The capacs 1, capacs 2, etr1-1, nptII and uidA genes and their associated regulatory sequences have been introduced into papaya (variety ‘Solo’) by microprojectile particle bombardment. This technique is a well-established method of plant transformation that uses compressed air to ‘shoot’ tiny tungsten or gold particles coated with the genes to be inserted into plant cells. The introduced genes become incorporated into the genome of the bombarded plant cells. The antibiotic kanamycin is used to select for plant cells containing the introduced genes and these cells are regenerated into whole plants in the laboratory.
Section 5 Characterisation of the inserted genetic material and stability of the genetic modification
The applicant intends to gather data on key information regarding the genetic modification and successful functioning of the inserted genes from the proposed limited and controlled release. It is indicated that these data cannot be generated without a field release.
Section 5.1 Characterisation by Southern blot and PCR analysis
Polymerase Chain Reaction (PCR) and Southern blotting can be used to demonstrate the presence of the introduced capacs 1, capacs 2, etr1-1, nptII and uidA genes. PCR detects the introduced genes in the GM plant but does not indicate the number of copies of the gene that are present. In contrast, Southern blotting indicates the number of copies of each inserted gene.
PCR has been used to demonstrate the presence of the introduced ACC synthase genes in both sense and antisense orientation in the GM papaya plants currently growing at the proposed release site under licence number PR-128 but to date, Southern blotting has not been employed. Using PCR, UQ has indicated that four plants contain the capacs 1 gene in the sense orientation, eight plants contain the capacs 1 gene in the antisense orientation and eight plants contain the capacs 2 gene in the antisense orientation. In addition, UQ has indicated that additional plants containing either sense and/or antisense copies of the capacs 1 and capacs 2 genes are in tissue culture. The applicant expects that these immature GM papaya plants will be ready for transplantation to the proposed release site in August 2003.
Neither PCR nor Southern blot data are available for the GM papaya plants containing the capacs 1 and capacs 2 hairpin constructs, the GUS reporter gene or the etr1 1 gene, as these GM plants are still being developed. As with the capacs 1 and capacs 2 genes, the applicant expects that GM papayas containing the capacs 1 and capacs 2 hairpin constructs, the GUS reporter gene or the etr1 1 gene will be ready for transplantation to the proposed release site in August 2003.
As a condition of licence, the applicant is required to confirm the presence of the inserted genes or hairpin constructs in all plants released into the field. More detailed characterisation of the inserted genes and how these affect the phenotype of the GM papayas is not required as a condition of the proposed licence, as the risk management measures given effect by the proposed specific licence conditions (see Appendix 5) effectively manage any risks. However, this additional information would be required by the Regulator if the same GM papayas are proposed for release in future applications (which would be subject to separate applications and assessments), before the application could be considered.
Assessing the stability of the introduced genes in progeny requires plants to produce fruit and seed and the resulting progeny to be analysed by Southern blotting. As the GM papayas that are currently released under PR-128 have not yet produced fruit, this is yet to be done. For the same reasons, the stability of the introduced genes has not been assessed in the other GM papaya plants proposed for release.
UQ has indicated that plants released into the field under PR-128 are currently flowering and starting to set fruit. Determining the stability of the inserted genes and genetic constructs in progeny of the GM papayas would be required by the Regulator if an additional or larger scale release of the same types of GM papayas are proposed for release in the future (which would be subject to separate applications and assessments).
Section 6 Expression of the introduced proteins
Northern blot analysis is a technique that can be used to determine expression levels and functioning of the introduced genes and gene constructs in the GM papaya plants. Mason and Botella (1997) used Northern blot analysis to study expression levels of capacs 1 and capacs 2 in non-GM papaya fruit. UQ has indicated that when fruit become available, expression levels of ACC synthase will be monitored in GM papaya fruit from various stages of ripening, to verify the functioning of the inserted genes or genetic constructs.
As the capacs 1 and capacs 2 sense and antisense genes and hairpin constructs are under the control of the 35S promoter, it is expected that they will be expressed in all plant tissues. Future applications to release the same types of GM papaya (which would be subject to separate applications and assessments) would require analyses of non-fruit parts of the GM papaya plants to ascertain expression levels of the introduced genes and hairpin constructs throughout the plant, before the application could be considered. Future applications would also require analyses of plants containing the GUS gene to evaluate the probable expression pattern and expression level of the introduced genes.
As the etr1-1 gene is under the control of a fruit-specific promoter, the applicant expects that etr1-1 will only be expressed in fruit. Future applications to release the same types of GM papaya would require measurement of the expression levels of the etr1-1 gene in fruit and non-edible plant parts such as leaves, the skin of fruit, seeds and roots, before the application could be considered.