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ARTICLE IN PRESSG ModelSL-8963; No. of Pages 8

Plant Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Plant Science

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recision breeding of grapevine (Vitis vinifera L.) for improved traits

ennis J. Graya,∗, Zhijian T. Lia, Sadanand A. Dhekneyb

Grape Biotechnology Core Laboratory, Mid-Florida Research and Education Center, University of Florida/IFAS, 2725 Binion Road, Apopka, FL 32703-8504SADepartment of Plant Sciences, Sheridan Research and Extension Center, University of Wyoming, 663 Wyarno Road, Sheridan, WY 82801 USA

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rticle history:vailable online xxx

eywords:rapevineitis viniferaisgenics

ntragenicsenetic engineeringisease resistance

a b s t r a c t

This review provides an overview of recent technological advancements that enable precision breedingto genetically improve elite cultivars of grapevine (Vitis vinifera L.). Precision breeding, previously termed“cisgenic” or “intragenic” genetic improvement, necessitates a better understanding and use of genomicresources now becoming accessible. Although it is now a relatively simple task to identify genetic ele-ments and genes from numerous “omics” databases, the control of major agronomic and enological traitsoften involves the currently unknown participation of many genes and regulatory machineries. In addi-tion, genetic evolution has left numerous vestigial genes and sequences without tangible functions. Thus,it is critical to functionally test each of these genetic entities to determine their real-world functionalityor contribution to trait attributes. Toward this goal, several diverse techniques now are in place, including

nthocyanin markerxpression analysis

cell culture systems to allow efficient plant regeneration, advanced gene insertion techniques, and, veryrecently, resources for genomic analyses. Currently, these techniques are being used for high-throughputexpression analysis of a wide range of grapevine-derived promoters and disease-related genes. It is envi-sioned that future research efforts will be extended to the study of promoters and genes functioning toenhance other important traits, such as fruit quality and vigor.

Published by Elsevier Ireland Ltd.

ontents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Advances in gene insertion technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Development of a grape gene-based marker system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Traditional marker genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Grapevine gene-derived anthocyanin marker system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Promoter mining and functional analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00A non-destructive anthocyanin-based promoter assay system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Constitutively active promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Tissue-specific, inducible and developmentally regulated promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Resistance/tolerance genes to biotic and abiotic stresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Antifungal genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Antibacterial genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Antiviral genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Abiotic stress tolerance genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Precision bred plants under field testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: D.J. Gray, et al., Precision breeding of grapevine (Vitis vinifera L.) for improved traits, Plant Sci. (2014),http://dx.doi.org/10.1016/j.plantsci.2014.03.023

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author. Tel.: +407 41 6946; fax: +407 814 6186.E-mail address: [email protected] (D.J. Gray).

ttp://dx.doi.org/10.1016/j.plantsci.2014.03.023168-9452/Published by Elsevier Ireland Ltd.

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ARTICLESL-8963; No. of Pages 8

D.J. Gray et al. / Plant

ntroduction

Genetic improvement of grapevine (Vitis vinifera L.) is one of theritical needs to enhance crop productivity and foster profitableine industries throughout the world [1]. Although numerousnique hybrids were developed over the years, genetic improve-ent of elite hybrids, the mainstays of worldwide production,

s deemed to be largely unsuccessful, especially in areas ravagedy severe disease/pest infestations and/or that require extensivehemical control to maintain. For example, the bacterial pathogenhat incites Pierce’s disease, Xylella fastidiosa, has no proven methodf durable control other than well-known genetic resistance andhe unsustainable mass spraying of pesticides to inhibit insect vec-ors, despite well over 50 million dollars expended, apparentlynsuccessfully, by Federal and State governments since 1999 [2].owever, genetic resistance (tolerance) among native Vitis speciesas identified by 1958 [3]. The practical use of genetic resis-

ance was subsequently confirmed through hybridization with V.inifera cultivars to instill durable and near complete control ofhe pathogen [4–6]. Particularly urgent now is the introduction ofpecific traits for durable tolerance to diseases, pests, and abiotictresses, while maintaining the essential quality of highly desiredlite cultivars [7,8]. However, it is not possible to rely on conven-ional breeding to improve elite cultivars so that they can adapt tohe production environment, while still meeting the strict expecta-ions of oenophiles [9,10]. Conventional breeding cannot practicallye used to add desired disease resistance traits to elite cultivarsf Vitis because of a long lifecycle, severe inbreeding depression,nd complex genetic control of enological qualities [11]. A major-ty of the relatively few elite grape cultivars currently cultivated

