1a. Objectives (from AD-416)
1. Improve temperate stone and pome fruit quality traits. 2. Improve host-plant resistance of temperate stone and pome fruit to plum pox virus, tomato ringspot virus, prunus necrotic virus, and prune dwarf virus. 3. Develop a model biotechnology risk mitigation system and efficient genetic transformation protocols for Rosaceae. PDRAM #R08 Program Increase Objective: 4. Initiate a FasTrack breeding project to develop, characterize, and select early and continual fruiting lines of plum, pear, and apple, and relevant rootstocks to accelerate fruit breeding programs.
1b. Approach (from AD-416)
This project proposes the development of genetic solutions to the major problems affecting temperate tree fruit production and consumer acceptance of tree fruits through a coordinated effort utilizing the most appropriate technologies. The improvement of fruit quality will be approached through traditional breeding of novel genetic material, and also through the isolation and transgenic manipulation of key genes that control fruit development, stone formation, and ripening. The use of these diverse technologies will be coordinated in order to develop tree fruit varieties with improved flavor and firmness that is maintained post-harvest and investigate the possibility of creating marketable stoneless varieties. Improvement of fire blight resistance in pear will be approached through hybridization of existing fire blight resistant germplasm to develop new resistant cultivars that have high fruit quality. Pathogen- and host-derived resistance to stone fruit viruses will be incorporated into commercial quality cultivars through genetic transformation. Improved tree form for high-density production systems in peach will be approached through hybridization of peach germplasm with desirable tree growth habits such as columnar and semi-columnar with the aid of molecular markers that improve breeding efficiency. Pear growth habits will be manipulated through genetic transformation. Foundational work in genetic engineering and risk mitigation will facilitate the development of enhanced technologies that will help alleviate industry concerns over marketing genetically engineered crops. Improved plant material generated from both the laboratory and field will be evaluated in collaboration with the industry and consumer groups to facilitate its utilization.
3. Progress Report
With a goal of making stoneless plums, microarrays were used to find genes involved in stone formation. Those genes showed the stoneless mutant in plum resulted from making fewer stone cells. New pear varieties are being developed for increases in sweetness, 'Bartlett' flavor, vitamin C and psylla resistance. Selections were analyzed for fruit quality, juvenility, flowering and fire blight resistance. A source of resistance to European pear sawfly was identified. Transgenic plums were created with a dwarfing gene to test for use in making dwarfing pear rootstocks. Two peach releases were made: 'SummerFest'-high quality, mid-season and upright growing and 'Flavrburst'-high quality, exceptional flavor and standard growth. Other high fruit quality upright and columnar selections have been transferred to our CRADA partner, for testing. New varieties are being developed for higher sugar and upright growth. Plums that fruit within one year were produced through genetic engineering. These are used to develop a 'FasTrack' breeding system to rapidly introduce traits and segregate out the transgene so trees are no longer genetically engineered. Plants are in the second generation. Studies on plant genes that interact with Plum pox virus (PPV) found that many were involved in photosynthesis. When genes were blocked or chemical inhibitors of chloroplast activity were applied, plants became more susceptible to potyvirus. Treatment with inducers of a plant defense pathway did not increase resistance, but if potyvirus and salicylic acid were applied, a resistance response was obtained. 'HoneySweet' plum, genetically engineered for resistance to PPV, was deregulated by FDA. Following approval by EPA, 'HoneySweet' will be the first genetically engineered disease resistant temperate fruit tree available to U.S. growers. The transgene insert in 'HoneySweet' was sequenced, revealing a rearranged part of the PPV-coat protein gene. This "hairpin" when transformed into plum was able to confer resistant to PPV. This demonstrated the resistance mechanism of 'HoneySweet' and the effectiveness of "hairpin" constructs for inducing resistance. To make directed gene improvements in plum cultivars, regeneration from leaves is necessary. A system for leaf derived regeneration of the major plum cultivar has been developed and is used for gene transfer studies for PPV resistance. Preventing unwanted gene flow from transgenic plants is being advanced through the development of the Tissue-Specific Transgene Removal and Containment System. New constructs were made that have a plant insulator sequence to prevent interactions that give unpredictable results. To contain gene flow, we made RNAi constructs that are successful in targeting silencing of meiosis-critical genes and generating male and female sterility. Gene expression, tissue section and cytogenetic analyses were done to verify observation. A new plant gene expression regulatory system was discovered, Enhancer-Mediated Gene Silencing System, which is very stable at low and high temperatures and produces male and female sterility in plants.
