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The transgenic pineapple expressing the traits of herbicide resistance and delayed flowering or reporter gene expression (OGTR 2002) were generated using gene technology. Firstly, tobacco acetolactate synthase gene (suRB) and 1-aminocyclopropane-1-carboxylate synthase gene (ACACS2) or ß-Glucuronidase (uidA) gene, derived from Nicotiana tabacum, and Ananas comosus or Escherichia coli (OGTR 2002), respectively, were isolated. Those interested genes were then introduced into plant cells of cultivated pineapple (Ananas comosus), named Smooth Cayenne, utilizing Agrobacterium tumefaciens-mediated transformation system (UQ 2003). Agrobacterium tumefaciens is a disarmed strain which carries a binary vector (UQ 2003). During inoculation of leaf base cells with A. tumefaciens, the T-DNA plasmid of A. tumefaciens carrying the genes of interest inserts into the plant genome (Glick and Jack 2003). Afterward, transformed tissues were selected on medium containing chlorosulfuron (UQ 2003). The expression of transgenes are under the regulation of several promoters, such as enhanced 35S promoter which is derived from the Cauliflower Mosaic Virus, ubi1 promoter from maize, or the chimeric promoter, SpMas, isolated from Agrobacterium tumefaciens Ti plasmid (UQ 2003).
ENZYME ACTIVITIES OR METABOLIC CHANGES
In this typical application, two types of GMOs were proposed in the field trial. One contains sequences encoding ACC (1-aminocyclopropane-1-carboxylate) synthase and tobacco acetolactate synthase, while the other carries the transgenes expressing tobacco acetolactate synthase, and ß-Glucuronidase (UQ 2003).
ACC synthase, known as pyridoxal enzyme (Davis and Metzler 1972), is derived from Ananas comosus (UQ 2003)
It is the major regulatory enzyme involving in the endogenous production of ethylene (Botella et al. 2000; Trusov and Botella 2006). ACC synthase converts S-adenosylmethionine (SAM), which is synthesized from the amino acid methionine using the enzyme SAM synthase (Yang and Hoffman 1984), into ACC (Adams and Yang 1979). ACC is then used as a metabolic intermediate in the synthesis of ethylene (Davey et al. 2007), one of compounds inducing flowering in pineapple (UQ 2003), via enzyme ACC oxidase (Yang and Hoffman 1984).
ACC synthase also converts SAM to 5-methylthioadenosine (MTA), which is used for the re-synthesis of methionine. Consequently, methionine cycle is maintained constantly in the cell (UQ 2003).
The addition of ACC synthase gene in pineapple genome results in transgenic pineapple carrying sense and antisense copies of the gene of ACC synthase (Cunha 2005). It leads to silencing of ACACS2 in transgenic pineapple plants, thereby suppressing natural flowering (Cunha 2005) until it is induced arti¬cially (Gomez-Lim and Litz 2004). Hence, the transgenic pineapple, conferred delayed flowering properties, does not produce ethylene in response to the reduction of temperature (Gomez-Lim and Litz 2004).
Tobacco acetolactate synthase, a vital enzyme in the synthesis of amino acids, is derived from Nicotiana tabacum (UQ 2003). Inserting an alternative form of the enzyme acetolactate synthase (ALS), less sensitive to herbicides, could confer resistance to sulfonylurea, imidazolinone, and triazolopyrimidine herbicide (Mazure and Falco 1989).
ß-Glucuronidase is a reporter gene which is derived from Escherichia coli (UQ 2003). It could be used to identify the transgenic plants in order to assess the activity of different regulatory sequences under field conditions (UQ 2003).
ADVANTAGES FOR PRODUCER AND CONSUMER
Application of transgenic pineapples which could delay natural flowering probably brings a number of benefits to producers as well as consumers. For instance, the harvesting cost, contributing 34% of growing costs (Thew 1990), could be reduced by as much as half because extra harvesting passes are not required (Botella et al. 2000). In addition, losses due to over-mature or immature fruit are probably minimized (Botella et al. 2000). Consequently, it reduces the cost of fruit production (Davey et al. 2007). Growing herbicide-tolerant pineapple plants also reduces the need to undertake frequent crop tillage, which is labour-costing (Sripaoraya et al. 2001). Furthermore, it leads to reduction of using chemicals, such as auxins and ethephon, for flowering control which has been used in the production of conventional pineapple (Min and Bartholomew 1996). Therefore, chemical costs and the labour costs for chemical application could be reduced (Rabie et al. 2000). As a result, customers could purchase pineapple at a lower price. Furthermore, skin texture and colour are important quality traits connected to the production of ethylene. Hence, producing transgenic pineapples, customers could be supplied fruits with better quality (Botella et al. 2000). In addition, blocking the ripening of the skin would provide a harder shell and better transport characteristics (Botella et al. 2000). Suppressing natural flowering could not only lead to the achievement of constant fruit production during the whole year (Rebolledo et al. 2000), but also allow more flexibility in planning cropping schedules. It thereby reduces overproduction which could cause low market price (Rabie et al. 2000). Another benefit could be the increase of fruit yields because improved weed control will lead to reduced competition (Sripaoraya et al. 2001). Therefore, producers may gain higher income (Sripaoraya et al. 2001).
