Methods for Risk Assessment of Transgenic Plants III. EcologICal risks and propspects of transgenic plants K. Ammann, Y Jacot, V. Simonsen and G. Kjellsson (eds) © 1999 Brrkhauser Verlag Basel/Switzerland

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Monitoring the environmental impact of transgenic sugar beet Beta vulgaris subspec. vulgaris altissima Doll - are we able to ask the right questions?

Matthias PoW-Orfl , Ulrike Brand2, Ingolf Schuphanl and Detlef Bartsch3

IDepartment of Biology V. Ecology, Ecochemistry and Ecotoxicology, RWTH-Aachen University of Technology, Worringerweg I, D-52056 Aachen, Germany

2 Institute for Developmental Biology, University Cologne, Gyrhofstr. 17, D-50923 K61n, German 3present address: University of California, Riverside, Department of Botany and Plant Sciences, Riverside, CA

92521, USA

Introduction

With the release and the commercialization of transgenic plants the spread of genetically mod­ified phenotypes in the environment seems certain. The remaining question is merely how long this process will take and what effects it will cause. The fundamental problem when talking about risk assessment is that we lack the necessary extensive knowledge about the ecology of the modified species and the function of the transferred genes. Only this knowledge makes it possible to look for the really relevant topics and to ask the right questions for an adequate risk assessment. To address ecologically important features like outcrossing [1], competitiveness [2], gene flow [3] or survival through the winter the following investigations were carried out. To make the aims of the investigation clearer it is useful to differentiate between two kinds of monitoring. Following Maas [4], there is on the one hand specific monitoring which exatnines possible cause-effect relationships, and on the other hand a general monitoring which focuses on investigations for example in natural populations without using the transgenic organism.

Our specific monitoring was primarily carried out in single organism tests with the trans­genic plants themselves to check the ecologically relevant winter survival. Our general moni­toring focuses on the virus infestation level in the potentially influenced habitats and on the genetic structure of the populations of the wild relatives. A definition of the actual state of pop­ulation dynamics using RAPD-PCR was necessary for an evaluation of possible future changes in natural populations due to transgene introgression. These data give information about whether a specific trait is able to cause a competitive advantage in a particular habitat and thus help to assess the potential risk of outcrossing and establishment of transgenic traits in wild beet habitats. Only precise knowledge about such transgene/environment interactions enables us to identify priorities for research.

Material and methods

Plant material

The breeding lines, cultivars and transgenic varieties were made available to us by KWSIPLAN­TA, Einbeck, Germany. The transgenic sugar beets we worked with were carrying the addi-

22 Matthias Pohl-Orf et al.

tional transgenic sequences of the c-DNA of the coat protein of beet necrotic yellow vein virus (BNYVV) [5], the nptII gene [6] as a resistance marker against kanamycin and the bar-gene [7] mediating resistance against the herbicide BASTA/LffiERTY with its active agent glufos­inate-ammonium. Some wild varieties were from FAL, Braunschweig, Germany, while others were from own collections.

Virus infestation

12 to 16 days after germination, the seedlings were placed in soil containing BNYVV. Thirty­two plants of each treatment were watered twice a week with sea salt water at two different concentrations. The first treatment was 1 % salt, the second 0.5% and the third was tap water as a control. After cultivation for 95 to 106 days, the roots were harvested and checked for the presence of BNYVV [8-10] with a specific antibody test (ELISA, enzyme linked immunosor­bent assay) [II, 12].

Survival of sugar beet in the winter

Between 1994 and 1997, field tests were performed at two sites, one with virus infestation (and pre-inoculation) in Mainz, Germany, the other at a virus free control site in Aachen, Germany. From 1994 to 1996 the tests were conducted with conventional plants done at different sites in Germany (Braunschweig, Dresden, Aachen, KOln, Stuttgart) and at the Dutch coast near Breskens.

Molecular analysis of population genetics

The RAPD-PCR (randomly amplified polymorphic DNA-Polymerase chain reaction) was based on Lorenz et al. [13], Uphoff and Wricke [14, 15] and Eagen and Goldman [16], opti­mized for the specific requirements like used enzymes or DNA-template. (PCR-conditions: 10 mM buffer, 1.5 mM MgClz, 0.2 mM dNTPs, lu taq, 0.001 % gelatin, 0.5 ~g primer, approx. 100 ng template, added to 50 ~ with water; amplification program: denaturation 30 sec, 94°C, annealing 1 min, 35°C, synthesis 2 min, 72 °C, 40 cycles, DNA-isolation: SDS-method, Taq­polymerase: EUROGENTEC Goldstar, Primer: MWG-Biotech).

Results

Virus infection under different soil conditions

The typical extinction value of an infected plant was about 2.5 after 60 min of reaction time. Transgenic plants expressing the virus coat protein and thus responding to the test showed val­ues as a positive control of approximately 0.5. The different salt concentrations in the water caused a decrease of infection with an increase of salt content in the water. As shown in Figure I, the decrease is significant except in wild beet population #1, which showed the highest infec­tion under 0.5% salt solution watering.

