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Effect of zeaxanthin potato on soil life and soil quality

(2005 – 2008) Technical University of Munich (TUM), Chair of Soil Ecology, Oberschleissheim

Topic

Organisms that live in the soil (bacteria and fungi) have a key position in the agricultural ecological system with regards to soil quality and plant health. They are also responsible for the decomposition of the residues from harvesting, mineralising organically bound nitrogen and stabilising the soil structure.

The aim of the project is to investigate the effect of transgenic potatoes with a modified carotenoid composition on the bacterial and fungal community in the root area. In addition, it was investigated whether the genetic modification had an influence on the phenotype of the plants, or whether the decomposition of the stubble of transgenic plants differed and so possible had an effect on subsequently planted wheat.

Summary

  • From the time point of flowering, the development of all varieties and lines, whether in the greenhouse or outside were similar.
  • The transgenic and non-transgenic potato plants showed no differences with regards to colour, size and shape of the tubers.
  • The modification of a gene in transgenic plants can lead to a change in the phenotype, beyond the change planned with the modified gene (pleiotropic effect). However, the differences in phenotypes between transgenic and wild-type plants are fewer than within the various species.
  • An altered phenotype has an influence on the composition of microorganisms in the soil around the roots and therefore also on the translation rates. The influence is determined in the first instance through the location, weather conditions and the stage of development of the plant.

Experiment Description

Greenhouse and field

Plant material was available from two transgenic lines, the non-transgenic parent line and another six conventional varieties. In the first year of the trial (2005) cultivation took place at the trial site in Roggenstein (Bavaria), and from 2006 also in Oberviehhausen.

Root windows are set up for observing root growth and taking samples.
Root window
Root window

Installation of “root windows” under field conditions.

Sampling at a root window Little balls of filter paper serve as catching containers on the root surface

Sampling of soil water close to the roots. Pellets made of filter paper serve as collectors at the root surface.

Plant growth and rhizosphere chemistry (University of Hohenheim, Institute of Plant Nutrition)

Genetic modification of the plant metabolism can potentially have unintentional effects on root exudation. Root exudates affect the nutrient ratios in the root area, i.e. they have an effect on nutrient mobilisation by micro-organisms in the rhizosphere and on the nutritional status of plants and therefore on plant growth.

Field trials. The nutritional status of the plants was determined at different stages of plant development using fully developed leaves (content analysis). Monitoring of root growth was done in two stages: the main focus was on investigations under outdoor conditions of “root windows” (see figure). Root exudates and soil solutions from the vicinity of the roots were examined chemically.

Pot trials. In parallel, pot trials were carried out in so-called root boxes under controlled conditions to determine the influence of possible stress factors such as drought, mineral deficiency or salinity. Potato plants were initially propagated in nutrient solution or peat-sand mixture up to the shoot axle and subsequently replanted in the root boxes. The cultivation of transgenic lines and control plants was carried out over a period of six weeks, with five repeats for the individual stress treatments.

Expression of zeaxathin epoxidase (Technical University of Munich, Institute of Plant Breeding)

The genetic modification causes a build-up of the carotenoid zeaxanthin in the tubers. This is achieved by blocking a specific enzyme (zeaxanthin epoxydase), which converts zeaxanthin to a different carotenoid. The genetic modification is based on the antisense strategy.

Since the idea is that the zeaxanthin should accumulate only in the tubers, the antisense mRNA for the relevant enzyme is transcribed under the control of a tuber-specific promoter. The amount of antisense mRNA formed was measured in the tubers, roots, berries and plant parts at various times (including using “real time” reverse transcriptase PCR).

Solanine content in transgenic potato berries (TU München)

In the event that a change in the level of toxic solanine is found in the potato berries of transgenic plants, the possibility of increased seed dispersal by wild animals will also be investigated.

Microbial conversion of nitrogen (TU München)

Mineralisation of organic nitrogen, nitrification and denitrification are very important processes in the soil and are carried out by micro-organisms. Organically bound nitrogen in harvest residues is converted into plant-available mineral nitrogen (mineralisation). The ammonium formed is either used directly by the plant or converted into nitrate by micro-organisms (nitrification). If the soil is lacking in oxygen, nitrate is converted into elementary nitrogen or into nitrous oxide (“laughing gas”) – a greenhouse gas (denitrification). It is possible to measure the activation of the genes responsible for these processes.

