The application of precision breeding (PB) for crop improvement is fully consistent with the plant life cycle: the utility of PB for grapevine

Transcription

1 The application of precision breeding (PB) for crop improvement is fully consistent with the plant life cycle: the utility of PB for grapevine D.J. Gray 1,a, Z.T. Li 1, N.L. Grant 1, D.A. Dean 1, R.N. Trigiano 2 and S.A. Dhekney 3 1Mid-Florida Research & Education Center, University of Florida/IFAS, Orlando, USA; 2 Department of Entomology and Plant Pathology, The University of Tennessee, Knoxville, Tennessee, USA; 3 Department of Plant Sciences, Sheridan Research and Extension Center, University of Wyoming, Sheridan, USA. Abstract Grapevine is unique among all crops because of its special sensory attributes. A relatively small number of well-known elite cultivars and their landraces account for the majority of world production. Those few cultivars are subject to significant disease pressures, making substantial chemical control and sanitation necessary in many regions. Although better genetic resistance is required to ease production losses, grapevine is difficult to improve by conventional breeding due to obstacles imposed by its lifecycle. Because of inbreeding depression, self-incompatibility and a long lifecycle, it is not feasible to add specific genetic traits to elite cultivars. However, technologies to bypass those obstacles are now available. Initially considered to be highly recalcitrant to advanced genetic engineering, the crucial cell culture and gene insertion systems now are well-established for a wide range of grapevine cultivars. The recently-published genomic sequence map of Pinot Noir is a significant achievement, providing invaluable insights into grapevine genetics and greatly accelerating the discovery of useful functional genetic elements. Application of precision breeding technology to cultivar development, in which only genetic fragments from sexually-compatible parents are utilized, is now attainable and is a logical extension of conventional breeding. A precision breeding approach is more predictable, much less disruptive, and more efficient than that of conventional breeding because only specific traits are transferred and key obstacles are avoided. However, as with all new varieties, substantial field evaluation, as is the norm for conventionally-bred crops, will be required to determine whether or not precision bred versions of elite cultivars will possess desirable attributes and/or otherwise be useful. Keywords: plant genetic improvement, disease resistance, Vitis INTRODUCTION Definition of precision breeding (PB): An approach to genetic plant improvement that transfers only specific desirable traits among sexually-compatible relatives without the genetic disruption imposed by meiosis. With the results from decades of highly productive molecular and functional genetic research in place, we have rounded a corner in the field of plant genetic improvement by being able to manipulate the natural genetic constitution of any given plant without introducing foreign DNA. However, it has been overlooked that, with attainment of in situ genetic manipulation, it is now proper to adopt teachings and discussions that focus within the well-established frameworks of botany, plant genetics and breeding. Concomitantly, certain key, but highly incorrect, terminologies that are in pervasive use today by both scientists and consumers (i.e., GM, GMO and non-gm ) must be discarded and replaced with terms that are scientifically accurate; that allow logic and fact-based discussions to occur. The present article will exemplify the PB approach to plant genetic improvement. a djg@ufl.edu Acta Hortic ISHS DOI /ActaHortic XXIX IHC Proc. IV Int. Symp. on Tropical Wines and Int. Symp. on Grape and Wine Production in Diverse Regions Ed.: P.E. Read 49

2 PB is a method of plant genetic improvement that augments and extends Conventional Plant Breeding (CB). However, PB differs from CB in that it transfers only specific desired traits, such as disease resistance, into existing outstanding cultivars. Functionally, PB transfers genetic sequences of tested, known expression between sexuallyrelated species in the same manner as with CB. However, in contrast to CB, which combines nearly half of the genes b from each of two parents through the variation-inducing meiotic (sexual reproductive) pathway, PB instead utilizes the significantly more stable and predictable mitotic pathway to transfer only DNA fragments with well-known functions. Thus, compared to CB, PB is significantly more precise, accurate, and results in far fewer unintended consequences. FOUNDATIONS OF PLANT GENETIC IMPROVEMENT Without exception, all crops used for food and fiber have been intentionally genetically-modified by humankind. It is a fact that every fruit, vegetable and grain, including all organic produce, that we purchase from the grocery store are significantly and purposely genetically modified. Table 1 reveals the diversity of crops, their origin, and documents when their domestication, i.e., genetic improvement began (Hirst, 2014). As early as the Neolithic period (approximately 10,000-to-4,500-2,000 BC), humans began to intentionally select seeds from only the best plants in their fields. They knew that, by planting only these, next year s yields would be better. In fact, domestication of plants from the wild has long been considered to be the very cornerstone of civilization (Purugganan and Fuller, 2009). The development of crop plants from wild species is a key reason why humans thrived and became successful. For example, humans from the Middle-East through Europe literally evolved with wheat (see Hirst, 2014). The modern corn found in North America by Europeans originated from a grass plant that was genetically modified by indigenous Americans (Iltis, 2000). The massive genetic modification that created corn has yet to be repeated by modern breeder/geneticists for any crop. Table 1. Examples of crop domestication (Hirst, 2014), i.e., purposely genetically modified. Plant Where domesticated Date Fig trees Near East 9000 BC Emmer wheat Near East 9000 BC Foxtail Millet East Asia 9000 BC Flax Near East 9000 BC Einkorn wheat Near East 8500 BC Barley Near East 8500 BC Chickpea Anatolia 8500 BC Bottle gourd Asia 8000 BC Bottle gourd Central America 8000 BC Rice Asia 8000 BC Potatoes Andes Mountains 8000 BC Beans South America 8000 BC Squash (Cucurbita pepo) Central America 8000 BC Maize Central America 7000 BC Water Chestnut Asia 7000 BC Perilla Asia 7000 BC Burdock Asia 7000 BC Broomcorn millet East Asia 6000 BC Bread wheat Near East 6000 BC b Note that about 3% of transferred DNA is of maternal mitochondrial and chloroplast origin. 50

