In preparing the risk assessment and risk management plan, the Regulator is required under Section 49 (2) of the Act to consider the properties of the parent organism and the effects of genetic modification.
This part of the document addresses these matters and provides detailed information about the GMOs for release, the parent organism, the genetic modification process, the genes that have been introduced, the information on genetic constructs, the new proteins that are expressed and altered phenotype of the papayas as a result of the genetic modification.
Section 1 Summary information about the GMOs
The University of Queensland (UQ) proposes a continued limited and controlled release of GM papaya (Carica papaya) plants with altered fruit ripening characteristics. UQ aims to evaluate the effect on fruit ripening of two genes involved in ethylene production and one gene involved in ethylene perception. Ethylene is a gaseous plant hormone that regulates many aspects of plant growth and development and is responsible for the timing of ripening in fruit (Alexander & Grierson 2002) (see Section 1.1).
In total, eight types of GM papayas are proposed for release (see Table 1). Seven of these have been modified to delay the process of fruit ripening and of these, six incorporate either sense, antisense or ‘hairpin’ (both sense and antisense genes linked in one construct) versions of two genes involved with ethylene biosynthesis. The seventh of these GM papayas with delayed fruit ripening contains a sense version of a gene involved with ethylene perception (see Section 1.2). The eighth type of GM papaya contains a reporter gene that helps to identify plant tissues in which the modified fruit-ripening traits are likely to be expressed.
Three types of GM papayas, with sense or antisense versions of two genes involved in ethylene biosynthesis, were released into the environment under limited and controlled conditions under licence number PR-128 in 2002, based on approval issued under the previous voluntary system administered by the Genetic Manipulation Advisory Committee (GMAC). Approval of licence application DIR 026/2002 would enable the continued limited and controlled release of the GM papayas licensed under PR-128, as well as the release of the five new types of GM papayas (with ‘hairpin’ versions of ACC synthase genes, a sense version of an ethylene perception gene or a reporter gene).
Section 1.1 The role of ethylene in plant biology
Ethylene is a plant hormone that plays a role in many different aspects of plant growth and development, including cell elongation, formation of root hairs, induction of seed germination and leaf and flower abscission (1999). Likewise, plant responses to stresses, such as wounding or pathogen attack, involve ethylene (Stepanova & Ecker 2000; Thomma et al. 2001). The process of ripening in many fruit, such as bananas, melons, avocados and tomatoes, is also controlled by ethylene, which is particularly important in the transition from nature green fruit to ripe fruit (Alexander & Grierson 2002).
Ethylene is a gas, which is produced in all plant tissues and is able to rapidly diffuse out of the tissue in which it is produced. ACC (1-amino-cyclopropane-1-carboxylic acid) is the immediate precursor of ethylene. The enzyme ACC synthase produces ACC from SAM (S-adenosyl methionine), which in turn is derived from the amino acid methionine via the action of the enzyme SAM synthase (see Figure 1). ACC synthase also converts SAM to 5-methylthioadenosine (MTA), which is used for the re-synthesis of methionine, thus maintaining a constant pool of methionine in the cell.
Figure 1 The biosynthesis of ethylene in plants. Intermediates in the pathway are in bold; enzymes responsible for each step in the pathway are in italics.
ACC synthase methionine
Papayas were genetically modified to delay fruit ripening by either down-regulating ethylene production or disrupting ethylene perception.
1.2.1 Down-regulation of ethylene production
The GM papaya plants will contain either sense or antisense copies (copies in the correct or inverse orientation) of the native papaya genes, capacs 1 and capacs 2, encoding the enzyme ACC synthase, or ‘hairpin’ constructs (sense and antisense copies linked in one construct) (Smith et al. 2002) of these genes. Introduction of an additional copy or copies of the native capacs 1 and capacs 2 genes in sense and/or antisense orientation is expected to silence the corresponding native genes, resulting in decreased production of ethylene by the plant due to inhibition of ACC production. The resulting reduction in ethylene is expected to prevent or delay ripening of mature papaya fruit.
Table 1 Summary of the introduced genes present in the eight types of GM papayas proposed for release and the expected phenotypes. Entries in bold refer to GM papaya types that were released under PR-128.
12 two different transformation vectors were used for generating the GM papaya plants. Consequently, the GM papaya plants will contain either two versions of the nptII gene, one of which is controlled by a bacterial promoter and will not be expressed in the GM papaya plants, or the nptII gene and the bla gene, which is also under the control of a bacterial promoter and not expressed in the GM papaya plants (see Section 3.4).
1.2.2 Disruption of ethylene perception
The proposed release also aims to evaluate the fruit ripening characteristics of GM papaya plants carrying a gene required for ethylene perception from Arabidopsis thaliana (mustard cress). Arabidopsis plants have been identified that carry a non-functional copy of the etr1 gene (etr1-1) (Chang et al. 1993; Bleecker et al. 1988). Plants carrying this non-functional gene are insensitive to ethylene and exhibit delayed fruit ripening and floral senescence.
The capacs 1, capacs 2 and etr1-1 genes are discussed in more detail in Section 3 of this Appendix. Potential hazards relating to transfer of these genes to other papaya plants are discussed in Appendix 4.
Section 1.3 Selectable marker and reporter genes
Some GM papaya plants proposed for release express the uidA gene from the bacterium, Escherichia coli, instead of the ethylene-related genes. This gene codes for the enzyme b-glucuronidase (GUS). Its expression enables visual identification of plant tissues in which this enzyme is produced and will provide a means of confirming the effectiveness of the regulatory sequence (promoter) that is used to drive expression of the fruit ripening genes. GUS activity will also be used to indicate the tissues in which the genes are expressed.
The GM papaya plants also contain antibiotic resistance genes. These genes were used as selectable marker genes in the early laboratory stages of development of the plants, to enable selection of bacteria or plant cells containing the desired genetic modification. The antibiotic resistance genes are the bacterial neomycin phosphotransferase type II (nptII) gene, conferring resistance to the antibiotics kanamycin and neomycin, and the beta-lactamase (bla) gene. The bla gene confers resistance to the antibiotic ampicillan and is linked to a bacterial promoter that does not function in plants, so the protein is not expressed in the GM papayas. The antibiotic resistance genes are discussed in more detail in Section 3 of this Appendix. Potential hazards relating to transfer of these genes to other organisms are discussed in Appendix 4.