Environmental impact of transgenic sugar beet Beta vulgaris ssp. vulgaris altissima 23

2

0 cuitlvar 2 Edda

I 3 4

_ 0.0"10 sail

0.5"10 sail 1.0% sail

I • 5

wild beet populations

6

Figure I. Infection of different wild populations of Beta vulgaris L. with BNYVV depending on salt concentra­tion in the soil (mean of 32 plants with standard error). The six different wild beet populations originated from the AdriatIc coast in Italy.

The different wild variants showed a different range of extinction values. One of the popu­lations even proved to be completely tolerant against virus infection. In this case the extinction never exceeded a value of 0.3 in the ELISA. Especially in the case of the cultivar Edda, mor­phological changes due to salt irrigation were noticed. The plants developed succulent charac­teristics such as thick leaves with a strong cuticle and more compact growth thus causing mor­phological similarity to their wild relatives.

Survival of transgenic and conventional sugar beet

As shown in Figure 2, correlation was found between survival rate and temperature in the tri­als when survival rate was plotted against cold sum. The independent variable cold sum is defined as the sum of every daily negative average temperature at 2 m above ground over the whole winter. Summing only average temperatures below -4 °C gave better correlations and this parameter was chosen as independent variable. The logistic regression with the formula f(x) = (100)/(1 + (X/C)b) and the parameters b = 4.213 and c = -32.461 had a correlation coeffi­cient of r = 0.931.

Significant differences between the genotypes at the same test site were not detected and so the different genotypes were combined and compared within the different sites. In the winter

24

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100

80

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20

0 -200

cold sum vs sUlVlval rate -- logistic regression

Malnz 96197 -J Aachen 96197 95196

4 -150 -100 -50

cold sum (m sum of

Matthias Pohl-Orf et al.

C95196 Cologne 94/95

Eschweiler 94/95

Braunschweig 94/95

Mamz95196

0 50

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Figure 2. Survival rates of Beta vulgaris L. at different sites in different test periods (mean over all genotypes). Transgenic plants were used in Mainz and Aachen, Germany. The tests in 1996/97 were carried out with hybrids between sugar beet and Swiss chard.

199411995 the temperature was moderate and the survival rates were high. At the field site in Cologne more than 90% of the beets survived. The winters of 1995/1996 and 1996/1997 were much harder and survival rates were so low that nearly all plants died (Fig. 2). Only in Breskens at the Dutch border were the temperatures milder and here survival rates were high (90%).

Characteristic RAPD marker for wild and cultivated beets

Four primers showed suitable banding patterns and produced 24 useful bands. To find particu­larly reproducible fragments, only amplification products of a specific size (depending on the primer, between 400 and 2000 bp) were evaluated. Five markers were found that occurred only in wild beets and two were found to be characteristic for cultivars.

Discussion

Specific monitoring

Even in very harsh winters, survival of sugar beet in North Europe is possible. Contrary to ear­lier investigations [17] according to which sugar beet will die at temperatures below -5°C, plants survived minimum temperatures of -10°C and less. The comparison of different geno-

Environmental impact of transgenic sugar beet Beta vulgaris ssp. vulgaris altissima 25

types in a species, especially in the case of the Swiss chard hybrids, may be matter of discus­sion. Swiss chard, being a leaf crop, is not like the other beet varieties selected towards big beet­roots. Small beetroots have a relatively higher content of osmotic compounds and less water causing a potentially higher frost resistance [18].

General Monitoring

Our investigations show that the infection of wild beets with BNYVV is low in natural, meso­haline habitats. This is very important due to the ecological relevance of transgenic rhizoma­nia resistance. The occurrence of Beta vulgaris ssp. maritima is limited to a small area along the coasts where a moderate salt concentration reveals. This could be the reason why BNYVV has not been found in wild beets grown in such natural habitats. In addition to this, wild beets are genetically less susceptible to rhizomania [8] and so wild beets carrying the transgenic virus resistance would not behave much differently from their unaffected relatives in salt free habi­tats where an infection could occur. Whether the decreasing infection at higher salt concentra­tion directly depends on the virus or on the disturbance of the vector Polymyxa betae KESKIN has not been clarified, but the important aspect is that a transgenic virus resistance is of minor ecological importance due to the lack of selection pressure. However, the fact that a tolerance against BNYVV in wild beet is found seems to be a hint towards an co-evolutionary process and thus the temporary occurrence of BNYVV in wild beet habitats.

The RAPD-PCR resulted in several specific marker bands which are characteristic either for wild beets or for cultivars. These markers are useful tools to investigate the interaction between wild and cultivated beets and the great heterogeneity within the species Beta vulgaris [19], and support the hypothesis of creation of the weed beets by hybridization during seed production near wild beet populations, as in Italy and France [20, 21].

The present investigation gives an impression of how many aspects have to be observed in an extensive monitoring program. It should be clear that all modified traits of a transgenic plant have to be checked towards their possible ecological consequences based on the qualities of the new gene. Moreover, the conditions in the habitats of the wild relatives are of great importance to evaluate the relevance of the selective advantage caused by the new traits.

