Genetic engineering of pineapples with blackheart resistance

• Malaysian Agricultural Research and Development Institute, Malaysia
• International Service for the Acquisition of Agro-biotech Applications, Philippines

In Australia and Asia, management of horticultural produce quality and minimisation of damage and losses have become increasingly important. Blackheart, an unsightly discolouration, is a major postharvest quality defect of pineapple that develops inside fruit during cool weather cultivation or cool storage, and affects the availability and quality of fruit for fresh market and processing. In some cultivars another disorder, crown deterioration, also occurs and it is possible that similar physiological mechanisms are involved. Earlier research under ACIAR project PHT/1994/007 (Pineapple quality improvement) saw advances in agricultural biotechnology to 'switch-off' the genes responsible for known as 'blackheart' that occurs inside pineapples at low temperature. That project developed the technology underpinning the research (including a novel transformation protocol), together with some of the transformed lines. Putative blackheart-resistant transgenic pineapples were developed in Australia and Malaysia.
The use of biotechnology for quality improvement in fruit has been recognised as a cost-effective and equitable means of reducing losses and improving returns to farmers. Project 1994/007 was a 'test case' for the application of biotechnology in horticultural crops in both countries. The next step, and the subject of this new project, was to select blackheart-resistant or immune lines with acceptable agronomic and postharvest characteristics and determine the chemistry of black heart symptom development. The project also developed intellectual property strategies to assist the commercial release and marketing of blackheart-resistant pineapples in Malaysia and Australia.

The project objectives were: to evaluate the agronomic characteristics and postharvest physiology of the putative blackheart-resistant transgenic pineapple plants; to develop early screening techniques based on any combination of molecular or biochemical techniques that correlated with blackheart resistance in fruit; to examine the effectiveness of alternative blackheart control strategies in the light of research that indicated a link to gibberellin biosynthesis; to test the effectiveness of fruit-specific promoters in transient assays and compare with the constitutive promoters currently being used; to develop a satisfactory intellectual property (IP) strategy to enable the commercial release and marketing of blackheart-resistant pineapples in Australia and Malaysia.

Scientists undertook field evaluation of transformed pineapple lines to assess plants and fruit for their agronomic and commercial suitability. They developed protocols to determine whether blackheart resistance resulted from cosuppression or enhanced cosuppression of polyphenol oxidase. They compared transformed and untransformed pineapples to determine whether premature abscission of some fruitlets was associated with blackheart symptom development. They also developed leaf assays and other techniques to allow early screening of material.
In addition, comparisons were made between transformed and untransformed pineapples to determine if susceptibility to crown deterioration was affected. Observations were also made on transformed pineapples with blackheart resistance to determine if they had any undesirable traits (such as increased susceptibility to disease) that would make them commercially uneconomic.
ACIAR and the project teams also worked to establish the linkages needed to broker intellectual property management plans with IP owners, to allow release and marketing of transgenic pineapples by Australia and Malaysia, with provisions to ensure access by smallholders in developing countries to planting material.

The project developed lines of commercially acceptable transgenic pineapple cultivars with demonstrated resistance to blackheart. The team then pursued an IP and commercial partnership strategy to allow the release to farmers of the transgenic pineapple cultivars in Australia and Malaysia and access of transformed pineapple fruit and products to some key markets.
The ground-breaking gene-silencing technology developed in the project to switch off the particular gene in pineapples responsible for 'blackheart' was also found to have the potential to deliver a new generation of precision pharmaceuticals and revolutionise the treatment of cancer. Dr Peter Young, director of biotechnology research for the Agency for Food and Fibre Sciences, the science and innovation arm of the Queensland Department of Primary Industries and Fisheries (DPIF), described the gene-silencing technology as 'arguably the most powerful biotechnology tool to emerge in the last decade'. Dr Young was part of the team that originally developed the technology to deal with pineapple blackheart.
'Basically we discovered how to switch the blackheart gene (the PPO gene) off-and when we worked out how to do that, it was immediately apparent that what we were researching had commercial applications far beyond agriculture,' Dr Young said.
'Gene silencing enables the activity of a specific gene in any organism, animal or plant to be shut down with pinpoint precision. If you can do that, you can get a complete understanding of what a particular gene does or doesn't do. And if you consider that most health problems and disease result from inappropriate gene activity, this incredibly powerful technology gives us, for the first time, the ability to tackle the problem at its source. No one has been able to do that before,' he concluded.
Queensland-based biotechnology company Benitec is currently commercialising the gene-silencing technology under a collaborative research agreement with DPIF. Malaysia and other ASEAN countries stand to benefit from the transfer of molecular and transformation technology from Australia, through strategic links to the International Service for the Acquisition of Agri-biotech Applications (ISAAA) for training and IP brokerage.