3 Table 1. Continued. Plant Where domesticated Date Manioc/Cassava South America 6000 BC Chenopodium South America 5500 BC Date Palm Southwest Asia 5000 BC Avocado Central America 5000 BC Cotton Southwest Asia 5000 BC Bananas Island Southeast Asia 5000 BC Beans Central America 5000 BC Opium Poppy Europe 5000 BC Chili peppers South America 4000 BC Amaranth Central America 4000 BC Watermelon Near East 4000 BC Olives Near East 4000 BC Cotton Peru 4000 BC Pomegranate Iran 3500 BC Hemp East Asia 3500 BC Cotton Mesoamerica 3000 BC Azuki Bean East Asia 3000 BC Coca South America 3000 BC Sago Palm Southeast Asia 3000 BC Squash (Cucurbita pepo ovifera) North America 3000 BC Sunflower Central America 2600 BC Rice India 2500 BC Sweet Potato Peru 2500 BC Pearl millet Africa 2500 BC Marsh elder (Iva annua) North America 2400 BC Sorghum Africa 2000 BC Sunflower North America 2000 BC Bottle gourd Africa 2000 BC Saffron Mediterranean 1900 BC Chenopodium China 1900 BC Chenopodium North America 1800 BC Chocolate Mexico 1600 BC Coconut Southeast Asia 1500 BC Rice Africa 1500 BC Tobacco South America 1000 BC Eggplant Asia 1 st century BC Vanilla Central America 14 th century AD SOURCES OF VARIATION IN CONVENTIONAL BREEDING Dictated by evolutionary processes, sexual reproduction causes significant, uncontrollable variation through the process of meiosis (Charlesworth and Wright, 2001). Meiotic variation is of critical importance in conventional breeding where it is used to develop new cultivars. Meiosis functions to create gametes, each with nearly ½ of the parental DNA b. Male & female gametes then fuse to reconstitute progeny with the full, but now rearranged, amount of DNA. The rearrangement in itself is uncontrollable but may have some predictability, depending on the amount of genetic homology between parents. The outcome is progeny that may or may not fit expectations. However, several other mechanisms operate implicitly with meiotic rearrangement to create significant additional variation. Crossing over, in which complete chromosomal arms, or segments of such, are 51

