Control of ripening in papaya and mango by genetic engineering

• Malaysian Agricultural Research and Development Institute, Malaysia
• Queensland University of Technology, Australia
• University of the Philippines at Los Banos, Philippines
• Queensland Department of Primary Industries and Fisheries, Australia

All the ASEAN countries, and Australia, produce papaya. It is a popular fruit, and exports to non-producing countries are increasing. It provides a cheap source of several vitamins and minerals. However, like most tropical fruits, papaya ripens quickly after harvesting and will then rapidly deteriorate and succumb to diseases. It is liable to soften and spoils easily. It is also sensitive to low temperatures and so cannot be stored in cool conditions. Hence, transporting the fruit within a country - let alone exporting it - causes large losses. Up to 40% of the harvest is lost in transit from the farmer to the consumer.
Postharvest ripening and senescence are controlled mainly by the gas ethylene - many fruits produce this gas in increasing quantities as they mature. Papaya and mango produce their own ethylene, and are therefore self-ripening and self-destroying. Attempts to control ripening based on removing or inhibiting ethylene have succeeded but are expensive and require sophisticated technology.
By contrast, genetic engineering can modify the genes responsible for ethylene production and other features of the ripening process and thereby create a cultivar that will by itself have a longer shelf life and better flavour retention, without the need for advanced storage equipment. This approach has worked with tomatoes, and genetically modified varieties are already on sale.
This project attempted similar genetic engineering techniques to create modified papaya and also started on tissue culture preparation of mango - a necessary precursor step to enable genetic modification to take place later.

The main aim of this work was to produce genetically modified strains of papaya that keep their quality for longer after harvesting. In a project extension the scientists also evaluated transgenic papaya trees derived from the earlier work. As well the scientists undertook research to harvest embryonic cells from mango varieties, in preparation for genetic modification experiments similar to those described for papaya.

The project was divided into three programs. In the first, genes involved in ripening in papaya were cloned and characterised. The scientists studied the expression patterns of each selected gene during papaya fruit ripening then applied transformation techniques to insert 'anti-sense' gene sequences that nullified the messages from the identified ripening genes.
In the second stage, these anti-sense genes were inserted into embryonic plants in tissue culture. The scientists determined the best conditions for inserting the new genes to ensure they remained stable, and once transformation had taken place successfully, plantlets were produced from the embryos then grown on. These modified plants were then analysed to confirm their genetic composition, and those showing the highest levels of anti-sense gene activity were selected and grown in secure glasshouses. These would form the starting point for delayed-ripening papaya cultivars.
The third program concentrated on obtaining cultures of embryonic mango plant cells. In other research this had been achieved for some varieties, and this program focused on others that had proved harder to culture. The project team studied development of embryos from the chosen varieties and noted the best stage for obtaining cells. This paved the way for later use of these cells in genetic modification experiments similar to those described for papaya.

Australia
The major outputs were the identification and cloning of two important genes in the ripening process and production of transgenic papaya trees. Approximately 100 transgenic lines were produced. Transgenic trees were evaluated in field trials and the main fruit characteristics analysed. A number of transgenic papaya lines were identified with fruits showing an increased shelf life. These lines show an increased fruit life but no other important changes in the quality of the fruits. In addition we developed tissue culture protocols for mango and produced transgenic tissues containing the 'Green Fluorescent Protein' gene that confers fluorescence upon illumination with UV light.

Philippines
Papaya
During the first three years of implementation of the project, we reported the cloning of the ripening-related ACC synthase (ACS2) gene from Philippine Solo papaya, preparation of the gene construct containing the ACS2 in reverse or antisense orientation, optimisation of somatic embryogenesis and regeneration of papaya, optimisation of particle bombardment conditions, transformation of somatic embryos via particle bombardment, selection and regeneration of putative transgenic papaya plants and growing out in the second-biosafety level screenhouse for evaluation.

For the extension phase, we selected nine papaya trees from six unique lines on the basis of molecular analysis and phenotypic traits. In general, the selected papaya trees had a good stand with normal sigmoidal growth and prolific fruiting habit, with 15 to 48 fruits upon reaching the first sign of ripening. The selected transgenic lines exhibited longer number of days from colour break to full yellow of 6 to 7 days compared with 5 to 6 days for control non-transgenic papayas. The difference in the number of days from full yellow to start of loss of firmness (and/or to rotting stage) was more pronounced: 6 to 14 days for selected transgenic lines compared with 2 days for control non-transgenic papayas. Selected transgenic papayas also were firmer5.5 to 7.3 kgf compared with 0.9 to 1.2 kgf for control papayas at 12 days after color break.

The presence of the transgenes, the kanamycin resistance gene and the antisense ACC synthase gene was detected by using appropriate primers and PCR-based analysis. The transgenes were also detected in the leaves, fruits and peduncles of the transgenic papaya trees, indicating the non-chimeric character of the transformed plants. Southern blot analysis showed a single copy of the transgene in the selected transgenic lines.

We subjected the new transgenic papaya lines to various biochemical analyses such as proximate chemical composition, vitamin C and beta-carotene, total soluble solids or total sugars, and benzyl isothiocyanate (BITC) contents. At all three stages of the fruit (green mature, color break or 10% yellow, and 100% yellow), the values obtained for the different lines including the control were quite close to each other and were within the range reported in the literature. The results indicate that the transgenic papayas are substantially equivalent to the nontransgenic control papayas.

The selected transgenic and control papayas are undergoing further screenhouse evaluation and preparations for field testing are being made. Plans are also being made for biosafety and food safety tests that may be required by the concerned biosafety regulatory agencies in the Philippines. These transgenic papaya plants are the first transgenic crop developed in the Philippines.

Mango
For mango, the ACC synthase gene from ripe mango var 'Carabao' has been isolated, cloned and sequenced. A major output of this project is the establishment of optimum conditions for the somatic embryogenesis of mango var 'Carabao'. The developed protocol can also be applied to other varieties of mango. Efforts now focus on identifying the conditions for complete plantlet regeneration from germinated somatic embryos. At this time, recovery of plantlets is very low and it takes a long period (about two years) to obtain plantlets with true leaves.