2.2—The use of tissue culture in plant breeding is not new

Plant tissue culture techniques are used by breeders to generate new varieties of crops

See Genetic Roulette’s False Claims at Bottom of Page

Analysis of Peer-Reviewed Research:

Artificial culture of plant tissues has been used routinely by plant breeders for many decades and has resulted in numerous food varieties that are grown and eaten as food all over the world.

These varieties have worked well in farmers’ fields, and have not caused any health problems for the numerous people in many countries who eat them. With this long history of safe use and practical value for farmers, it is rather difficult to understand why Genetic Roulette has bought up this issue as a serious reason to worry.

Genetic Roulette mentions mutations can occur in plant tissue culture, but the amount of genetic variation coming from tissue culture is modest and manageable, and mutations are an integral part of crop breeding. They occur all the time, and can be either positive or detrimental in terms of the value of a crop. It is the specialist plant breeder’s job to choose among many thousands of plants for favorable combinations of traits, eliminate deleterious mutations and select for favorable mutations. Breeders know how to select mutants that have improved traits and that are safe to consume, and have been doing so for decades.

In Genetic Roulette, Smith incorrectly claims that only genetic engineering can produce mutations when in fact all breeding aims to create, find and utilize genetic variation (also known as mutation). Genetic variation occurs even when two plant varieties are cross-pollinated. In fact, we ourselves are mutants! If the prospect of unintended mutation were deemed unacceptable, then all plant breeding would be unacceptable.

Transgenic breeding has been shown to produce less unintended disruption of the DNA than other methods of creating genetic change.  Genetic engineering is more precise than many other methods of breeding.  A further insurance against unintended consequences is that transgenics are produced in a known, high-performing, cultivated variety. Compared to conventional breeding a much higher level of scrutiny is applied to ensure that only the intended change is achieved. Furthermore, the researcher can eliminate unintended changes through a series of backcrosses to the parent cultivated variety. Genetic Roulette has not told the full story.

1. The use of tissue culture in plant breeding is not new. Regeneration of plants on artificial media has long been used by plant breeders, and it is widely recognized that this technique can generate mutational changes in plants.  Plant breeders call the phenomenon “somaclonal variation,” and it is a valuable tool in the breeders’ kit. (Kaeppler SM and others 1998; Kaeppler SM and others 2000; Larkin and Scowcroft 1981; Larkin 2004, van Harten 1998)

2. There are many varieties of conventionally-bred plants that have been produced using tissue culture. Plants produced from tissue culture have frequently been introduced into our food supply in the past and do not cause any health problems. Many widely grown barley and canola cultivated varieties (usually called cultivars by breeders) were produced with a tissue culture step for obtaining true-breeding lines. (Speaking technically, this involved another microspore culture for doubled haploids.) Commercial cultivars with improvements through tissue culture are documented in barley, wheat, potato, blackberry, flax, celery, tomato, rice (Larkin 2004). Virus resistance was introduced to bread wheat through tissue culture and released as variety Mackellar that has been grown in Australia since 2003 (CSIRO 2003, CSIRO n.d.)). The technique has been used to make celery and tomato varieties that are resistant to fungal attack (Heath-Pagliuso, Rappaport 1990; Evans 1989). It is used to introduce wild nightshade family genes into tomatoes (Rick and others 1986; Rick, Chetelat 1995). It has also been used to breed linseed flax, and commercial canola varieties used to produce vegetable oils (McHughen 2000). Specialized cell culture methods such as anther culture and embryo culture are used in conventional breeding to assist in wide-crosses and to enable faster fixing of desired traits (Goodman and others 1987).

3. Plant breeders have found that the amount of genetic variation coming from tissue culture is modest and manageable. When plant tissue culture is used in making transgenic plants the appearance of mutations is minimized by optimizing the plantlet regeneration method and rejecting any regenerated plantlets that have unwanted changes to them (e.g. see Lakshmanan P and others 2006). Transgenic plants are extensively analyzed in various stages through the development process. The analysis takes place over several years (on average about 10 years) before they can be ready for market. Typically researchers will begin analysis on a large number of plants and over time the numbers are reduced if the plants are not suitable.  At no stage would a plant with an undesirable quality make it through this process. Researchers obtain large amounts of information on their products — much more than required for any crop that is produced by conventional methods. Backcrossing (cross-pollination with a parent) is often used to separate the transgenic trait from unintended changes; a recent study with transgenic barley showed a single backcross is usually sufficient (Bregitzer and others 2008). Many years of experience have shown that incidental mutations in the background of transgenic plants are usually of no significance and are easily dealth with, in any case (Filipecki, Malepszy, 2006; Strauss and others 2004). That plants derived from tissue culture are commonly used in commercial conventional breeding operations is the best evidence that the technique does not cause problems

4. Conventional breeding uses many disruptive techniques to create mutations in crops. Existing methods for creation of mutations in plants such as radiation or treatment with harsh chemicals are widely used in conventional breeding and are known to produce extensive changes to the chromosomes of plants. These processes are less refined than genetic engineering techniques and in some cases have produced extensive changes to the chromosomes of plants.  It is simply incorrect to claim, as Smith does in Genetic Roulette, that these changes are not used.