Decomposition of litter – microbial, chemical and molecular biological investigations (TU München)

Genetic modification of the plant can lead to changes in the levels of plant secondary metabolites in all plant parts, which then alter the breakdown of plant litter and therefore the release of nutrients for the following crop.

The litter (stem, leave and root) of the different potato plants were collected and filled into litter bags. These were dug 20 cm deep into the soil at both locations in eightfold repeats along the fields. After 2, 8 or 19 weeks, the decomposition rates of the contents of the bags was determined and, in parallel, the bacterial and fungal communities involved in the decomposition and the colonisation of the litter were ascertained using DNA analyses.

Results

The first field trials took place in Roggenstein in 2005. The test plants were cultivated in randomly distributed tracts, with eight repetitions for each type. Since the approval for release for this location was received too late in 2005, conventional strains were cultivated to be able to make a comparison of the location.

In 2006, the Oberviehhausen location also became available. The trial approach followed the same pattern as the year before in Roggenstein. Sampling was carried out three times within the growing season dependent on the development stage of the potato.

Quality and volume of yields

In terms of the colour, size and shape of the tubers, there were no differences between the transgenic and non-transgenic potato plants. The firmness of the skin was also judged to be the same. As expected, the flesh of the transgenic potato variety was a more intense yellow because of the higher zeaxanthin content. The average yield for all strains from the field trials in Roggenstein (2005 and 2006) were slightly higher than the average for Southern Germany, whereas in 2007 all yields are lower because of the unfavourable weather conditions. The yields of the two transgenic lines were not significantly different from those of the reference strain Baltica.

Plant growth and rhizosphere chemistry

Field trials. The investigated parameters – nutritional status of the plants, root growth, pH value at the root surface and the concentration of the root exudate – obtained at Roggenstein and Oberviehhausen did not show any difference between the transgenic and non-transgenic plants, and the variability was comparable to that seen with the conventional plants. However, for some of the parameters a difference was seen between the two trial years. The root growth, for example, generally higher in 2006, which was probably due to the damp, and therefore better growing, conditions before the specific sampling dates.

The concentration of organic root exudates in the rhizosphere soil solution was generally comparable between the varieties and the transgenic lines, although there appeared to be a shift towards more organic acids in 2006, possibly in conjunction with a lower phosphate nutritional status.

Container experiments. In the first year of the project a cultivation system was established for propagating potatoes through side shoots. This was used to determine the root exudation of the different species under stress conditions.

The starting species Baltica and both of its transgenic lines were subjected to different stress factors, such as deficiency of nitrogen, phosphate or oxygen, or an increased aluminium concentration. The root exudate was measured in the root washings. No significant differences in the root exudations were for the examined species – except with the treatment with aluminium ions (Al3+) in toxic concentrations, which caused a stronger secretion of citrate in the area of the root tips. The reason for this needs to be clarified in further investigations.

Expression of zeaxanthin epoxidase

In 2005 an analysis method was developed for quantifying the antisense mRNA from the different plant organs. The method was then applied to the samples from all three root growth stages in 2006. In the leaves and roots, the transgenic lines were found to have the same enzyme activity as the parent variety Baltica at all three sampling times. Considerable amounts of Zeaxanthin epoxidase in the tubers were, however, as expected, only found in the two transgenic lines, with one of these transgenic lines showing a higher amount of the enzyme.

Construction of a transcription profile for the greenhouse trials

In the examined leaf samples, differing transcription rates were seen for some of the genes investigated. In contrast, no differences in the transcription rates were measured in the tubers of both transgenic lines in comparison with the wild-type Baltica.

Solanine content of the transgenic potato berries

Since the berries, after consultation with the local authorities, were removed before they were ripe, the analysis of their solanine content could not be performed.

Microbial nitrogen conversion

Genes involved in the microbial conversion of nitrogen in the rhizosphere of the transgenic and non-transgenic potato plants were detected and their number recorded. For both nitrification and denitrification, the differences between the wild type and the transgenic species were below that observed for conventionally breed species. There was, however, a clear influence of location and weather conditions on the behaviour of the gene involved in the microbial nitrogen conversion.

Litter decomposition

Litter decomposition varied between the two locations. In Oberviehhausen, the litter of the wild type Baltica decomposed the slowest at all three time points, whereas the litter from both transgenic species decomposed noticeable quicker. At the Roggenstein location these differences were very small and not statistically significant.