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ISIS Report 01/03/05
Terminator Trees
Sterile GM trees cannot contain transgenes, instead, they raise
special safety concerns for health and biodiversity
Prof. Joe Cummins and
Dr. Mae-Wan Ho
Transgenic or genetically modified (GM) trees have been tested
extensively in large open plots with little concern over the spread of
transgenes. Studies on the dispersal of pollen and seeds from forest trees have
shown that gene-flow can be measured in kilometres [1,2]. It is clear that the
transgenes from GM trees cannot be contained once released into the
environment. For that reason, a great deal of effort has been devoted to
developing genetic modifications – commonly referred to as terminator
techniques - that prevent flowering or pollen production.
In view of the serious threats posed by GM forest trees to the forest
ecosystems of the world (see "GM
forest trees – the ultimate threat", this series), commercial release
of transgenic trees is widely rejected unless strict containment of transgenes
can be assured, it is hoped, through engineering such ‘terminator
trees’.
For the most part, the methods used to control flowering or pollination
involved interfering with the genetic programme for floral development or for
deleting cells involved in floral development. A group of genes - MADS-box
genes - code for the protein transcription factors that recognize DNA binding
domains (See "View from MADS
house", this series). The plant MADS genes are related to the extensively
studied animal homeotic (HOX) genes that regulate developmental pathways [3].
Unraveling the functions of MADS genes has allowed flower development to be
manipulated.
Flowering is prevented by anti-sense genes, or small regulatory RNA to
prevent active gene products such as the MADS box transcription factor from
being formed. Also deployed is a kind of genetic abortion using a suicide gene.
The preferred suicide gene is the barnase ribonuclease from the soil bacterium
Bacillus amylolquefaciens. The ribonuclease is placed under the control
of a promoter specific to floral or pollen development. When activated, the
gene product effectively kills the cells in which the gene is expressed.
Another suicide gene used is the diphtheria toxin from the bacterium
Cornyebacterium diphtheria or related ADP-Ribosyltransferase toxins from
other bacteria; but these toxin genes are less commonly used than the barnase
gene. The preferred barnase gene is a part of the genetic construction that
first attracted the label "terminator" for engineered sterility, designed to
place seed production under corporate control [5,6].
Professor Steven Strauss of Oregon State University pioneered flower and
pollen control in poplar. He and his colleagues have led in the area of
flowering control in forest trees. Strauss pointed out that when complete
floral sterility is achieved, the plant would require vegetative propagation
[7]. Floral sterility has begun to be extended from poplar to shade trees [8].
Strauss has argued that management of GM poplar is comparable to conventional
poplar even though he is well aware of the seed and pollen dispersal with
transgenic poplar [9]. Along with the exploration of floral sterility, Strauss
has investigated speeding flower development (trees normally take years to
develop sexually) to allow rapid breeding and selection cycles [10]. Of course
the rapid breeding cycle is fraught with uncertainty regarding the subsequent
development of the mature tree. Strauss has pioneered the use of the poplar
homologue to the floral MADS box genes, the poplar promoter gene PTD [11]. The
PTD promoter was combined with the diphtheria-toxin gene, DTA, to produce
sterile polar without the detrimental effects on yield encountered earlier
[12]. The problem of somaclonal variation is hardly mentioned in the discussion
of flower control in poplar even though the problem was discussed in a report
on a four-year field trial of herbicide tolerant poplar carried out by the
Strauss group [13]. Somaclonal variation results from the cell culture
technique used to select and propagate transgenic plants. It results in
extremely high levels of mutation and chromosome instability, which could
reverse floral sterility. Earlier reports showed that poplar cell culture
resulted in extremely high levels of somaclonal variation [14,15].
In Finland, investigators from Sopanen University have studied the
control of flowering in silver birch. Those investigators identified the MADS
box genes controlling flowering in the birch tree [16,17]. When a flower
specific birch promoter gene BpMADS1 was used to drive the barnase gene, floral
cell ablation prevented flowering but there were marked side effects affecting
leaves and branching [18]. The side effects were likely a pleiotropic effect of
the gene insertion but could, as well have been affected by somaclonal
variation from cell culture. A recent report altered the name of the MADS box
gene from BpMADS to BpFULL1. As in the previous study flowering was prevented
but the gene modification affected leaves and branching [19]. The pleiotropic
effects observed may extend into areas not yet detected and they require more
extensive study.
Ecological and health hazards of terminator trees
Trees that do not flower and fruit will provide no food for the
multitude of insects, birds and mammals that feed on pollen, nectar, seed and
fruit, and will inevitably have huge impacts on biodiversity. The ablation
toxins used to create sterile trees are themselves an additional hazard.
Barnase ribonuclease proved toxic to the kidneys of rats [20] Barnase was
cytotoxic in mice and in human cell lines [21]. Animals may not find the GM
forests welcoming. Diphtheria toxin has been associated with anaphylactic
response [22]. As the song goes: "If you go down in the (transgenic) woods
today, You’re sure of a big surprise."
Even if these trees are sterile, they can still spread by asexual means
and certainly, the genes can spread horizontally to soil bacteria, fungi and
other organisms in the extensive root system of the forest trees, with
unpredictable impacts on the soil biota and fertility. There is a remote chance
that such genes could also spread horizontally to other forest trees, making
those also infertile.
