2010 Annual Report
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.
Towards a goal of making stoneless plums, expression of the genes involved with stone hardening was measured, both within developing fruit and throughout the whole tree. Combinations of regulatory elements and these genes were designed to prevent stone formation/hardening while not affecting other processes, such as wood development.
Studies on plant genes that interact with Plum pox virus (PPV) found that many of these genes are also involved in photosynthesis. When these genes were blocked or chemical inhibitors of photosynthesis were applied, plants became more susceptible to virus infection. This shows that disruption of photosynthesis by chemicals or dark leads to a response that is known to inhibit the plant defense signals. When these signals are inhibited, plants are more susceptible to virus infection.
Horticultural management experiments to improve fruit production of early flowering plums in order to speed up the breeding cycle showed that not only do these plums flower early and continually, but they also have no cold or dormancy requirement for growth and fruiting.
To develop high quality disease and insect resistant pears, seedlings were evaluated for fruit quality and resistance to fire blight bacteria, Fabraea leaf spot fungus, and pear psylla insects. Further crosses were made to introduce into the best selections improved sweetness, acidity, and increased vitamin C content.
Asian and European pear species were screened for effects on psylla insect pest larval feeding, larval development, and mortality.
To understand the inheritance of fruit quality traits in psylla-resistance pear tree populations and in peach trees with novel growth habits, fruit quality traits of seedlings were evaluated to use for linking molecular markers to fruit quality traits.
Progress was made on improving the rates of regeneration of plants from leaves of plum and pear to enable genetic engineering of improved traits.
The susceptibility of pears to the new pest, European pear sawfly, was studied, and several possible sources of genetic resistance were identified in Asian pears and hybrids.
To better engineer plant sterility in transgenic plants, genetic enhancers for gene silencing have been developed and tested. Enhancer DNA is a powerful gene silencer. Silencing the genes that lead to pollen and seed development holds promise as a method for preventing the spread of genes from transgenic plants (transgenes) into the environment where they may not be wanted.
The 'HoneySweet' plum genome was sequenced using NextGen, an advanced, rapid and cost efficient sequencing method.
Continued evaluation of pollen movement (year 10) from a block of transgenic plums shows very limited movement of trangenes with the highest rate of gene transfer at 2% within 33-77 meters from the block rapidly decreasing to zero at most sites over 300 m from the block.
Method to enhance propagation of plants. Shoot regeneration from leaves of known fruit varieties is a preferred method of regeneration of transgenic plants, because the transgenic plant is identical to the variety except for the inserted gene. Genetically engineered stone fruit plants are currently made from seedlings, so they are all genetically different from the original variety. The seedlings are used, because leaves from varieties of plum and other stone fruits generally will not regenerate plants. ARS researchers at Kearneysville, WV, found that when plum leaves express an inserted corn gene, KNOX1, they produced shoots in high frequency. This is the first report of plant regeneration from leaves of a woody perennial fruit tree, such as plum, using a regeneration-inducing gene from corn.
'Orablue' plum was released to growers. 'Orablue' provides U.S. growers and gardeners with an exceptionally large, sweet, and highly flavored plum that grows and fruits well in the mid-Atlantic and northeastern U.S., and other plum growing areas.
'HoneySweet' plum conditionally registered by EPA. There are limited sources of Plum pox virus (PPV) resistance in stonefruits. 'HoneySweet', a plum genetically engineered for resistance to PPV, which had previously been deregulated by APHIS and FDA, is now conditionally registered by EPA. 'HoneySweet' is the result of over 20 years of research by ARS researchers at Kearneysville, WV, and collaborators at other ARS locations, as well as European collaborators. It has been found to be resistant to PPV in test plots in Europe over the last 10 or more years. It will be the first genetically engineered disease resistant temperate fruit tree available to U.S. growers and will provide U.S. growers with a high quality fresh market, PPV resistant plum. It can also serve as a breeding parent to reliably and efficiently introduce the resistance trait into additional new plum varieties.
Dardick, C.D., Callahan, A.M., Chiozzotto, R., Schaffer, R., Piagnani, M., Scorza, R. 2010. Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence. BioMed Central Biology. 8:13.
Singer, S.D., Hily, J., Liu, Z. 2009. A 1-kb bacteriophage lambda fragment functions as an insulator to effectively block enhancer-promoter interactions in Arabidopsis thaliana. Plant Molecular Biology Reporter. 28:69-76.
Yang, Y., Singer, S., Liu, Z. 2010. Two similar but distinct second intron fragments from tobacco AGAMOUS homologs confer identical floral organ-specific expression sufficient for generating complete sterility in plants. Planta. 231:1159-1169.
Petri, C., Scorza, R. 2010. Factors affecting adventitious regeneration from in vitro leaf explants of 'Improved French' plum, the most important dried plum cultivar in the USA. Annals of Applied Biology. 156:79-89.
Petri, C., Scorza, R., Dardick, C.D. 2009. Genetic engineering of plum (Prunus domestica L.) for plant improvement and genomics research in Rosaceae. In: Folta, K.M. Gardiner, S.E., editors. Genetics and Genomics of Rosaceae. New York, NY: Springer. p. 277-290.
Yang, Y., Singer, S., Liu, Z. 2010. Petunia AGAMOUS enhancer-derived chimeric promoters specify a carpel-, stamen- and petal-specific expression pattern sufficient for engineering male and female sterility in tobacco. Plant Molecular Biology Reporter. DOI 10.1007/s11105-010-0215-z
Sun, Q., Sun, H., Li, L., Bell, R.L. 2009. In vitro colchicine-induced polyploid plantlet production and regeneration from leaf explants of the diploid pear (Pyrus communis L.) cultivar, 'Fertility'. Journal of Horticultural Science and Biotechnology. 84(5):548-552.
Hu, D., Scorza, R. 2009. Analysis of the 'A72' peach tree growth habit and its inheritance in progeny obtained from crosses of 'A72' with columnar peach trees. Journal of the American Society for Horticultural Science. 134(2):236-243.