produce non-transgenic pollen, thereby preventing outcrossings of genetically modified DNA.

Effects on micro-organisms

The leaves of apple trees provide a natural habitat for both endophytic bacteria, which grow within the plant, and epiphytic bacteria, which live on the plant. Little is yet known about their lifestyle, function and interaction with the host plants and other micro-organisms. Some of them help the plant fight off pathogens. For example the presence of the bacterium Pseudomonas fluorescens in apple trees has an antagonistic effect on the pathogen which causes apple scab. Other micro-organisms damage the host plant, causing the plant tissue to die off. To control these plant-pathogenic bacteria, genes which confer a bacterial resistance are inserted into apple tree plants.

The aim was to clarify the following questions:

  • What effect does the expression of antibacterial genes have on the species spectrum of plant-associated bacteria?
  • Does a potential change in the species spectrum of the bacteria affect the growth and fertility of transgenic trees?
  • How great is the risk of a horizontal gene transfer between transgenic apple trees and plant-associated bacteria?
  • What effect does the grafting of non-transgenic apple varieties onto transgenic rootstocks have on the species spectrum of micro-organisms?

Systemically induced gene silencing

It is already known from experiments with tobacco plants that targeted gene silencing can be triggered in grafted non-transgenic tobacco plants. The underlying mechanism is referred to as RNA interference.

This project aimed to clarify the following question:

  • Can targeted gene silencing be triggered in non-transgenic apple varieties by grafting them onto transgenic apple varieties?

Summary

  • The spectrum of the species of endophytically living bacteria was very similar in transgenic and non-transgenic outdoor-growing apple trees. Differences were seen primarily in the frequency of the types of bacteria found. In addition, the methods of cultivation and plant breeding (on its own root, grafted onto a stock) had no major effect on the spectrum of endophyte species seen. Differences were observed in particular between plants grown in vitro or in a greenhouse.
  • No horizontal gene transfer of transgenic DNA from apple plants to the endophytic bacteria was detected.
  • The results of systemically acquired gene silencing were contradictory. Some investigations indicated the existence of a transport of silencing. More studies are needed to investigate this.

Experiment description

All experiments were conducted on laboratory and greenhouse plants of various transgenic and non-transgenic apple varieties, some of which have been grafted.

Small apple leaves in bacterial solution: The genetic transformation of the apple trees was carried out with the help of Agrobacterium tumefaciens.

Genetic transformation with the help of Agrobacteria. The genes to be transferred are inserted into the bacterium, which is then used to infect the plant.

Genetically modified apple trees growing in a special safety tent under field-like conditions.

Transgenic apple trees growing in a special safety tent under field-like conditions.

Transgenic apple trees in the safety tent

Transgenic apple trees in the safety tent

In apple growing as in apple breeding, twigs from the desired variety are grafted onto a rootstock. This is the only way to keep the variety intact.

Isolation and characterisation of endophytic bacteria

For these experiments, apple plants were transformed with genes to increase bacterial resistance. The transgenic plants also contain the genetic marker nptII.

Endophytic bacteria were isolated and characterised using both culture-dependent and culture-independent methods.

  • Culture-dependent method: The culture-dependent method is used to identify bacteria which can be cultivated on a culture medium, where they form colonies. These are then analysed more closely by sequencing individual gene fragments.
  • Culture-independent method: The culture-independent method is used to characterise bacterial communities on the basis of DNA fingerprinting techniques. This enables assertions to be made about the species spectrum of the bacterial community, as well as any changes within a spectrum.

Investigating epiphytic bacteria

Transgenic and non-transgenic plants were infected with the bacterium Pseudomonas fluorescens. The grafted plants were infected via the rootstock. The spread of the bacterial strain is measured every two days over a period of two weeks. In addition, leaves were analysed at different leaf stages.

Investigations of horizontal gene transfer (HGT)

To investigate HGT from transgenic apple plants to endophytically living bacteria, leaf material from the transgenic plants were ground with a pestle and placed in culture medium with or without kanamycin onto agar plates. Only those bacteria with a natural resistance to kanamycin or that have gained the nptII gene as a result of an HGT can grow on plates containing kanamycin. Any bacterial colony that survives culture on kanamycin plates should be tested by PCR for the nptII gene.

