Transformation has played an important role in the transgenesis of B. napus, B. rapa and B. juncea since the late eighties. Agrobacterium tumefaciens is commonly used, although A. rhizogenes also has its own applications. The success of transformation is generally determined by the plant genotype and the characteristics of the vector constructs. Although A. tumefacien mediated transformation is preferred, there is still interest concerning DNA uptake by protoplasts and by micro-projectile bombardment.
From the common methods of transformation only a small percentage of cells become transformed so it is important to use a selectable marker gene. The most frequently used marker gene is nptII, conferring resistance to kanamycin (antibiotic). However there is evidence both for and against having the nptII gene in the finished plant varieties. The protein product of the nptII gene is of little or no consequence to human health or the environment, but the questions are still asked about the advisability of including the nptII gene in commercialised transgenic plant varieties as this antibiotic resistance gene may become integrated into other plant systems. In the USA, 'Flavr Savr' tomatoes carrying the nptII have been deemed safe for commercial use and human consumption. A few years ago in the UK the Advisory Committee on Novel Foods and Processes (ACNFP) stated that researchers developing food GMO's should be encouraged to develop and use alternatives to antibiotic resistance markers.
One method utilised for elimination of the nptII gene from transgenic varieties is to transform the plant with a combination of two cell lines of Agrobacterium:
The requirement is for the plant to become transformed with both Agrobacterium strains so that the two T-DNA's (transforming DNA's) are inserted at different chromosomal locations. Back-crossing to non-transgenic lines should give rise to some progeny containing the gene of interest, but without the nptII gene.
An example of marker elimination was shown by Bade and Damm (1995) when nptII was used as the selectable marker gene and gus as the reporter gene. Following transformation with an inoculum of two Agrobacterium lines and after selection for kanamycin resistance, 30-50% of the regenerated plants were gus positive and therefore co-transformed. More than 50% of these co-transformed lines gave rise to progeny that were both gus positive and kanamycin susceptible. This suggests that in about half of the lines the gus and nptII genes were unlinked (or not tightly linked) and could therefore be separated by sexual genetic recombination.
Transgene instability is commonly observed in primary transformants (TO) and occasionally during early sexual generations. There is also variation between independently transformed plants in transgene expression.
For example, there has been research into transgene stability in B. napus using a range of vector derivatives containing constitutive promoters (35S from cauliflower mosaic virus, nos from Agrobacterium) and embryo specific promoters (oleosin and cruciferin from B. napus) that regulate the coding sequences for gus (Beta-glucuronidase) and bar (resistance to the herbicide, 'Basta'). For all constructs, transgenic plants were observed that had some loss of expression and some physical loss of the construct. Some transgenic lines containing the embryo specific promoters showed an alteration in tissue specificity of transgene expression, which was also observed in the T1 generation.
In other studies with B. napus there is evidence that the background genotype can affect the expression and structural stability of specific vector constructs.
Importantly, the long term stability and utility of transgenes in transgenic plant varieties can only really be assessed during commercial production. However, it is expected that transgenes will be as stable as their resident native genes once the transgenic lines have gone through the standard selection and evaluation trials used in conventional breeding. Subsequently, in practise it is usual to create 100 independently transformed plants with any given construct to provide enough transgenic lines to be able to select for the desired transgene:
