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GM gene stacking is a bad idea



January 3, 2004

Prof. Joe Cummins

e-mail: jcummins@uwo.ca <mailto:jcummins@uwo.ca>

“Homologous recombination: a problem with gene stacking”

Recently the United States (US) and Canada approved genetically modified
(GM) crops that contained two different Bacillus thuringiensis (Bt) Cry
genes to enhance insect bio-control. Both maize and cotton were modified
with two genes using a procedure called “stacking” in which GM strains
of the crop that had been approved for commercial release were mated to
produce the stacked variety. Since both parental lines were approved it
was presumed that the stacked product of the mating would simply produce
both forms of the insect toxin produced by the stacked variety with no
further complications. However, the two genes and their associated
promoters, transcription terminators, and introns along with their
source plasmid associated genes including the selectable markers
(antibiotic resistance genes) have considerable DNA sequence homology.
Genes with sequence homology within a set of chromosomes may trigger
recombination leading to translocations , duplications and deficiencies
in both pollen and egg formation and in somatic tissue. Such chromosome
abnormalities lead to loss of fertility And tissue disruption.

A simple description of recombination may help explain why DNA sequence
homology triggers chromosome instability. Recombination is the exchange
of genes between related organisms. Its purpose is to generate optimum
gene combinations to cope with a changing environment. Homologous
recombination is the gene exchange that depends on DNA sequence homology
to initiate gene change at a locus (chromosomal address of a gene in
which small alterations in DNA sequence are called alleles). The process
leads to mixing of different alleles to provide variation upon which
natural selection can act. In meiosis (formation of egg or sperm)
parental chromosomes align and exchange strands initiated at exchange
points (chiasmata) starting at a point of homologous recombination.
Homologous recombination is most active at meiosis but also takes place
in somatic tissue both spontaneously at low frequency or following
environmental gene damage at elevated frequency. Homologous
recombination is the process providing genetic variability for selection
agricultural crops in the long history of agriculture. GM crops,
however, are created by a process called illegitimate recombination
which does not depend on homologous recombination (legitimate
recombination). GM crops are created by the insertion of non-homologous
DNA into gaps produced in broken chromosomes. The original DNA insert
has no allelic counterparts in the chromosome (the insert is hemizygous)
but alleles of the illegitimate insertions can be selected by crossing
(for example selfing a hemizygous plant yields 25% homozygous transgenic
plants). In homozygous transgenic plants homologous recombination may
take place at the transgene or if two homologous transgenes are inserted
present on different chromosomes (or loci) these transgenes may
participate in homologous recombination leading to chromosome disruption.

Homologous recombination in somatic tissue is also called mitotic
recombination. Mitotic recombination was initially studied in fungi in
which homologous chromosomes were associated in pairs during vegetative
growth. Later, as genetic analysis became more sensitive, mitotic
recombination was observed in mammals and in higher plants. Recently,
both somatic recombination and chromosome rearrangements were observed
in plants infected with pathogens (viral and fungal pathogens) and that
increase was observed in the progeny of infected plants and could be
transmitted through graft unions (indicating that the recombination
stimulus was likely to activate a signal pathway)1,2,3. The stress
related recombination response provides a pool of genetic variability
from which resistant forms may be selected. However, transgenic
modification of crops may lead to additional homologous sequences that
would lead to extensive chromosome rearrangement causing loss in
fertility of the plant. Multiple Agrobacterium tumefaciens T DNA
insertions in Arabidopsis caused multiple gene duplications and
chromosome translocations in the cells of somatic tissues (4). Bacillus
thuringiensis (Bt) insect toxin gene Cry1Ac which were inserted at
different loci in the same cotton line were found to have unstable
non-Mendelian patterns of inheritance in early generations but the
patterns were stabilized after multiple generations (5). The authors
argued that the instability in Bt genes needed time for the gene to
“compatibilize”. Finally, somatic inter-chromosomal recombination led to
the formation of a new zein allele in hybrid Maize. The somatically
derived recombinant gene can give rise to a new allele because plant
reproductive tissue is directly derived from somatic tissue (6).Addition
of homologous transgenes at separate loci (gene stacking) predictably
leads to chromosome instability and the impact of stress leading to
enhanced mitotic recombination has not been evaluated by regulators.