orldwide are centuries-old and maintained primarily through stringently managed system of vegetative propagation [12,13].owever, elite cultivars often lack other desirable traits such asurable disease and pest resistance that are demanded by today’s

ntensive agricultural conditions. As such, producers rely on fre-uent use of pesticides to control diseases, particularly in areas ofigher humidity; this is in spite of increasing public outcry againstuch practices and resulting environmental issues [14]. To mitigateuch increasingly crucial agricultural and health concerns, moderniotechnology has advanced to the point where it is now possible toxpedite genetic improvement of existing elite cultivars via preci-ion breeding [7,15]. This review is intended to provide an overviewf current technological advancement, particularly genomic analy-es, for the development of resistance to biotic and abiotic stresses,s well as other traits, via precision breeding of elite cultivars.

dvances in gene insertion technology

The methodology to insert specific genes into plants withoutnducing significant genetic rearrangement has been in develop-

ent for over thirty years. Such technology is particularly attractiveor perennial crops like grapevine that have severe genetic obsta-les to conventional breeding and require multi-year evaluation forurability of desired traits due to their long lifecycle and longevity.

n order to provide a reliable working platform for genetic test-ng, efficient cell regeneration systems were developed [16–20].n increasing number of scientists used such regeneration systems

o document insertion of single or few genes into grapevine [11].he precise methods of gene insertion employed either biolisticarticle bombardment [21,22] or, more commonly, Agrobacterium-ediated gene insertion into regenerative cells, followed by plant

Please cite this article in press as: D.J. Gray, et al., Precision breeding ohttp://dx.doi.org/10.1016/j.plantsci.2014.03.023

ecovery [11,23–25]. Both methods have been meticulously refinednd optimized over the years and are now capable of produc-ng hundreds of genetically modified plants. The majority oflants modified via the Agrobacterium approach tended to harbor

PRESSe xxx (2014) xxx–xxx

low-gene copy number and defined gene insertion [24,26]. A largenumber of modified plants is critical for identification of lines witha desirable level of gene expression and performance to meet over-all improvement objectives [27]. The need to test many plant lines,as is the norm with conventional breeding, is critical in order toselect outstanding individuals.

During the early years of technology development aimed towardprecision breeding, it was necessary to test genetic elementsfrom non-plant hosts, including animals and bacteria, due to therelatively primitive state of biotechnology. This approach was gen-erally referred to as “transgenic” modification. This early discoveryresearch was absolutely essential so that cell culture and geneinsertion methods could be refined to the point of being fullyfunctional [28]. Subsequently, as pointed out by Rommens [29],many non-plant genes and promoters with known functionalitywere utilized to display the technological marvel of biotechnology.This approach to crop improvement inevitably invited argumentsand ongoing worries as to whether such plants with foreign genesand promoters were healthy, represented an environmental threatand/or were otherwise dangerous in some way. The use of foreigngenetic material in food crops including grapevine remains to bethe pivot of social and ethical public debate [7,29,30].

Along with the refinement of cell culture and gene insertionmethods, the final technology required to enable precision breed-ing was completion of the draft genome of V. vinifera ‘Pinot Noir’in 2007 and the relatively new-found and simplified availabilityof computational analysis [31,32]. It is now possible to identifygrapevine genes, along with their associated genetic elements, iso-late them, from sexually-compatible disease-resistant relatives,and insert them into elite cultivars. While still in its infancy, theapplication of precision breeding to grapevine improvement is wellunderway, with a number of modified plants in approved field trialsand more on the way [1,33–35].

Application of precision breeding is the logical and biologicallyconservative extension of conventional breeding, made possibleonly by long-term scientific research. Studies have suggested thatapplication of precision breeding will boost consumer’s confidenceand acceptance of improved crop products as well [36–38].

As we continue to refine precision breeding, more remains to bediscovered. We require a better understanding of genome structureorganization and sequence/function associations. It is estimatedthat grape genome contains over 30,400 genes, which is morethan that found in most animals [39]. Many important agronomictraits are controlled by a complex network of regulatory sequencesand factors and often influenced by dynamic sequence alterations,such as gene duplication, transposon insertion and loss- or gain-of-function mutations [40]. Environmental factors also play animportant role in gene expression and interaction [41]. Thus, theactual function and sustainability of any isolated genetic mate-rial, whether a gene or a promoter, has to be confirmed withinits intricate genetic milieu and then rigorously tested over a pro-longed time in the environment; this is the fundamental way todetermine durable structure/function relationships. Although weare making rapid progress in sequence analysis and functionalannotation of the grapevine genome [31,32,42–44], progress infunctional characterization of important genes/promoters is slow,creating a significant obstacle to the practical utilization of thegenetic resources already available. Since precision breeding is bio-logically consistent with conventionally bred crops and, indeed, theentire plant lifecycle, it constitutes a technical refinement of exist-ing breeding methodologies. The solution to accelerating the crucialfunctional analyses needed to both test the technology and produce

f grapevine (Vitis vinifera L.) for improved traits, Plant Sci. (2014),

improved cultivars is to not regulate evaluation of precision bredgrapevine, so that individual lines can be tested in quantity andin grower fields, as has always been the manner for convention-ally bred crops. As with all crop breeding, many progeny must be


