Efficient screening of antibacterial genes by juvenile phase free technology for developing resistance to fire blight in pear and apple trees

Abstract

Objectives: The original objectives of this project were to: Produce juvenile-free pear and apple plants and examine their sensitivity to E. amylovora; Design novel vectors, for antibacterial proteins and promoters expression, combined with the antisense TFL1 gene, and transformation of Spadona pear in Israel and Galaxy apple in USA. The original objectives were revised from the development of novel vectors with antibacterial proteins combined with the TFL-1 due to the inefficiency of alternative markes initially evaluated in pear, phoshomannose-isomerase and 2-deoxyglucose-6-phosphate phosphatase and the lack of development of double selection system. The objectives of project were revised to focus primarily on the development additional juvenile free systems by the use of another pear variety and manipulation of the FT gene under the control of several promoters. Based on the results creation of fire blight resistance pear variety was developed by the use of the juvenile free transgenic plant. Background: Young tree seedlings are unable to initiate reproductive organs and require a long period of shoot maturation, known as juvenile phase. In pear, juvenile period can last 5-7 years and it causes a major delay in breeding programs. We isolated the TFL1 gene from Spadona pear (PcTFL1-1) and produced transgenic ‘Spadona’ trees silencing the PcTFL1 gene using a RNAi approach. Transgenic tissue culture ‘Spadona’ pear flowered in vitro. As expected, the expression of the endogenous PcTFL1 was suppressed in the transgenic line that showed precocious flowering. Transgenic plants were successfully rooted in the greenhouse and most of the plants flowered after only 4-8 months, whereas the non-transformed control plants have flowered only after 5-6 years of development. Major achievements: Prior to flower induction, transgenic TFL1-RNAi ‘Spadona’ plants developed a few branches and leaves. Flower production in the small trees suppressed the development of the vegetative branches, thus resulting in compact flowering trees. Flowering was initiated in terminal buds, as described for the Arabidopsis tfl1 mutant. Propagation of the transgenic TFL1-RNAi ‘Spadona’ was performed by bud grafting on 'Betulifolia' rootstock and resulted in compact flowering trees. The transgenic flowering grafted plants were grown in the greenhouse under a long photoperiod for one year, and flowered continuously. Pollination of the transgenic flowers with ‘Costia‘ pear pollen generated fruits of regular shape with fertile F1 seeds. The F1 transgenic seedling grown in the greenhouse formed shoots and produced terminal flowers only five months after germination. In addition, grafted F1 transgenic buds flower and fruit continuously, generating hybrid fruits with regular shape, color and taste. Several pear varieties were pollinated with the transgenic TFL1-RNAi ‘Spadona’ pollen including `Herald Harw` that was reported to have resistance to fire blight diseases. The F-1 hybrid seedlings currently grow in our greenhouse. We conclude that the juvenile-free transgenic ‘Spadona’ pear enables the development of a fast breeding method in pear that will enable us to generate a resistance pear to fire blight. Implications: The research supported by this grant has demonstrated the use of transgenic juvenile free technology in pear. The use of the juvenile free technology for enhancement of conventional breeding in fruit tree will serve to enhance fast breeding systems in pear and another fruit trees.