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December 2012

Introduction


There are a number of methods for genetically modifying (or 'transforming') plants. The most common three methods are outlined below. More detail about these and other techniques can be found in a number of recent reviews (eg Barampuram & Zhang 2011; Hooykaas 2010; Rivera et al. 2012).

Methods for introducing new DNA into plant cells can be divided into two major categories: indirect and direct DNA delivery. In the former approach, genes of interest are introduced into the target cell via bacteria, usually Agrobacterium tumefaciens or (less commonly) Agrobacterium rhizogenes (Tzfira & Citovsky 2006). Direct transformation, introduces genetic material without an intermediate host. The most commonly used direct transformation methods are biolistic transformation and protoplast transformation (reviewed by Rivera et al 2012).

Agrobacterium-mediated transformation


Agrobacterium-mediated transformation is well-established for dicotyledonous plants (flowering plants whose seeds produce two seed leaves when germinating; most herbaceous plants, trees, and bushes are dicotyledonous). However, most monocotyledonous plants (flowering plants that produce one seed leaf, eg grasses) are not natural hosts of A. tumefaciens (De Cleene & De Ley 1976), and until recently transformation of monocots using Agrobacterium was difficult and unreliable (Sood et al. 2011).

Agrobacterium tumefaciens is a common soil bacterium that naturally causes gall formation on a wide range of plant species, including most dicotyledonous and some monocotyledonous species (Van Larebeke et al. 1974). The gall is induced by transfer of hormone-producing genes from the bacterial cell into the plant genome. The genes are carried on a circular DNA molecule found within the bacterial cell called a Tumour-inducing (Ti) plasmid. During the infection process, only a section of the Ti plasmid known as the Transfer DNA (T-DNA) is transferred to the plant. The infection and T-DNA transfer process of A. tumefaciens has been extensively studied. This natural process has been used to facilitate genetic modification of plants.

A. tumefaciens Ti plasmids have been produced that lack the genes responsible for gall formation (disarmed plasmids). Genes to be inserted into the plant are put into the T-DNA section of these disarmed plasmids. A. tumefaciens cells carrying such plasmids cannot produce a gall in an infected plant but will transfer the T-DNA sequence carrying the genes of interest into the plant cell where they stably integrate into the plant genome (Bevan 1984; Klee & Rogers 1989).

Transformation with Agrobacterium can be achieved using a variety of plant tissues including protoplasts (isolated cells with their cell walls removed) and leaf discs. The plant tissue is incubated with the bacterium for a variable period to allow infection to occur and then transferred to a synthetic medium containing nutrients and hormones that promote the growth of plants from single transformed cells, as well as a selective agent such as an antibiotic to eliminate untransformed cells1. During this regeneration process, transformed cells usually first divide to form a mass of tissue called callus (callus normally forms at a wound or graft site on a plant) before formation of plantlets. Independent of the process of Agrobacterium-mediated transformation, the regeneration of callus can induce mutations or other changes in the plant cell genome, known as somaclonal variation.

Alternatively, methods have been developed for Agrobacterium-mediated transformation using whole plants, such as vacuum infiltration and floral-dip. These methods minimise somaclonal variation and result in more genetically uniform progeny. For the floral dip method (Clough & Bent 1998) flowers are dipped into an Agrobacterium culture and the bacterium transforms the germline cells that make the female gametes. The seeds from these plants are grown and screened for the genetic modification, eg using an antibiotic resistance or herbicide tolerance marker.

For plants transformed via Agrobacterium, residual bacteria have been shown in some cases to survive through the transformation process and remain within regenerated GM plants (Barrett et al. 1997; Yang et al. 2006). Agrobacteria are usually eliminated when GM plants are grown from seed but may remain in plants that are propagated vegetatively, potentially allowing residual GM Agrobacterium to transfer genes to other bacteria. It has also been suggested that if Agrobacteria and fungi were both present at plant wound sites, natural gene transfer may occur from bacterium to fungus (Knight et al. 2010). For these reasons, Agrobacterium-mediated transformation techniques usually include stringent methods to prevent Agrobacteria surviving, as well as steps to screen for and eliminate any GM plants with remaining Agrobacteria, to minimise potential for gene transfer occurring in the environment.

