Control and transgenic Royal gala fruits after 90 days of storage at room temperature and humidity.

Apples are an important crop in the United States, annual production is approximately 251.5 million bushels (1 bushel = ca. 42 lb =ca. 20 kg), that is approximately 5 million tons. Fom this New York State produced last year 25.5 million bushels, or nearly 0.5 million tons. The cultivar produced in largest amount is McIntosh. McIntosh apples have a deep red color, pleasant aroma, flavor and firm texture. However, the fruits soften rather soon after harvest and have a relatively short storage time. Present practice to extend shelf life is the use of controlled atmospheric storage, where gas composition and temperature is kept under strict control. This storage method is effective for up to four months for McIntosh, but after this time fruit texture rapidly deteriorates.

The softening of apples, and in general other fruits, is the result of cell wall degrading enzymes the activity of which is under the control of ethylene. Ethylene production, in turn depends on the generation of its precursor, 1-aminocyclopropane carboxylic acid (ACC) by the enzyme ACC-synthase. Transforming apple with either the sense or anti-sense versions of the ACC-synthase gene is expected to reduce the activity of ACC-synthase in apple fruit, and thereby reduce ethylene production and consequent softening. Our objectives in this project were to transfer the ACC-synthase gene from apple in sense and anti-sense forms into Royal Gala and McIntosh apples, and to test the transformed plants and eventually their fruit for ethylene production, ACC synthase activity and softening.

We have isolated mRNA from the cortical tissue of ripening McIntosh apples and using primer sequences that have been conserved in ACS genes isolated from tomatoes and apples we synthesized a 1093 bp DNA fragment by RT-PCR. This DNA fragment had an 82% sequence homology to an ACS gene reported from peppers, a 67% homology to the ripening and wound induced ACS gene from tomato (LeACS4) and a 56% homology to the ACS gene reported from apple. This gene fragment was inserted in reverse orientation between the CaMV-35S promoter and the kanamycin resistance selectable marker sequences into the vector pBI101. Young leaves from Royal Gala and McIntosh were transformed with Agrobacterium tumefaciens strain EHA101, and transformants selected on kanamycin containing medium. Shoot formation was initiated on shooting medium and root formation on rooting medium. Transgenic explants were propagated in glass jars and their ethylene production measured. The explants were micrografted onto M7 rootstock, propagated in the greenhouse and after one year transplanted to the field. No morphological differences were visible between control and transgenic plants. Flowers were observed after three years in the field in transgenic Royal Gala, and one plant produced two transgenic fruits. McIntosh produced the first fruit three years later.

We characterized fruit from four control Royal Gala trees, 40 transformant RG trees, two control McIntosh trees and all twenty transgenic McIntosh trees that bore fruits. Characterization of the fruit in the initial phase consisted of measuring ethylene. We found seven transgenic Royal Gala plants the fruit of which produced significantly lower amounts of ethylene then the controls, falling between 0.3 and 87% of the control fuits. From the twenty McIntosh trees measured, only two showed significant ethylene down regulation in the fruit, ranging between 59 and 71%. All transgenic fruits with significantly altered ethylene production were also evaluated for total soluble solids that indicated the state of ripeness, firmness of flesh, ACS and polygalcturonase enzyme activities. The data were in agreement with ripeness stage and ethylene production. Fruit from the extremely down regulated tree (99.7%) were very hard, smaller then fruits from other transgenic trees, indicating that ethylene interfered with cell elongation/expansion also. Taste testing of the transgenic fruits indicated a sweeter taste of the ethylene down regulated fruits.

Transgenic fruits from both Royal Gala and McIntosh trees soften slower than the non-transgenic fruits, thereby extending their shelf life. Also, the ethylene down regulated transgenic fruit does not need controlled atmospheric storage conditions, simple cold room storage is sufficient. The ethylene down regulated fruit stores better, for a longer time and cheaper then the non-transgenic fruit. This results in better fruit quality, cheaper storage costs, easier transportation and a longer shelf life, benefiting both growers and consumers alike. This is a long project that we started in 1990 in collaboration with the Aldwinckle lab. We did the molecular biology part of the project, while the Aldwinckle lab did the transformations (see their website: http://www.nysaes.cornell.edu/pp/faculty/aldwinckle/).