Location: Innovative Fruit Production, Improvement, and Protection
2021 Annual Report
Objectives
Objective 1. Identify and functionally analyze genes regulating plant architecture, abiotic stress tolerance, fruit quality, and disease resistance. [NP301, C1, PS1A, PS1B; C3, PS3A, PS3B]
Sub-objective 1a. Identify and functionally analyze genes regulating plant architecture in temperate, deciduous fruit crops and rootstocks.
Sub-objective 1b. Identify and functionally analyze genes regulating dormancy, cold hardiness, drought, disease resistance in apple and stone fruit crops.
Sub-objective 1c. Identify and functionally analyze genes regulating fruit development traits in stone fruits.
Objective 2. Develop and optimize advanced methods for tissue culture propagation and genetic transformation of temperate, deciduous fruit crops. [NP301, C1, PS1A, PS1B; C3, PS3A, PS3B]
Sub-objective 2a. Optimize tissue culture production of apple, pear, and stone fruit scion and rootstock genotypes.
Sub-objective 2b. Develop transgenic and gene edited lines and field plantings of fruit crops for functional analysis of genes that regulate important traits identified in Objective 1.
Sub-objective 2c. Develop CRISPR technologies for modifying important functional traits in fruit crops.
Objective 3. Use standard and rapid cycle breeding systems to generate advanced lines of germplasm for the apple and stone fruit breeding community and industry. [NP301, C1, PS1A, PS1B; C3, PS3A, PS3B]
Sub-objective 3a. Use early-flowering apple and stone fruit rapid breeding systems to introgress and or pyramid economically-important traits, such as disease resistance, from wild species and known sources of established cultivars, into commercial germplasm.
Sub-objective 3b. Utilize rapid breeding system to eliminate transgenes from Agrobacterium-based-CRISPR transformation of fruit crops.
Sub-objective 3c. Establish field plantings of select lines of stone fruit and apple germplasm developed through classical and transgenic technologies that exhibit economically-desirable traits.
Approach
This project leverages plant breeding, genomics, genetics, molecular biology, and biotechnology strategies to address fundamental problems facing tree fruit production. The variety development and basic research activities are synergistic as the germplasm developed through the breeding efforts serve as a critical resource for identifying the genetic basis for complex traits. Many of the objectives proposed will use the unique transformation technologies developed by the investigators coupled with available genome sequences for several tree fruit species. These transformation systems have been used to develop FasTrack technology to shorten the breeding cycle for fruit tree species. Plums and pears transformed with the poplar FLOWERING LOCUS T produce flowers within the first year of growth and can be hybridized to achieve generation cycles of one to two years. Biological processes under study will include flowering time/dormancy, tree architecture, and fruit development. Regulation of flowering time in peach will be investigated using genetic, molecular and deep sequencing-based strategies. An extreme late blooming trait that avoids spring frost will be combined with commercial quality traits through conventional breeding. Tree architecture, specifically the regulation of TAC1, LAZY1, and LAZY2 expression by light, gravity, and the circadian clock, will be carried out via gene expression studies along with promoter swap experiments to determine the functional consequences of mis-expressing each gene. Collectively, these data will provide important practical information about how light regulation of IGT genes contributes to tree shape. We will continue to characterize previously created plum and apple PpeDRO1 over-expression transgenic lines and RNAi silencing lines to evaluate the impacts of over- or loss- of DRO1 function on root system architecture. In pear, we will leverage our biotechnology system to functionally characterize putative apple dwarfing/precocity genes and assess their potential to confer these traits in pear rootstocks. To study how fruit tissue determination is achieved, we have begun transcriptome-based comparisons of different fruit types, tissues, and developmental times to identify gene networks that specify properties of fleshy versus non-fleshy tissues during and after fruit set. Technology to engineer and breed for stoneless fruits will be tested using a combination of biotechnology and conventional breeding. A novel super sweet trait in peach/nectarine that confers extremely high brix (20o–30o) will be bred to develop commercial quality super sweet varieties. Methods for gene editing will be developed for plum and pear via isolation and use of novel promoters. Lastly, the research unit will continue to pursue national and international release of the transgenic plum ‘HoneySweet’ that is resistant to Plum Pox Virus (PPV). Collectively, these efforts will fill in key knowledge gaps about fundamental fruit tree developmental processes, provide new technologies for developing fruit tree germplasm with economically important traits, and lead to the development of new fruit varieties with superior traits.
Progress Report
Hundreds of transgenic lines that affect the expression of genes that control dormancy (DAM genes) were created to further the understanding of the dormancy process (Objective 1A). Approximately 1,000 trees have been transferred for greenhouse evaluation. Field planting permits were initiated to establish blocks for long-term phenotyping and evaluation. Progress has been made on advancing breeding lines for the extremely late-blooming peach and nectarine selections and high sugar traits and combinations of both with desired architecture and disease resistance traits (Objective 1B and 1C). A handful of selections were made for further study. Trees were evaluated for bacterial spot resistance, with some showing resistance segregation. Additional peach and nectarine crosses were made with new parental genotypes in an attempt to obtain higher-quality offspring.
