Blackheart occurs when the pineapple fruit is exposed to lower than optimum temperatures. In Malaysia and other tropical pineapple-growing countries the continuous cool storage of fruit after harvest (3 days at < 21C) appears to injure the fruit, particularly undermining the fresh fruit export industry.
In Australia and other more temperate countries blackheart occurs before harvest when growing temperatures fall below 25/20C day/night with low light. It undermines both the fresh fruit and the canning industries. Pineapples with blackheart appear good until they are cut open. Immature fruit are less susceptible, so some Australian growers are harvesting early to try to avoid blackheart, but pineapples must ripen on the plant to develop full flavour.
The problem of crown deterioration, a condition that attacks the crown leaves before it attacks the fruit, was also tackled in this project, chiefly by the Malaysian collaborators. The roles of water loss, ethylene production, temperature in storage and pathogen invasion as the causes of crown deterioration were studied and appropriate strategies devised to counteract the problem.
This project aimed to restore consumer confidence in the quality of fresh pineapples, and to sustain and expand the pineapple-growing industries in Malaysia and Australia. The project's twin targets were blackheart and crown deterioration. The project addressed the problems through the use of molecular biological techniques.
The project involved 18 research scientists from DPI Queensland, CSIRO and MARDI (Malaysia) working on aspects of molecular biology, tissue culture, transformation, plant biochemistry and postharvest physiology
For blackheart control, step one was to characterise the enzymes, polyphenol oxidase (PPO) and peroxidase (POD), thought to initiate the condition. Pineapple cultivars that partially resist blackheart were studied both in Malaysia and Australia, as well as the susceptible commercial cultivar Smooth Cayenne.
When the more important of the two enzymes was identified the gene responsible was extracted from pineapple cells and cloned. Researchers sought to modify the gene so it eliminated browning. A marker gene, which confers a recognisable new characteristic on pineapples, was also incorporated. The newly-modified gene was then substituted for the corresponding section of pineapple DNA via a transformation system, so the host acquired the desired non-browning characteristics.
Scientists tested for a suitable transformation system by bombarding the host DNA with microprojectiles, and by letting Agrobacterium invade the host cell carrying the gene construct with it. The cells resulting from transformation were grown through micropropagation into plants that subsequently set fruit. Plants and fruit were tested for resistance to blackheart, and to ensure they retained the commercially desirable characteristics of the Smooth Cayenne cultivar.
As a consequence of this project, the role of the PPO enzyme in blackheart was confirmed and a deeper understanding of factors involved in the induction of blackheart obtained. This research also led to a greatly increased understanding of the physiology of crown deterioration. In addition pineapple PPO genes were extensively characterised and constructs, designed to inactivate PPO gene expression in transgenic pineapple, were prepared.
The results showed the importance of fruit maturity on blackheart development, which is a critical factor in harvesting and organoleptic (sensory evaluation such as taste, colour and smell) issues. The results also indicated that ascorbic acid levels in healthy fruit do not contribute to the potential resistance or susceptibility of the fruit to blackheart. This is significant in light of suggestions that pineapples bred for high ascorbic acid levels may have some level of resistance to blackheart.
Efficient pineapple transformation systems were also developed and the production of transgenic plants, in which PPO gene expression might be inactivated, was well advanced at the end of the project.
In Malaysia reduced losses due to blackheart will allow increased productivity per unit land, water and Agri-dollar invested. Conversely, an increased demand for fresh pineapples could facilitate an expansion of planting within individual farms as well as an increase in the total number of farms. There are considerable areas of land that previously sustained pineapple production that could be utilised to service and increased product demand, however competition with other land uses (such as housing) may limit this.
In Australia, resolution of the blackheart problem will save the industry $2.6m a year. It will also help increase consumption, particularly during the winter, and therefore increase sales. Considering the support shown by the sole processor, Golden Circle, project members anticipate a high level of industry adoption if the transgenic plants pass independent evaluation, which would be expected 6-8 years after the project finished. The fact that tissue culture has been used previously for the introduction of new pineapple varieties also suggests that the adoption process could be completed relatively quickly.
Links:
[1] http://www.aciar.gov.au/country/Malaysia
[2] http://www.aciar.gov.au/programarea/Horticulture