One example of a non-GM technique used by conventional breeders is chemical mutagenesis. The chemical ethyl methane sulfonate was used to make Linola™, a special kind of food linseed oil developed in Australia. Another toxic chemical, sodium azide, has been used to make mutants of the malting barley used in beer production. These mutant barleys make lower amounts of beer haze and they include barley variety Caminant used in Denmark (van Harten 1998). It is likely that part of the success of Danish lager is dependent on the use of this mutation in barley varieties used in its production.

Another conventional breeding technique is radiation mutagenesis, which was used to create the durum wheat variety Creso used to make pasta in Italy, the rice variety Amaroo sown to more than 60 percent of the rice area in Australia, and the Rio Star ® grapefruit popular in the USA (Ahloowalia and others 2004; IAEA 2008; McHughen 2000; NAS 2004). For rice crops alone, hundreds and hundreds of different mutant varieties have been developed mostly made using ionizing radiation. One of these, called Calrose 76, was produced in California and is a short stature mutant made by intense exposure of rice to gamma rays from radioactive cobalt, used also to sterilize medical instruments (van Harten 1998).

Extensive changes to chromosomes caused by radiation treatment are thoroughly documented in the peer-reviewed scientific literature (NAS 2004, Shirley and others 1992) and are known to be similar to those caused by insertion of transgenic DNA (Cellini and others 2004; Gorbunova and Levy 1999). A recent detailed analysis in rice shows the frequency of altered gene expression is higher following conventional radiation treatment to induce mutations than following genetic engineering to introduce a transgene (Batista and others 2008).

Most of these basic facts about practical breeding of crops using mutation techniques are widely known by plant breeders, geneticists and well-educated biologists.  The errors at best display a lack of diligence by Smith to check the facts. Genetic Roulette explicitly claims that mutagenic agents are not used in plant breeding. It also claims incorrectly that mutagenesis techniques don’t cause the same kinds of changes as genetic engineering.

5. Radiation treatment has been used to create thousands (ca 3000 or more) of new plant varieties.  These varieties are cultivated as food and feed.   Radiation is known to be much more disruptive of chromosomal structure than the manipulations used to make transgenic plants. Such radiation treatment of crop plants has caused no documented instances of ill-health among consumers despite having been used commercially for several decades. Genetic Roulette ignores or denies published scientific papers that show that conventional plant breeding causes mutations (Ahloowalia and others 2004, IAEA 2008).

References

Ahloowalia BS, Maluszynski M, Nichterlein K (2004) Global impact of mutation-derived varieties. Euphytica 135:187–204. Reports at least 2250 mutant varieties of crop have been released. Most frequently they were created by gamma rays or X-rays. Both are known to scramble DNA.

Batista R, Saibo N, Lourenco T, and Oliveira, MM. (2008) Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion. Proceedings of the National Academy of Sciences 105, 3640-3645.

Bregitzer, P., Dahleen, L. S., Neate, S., Schwarz, P., and Mancharan, M. (2008). A single backcross effectively eliminates agronomic and quality alterations caused by somaclonal variation in transgenic barley. Crop Science 48, 471-479. “… an abbreviated breeding scheme involving a single backcross to the wild-type parent used to produce a transgenic line, which would replace 75% of the variant alleles, should produce transgenic lines with improved performance… It is concluded that a single backcross is an effective, rapid, and inexpensive method for creating superior transgenic lines.”

Cellini F, Chesson A, Colquhoun I, and others (2004). Unintended effects and their detection in genetically modified crops. Food and Chemical Toxicology 42:1089-1125. Unintended effects are those outcomes that are totally unexpected and not predictable. This paper focuses on technology for detection of such unexpected outcomes, and this includes metabolomics, proteomics, and transcriptomics. These current scientific buzzwords correspond to various forms of chemical fingerprinting.

CSIRO (2003). Wild grasses lead to first BYDV resistant wheat. www.pi.csiro.au/enewsletter/previousEditions/002story3.htm

CSIRO (n.d.). MacKeller –virus beating wheat.

www.pi.csiro.au/enewsletter/PDF/MacKellar.pdf accessed Dec 11 2008. How an improved virus resistant transgenic wheat named MackKellar containing genes from a wild grass introduced using plant tissue culture methods was developed and released to Australian wheat farmers.