In addition to transfer of the T-DNA sequence, small segments of flanking Ti plasmid sequence and A. tumefaciens chromosomal sequence may be transferred into the plant genome at a low frequency (Smith 1998; Ulker et al. 2008).

Biolistic transformation (particle bombardment)


Biolistic transformation was developed in the 1980s to genetically modify plants that were not amenable to transformation with Agrobacterium. In this technique, DNA is delivered into plant cells on small tungsten or gold carrier particles, approximately 2 microns in diameter. The particles are coated with the gene(s) of interest and fired into plant cells or tissues, usually using pressurised helium. Some of the particles penetrate the cell nucleus, where the introduced genetic material is incorporated into nuclear DNA (Sanford 1990). The bombarded cells or tissues are then transferred to a synthetic growth medium containing a selective agent, such as an antibiotic, to eliminate untransformed plants.

In recent years, biolistic transformation has become a very common method to genetically modify plants, and has been shown to be applicable to virtually all species investigated. It can also be used to deliver DNA to specific parts of plant cells (eg chloroplasts), as well as to bacteria and fungi. The major factor limiting the use of biolistic transformation is that it often results in insertion of multiple copies of the introduced genes at multiple sites within the genome, which can lead to gene silencing or altered gene expression (Rivera et al 2012).

Protoplast direct transformation


The plant cell wall forms a major obstacle to transformation, so early direct transformation methods relied on protoplasts (plant cells with the cell wall removed typically by enzymatic digestion). Protoplast technology was initially restricted to certain dicotyledonous plants, but subsequently became feasible for important cereal crops (Hooykaas 2010).

A number of methods have been developed for transformation of protoplasts (reviewed in Rivera et al 2012), most of these being variations of methods used for animal cell transformation. For reasons of low cost and ease of use, the two most commonly used methods are chemical treatment with polyethylene glycol (in combination with calcium ions), and electroporation. Both of these methods change the permeability of the cell membrane, allowing entry of exogenous DNA. The use of protoplasts for transformation requires elaborate tissue culture steps to regenerate fertile adult plants and may introduce genetic instability and resulting somaclonal variation.

Risk assessment


The techniques outlined above are routinely employed for plant transformation and the mechanisms are well described. Any discussion of potential risk associated with these techniques has focused largely on the possibility of insertional mutagenesis (Wilson et al. 2006). Biolistic transformation can lead to multiple insertions and complex integration patterns that could potentially influence the traits of GM plants, for example through silencing of introduced or endogenous genes, or creation of new open reading frames. Agrobacterium-mediated transformation usually only results in one or a few insertions into the plant genome, however small segments of Agrobacterium chromosomal DNA may also be transferred at a very low frequency. The likelihood of the A. tumefaciens chromosomal DNA having an adverse impact on the GM plants is small because Agrobacterium chromosomal genes do not contain regulatory elements required for expression in plants are therefore are unlikely to be expressed.

The range of possible unintended effects produced by genetic modification is not likely to be greater than that from accepted traditional breeding techniques such as hybridisation, mutagenesis and somaclonal variation (Bradford et al. 2005; Committee on Identifying and Assessing Unintended Effects of Genetically Engineered Foods on Human Health 2004). New varieties produced by traditional breeding techniques have rarely had traits that are undesirable for human health, safety or the environment (Hajjar & Hodgkin 2007). Plants produced by genetic modification rarely have unintended changes that are advantageous to the plant (Kurland et al. 2003). Any unintended changes that might arise are usually eliminated before reaching a commercial product.