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evelopment of a grape gene-based marker system

raditional marker genes

Marker genes are utilized to facilitate identification and selec-ion of engineered cells; which is akin to finding a needle in aaystack. Such genes may provide growth advantages for mod-

fied cells to outgrow the large number of non-modified cellsnd/or signify the successful integration of new genetic elements45]. Presently, the markers in use are not derived from therapevine genome. Among these genetic marker genes, some con-er resistance/tolerance to antibiotics or cytotoxic chemicals suchs herbicides. Others encode selectable metabolic enzymes suchs visible green fluorescence protein (GFP) and the assayable �-lucuronidase (GUS) enzyme [30,46,47]. These marker genes werelso successfully tested in grapevine [21,25,48]. Thus far, the major-ty of previous genetically modified grape plants were generatedrimarily by using the NPTII gene for kanamycin-based selectionnd GFP for visual selection; this allowed for the identification andelection of modified cells, which typically are present in very lowumbers.

Although current regulatory guidelines recognize NPTII, amongther microbe-derived marker genes used in modified plants, to beafe, they are no longer needed and their use continues to remain anssue causing polarized public debate and governmental regulation45].

Recently, a co-transformation system was tested to achievearker-free genetically modified grapevine [26]. In this system,

wo strains of Agrobacterium were used to treat target explants: onetrain harboring a binary vector containing the target gene expres-ion unit only, whereas the other strain contained a binary vectorith a dual gene codA/nptII selection marker expression unit. Bysing a brief exposure to kanamycin, modified cells with the tar-et gene are encouraged to grow along with cells containing theual gene cassette. Upon applying negative selection based on theodA function, the codA/nptII-containing cells cease to grow and thenly survival is from target gene-containing cells. Although the effi-iency of plant recovery needs further improvement, this systemffers a working example of marker gene elimination for perenniallants such as grapevine.

rapevine gene-derived anthocyanin marker system

A grapevine gene, VvMybA1 isolated from ‘Merlot’ was recentlyested as a new visible reporter marker for non-destructivend quantitative analysis of gene expression [49,50]. VvMybA1elongs to the MYB transcription factor superfamily functioning

n the grapevine anthocyanin biosynthesis pathway. It activateshe expression of UDP-glucose flavonoid 3-O-glucosyltransferaseUFGT), an enzyme catalyzing the final reaction in the modifica-ion and stabilization of anthocyanin [51]. The ectopic expression ofvMybA1 results in production of anthocyanin, an easily discernibleoloration in otherwise non-pigmented cells/tissues.

VvMybA1 can serve as visual reporter to identify modified cellsnd subsequent regenerants, which was first demonstrated byhe recovery of stably transformed grapevine without relying onhe use of NPTII expression and kanamycin selection [49]. How-ver, the VvMybA1-expressing grapevines (‘Thompson Seedless’)


hat were recovered lacked vigor and did not persist in the fieldnvironment. These plants produced intensely pigmented, curlynd highly brittle leaves as a result of the over-accumulationf non-recyclable anthocyanin in cell vacuoles [49]. Such

PRESSe xxx (2014) xxx–xxx 3

undesirable phenotypic consequences of VvMybA1 as a reportermarker now are being mitigated by employing promoters withtissue-specific and/or developmentally regulated expression capa-bility to avoid unwanted hyper-accumulation [52]. Recently,VvMybA1 was placed under control of a late embryogenesis abun-dant protein Dc3 gene promoter of carrot (Daucus carota) andshown to be capable of rendering pigment production exclusivelyin embryos but not in vegetative tissues of citrus [53,54]. Thetissue-specific expression strategy may provide a solution to utilizeVvMybA1 as an indicator marker. Current endeavors in the discov-ery and mining of grapevine promoters should take such findingsinto consideration. An effective plant-based marker system elim-inates reliance on microbe-derived genes and/or complicated andinefficient gene elimination technologies for the development ofgenetically modified grapevine [46].

