Genetic Engineering of Canola

1

Introduction Biotechnology is an all-encompassing term that describes the use of any living thing

or its parts (e.g. proteins, DNA, fungus, or bacteria) to make a product or to run a

process. Examples of traditional biotechnology include using yeast to make bread or

using bacteria to make yogurt. It also covers the bacterial and fungal inoculants that

farmers now use to provide nitrogen and phosphorus to crops.

The area of biotechnology that has been the focus of much attention and

controversy is genetic engineering. This technology involves taking DNA from one

organism and splicing it into the DNA of another organism. For herbicide resistance,

genetic material from common soil bacteria was introduced into canola. Varieties

using the Liberty Link and Roundup Ready systems were created this way. Another

system based on the herbicide bromoxynil is also in the works. Improvement on

“good fat” content was also taking place. This assignment will discuss on process,

problems, and progress involving genetically modified canola.

The Starting Point

Hundreds of years ago, Asians and Europeans used rapeseed oil in lamps. As time

progressed, people employed it as cooking oil and added it to foods. Its use was

limited until the development of steam power, when machinists found rapeseed oil

clung to water or steam-washed metal surfaces better than other lubricants. High

demand for rapeseed oil as a lubricant for the rapidly increasing number of steam

engines in naval and merchant ships happens during World War II. When the war

blocked European and Asian sources of rapeseed oil, a critical shortage developed

and Canada began to expand its limited rapeseed production.

After the war, demand declined sharply and farmers began to look for other uses for

the plant and its products. Edible rapeseed oil extracts were first put on the market

in 1956-1957, but these suffered from several unacceptable characteristics.

Rapeseed oil had a distinctive taste and a disagreeable greenish colour due to the

presence of chlorophyll. It also contained a high concentration of erucic acid.

Genetic Engineering of Canola

2

Experiments on animals have pointed to the possibility that erucic acid if consumed

in large quantities, may cause heart damage (Bellenand et al., 1980), although Indian

researchers Achaya K.T. (1987) have published findings that call into question these

conclusions and the implication that the consumption of mustard or rapeseed oil is

dangerous. Feed meal from the rapeseed plant was not particularly appealing to

livestock, due to high levels of sharp-tasting compounds called glucosinolates.

Plant breeders in Canada, where rapeseed had been grown (mainly in Saskatchewan)

since 1936, worked to improve the quality of the plant. In 1968, Dr Baldur Stefansson

of the University of Manitoba used selective breeding of two cultivars of rapeseed or

Brassica campestris (Brassica napus and B. campestris) (Brown et al., 1996) to

develop a variety of rapeseed low in erucic acid. In 1974, another variety was

produced low in both erucic acid and glucosinolates; it was named canola, from

Canadian oil, low acid (Stoorgard, 2008). Their seeds are used to produce edible oil

that is fit for human consumption because it has lower levels of erucic acid than

traditional rapeseed oils and to produce livestock feed because it has reduced levels

of the toxin glucosinolates.

The Process

Each canola plant produces yellow flowers that, in turn, produce pods; similar in

shape to pea pods about one-fifth the size. Within the pods are tiny round seeds that

are crushed to obtain canola oil. Each seed contains approximately 40 per cent oil.

Genetic Engineering of Canola

3

Genetically-modified canola

Genetically modified (GM) or transgenic canola varieties made by utilizing modern

plant breeding or genetic engineering have been developed to be resistant to

specific herbicides. They are called herbicide-resistant varieties. The plants are

modified, but the oil is not modified.

Agriculture giant, Monsanto introduced GM canola variety GT73 that are herbicide-

resistant in 1997. It was produced by inserting the EPSPS gene encoding the enzyme

5-enolypyruvylshikimate-3-phosphate synthase (EPSPS) from the CP4 strain of

Agrobacterium tumefaciens and glyphosate oxidase (gox) gene isolated from strain

LBAA Ochrobactrum anthropi. Agrobacterium-mediated transformation method was

used, utilizing single insertion event containing one copy of the T-DNA from plasmid

PV-BNGT04 (Batista and Oliviera, 2009). The T-DNA consists of CmoVb promoter

(modified figwort mosaic virus), the CP4 EPSPS gene, the gene coding GOX v247 and

the E9 3' polyadenylation signals. Only the DNA required to confer the glyphosate-

tolerance phenotype was transferred and inserted at a single locus in the canola

genome. No genetic elements from outside of the right and left borders of the

plasmid were transferred into or are present in the genomic DNA of Roundup Ready

canola.