The US Environmental Protection Agency (EPA)approved for commercial
release a stacked variety of maize called Yieldgard Plus Corn consisting
of Bt Cry 1Ab, to control corn borer and Bt Cry 3Bb1, to control corn
rootworm (7).The stacked hybrid is produced by mating inbred lines one
bearing the gene Bt Cry11Ab and the other Bt Cry 3Bb1, for that reason
the parental lines will not be effected by somatic recombination between
the Cry genes or associated genes such as promoter, intron, antibiotic
resistance gene , etc. but the marketed hybrid will be strongly
influenced by mitotic recombination between the two inserts. Of course,
since the hybrid is not a source of seed for planting somatic
recombination will not have a major impact but any seed corn pollinated
by the crop will show detrimental side effects. EPA does not seemed to
have considered the probable complication of somatic recombination
between the two genes in their evaluation of the stacked corn line. EPA
approval of the original Bt corn lines was also criticized on the basis
that the mammalian and environmental safety testing of the Cry gene
toxins accepted by EPA was done with toxin proteins that were produced
in bacteria and the toxin proteins tested were clearly different from
the protein toxin produced in the commercial maize crop (8).

EPA approved for commercial release Bollgard II cotton modified with Bt
Cry 2Ab at one locus and Bt Cry1Ac at a second locus to produce high
levels of the two toxins to control lepidopteran pests (9). The Canadian
government promptly provided for commercial release of the modified
cotton (Canada does not grow cotton but imports cotton seed meal for
cotton seed oil for human and animal consumption)(10). Unlike corn,
which is commercially produced as hybrid from inbred lines, cotton is
self pollinating but also subject to insect pollination (11). The
presence of two separate Cry toxin loci with a high level of homology
along with the associated transgene promoter, intron, transcription
terminator etc. along with the parental insertion of antibiotic marker
from the inserted bacterial plasmid must confer upon the stacked cotton
a fundamental genetic instability and that instability will be enhanced
by a number of environmental stresses. It seems bizarre that both North
American regulatory agencies have ignored the implications of
fundamental genetic instability. Cotton crops inadvertently pollinated
by the transgenic cotton will suffer significant genetic insult.
Furthermore, the primary seed stocks of the stacked cotton lines will
require constant screening to eliminate recombinant individuals.

In conclusion, gene stacking might appear to be a reasonable approach to
improving approved GM crops by combining the approved genes and gene
products to make a bargain bundle for the grower. However, the
regulators seem to have ignored the fundamental genetic instability of
the stacked lines and their potential to rapidly recombine to create
novel toxins attacking those who consume the crop. They have neglected
current findings showing that stress such as virus or fugal attacks can
greatly amplify gene rearrangement leading to consequences that should
have been evaluated before the stacked crops were commercialized The
stacked crops should not have been released without adequate safety testing.

References

1.Dong,X “Pathogen induced systemic DNA rearrangement in plants” 2004
Trends in Plant Science (in press).

2 Luch,J.,Mauch-Mani,B.,Steiner,H.,Metraux,J.,Ryalsi,J., and Hohn,B.
“Pathogen stress increases somatic recombination frequency in A
rabidopsis” 2002 Nature Genetics 30,311-5

3.
Kovalchuck,I.,Kavaichuck,O.,Kalck,V.,Boykot,V.,Filkowski,J.,Heinline,M.
and Hohn,B. “Pathogen induced systemic plant signal triggers DNA
rearrangements” 2003 Nature 423, 761-5

4. Tax,F. and Vernon,D. “T-DNA-Associated Duplication/Translocations in

Arabidopsis. Implications for Mutant Analysis and Functional Genomics”
2001 Plant Physiology 126,1527-38

5.Wu,J.,Zhang,X.,Lou,X. and Tian,Y. “Inheritance and segregation of
transformants in cotton with two types of insect resistance genes” 2003
YiChuan XueBao 7,631-6

6.Hu,W.,Timmermans,M.,and Messing,J. “Interchromosomal recombination in
Zea mays” 1998 Genetics,150,129-37

7.US Environmental Protection Agency “Pesticide Products;Registration
Applications” 2003 Federal Register 68,16036-38

8.Cummins,J. “Regulatory sham on Bt-crops”2003 pp1-4
http://www.i-sis.org.uk <http://www.i-sis.org.uk/>

9.Department of Agriculture Animal and Plant Health Inspection Service
“Monsanto Co.; Availability of determination of non regulated status for
cotton genetically engineered for insect resistance” 2002 Federal
Register 67, 70391-2

10. Canadian Food Inspection Agency “Decision Document DD2003-45
Determination of the safety of Monsanto’s insect resistant bollgard II
Cotton” 2003
http://www.inspection.gc.ca/english/plaveg/bio/dd/dd0345e.shtml pp1-13

11. Lasky,J and .Department of Agriculture Animal and Plant Health
Inspection Service “Biotechnology Permits-Biology of Cotton” 2003
http://www.aphis.usda.gov/bbep/bp/cotton.html

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