Systemically induced gene silencing

The following experimental model was to be used to test whether gene silencing is triggered in apple plants: The gene that expresses the red dye anthocyan in the leaves was switched off in a transgenic rootstock. Two methods of transformation were used: antisense and the introduction of transformation vectors, which enable plants to express double-stranded RNA molecules. Non-transgenic, red-leaved apple plants were then grafted onto these plants. Starting from the transgenic rootstock, the silencing information should be relayed via the graft union to the non-transgenic scion, where it will decolourise the red-leaved apple variety.

Results

Endophytic bacteria isolated from in-vitro cultures of the “Pirella” apple variety

Isolation and characterisation of endophytic bacteria

At the start of the project, methods for recording the species spectrum of endophytic bacteria had to be developed and established. Appropriate trees were selected on the trial site of the Institute of Fruit Breeding (IOZ) and the State Research Centre for Agriculture in Saxony (LfL).

The areas were cultivated according to the principles of integrated and controlled production or ecological pomiculture. The trees were sampled in summer 2007 at three time points relevant for fruit growing. The spectrum of bacterial species in the examined trees grown outdoors was similar. Differences were mainly seen in the frequency of the species of bacteria found. No major differences were found between trees of one particular genotype grown on differently cultured areas (ecological or integrated).

For greenhouse trials both transgenic and non-transgenic plant material was propagated and rooted. These plants were also grafted, i.e. the scion is grafted onto a stock. The plants were then samples three times during summer 2007. No influence of either the transgene or the method of plant breeding (on own roots, grafted onto a stock) was detected on the spectrum of endophyte species. Differences in the species spectrum were mainly found between plants of a particular type grown in vitro or in a greenhouse. Here the type of cultivation appeared to have a major influence.

Investigation of an epiphytically living bacterial strain

Investigations of the spread of P.fluorescens were carried out in 2007 in early summer. It was observed that the strain of P.fluorescens was not able to colonise certain apple plants. This result was unexpected since this particular strain of P.fluorescens had been isolated form apple plants. In addition, another Pseudomonas strain that had also been isolated from apple plants was unable to propagate. The reasons for this are still not clear. However, this meant that it was not possible to obtain analysable results for this question.

Leaf material from the transgenic plants were crushed with a pestle and plated onto agar plates with or without kanamycin. On plates with kanamycin (right) only those bacteria can grow that are resistant to the antibiotic. These could be with naturally resistant bacteria or bacteria that had taken up the genetic marker from the transgenic plants by gene transfer.

Early flowering: In this type of apple plant the gene that inhibits flowering is disabled. “switched off”.

Investigations of horizontal gene transfer (HGT)

In a first step, the leaves from in vitro shoots of various kinds of apple and wild strains of cultivatable endophytes were documented and then assessed according colony colour and form. Using PCR, proteobacteria, bacilli and actinobacteria were identified. Subsequently, any transgenic lines of cultivatable endophytes growing on the kanamycin-containing medium should be analysed. However, no kanamycin-resistant colonies were found. Thus there was no evidence of a horizontal transfer of transgenic DNA to these bacteria.

Systemically acquired gene silencing

Transgenic apple plants were transferred to a greenhouse and grafted with scions from a red-leaved wild type of apple. If there is a systemic transport of gene silencing a decolouring of the wild type should occur. A first appraisal of these plants indicated the existence of silencing transport. Surprisingly, however, the effect was markedly less than expected. The leaves of the grafted shouts on the transgenic stock showed decolouration at the beginning of the growing season, but this effect decreased during the course of the growing season.

In addition, investigations on a gene that inhibits flowering were included in the experimental design. Gene silencing of this gene should lead to premature flowering. Here it could be shown that silencing already takes place under in vitro culturing conditions and early flowering begins. Subsequently, some of these plants were rooted and transferred to the greenhouse. Under these conditions the transgenic plants also began to flower after a few weeks.

In the spring of 2009, grafting trials were carried out on individual plants. In this, non-transgenic shoot pieces were grafted onto transgenic stocks. Until now, however, no flower formation has been observed on the non-transgenic shoots. In summary, the results for a systemically acquired gene silencing have been very contradictory. No clear statement is possible on the basis of the existing data, and further investigations are necessary.