Despite the widespread employment of these methods, there have been no reports of adverse effects on human health and safety or the environment as a result of their use. Nevertheless, irrespective of which method of transformation was used, the potential for adverse unintended effects as a result of gene technology is assessed on a case-by-case, in the context of the proposed dealings, in each risk assessment for release of a GM plant.

References

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Barampuram, S., Zhang, Z.J. (2011). Recent advances in plant transformation. Methods in Molecular Biology 701: 1-35

Barrett, C., Cobb, E., McNicol, R., Lyon, G. (1997). A risk assessment study of plant genetic transformation using Agrobacterium and implications for analysis of transgenic plants. Plant Cell, Tissue and Organ Culture 47: 135-144

Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12: 8711-8721

Bradford, K.J., van Deynze, A., Gutterson, N., Parrott, W., Strauss, S.H. (2005). Regulating transgenic crops sensibly: lessons from plant breeding, biotechnology and genomics. Nature Biotechnology 23: 439-444

Clough, S.J., Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743

Committee on Identifying and Assessing Unintended Effects of Genetically Engineered Foods on Human Health, N.R.C. (2004). Unintended Effects from Breeding. Chapter 3. In: Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects. The National Academies Press pp 39-71.

De Cleene, M., De Ley, J. (1976). The host range of crown gall. Botanical Review 42: 389-466

Hajjar, R., Hodgkin, T. (2007). The use of wild relatives in crop improvement: a survey of developments over the last 20 years. Euphytica 156: 1-13

Hooykaas, P. (2010). Plant transformation. In: Encyclopedia of Life Sciences (ELS). John Wiley and Sons, Ltd

Klee, H.J., Rogers, S.G. (1989). Plant gene vectors and genetic transformation: plant transformation systems based on the use of Agrobacterium tumefaciens. Cell Culture and Somatic Cell Genetics of Plants 6: 1-23

Knight, C.J., Bailey, A.M., Foster, G.D. (2010). Investigating Agrobacterium-mediated transformation of Verticillium albo-atrum on plant surfaces. PLoS ONE 5: 1-5

Kurland, C.G., Canback, B., Berg, O.G. (2003). Horizontal gene transfer: a critical view. Proceedings of the National Academy of Science of the United States of America 100: 9658-9662

Rivera, A.L., Gomez-Lim, M., Fernandez, F., Loske, A.M. (2012). Physical methods for genetic plant transformation. Physics of Life Reviews 9: 308-345

Sanford, J.C. (1990). Biolistic plant transformation. Physiologia Plantarum 79: 206-209

Smith, N. (1998). More T-DNA than meets the eye. Trends in Plant Science 3: 85

Sood, P., Bhattacharya, A., Sood, A. (2011). Problems and possibilities of monocot transformation. Biologia Plantarum 55: 1-15

Tzfira, T., Citovsky, V. (2006). Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Current Opinion in Biotechnology 17: 147-1

Ulker, B., Li, Y., Rosso, M.G., Logemann, E., Somssich, I.E., Weisshaar, B. (2008). T-DNA-mediated transfer of Agrobacterium tumefaciens chromosomal DNA into plants. Nature Biotechnology 26: 1015-1017

Van Larebeke, N., Engler, G., Holsters, M., Van den Elsacker, S., Zaenen, I., Schilperoort, R.A., Schell, J. (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252: 169-170

Wilson, A.K., Latham, J.R., Steinbrecher, R.A. (2006). Transformation-induced mutations in transgenic plants: analysis and biosafety implications. Biotechnology and Genetic Engineering Reviews 23: 209-234
Yang, M., Ewald, D., Wang, Y., Liang, H., Zhen, Z. (2006). Survival and escape of Agrobacterium tumefaciens in triploid hybrid lines of Chinese white poplar transformed with two insect-resistant genes. Acta Ecologica Sinica 26: 3555-3561


1Information on use of antibiotics and antibiotic selection markers in GM plants can be found in the risk assessment reference document Marker Genes in GM Plants available on the OGTR website at the "Documents relating to the Risk Assessment process" page