It seems to be a never ending story - research will only be done if effects of a change can be imagined. Who, for example, thought of CO2 production and global change when introduc­ing automobiles instead of horse carriages?

The question remains

Do we know enough about plant ecology to be sure we have considered all the possible impli­cations of releasing transgenic plants? Thus, the reason for many of the "reassuring" results could be that we have not looked for the right topics and have not asked the right questions.

Acknowledgement We want thank all who made the investigations possible due to their cooperation especially: KWSIPLANT, Einbeck, Dr. L. Frese, FAL-Braunschweig, Dr.Posselt and Mr. Stelz University Stuttgart-Hohenheim, MPI­Ziichtungsforschung, KOln, Dr. E. Biancardi, Instituto Sperimentale per Ie culturi, Rovigo. The work was funded by the German Ministry of Education and Science (No. 0310532).

26 Matthias Pohl-Orf et aI.

References

1. Bartsch D, Pohl-Orf M (1996) Ecological aspects of transgenic sugar beet: Transfer and expression of herbi­cide resistance in hybrids with wild beets. Euphytica 91: 55-58

2. Bartsch D, Schmidt M, Pohl-0rf M, Haag C, Schuphan I (1996) Competitiveness of transgenic sugar beet resistant to beet necrotic yellow vein virus and potential impact on wild beet populations. Molec Ecol 5: 199-205

3. Bartsch D, Schmidt M (1997) Influence of sugar beet breeding on populations of Beta vulgaris ssp. maritima in Italy. J Veg Sci 8: 81-84

4. Maas D (1996) Moglichkeiten des Biomonitorings bei der Langzeitbeobachtung transgener Organismen. - In: Langzeitrnonitoring von Umwelteffekten transgener Organismen. UBA-Texte 58/96: 47-60

5. Meulewaeter F, Soetaert P, Emmelo Van J (1989) Structural analysis of the coat protein gene in different BNYVV Isolates. Medelingen Faculteit Landbouwwetenschap Riijksuniversiteit Gent 54(2): 465-468

6. Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotid sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19: 327-336

7. Thompson CJ, Mova NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J 3: 2723-2730

8. Whitney E, D (1989) Identification, distribution, and testing for resistance to rhizomania. Beta maritima. Plant Dis 73(4): 287-290

9. Alderlieste M F J And Van Eeuwijk FA (1992) Assessment of concentrations of beet necrotIc yellow vein virus by enzyme-linked immunosorbent assay. J Virol Meth 37: 163-176

10. Geyl L, Garcia Heriz M, Valentin P, Hehn A, Merdinoglu D (1995) Identification and characterisation ofresis­tance to rhizomania in an ecotype of Beta vulgaris ssp. maritima. Plant Pathol44: 819-828

11. Konig R, Burgermeister W, Leseman D-E (1987) Methods for Detection and identification of Beet Necrotic Yellow Vein Vtrus. Proc. 50th Winter Congress, /.l.R.B., Brussels, 17-22

12. Kaufmann A, Konig R, Lesemann D-E (1992) Tissue print-immunoblotting reveals an uneven distribution of beet necrotic yellow vein and beet soil-born viruses in sugarbeets. Arch Viro1126: 329-335

13. Lorenz M, Weihe A, Borner T (1994) DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.). Theor Appl Genet 88: 775-779

14. UphoffH, Wricke G (1992) Random Amplified Polymorphic DNA (RAPD) Markers In Sugar Beet (Beta vul­garis L.): Mapping the Genes for Nematode Resistance and Hypocotyl Color. Plant Breeding 109: 168-171

15. Uphoff H, Wricke G (1995) A genetic map of sugar beet (Beta vulgaris) based on RAPD markers. Plant Breeding 114: 355-357

16. Eagen K A, Goldman I L (1996) Assessment of RAPD marker frequencies over cycles of recurrent selection for pigmnet concentrtion and percent solids in red beet (Beta vulgaris L.). Mol Breeding 2: 107-115

17. Anonymous (1993) Report results from frost resistance trials with sugar beet (Beta vulgaris L.) transformed with glyphosate resistance genes. Reports to the National Agency for Environmental Protection. Denmark

18. Barocka KH (1985) Zucker- und Futterriiben, In: Lehrbuch der PJlanzenzuchtung landwirtschaftlicher Kultuiformen, Bd. 2, Spezieller Teil. Paul Parey Verlag, Berlin-Hamburg, 245-287

19. Jung C, Pillen K, Frese L, Flihr S, Melchinger AE (1993) Phylogenetic relationship between cultivated and wild species of the genus Beta revealed by DNA "fingerprinting". Theor Appl Genet 86: 449-457

20. Boudry P, Morchen M, Saumitou-Laprade P, Vernet P, Van Dijk H (1993) The origin and evolution of weed beets: consequences for the breeding and release of herbicide-resistant transgenic sugar-beets. Theor Appl Genet 87: 471-478

21. Santoni S, Berville A (1992) Evidence for gene exchange between sugar beet (Beta vulgaris L.) and wild beets: consequences for transgenic sugar beets. Plant Mol Bioi 20: 578-580