Molecular characterisation of the parental GM canola lines included Southern blot and PCR analyses, as well as molecular cloning and sequencing of the site of insertion. Stable integration and inheritance of the inserted DNA was demonstrated in all of the lines. DNA sequencing was used to verify the inserted genes and to determine the regions flanking all of the insertions sites.
In lines T45, MS1, MS8, RF1, RF2 and GT73, a single insertion event occurred resulting in transfer of a single copy of the T-DNA. In line RF3, a single insertion event occurred that resulted in the integration of one complete copy and a second, incomplete T-DNA copy that included a second copy of the barstar gene. In line Topas 19/2, there is a single insertion event that resulted in a head to head inverted repeat of the T-DNA, such that there are two copies of each of the inserted pat and nptII genes.
Field trials of InVigor® canola and Roundup Ready® canola began in Australia in 1996 and 1997, respectively. Roundup Ready® canola has been grown commercially in NSW and Victoria since 2008, and in WA since 2010. In addition, events MS8, RF3 and GT73 have been commercialised for more than 10 years in Canada. In the multiple breeding programs and seed production, there have been no reports of aberrant segregation and instability.
InVigor® x Roundup Ready® canola
Southern blot analysis was used to demonstrate the molecular equivalence of the MS8, RF3 and GT73 events in InVigor® x Roundup Ready® canola to the same events in the individual parental lines. Identical Southern hybridisation patterns were observed for InVigor® x Roundup Ready® canola compared to InVigor® canola lines MS8 and RF3 and to Roundup Ready® canola GT73. These findings confirm the intactness of the GM loci and their flanking regions in InVigor® x Roundup Ready® canola, indicating that no rearrangement occurred during conventional breeding (Moens 2009b).
Expression of the encoded proteins in the GM canola
Parental GM canola lines
The expression of each of the introduced genes in each of the GM canola lines authorised under DIR 021/2002 (Topas 19/2, T45, RF1, RF2, RF3, MS1 and MS8) was determined using a variety of techniques including plant phenotype, mRNA expression, and detection of the novel protein by enzyme activity or Enzyme Linked ImmunoSorbent Assays (ELISA). The patterns and levels of expression of the introduced proteins in the GM canola lines were as predicted on the basis of the promoters controlling expression, and a summary of these data is given in Table .
Table Summary of expression of the introduced proteins in GM canola lines included in licence DIR 021/2002
Introduced Protein
(GM lines assayed)
Leaves
Seed
Other tissues
PAT
(All lines)
Low levels
Very low levels
Very low levels
BARNASE
(MS1, MS8 or MS x RF)
Not expressed
Not expressed
Flower buds only: tapetum layer of developing anthers
BARSTAR
(RF1, RF2, RF3 or MS x RF)
Not expressed
Not expressed
Flower buds only: tapetum layer of developing anthers
NPTII
(Topas 19/2, RF1, RF2, MS1)
Very low levels
Not detected
Not detected
The level of PAT in oil and meal derived from processing of seed from lines T45 and Topas 19/2 was investigated by ELISA. No PAT protein was detected in canola oil derived from the GM canola lines. While PAT protein could be detected by ELISA at less than 0.005% of total protein in toasted canola meal, the processing of canola seed to produce edible oil and meal for animal feed denatures the PAT protein and destroys the enzymatic activity (FDA 1997; ANZFA 2001b).
Expression of the bar, barnase, barstar, nptII genes was also investigated by Northern analysis. Expression patterns were as predicted for the promoters used, and no mRNA from any of the genes was detected in pollen or dry seed.
The levels of expression of the CP4 EPSPS and GOXv247 proteins in leaf tissue and seeds of the parental Roundup Ready® canola were measured by ELISA (see DIR 020/2002). Results from several field trials conducted overseas demonstrate that the CP4 EPSPS and GOXv247 proteins are expressed at very low levels in leaves and seeds. The level of expression of CP4 EPSPS and GOXv247 constitutes less than 0.02% and 0.07%, respectively, of the seed on a fresh weight basis. Expression levels of the introduced proteins in Roundup Ready® canola were not affected by application of glyphosate.
InVigor® x Roundup Ready® canola
The expression levels of PAT, CP4 EPSPS and GOX proteins in leaf and seed tissues of InVigor® x Roundup Ready® canola and its parental lines MS8, RF3 and GT73 were measured by ELISA. Prior to sampling, MS8 and RF3 plants were treated with glufosinate ammonium, GT73 plants were treated with glyphosate, and MS8 x RF3 x GT73 plants were treated with both herbicides.
Table provides a summary of the zygosity of the herbicide tolerance genes in the GM canola plants analysed in this study. GM canola line MS8 is hemizygous for the bar gene. Due to segregation, only 44% of the MS8 x RF3 x GT73 plants generated contained a copy of this gene. However, only MS8 x RF3 x GT73 plants containing two copies of the bar gene were used in this study.
Table Zygosity of the herbicide tolerance genes in the GM canola plants
Event/stack
bar gene
CP4 EPSPS gene
gox gene
MS8
hemizygous (1 copy)
–
–
RF3
homozygous (2 copies)
–
–
GT73
–
homozygous (2 copies)
homozygous (2 copies)
MS8 x RF3 x GT73
hemizygous for both MS8 and RF3 (2 copies)
hemizygous (1 copy)
hemizygous (1 copy)
For each protein to be analysed, 10 separate leaf or seed samples were assayed. The average expression levels of the PAT, CP4 EPSPS and GOX proteins in samples of InVigor® x Roundup Ready® canola and the parent lines are given in Table . Differences in protein expression levels between the parent lines and the MS8 x RF3 x GT73 stack correlated with the zygosity of the plants. Protein expression levels in InVigor® x Roundup Ready® canola are either similar to or lower than the low levels observed in the parental lines. This analysis showed no evidence for any interaction between the three events when combined in InVigor® x Roundup Ready® canola (Moens 2009a).
Table Average amount of protein per gram fresh weight in leaf and seed tissue samples from GM canola plants