The EPSPS gene codes for the enzyme 5-enolpyruvylshikimate-3-phosphate synthase

that is present in all plants, bacteria and fungi. The EPSPS enzyme is part of an

Genetic Engineering of Canola

4

important biochemical pathway in plants called the shikimate pathway, which is

involved in the production of aromatic amino acids and other aromatic compounds.

When conventional canola plants are treated with glyphosate, the plants cannot

produce the aromatic amino acids needed to grow and survive. EPSPS is not present

in mammals, birds or aquatic life forms, which do not synthesize their own aromatic

amino acids. For this reason, glyphosate has little toxicity to these organisms. The

EPSPS enzyme is naturally present in foods derived from plant and microbial sources.

Glyphosate oxidase (gox) enzyme accelerates the normal breakdown of the herbicide

glyphosate into two non-toxic compounds, aminomethylphosphonic acid (AMPA)

and glyoxylate. AMPA is the principal breakdown product of glyphosate and is

degraded by several microorganisms, while glyoxylate is commonly found in plant

cells and is broken down by the glyoxylic pathway for lipid metabolism.

Plasmid map for PV-BNGT04

Genetic Engineering of Canola

5

Main stages required for production of GM canola plants: EPSPS gene and gox gene isolated from donor organism (a), cloned in vector plasmid PV-BNGT04 with CmoVb as promoter and E9 3' polyadenylation signals (b) and transferred into the receptor organism (c). The receptor plant cells are then selected and regenerated to obtain complete GM plants (d). Finally, the obtained GE plants are crossed with other, already improved plants (e). The regenerated plants and their progenies are analysed at various levels throughout this process.

Australian scientist J.M. Manners and K. Kazan are undertaking studies aimed at the

development of transgenic canola plants expressing novel genes for resistance to

fungal pathogens such as Leptosphaeria maculans, Sclerotinia sclerotiorum,

Rhizoctonia solani and Alternaria brassicicola. The genes being tested include those

encoding novel antimicrobial peptides derived from Australian native plants and

from macadamia in particular. So far, transgenic plants expressing three peptides

have been developed and advanced lines expressing one peptide at high levels are

under evaluation for improved disease resistance. Other related research aimed at

the development of transgenic canola plants, expressing genes encoding antifungal

proteins from Brassicaceae seeds, for resistance to L. maculans and S. sclerotiorum.

Transgenic approaches employed to enhance yield and to develop novel inducible

male sterility systems for hybrid seed production.

Varieties with very high oleic acid types was also developed based on elite Australian

B. napus breeding lines and advanced double-low B. juncea germplasm.

Genetic Engineering of Canola

6

Modifications to existing Brassica transformation protocols and the use of an intron-

interrupted hygromycin-resistance gene as the selectable marker have resulted in

improved transformation efficiencies. Silencing of the endogenous oleate desaturase

genes have resulted in substantial increases in oleic acid levels, up to 89% in B. napus

and 73% in B. juncea (Graef et al., 2007).

Current research also involves development of canola with new trait such as

photoperiod insensitivity, modified plant architecture, and with reduced yield loss.

Various modern genetic engineering methods have been employed such as

protoplast fusion and microspore technologies. Embryo rescue method used to

widen genetic base of rapeseed by hybridization of B. oleracea and B. campestris to

resynthesize B. napus (Inomata, 1978).

Benefits

Compared to conventional selection and breeding of living organisms, the

combination of genetics, molecular biology, and cell biology that underlies genetic

engineering of plants allows for: reduced time to achieve genetic improvement;

more precise genetic manipulation (a single gene or set of genes of known function

can be transferred from one organism to another); and a wider range of genetic

changes, as the genetic transfers facilitated via genetic engineering are often ones

that would not normally occur in nature.

In conventional canola, there are some weeds that are difficult to control such as

wild mustard, stinkweed, cleavers and shepherd's purse. Growers choose herbicide-

resistant canola varieties primarily because weed control is easier, cost effective (up

to 30% saving on pesticides), and better (Brown et al., 1996). They allow farmers to

produce canola on land where it was previously hard to get good yields because of

high broadleaf weed populations. These varieties have also been effective at

reducing dockage in canola, and at reducing future weed problems and pesticide use.

Crop establishment and low plant populations are often problems with canola

because of soil crusting and poor emergence. Herbicide-tolerant varieties provide

Genetic Engineering of Canola

7

good weed control and allow producers to leave thinner crop stands to still obtain

acceptable yields. Reseeding can be avoided and the farmer’s bottom line is

improved. Herbicide resistant canola can be used in direct seeding systems to reduce

erosion and lower the carbon content in the air. Nearly 90% of canola cultivation at

Canada is herbicide-tolerant (Brown et al., 1996).

Genetic engineering is also being directed toward products that benefit consumers:

more nutritious foods, foods with medicinal value, and foods with improved cooking

characteristics. Canola oil has been claimed to promote good health due to its very

low saturated fat and high monounsaturated fat content, and beneficial omega-3

fatty acid profile. The Canola Council of Canada states that it is completely safe and

is the "healthiest" of all commonly used cooking oils. It has well established heart

health benefits and is recognized by many health professional organizations

including the American Dietetic Association and American Heart Association (Lorgeril

and Salen, 2006). Canola oil has been authorized a qualified health claim from the US

Food and Drug Administration based on its ability to reduce the risk of coronary

heart disease due to its unsaturated fat content.

Concerns and Challenges

Various studies confirm that outcrossing, or gene transfer, does happen, albeit at a

rate of less than 0.25% in a typical 160 acre field. Research has shown that most of

the outcrossing happens within 100 metres of the field edge (Kott et al., 1990; Linder,

1994). Crawley et al. (1993), who staged one of the largest-scale plant population

studies of all time, to evaluate the invasiveness of conventional canola and canola

genetically engineered to express resistance to the antibiotic kanamycin or the

herbicide glufosinate. Crawley et al. measured the finite rate of increase of these

lines of canola in four habitat types in each of three sites, with a barrage of

treatments that included cultivation/no cultivation, fencing, molluscicide, insecticide,

and fungicide. They found that only in cultivated plots can canola replace itself, and

in no case did the transgenic plants exhibit a higher finite rate of increase than the

conventional plants.

Genetic Engineering of Canola

8

Canola Council of Canada refers this concern as isolated case and urging the farmers

to develop herbicide use pattern to manage the problem of outcrossing when

different types of canola, such as Roundup Ready, Liberty Link, Smart or

conventional varieties are grown side by side. There are many broadleaf herbicides

on the market that will control this problem. The familiar 2,4-D or MCPA, used in

rotational crops such as cereals, are effective.

GM canola facing similar challenges like other GM crops such as corn, cotton, and

soybean due to strong objections about some aspects of genetic engineering.

Opponents range from those who believe the techniques should not be used at all to

those who believe products based on the techniques should be labelled as such. The

fierce debate continues even though supporters of genetic engineering have

tempered, and in some cases refuted, the arguments advanced by the opposition.

Those who oppose genetic engineering argue that these techniques are largely

untested. They assert that genetically engineered crops and foods are not

“substantially equivalent” to those produced using traditional means and should not

be expected to have similar health and environmental impacts. Among the major

health concerns is the potential for creating allergenic properties in foods. One

widely reported case involved the development of a genetically transformed

soybean variety that, in the course of field trials, was found to contribute to allergies

(Bailey, 2000). The long-term environmental impacts of growing genetically

engineered canola are also being questioned (Weick and Walchli, 2002)

Genetic engineering has also been opposed on ethical and religious grounds because

the techniques allow for cross-fertilizing that would not occur in nature (Moseley,

1999). The role of business - particularly large multinationals - in the research,

development, and marketing of genetically engineered crops and foods also has

caused some scepticism. Critics argue that agricultural biotechnology is dominated

by large companies that may allow profits to blind them to potential risks. Some are

concerned that these companies will tend to favour applications with large markets