Evans, D. A. (1989). Somaclonal variation – genetic basis and breeding applications. Trends in Genetics 5, 46-50.

Filipecki, M. and Malepszy, S. (2006). Unintended Consequences of Plant Transformation: a Molecular Insight. Journal of Applied Genetics 47, 277-286. “The ability to inspect large portions of genomes clearly shows that tissue culture contributes to a vast majority of observed genetic and epigenetic changes. Nevertheless, monitoring of thousands transcripts, proteins and metabolites reveals that unintended variation most often falls in the range of natural differences between land races or varieties”

Goodman RM and others (1987). Gene transfer in crop improvement. Science 236:48-54.”Transfer of genes between plant species has played an important role in crop improvement for many decades. Useful traits such as resistance to disease, insects, and stress have been transferred to crop varieties from noncultivated plants.”

Gorbunova V and Levy AA (1999) How plants make ends meet: DNA double-strand break repair Trends in Plant Science 4(7):263-269. Plants have particularly error-prone mechanisms that join together bits of broken chromosomes. These repair mechanisms scramble the DNA at the site at which the chromosomes are joined together during their repair. Radiation is a common cause of broken chromosomes and triggers these processes which scramble plant DNA and cause mutations.

Heath-Pagliuso S, Rappaport L (1990). Somaclonal variant UC-T3: the expression of Fusarium wilt resistance in progeny arrays of celery, Apium graveolens L. Theor Appl Genet (1990) 80:390-394. Disease-resistant varieties made by conventional (non-GM) breeding produce a dangerous chemical.

IAEA (2008). Mutant plants can boost yields, resistance: IAEA conference (Vienna, Austria). Reports that some 3000 mutant plant varieties from 170 plant species are catalogued by the International Atomic Energy Agency. www.terradaily.com/2007/080812145530.x6uv6k68.html accessed Dec 11 2008

Kaeppler SM and others (1998). Molecular basis of heritable tissue culture-induced variation in plants. p. 465–484. In D.S.B.J.S.M. Jain and B.S. Ahloowalia (ed.) Somaclonal variation and induced mutations in crop improvement. Current Plant Science and Biotechnology in Agriculture, vol. 32. Kluwer Academic, Dordrecht, The Netherlands.

Kaeppler SM and other  (2000). Epigenetic aspects of somaclonal variation in plants. Plant Mol. Biol.43:179–188.

Lakshmanan P and others (2006) Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture. Plant Cell Rep. 2006 Oct;25(10):1007-15. One example of how somataclonal variation in plant cell culture can be minimized.

Larkin PJ and Scowcroft WR (1981) Somaclonal variation — a novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetics 60 (40) 1432-2242. Conventional breeding technique used to promote genetic variability in plant cells. This causes more unintended changes but is subject to far less regulatory scrutiny than are more precise modern methods. Despite this, wheat MacKeller developed by Dr Larkin was released to food markets without fuss.

Larkin, P. J. (2004). Somaclonal variation: origins and causes. In “Encyclopedia of Plant and Crop Science” (R. M. Goodman, Ed.), pp. 1185-1161. Marcel Dekker, New York.

NAS (2004). NAS Report- Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects (2004) by Food and Nutrition Board (FNB) Institute of Medicine (IOM) Board on Agriculture and Natural Resources (BANR) Board on Life Sciences (BLS)) books.nap.edu/openbook.php?isbn=0309092094 accessed Dec 12 2008. The executive summary is a superb brief overview, and the whole report discusses the possible risks of all forms of genetic manipulation including conventional breeding as a coherent topic.

Rick CM and others (1986) Meiosis in sequidiploid hybrids of Lycopersicon esculentum and Solanum lycopersicoides. Proceedings of the National Academy Of Sciences of the USA 83(11):3580-3583.

Rick, CM, Chetelat RT (1995). Utilisation of related wild species for tomato improvement. Acta Hort. (ISHS) 412:21-38 www.actahort.org/books/412/412_1.htm

Shirley B W Hanley S and Goodman H M 1992. Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations. The Plant Cell 4, 333-347. Demonstration that mutations in plants induced by radiation contain radically scrambled DNA.

Strauss, S. H., Brunner, A. M., Busov, V. B., Ma, C. P., and Meilan, R. (2004). Ten lessons from 15 years of transgenic populus research. Forestry 77, 455-465. “The key lessons from our experience are: (1) stable gene expression is the rule in vegetatively propagated transgenic poplars; (2) somaclonal variation is modest and manageable; (3) transformation and field tests are extraordinary functional genomics methods”…”

Tribe D (2008). Blog posting. Gene-chips prove transgenes are clean genes. gmopundit.blogspot.com/2008/07/gene-chips-prove-transgenes-are-clean.htmlaccessed Dec 11 2008.