As transgenic traits tend to be unstable, they could break down and
revert to flower-development, thereby spreading transgenes to native trees, or
create pollen that poison bees and other pollinators as well as causing
potential harm to human beings.
Finally, the effect of preventing sexual reproduction is to drastically
reduce genetic recombination that generates genetic diversity and evolutionary
novelty in nature. The sterile monocultures are much more likely to succumb to
disease or senescence, which could potentially wipe out entire plantations.
References
- DiFazio S, Slavov G, Burczyk J, Leonardi S and Strauss S. Gene flow
from tree plantations and implications for transgenic risk assessment.
Plantation Forest Biotechnology for the 21st Century eds.
Walter,C. and Carson,M. 405-22, 2004.
- Slavov G, DiFazio S. and Strauss S. Gene flow from forest trees: from
empirical estimates to transgenic risk assessment. Scientific methods
workshop: ecological and agronomic consequences of gene flow from transgenic
crops to wild relatives Columbus, Ohio, 2002.
- Ng M. and Yanofsky M. Function and evolution of the plant MADS
–Box gen family Nature Reviews Genetics 2001, 2,186-96
- Skinner J, Meilan M, Brunner A. and Strauss S. Options for genetic
engineering of floral sterility in forest trees. Molecular Biology of Woody
Plants, Jain S and Minocha S. eds, Kluwer Academic publishers Netherlands,
2000.
- Ho MW and Cummins J. Terminate the terminators! ISIS Report July 12,
2001 http://www.i-sis.org.uk/terminator.php
- Ho MW and Cummins J. Chronicle of an ecological disaster foretold.
ISIS report 2/02/03 http://www.i-sis.org.uk/CEDF.php;
also Science in Society 2003, 18, 26-27
http://www.i-sis.org.uk/isisnews.php
- Strauss S, Rottman W, Brunner A and Sheppard L. Genetic engineering
of reproductive sterility in forest trees. Molecular Breeding 1995, 1,
5-26.
- Brunner A, Mohamed R, Meilan R, Sheppard L, Rottman W and Strauss S.
Genetic engineering of sexual sterility in shade trees. J. Arboculture
1998, 24, 263-73.
- Strauss S, DiFazio S and Meilan R. Genetically modified poplars in
context. The Forestry Chronicle 2001, 77, 271-80.
- Strauss, S and Brunner A. Tree biotechnology in the 21st century:
Transforming trees in the light of comparative genomics. In S.H. Strauss and
H.D. Bradshaw (Eds.), The BioEngineered Forest: Challenges to Science and
Society, Pp 76-97. Resources for the Future, Washington, D.C., USA,
2004.
- Meilan R Brunner A, Skinner J and Strauss S. Modification of
flowering transgenic trees. Molecualr Breeding of Woody Plants
(Morohoshi M and Komamine A. eds.), Elsevier Science B.V. 247-57, 2001.
- Skinner J, Meilan R, Ma C and Strauss S. The poplar PTD promoter
imparts floral predominant expression and enables high levels of floral organ
ablation in Populus, Nicotiana and Arabidopsis.
Molecular Breeding 2003, 12,119-32.
- Meilan R, Auerbach D, Ma C, DiFazio S and Strauss S. Stability of
herbicide resistance and GUS expression in transgenic hybrid poplars during
four years of field trials and vegetative propagation. Hort Sci 2002,
37, 277-80.
- Antoneti L and Pinon J. Somaclonal variation within poplar. Plant
Cell, Tissue and Organ culture 1993, 35,99-106.
- Wang G, Castiglione S, Chen Y, Li L, Han Y and Tian Y. Poplar
(Populus nigra L.) plants transformed with a Bacillus
thuringiensis toxin gene: insecticidal activity and genomic analysis.
Transgenic Res 1996, 5, 289–301.
- Lemmetyinen J, Hassinen M, Elo A, Porali I, Keinonen K, Makela H and
Sopanen T. Functional characterization of SEPALLATA3 and AGAMOUS orthologues in
silver birch. Physiologia Plantarum 2004, 121,149-62.
- Lannenpa M, Janonen I, Holtta-Vuor M, Gardemeister M, Porali I and
Sopanen T. A new SBP-box gene BpSPL1 in silver birch (Betula pendula).
Physiologia Plantarum 2004, 120,491-500.
- Lemmetyinen J, Keinonen K and Sopanen T. Prevention of the flowering
of a tree, silver birch. Molecular Breeding 2004, 13, 243-9.
- Lännenpää M, Ranki A, Hölttä-Vuori M,
Lemmetyinen J, Keinonen K and Sopanen
- T. Prevention of flower development in birch and other plants using a
pFULL1::BARNASE construct 2005 Plant Cell Reports in press doi
10.1007/s00299-004-0903-y
- Ilinskaya,O. and Vamvakas,S. Nepherotoxic effects of bacterial
ribonuclease in the isolated perfused rat kidney. Toxicology 1997, 120,
55-63.
- Prior T, Kunwar S. and Pastan I. Studies on the activity of barnase
toxins in vitro and in vivo 1996 Bioconjugate Chemistry 1996, 7, 23-9.
- Rosenberg A. Immunogenicity of biological therapeutics: a hierarchy
of concerns Developmental Biology 2003, 112,15-21.
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