4 transferred between chromosomes, commonly occurs during chromosome replication (Harrison et al., 2010). These chromosomal pieces can either duplicate, sending a copy to a new position or they can simply excise and move to another site. Regardless, the mechanism of crossing over causes additional genetic variation during meiosis. Transposition, in which random fragments of DNA move from place to place in an enzyme-driven process (Huang et al., 2012), is common as well. It is considered to be an evolutionary survival mechanism meant to allow the rapid generation of variant individuals. Transposition is extremely common: It is estimated that 85% of the corn genomic organization occurred exclusively as a result of transposition (Vicient, 2010). An early analysis of Vitis reported over 1,000 complete transposons and over 2,000 incomplete versions (Benjak et al., 2008). While generation of variants is necessary for many crops in order to effect improvement, such variation is not desirable for others and blocks our ability to satisfactorily genetically improve them at all via CB. Grape is one such crop. OBSTACLES TO GENETIC IMPROVEMENT OF GRAPEVINE As previously discussed (Gray et al., 2014), genetic improvement of grapevine (Vitis spp.) is critically needed to enhance sustainable crop productivity and foster profitable wine industries throughout the world (Vivier and Pretorius, 2002). Although numerous unique hybrids were developed over the years, the application of CB to genetic improvement of elite cultivars, the mainstays of worldwide production, is deemed to be largely unsuccessful due to genetically-based obstacles. The importance of improving elite cultivars cannot be overstated in that only 35 elite cultivars accounted for 66% of world grape acreage in 2014 (Anderson and Aryal, 2014); a majority of these are centuries-old and maintained primarily through a stringently managed system of vegetative propagation (Bowers et al., 1999; Myles et al., 2011). However, these elite cultivars lack other very important traits, particularly durable disease and pest resistance, that are demanded by today's intensive agricultural conditions. As such, producers currently rely on frequent use of pesticides to control diseases, particularly in areas of higher humidity; this occurs despite the increasing public outcry against such practices and resulting environmental issues. Unfortunately, grape is now considered to be one of the most pesticide-contaminated fruits (C us et al., 2010). For example, the bacterial pathogen that incites Pierce s disease, Xylella fastidiosa, has no proven method of durable control other than well-known genetic resistance and the unsustainable mass spraying of pesticides to inhibit insect vectors. Despite well over $ 50 million expended, by Federal and State governments since 1999 (Alston et al., 2013), the critical fight against Pierce s disease remains far from finished. Furthermore, it is not possible to rely on CB to improve elite cultivars while still meeting the strict expectations of oenophiles (Bisson et al., 2002). Like many woody perennial crops, grape has a long lifecycle, exhibits severe inbreeding depression, and, unique to grape, complex genetic control of highly prized enological qualities; this makes useful selfing and backcrossing, typically used in CB, impossible (Gray et al., 2005). PB provides an unparalleled opportunity to insert only the pre-tested grape-derived DNA needed to control specific traits like disease resistance without disturbing prized oenological characteristics (Gray et al., 2014). THE GENESIS OF PRECISION BREEDING Precision breeding technology is only recently becoming available for an increasing number of crop species. It is a biotechnology that stems from decades of research aimed at improving crop plants. Its implementation, crop-by-crop, depends on development and merger of the following three technologies: 1) Tissue and cell culture methods aimed at regeneration of whole plants from single (totipotent) cells, 2) Insertion of DNA fragments, which express desired traits, into such cells, and 3) Advancements in computational technology that allow exact genomic sequences to be identified, retrieved, and tested for expression of specific traits. In this way, such DNA sequences, when inserted into totipotent cells, result in plants that express the desired traits. 52

5 For grape, we began to develop totipotent somatic embryogenic cultures in 1984, publishing the first report in 1987 (Gray and Mortensen, 1987). It was not until 1996 that, in collaboration with R. Scorza and J.M. Cordts, we reported the first instance of gene insertion and stable expression in V. vinifera Thompson Seedless syn. Sultania, a major elite cultivar (Scorza et al., 1996). In 2007, the genomic sequence of V. vinifera Pinot Noir was elaborated (Velasco et al., 2007), thus, fully enabling our approach to utilize PB for improvement of grapevine. APPLYING PRECISION BREEDING TO GRAPE The actual procedure to create PB grape cultivars appears to be deceptively simple, but it is made significantly more difficult and time-consuming by the relatively slow growth propensity typical of grape, plus the requirement of abundant somatic embryogenic cultures in order to fluidly test many genetic elements. Research designed to optimize methodology for induction and maintenance of somatic embryogenic cultures for many grape species, hybrids and cultivars was conducted for over three decades and was summarized in 2009, resulting in the disclosure of reliable improved procedures (Dhekney et al., 2009a, 2011a). Similarly, our research to effect gene insertion into many different grapevines also was summarized in 2009 and an optimized gene insertion protocol was presented that documented its use for a number of elite cultivars, among other grape species and hybrids.(dhekney et al., 2009b). Procedures for genetic analyses, including identification, recovery and functional analyses of genes and promoters, were described through a series of reports (Dhekney et al., 2011b; Gray et al., 2014; Li et al., 2006, 2007, 2010, 2011a, b, 2012; Li and Gray, 2005) and this research is ongoing. PERFORMANCE OF PB GRAPEVINES Research to produce prototype plants designed to study the real-world performance of various genetic elements, including putative grape-derived disease resistance genes and an array of promoters, began in 2006 and continues through to the present. Prototype PB plants are those that contain a particular grape-derived gene and/or promoter to be tested, but also contain non-grape genetic elements, such as genetic markers and antibiotic resistance genes to facilitate rapid selection and development; these are not considered to be the final PB constructions. Research designed to determine the real world performance of grapevines that contain various grape-derived disease resistance genes continues (Figures 1-3). CONCLUSIONS 1) Precision breeding circumvents the variation-inducing process of meiosis by instead utilizing the mitotic developmental pathway. 2) Precision breeding is akin to the selection of useful random somatic mutations from axillary buds, but differs in that it is highly controlled and predictable. Recognition and selection of mutated branches (sports) have resulted in strains of elite grape varieties, as well as useful traits in other crops (ex. all nectarines & pink-fruited citrus). 3) Compared to conventional breeding, precision breeding causes far less perturbation of the crop plant s genome, resulting in fewer unintended consequences. 4) Due to its clearly-documented benign nature compared to conventional breeding, we expect that plants produced via precision breeding will require no more regulation than those derived from conventional breeding. 5) However, as with conventional breeding, precision bred varieties must undergo rigorous field testing, ultimately in grower fields. 53