Genetic Engineering of Canola

9

where the products can be afforded, i.e. mostly in industrialized countries - rather

than farmers with smaller holdings in developing countries. Others fear that

developing countries will be used as a “dumping ground” for untested technology. In

fact, the very need for greatly increased amounts of food provided by genetic

engineering has been questioned. The world food shortage, opponents argue, is not

a matter of quantity but of distribution and affordability, and they argue that these

problems will not be solved via genetic engineering

The Future

Canola is the workhorse of the agricultural biotechnology industry. Compared to

other crops, its DNA is relatively easy to modify. Canola can be engineered to

produce medicines, or to produce a specific fatty acid for speciality markets or

industrial uses. The quality of the oil and protein can be improved, to produce better

products for frying and salad oil, or high quality livestock feed. Plant biotechnology is

adding value to canola. In this first generation of genetically modified crops, the

benefits have largely gone to the seed companies and producers. In the future,

consumers will see direct benefits as well.

References Achaya, K.T. 1987. Fat Status Among Indians - A Review. Journal of Scientific and

Industrial Research. 46: 112-126. Bailey P. 2000. Promise or Peril? Are Genetically Engineered Foods Cause for

Concern? UC Davis Magazine. 23(5): 23-26. Batista, R. and Oliviera, M.M. 2009. Fact or Fiction of Genetically Modified Food.

Trends in Biotechnology. 27(5): 277-286. Bellenand, J.F., Baloutch, G., Ong, N., and Lecerf, J. 1980. Effects of Coconut Oil on

Heart Lipids and on Fatty Acid Utilization in Rapeseed Oil. Lipids. 15(11): 938-943.

Brown, J, Thill, D.C., Brown, A.P., Mallory-Smith, C., Brammer, T.A., and Nair, H.S.

1996. Gene Transfer Between Canola (Brassica napus) and Related Weed Species. Annals of Applied Biology. 129(3): 513-522.

Genetic Engineering of Canola

10

Crawley, M.J., Hails, R.S., Rees, M., Kohn, D., and Buxton, J. 1993. Ecology of Transgenic Oilseed Rape in Natural Habitats. Nature. 363: 620-623.

Graef, F., Stachow, U., Werner, A., and Schutte, G. 2007. Agricultural Practice

Changes with Cultivating Genetically Modified Herbicide-tolerant Oilseed Rape. Agricultural Systems. 94(2): 111-118

Inomata, N. 1978. Production of Interspecific Hybrids of B. campestris x B. oleracea by Culture In Vitro of Ovaries. Japan Journal of Genetics. 53: 161-173.

Kott, L.S., Erickson, L.R., Beversdorf, W.D. 1990. The Role of Biotechnology in Canola/Rapeseed Research. In Shahidi, F. (ed), Canola and Rapeseed: Production, Chemistry, Nutrition, and Processing Technology. pp 47-80. Van Nostrand Reinhold, New York, USA.

Linder, C. R. 1994. The Ecology of Population Persistence for Wild, Crop, and Crop-

wild Hybrid Brassica and its Implications for Transgenes Escaped from Canola. PhD Thesis. Brown University, Providence, USA.

Lorgeril, M, and Salen, P. 2006. The Mediterranean-style Diet for the Prevention of Cardiovascular Diseases. Public Health Nutrition. 19(5): 118-123.

Moseley, B.E.B. 1999. The Safety and Social Acceptance of Novel Foods. International Journal of Food Microbiology. 50(6): 25-31.

Salisbury, P. 1998. Genetically Modified Oilseeds – The Impact of Gene Technology on

the Australian Oilseeds Industry. Australian Oilseeds Federation Innovations and Technology Committee, Sydney, Australia.

Storgaard, A.R. 2008. Stefansson, Baldur Rosmund. In The Editors, The Canadian

Encyclopedia, pp 600-602. Hurtig Publisher, Edmonton, Canada. Weick, C.W. and Walchli, S.B. 2002. Genetically Engineered Crops and Foods: Back to

the Basics of Technology Diffusion. Technology in Society. 24(3): 265-283 http://dictionary.infoplease.com/canola (131009) http://www.regional.org.au/au/gcirc/canola/p-09.htm (141009) http://www.fda.gov/Food/Biotechnology/Submissions/ucm161144.htm (171009) http://www.canola-council.org/cooking_myths.html (181009)