THE SAFETY MEASURES AND RISK REDUCTION MEASURES
In order to manage and minimize the potential risk that might occurs to humans, other organisms as well as environment, the developers proposed the following actions (UQ 2003)
GM pineapples will be grown in limited zone of 0.1 hectares in the Shire of Redlands. The field trial is surrounded by fence with a lockable gate, to prevent the unauthorized access. It will decrease likelihood of exposure, persistence as well as spread of transgenic pineapple.
In release site, a number of signs indicating that GM pineapples are being planted in the site will be placed, to limit the risks posed by any potential toxicity or allergenicity or weediness of the GM pineapple and to decreases probability of spread of the transgenic pineapple.
The GM pineapples and its material must not be removed from the release sites without authorization to avoid the unaware spread of GM materials.
None of GM pineapple parts is used for human and animal consumption or therapeutics to prevent exposure.
The GM pineapple and its materials will be harvested and stored segregatedly to prevent the spread and persistence of the transgenic pineapple in the environment after the release. Unrequired viable materials or harvested GM pineapples will be destroyed to prevent the spread of the GMO.
After being used in dealing with genetically modified pineapples, all equipments will be cleaned immediately at the release site to prevent the spread and persistence of the transgenic pineapples outside the release site.
Transportation of the GM pineapple or its materials will comply with the OGTR Guidelines for Transport of GMOs in order to decrease likelihood of exposure and to prevent any escape and the spread or persistence of the genetically engineering pineapples outside the release site.
After completing the trial, applicant will clean the release site. They will then inspect periodically (every 30 days) the release site for at least 6 months. If there are regrowing pineapples from plant materials remaining on the ground after harvesting, they will be destroyed to prevent the dissemination and persistence of the GMOs in the environment.
Applicant must submit a report to the Regulator when there are any adverse effects on human health and safety. It probably allows quick intervention by the Regulator in response to adverse information regarding human health and safety and environmental safety.
Applicant will develop a research program in consultation with the OGTR in order to provide more information on the genetically modified pineapples and the research results to the Regulator in the annual report.
COMMENT ON THE RISK MANAGEMENT MEASURES
Toxicity and allergenicity
Although GM pineapples are unlikely to be more toxic than non-GM pineapples (UQ 2003), the applicant established an adequate risk management plan to protect human and other organisms against the exposure of GM pineapple. Those strategies includes placing signs at the release site, restricting access to the trial field, not using GM pineapple as human food or animal feed, reporting side effects of GM pineapples on human and animal safety to the regulator (UQ 2003). According to OGTR (2009), the occupational health and safety of people who deal with genetically modified organisms and the general public who could contact with GM organisms are one of the aspects regarding to GMO risks which need to be considered. Therefore, the typical strategies involving food and occupational exposure applicant set fit neatly into the protection goals which OGTR (2009) has established to protect the health and safety of human.
Researcher observed that bromelain, a pineapple protease, could cause respiratory and gastrointestinal allergic reactions (Baur and Fruhmann 1979). It means workers dealing with GM pineapple and customers consuming pineapple have potential of allergenicity when being exposure of the non-transgenic and transgenic pineapple. In addition, the unripe pineapple is inedible and poisonous, irritating the throat and acting as a drastic purgative (Morton 1987). Therefore, the strategies "not using GM pineapple as food for human", "restricting access to the release site" are reasonable. They will help to reduce the risk regarding to toxicity and allergenicity.
Gene transfer and weediness
Other concerns which have been raised in the development of genetically modified crops are probably gene flow and weediness (Craig, Tepfer et al. 2008).
The applicant did not propose the risk management plan in term of gene transfer. It could be acceptable due to several reasons. Firstly, although Smooth Cayenne could hybridize theoretically with it relatives (Sanewski, 2003 cited on (OGTR 2003)), in nature this phenomenon is relatively rare because pollination does not occur in pineapples (Collins 1960). If the pollination occurred successfully, the seed would stay in the fruit because commercial pineapples have no seed releasing mechanisms (Collins 1960). Furthermore, commercial pineapple is propagated using through vegetative shoots instead of seeds (Collins 1960). According to OGTR (2003), gene transfer between pineapples and other organisms is extremely rare and has not been reported. Given those facts, the risk management plan for gene transfer is probably not necessary.
It is reported that the pineapple species has not become as weeds in Australia (OGTR 2003). It is probably because they grows slowly, and are susceptible to common fungi like Phytophthora cinnamomi and sensitive to common herbicides (OGTR 2003). However, pineapple occurs as a managed cultigen exclusively (OGTR 2003). Although this phenomenon is not recognised as weediness, pineapple could become an agricultural weed by reshooting from large pieces of stem that are left intact and buried within a commercially cultivated field of pineapples if the plants from the previous crop are not completely destroyed (OGTR 2003). Therefore, applicant has established an adequate risk management plan to reduce the potential effects in regard to weediness. Implementing management strategies, such as destroying the unused viable materials, cleaning the release site after the field trials, doing not allow to remove the GM pineapple unauthorizedly, could prevent the possibility of the spread and persistence of GM pineapples in the environment (UQ 2003).