Transgenic plum trees altered in architectural traits resulting in a pillar, weeping, and horizontal-Lazy-trees were evaluated for responses to different training systems to maximize production with minimal labor. Trees did not fruit in 2020 due to frost damage. Fruit quality will be evaluated in the summer, 2021 (Objective 2B and 2C).
Additional constructs were made that target expression of three transcription factors involved in early fruit development and are being used to transform plum (Objective 4A). The fruit was obtained for the first time from a block of transgenic plum trees targeting lignin production in the fruit endocarp. The intent of the gene manipulations was to form softer stones. Very few fruits were obtained in 2020; however, there was a strong indication that some of the lines displayed stone defects such as loss of endocarp tissue. Evaluations are being repeated in summer 2021 (Objective 4B). Stone defects in two F1 populations are also being evaluated that came from ‘Stoneless’ and a second related parent, ‘Sans Noyau’ plum, both of which had partial stone formation.
An import petition for ‘HoneySweet’ fruit to Canada was filed with Health Canada (Objective 2b). The Canadian agency sent two requests for additional information, which were provided. Additional consultation was held, and the process is continuing.
Additional work was performed to understand the action of a common harmless fungus (TC09), whose volatile production accelerates the growth of many different plants (Objective 2b). Two commercial evaluation licenses were issued to private companies. Assistance was provided in the form of protocols, data, and consultations to help troubleshoot issues as they arose. Work with NASA continued, focusing on the role of fungal-produced CO2. The results were inconclusive, and experiments continue.
Previous work on plum genomes brought about a collaborative effort to sequence three citrus greening (HLB) resistant wild Australian lime citrus genomes (in collaboration with the University of California, Riverside). The three genomes were sequenced, assembled, and annotated. Based on the results, additional long-read sequencing data was performed for all three genomes to improve the assembly. Potential resistance genes from the genomes were identified and analyzed (USDA ARS Citrus Greening Grand Challenge Synergies program).
Accomplishments
1. Bell pear. A pear selection with excellent fruit quality and commercial characteristics was submitted for release. The pear was named ‘Bell’ after its developer, retired ARS scientist Richard Bell. ‘Bell’ is a medium-sized pear with an attractive blush and superior flesh texture. A Cooperative Research Development Agreement (CRADA) partner Adam’s County Nursery requested that ARS release due to demand and requests by regional growers. ‘Bell’ was submitted to the ARS Plant Variety Protection Panel for approval.
Review Publications
Wisniewski, M., Artlip, T.S., Liu, J., Ma, J., Burchard, E.A., Norelli, J., Dardick, C.D. 2020. Fox hunting in wild apples: searching for novel genes in Malus sieversii. International Journal of Molecular Sciences. http://dx.doi.org/10.3390/ijms21249516.
Abdelfattah, A., Freilich, S., Bartuv, R., Zhimo, Y., Kumar, A., Biasi, A., Salim, S., Feygenberg, O., Burchard, E.A., Dardick, C.D., Liu, J., Khan, A., Amiri, A., Ellouze, Q., Ali, S., Spadaro, D., Torres, R., Teixido, N., Ozkaya, O., Buehlmann, A., Vero, S., Berg, G., Whitehead, S., Wisniewski, M., Droby, S. 2021. Global analysis of the apple fruit microbiome: are all apples the same. Environmental Microbiology. https://doi.org/10.1111/1462-2920.15469.
Kappagantu, M., Collum, T.D., Dardick, C.D., Culver, J.N. 2020. Viral hacks of the plant vasculature: the role of phloem alterations in systemic virus infection. Annual Review of Virology. 7:10.1-10.20. https://doi.org/10.1146/annurev-virology-010320-072410.
Tamukong, Y.B., Collum, T.D., Stone, A.L., Kappagantu, M., Sherman, D.J., Rogers, E.E., Dardick, C.D., Culver, J.N. 2020. Dynamic changes impact the plum pox virus population structure during leaf and bud development. Virology. 548:192-199. https://doi.org/10.1016/j.virol.2020.06.014.
Ravelonandro, M., Briard, P., Scorza, R., Callahan, A.M., Zagrai, I., Kundu, J., Dardick, C.D. 2021. Robust response to plum pox virus infection via plant biotechnology. Genes. 12(6). https://doi.org/10.3390/genes12060816.
Liu, J., Islam, T., Sapkota, S., Ravindran, P., Kumar, P., Artlip, T.S., Sherif, S. 2021. Ethephon-mediated modulation of bud phenology, cold hardiness and hormone biosynthesis in peach. Plants. 10(7). https://doi.org/10.3390/plants10071266.
Livingston, D.P., Bertrand, A., Wisniewski, M., Tisdale, R.H., Tuong, T.D., Gusta, L., Artlip, T.S. 2021. Factors contributing to ice nucleation and sequential freezing of leaves in wheat. Planta. 253:124. https://doi.org//10.1007/s00425-021-03637-w.