1. Early flowering genes allow rapid tree fruit breeding. Tree fruit breeding is a long term and expensive process due to the long juvenile non-flowering stage. Introduction of a gene that promotes early flowering and fruiting shortened the juvenile stage in plum from three to four years to less than one year. The genetically engineered early flowering trait is removed in the last generation resulting in a non-genetically engineered plant. Early flowering and fruiting will allow for the very rapid development of new and improved varieties of plum and other fruits, and forest and woody ornamental species.
2. Release of an exceptional 'Flavrburst' peach variety. Peach consumption in the U.S. is falling in large partly due to poor fruit quality. 'Flavrburst' peach was developed with an exceptional flavor – sweet and only mildly acidic. 'Flavrburst' peach provides a highly desirably flavored peach for the consumer and can help to reverse the downward trend in peach consumption.
3. Release of a high quality 'SummerFest' peach variety. Peach growers are facing increased costs for orchard operations (pruning, thinning, and harvesting). 'SummerFest' peach is an upright growth habit that has been shown to be more productive and easier to manage than standard growth habit peaches. It is also a very high quality peach. 'SummerFest' provides growers another in a series of upright growth habit peaches that may be used to reduce the costs of the production of peaches.
4. The stoneless trait in plum is identified. Pits (seed and the hard surrounding woody "stone") reduce the convenience of eating stone fruits (peaches, cherries, apricots and plums). Pit removal for processed fruit represents a major cost of production and polluting waste. We found that partially stoneless fruits developed in the early 1900’s, while having the gene that forms the woody stone tissue, have fewer cells that express the stone producing genes than normal stone fruits. This information can be used by plant molecular biologists and breeders to develop fruit without the stone and seed. Pitless plums would be more convenient to eat and easier to process increasing the consumption of healthy fruits in the diet.
5. European pear sawfly resistance identified in pear. The European pear sawfly has emerged as a new significant pest of pear in the Eastern U.S., and current cultivars are all very susceptible. Resistance to European pear sawfly was identified in pear germplasm with either Pyrus ussuriensis or Pyrus pyrifolia pedigrees. Early flowering data was identified as a factor in the escape from infestation. The highly resistant germplasm will be available to breeders to develop new cultivars resistant to this new pest.
6. Viral symptoms tied to damage of the photosynthetic system in plants. Viruses cause destructive symptoms to crop plants, the most serious being leaf and fruit yellowing, and death. We found that chloroplast impairment leads to increased virus susceptibility suggesting that viruses purposefully target chloroplast function to defeat plant defenses. This finding provides critical insight into the process of plant virus infections and provides a new foundation for developing new virus control strategies.
7. Precise engineering developed to genetically improve the quality of plants. Improvement of multiple traits through genetic engineering often requires the co-introduction of multiple genes controlled by different promoters. However, multiple gene promoters can cause unexpected problems with transgene expression. We found gene insulator sequences that prevent the interactions of the multiple gene promoters. The plant insulator sequences will be instrumental for transgenic research especially for co-expressing or precisely engineering multiple genes or traits.
Elkins, R.B., Turner, J.D., Castagnoli, S., Seavert, C.F., Mitcham, E.J., Biasi, W.V., Colonna, A., Bell, R.L. 2008. Evaluation of potential alternative European pear cultivars for U.S. West Coast growers. Acta Horticulturae. 800:483-489.