6 Figure field trial of PB prototype vine Thompson Seedless expressing the V. vinifera thaumatin-like (VvTL-1) protein gene. PB lines selected via greenhouse screening for resistance to powdery mildew disease (Erysiphe necator) displayed a two-week delay in black rot disease (Guignardia bidwellii). This suggests that lines constitutively expressing VvTL-1 protein may possess resistance to a broad range of fungal diseases (Dhekney et al., 2011b). Figure field trial of PB prototype vine Seyval Blanc expressing a V. vinifera pathogenesis-related protein gene (VvPR-1), demonstrating durable multi-fungal disease resistance and vigorous growth compared to control vines. 54

7 Figure 3. Demonstration of fruit rot resistance. A. PB prototype vine Syrah expressing the V. vinifera 2S albumin gene (VvAlb) in greenhouse screening demonstrates resistance to powdery mildew (Erysiphe necator). Fruit from control vines did not ripen due to heavy disease pressure, whereas PB vines remained disease-free and their fruit ripened normally (Gray et al., 2008). B. Fruit clusters of Thompson Seedless PB prototype produced fruit in the field that exhibited remarkable resistance to grape fruit rot disease, which is caused by a syndrome of pathogenic fungi and bacteria (Dhekney et al., 2011b). Literature cited Alston, J.M., Fuller, K.B., Kaplan, J.D., and Tumber, K.P. (2013). The costs and benefits of Pierce s disease research in the California winegrape industry. Working Paper 1303 (Robert Mondavi Institute, Center for Wine Economics). Anderson, K., and Aryal, N. (2014). Database of regional, national and global winegrape bearing areas by variety, 2000 and 2010 (Adelaide: Wine Econ. Res. Ctr., Univ.), Dec. 2013, rev. July Benjak, A., Forneck, A., and Casacuberta, J.M. (2008). Genome-wide analysis of the "cut-and-paste" transposons of grapevine. PLoS ONE 3 (9), e PubMed Bisson, L.F., Waterhouse, A.L., Ebeler, S.E., Walker, M.A., and Lapsley, J.T. (2002). The present and future of the international wine industry. Nature 418 (6898), PubMed Bowers, J., Boursiquot, J.M., This, P., Chu, K., Johansson, H., and Meredith, C. (1999). Historical genetics: the parentage of Chardonnay, Gamay, and other wine grapes of northeastern France. Science 285 (5433), PubMed Charlesworth, D., and Wright, S.I. (2001). Breeding systems and genome evolution. Curr. Opin. Genet. Dev. 11 (6), PubMed C us, F., Cesnik, H.B., Bolta, S.V., and Gregorcic, A. (2010). Pesticide residues and microbiological quality of bottled wines. Food Contr. 21 (2), Dhekney, S.A., Li, Z.T., Compton, M.E., and Gray, D.J. (2009a). Optimizing initiation and maintenance of Vitis embryogenic cultures. HortScience 44, Dhekney, S.A., Li, Z.T., Dutt, M., and Gray, D.J. (2009b). Factors Influencing genetic transformation and plant regeneration of Vitis. Am. J. Enol. Vitic. 60, Dhekney, S.A., Li, Z.T., and Gray, D.J. (2011a). Factors influencing induction and maintenance of Vitis rotundifolia Michx. embryogenic cultures. Plant Cell Tissue Organ Cult. 105 (2), Dhekney, S.A., Li, Z.T., and Gray, D.J. (2011b). Grapevines engineered to express cisgenic Vitis vinifera thaumatinlike protein exhibit fungal disease resistance. In Vitro Dev. Biol. Plant. 47, Gray, D.J., and Mortensen, J.A. (1987). Initiation and maintenance of long term somatic embryogenesis from anthers and ovaries of Vitis longii Microsperma. Plant Cell Tiss. Org. Cult. 9 (1),

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