In addition to its potential as a visible selectable marker,VvMybA1 currently is used for real-time monitoring of transgeneexpression at the whole plant level. Quite often the expressionof previously-used marker genes varied greatly between plantsand throughout the entire course of plant development due tofactors associated with gene integration and environmental inter-actions. Using multiple modified plant lines with a consistent levelof gene expression, facilitates the accurate assessment of trait anal-yses. Reporter markers such as GUS and GFP are widely utilized tomonitor transgene expression [55,56]. However, there has been nosuitable marker for expression analysis at the whole plant level thatdoes not require special equipment, detection reagents or destruc-tive assays, and that can be readily adapted to high-throughputexperiments involving selection of hundreds of genetically modi-fied grapevines.

Anthocyanin is a self-revealing pigment that can be easily dis-cerned with the naked eye due to its vibrant pink-to-red color. Italso remains relatively stable in vivo and accumulates in insolubleanthocyanic vacuolar inclusions (AVIs) [57]. As illustrated previ-ously, pigmented inflorescences as a result of VvMybA1 expressionwere consistently observed in modified tobacco [49,50]. Thisunique utility demonstrated that VvMybA1 provides a highly effi-cient and reliable monitoring system for gene activity in grapevine.Currently VvMybA1 has been incorporated into transformationvector platforms controlled by a constitutively active grapevine-derived ubiquitin gene promoter for high throughput analysis ofgenes and promoters [50].

Promoter mining and functional analysis

A non-destructive anthocyanin-based promoter assay system

Promoters play an indispensable role in the regulatory process ofgene expression [58]. The employment of appropriate native pro-moters for precision breeding dictates the final outcome of traitdevelopment and durability. In recent years, extensive analysisof the genome and transcriptome uncovered a plethora of func-tional genes. For instance, the sequence identity of at least 18,725grapevine genes have been confirmed [44]. The functional anno-tation of these genes also provided a significant opportunity toevaluate their promoters, which direct gene expression in responseto developmental, environmental and regulatory network cues.For better utilization, it is critical to acquire a thorough under-standing of the important features of these promoters includingtheir sequences, activation capacity, regulatory interaction and sta-bility throughout plant development. However, over the years,


progress in functional analysis of promoters of grapevine has laggedbehind other academically or economically important plant speciessuch as Arabidopsis and rice. Some major reasons for such stag-nation include the limited ability to initiate and maintain a large


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uantity of suitable explants and the lack of reporter systems forfficient expression analysis. Currently, only a few labs worldwidere capable of transforming grapevine, but many are still facingajor obstacles in efforts to expand the efficacy to the level com-

arable to that in other model plant species. Furthermore, usef non-grapevine-derived reporter markers like GFP and GUS areampered by requirement of a large quantity of explant materialsnd by laborious destructive assay procedures.

In order to expedite progress, the nondestructive reporterarker VvMybA1 in combination with an efficient Clonase cloning

ystem (Life Technologies, CA, USA) significantly facilitates char-cterization of promoters from grapevine [49]. The amount ofnthocyanin accumulation in grapevine or tobacco explants dueo VvMybA1 expression correlates with the expression activity of aontrolling promoter. Such correlation is readily determined using

non-destructive, quantitative color histogram analysis method inoth transient and stable expression materials [49]. The expres-ion reporting capability of VvMybA1 conforms to the commonriteria for previously used quantitative reporter markers [45,59].he VvMybA1-based expression assay system offers an efficientnd versatile alternative for high-throughput promoter analysis inrapevine and other plants.

onstitutively active promoters

Early studies inserted useful genes into grapevine under con-rol of constitutive viral promoters to provide high levels of genexpression at the whole explant/plant level [18,23,55,56]. Theseromoters were used to drive the expression of either marker genesor identification of genetically modified plants or of target genesor trait development, mostly aimed at increasing disease resis-ance in modified plants. To eliminate the need for such constitutiveiral promoters, a number of putatively constitutive ubiquitin generomoters were identified and their expression capabilities charac-erized using the anthocyanin-based assay method [50]. The studyevealed that among 7 VvUb promoters analyzed, many were inac-ive and appeared to be older in evolutionary lineage, with a higherevel of sequence abnormality, whereas two highly active pro-

oters, VvUb6 and VvUb7, were identified that possessed fewerucleotide deletions or substitutions between cultivars and con-ained more cis-acting elements, which are commonly involvedn up-regulation of gene expression. These latter promoters sup-orted an activity level equivalent to or higher than that of theommonly-used double-enhanced CaMV 35S promoter in bothransient and stable expression analyses [50]. Such constitutivelyctive grapevine promoters provide a key tool needed to addurable traits via precision breeding technology.

issue-specific, inducible and developmentally regulatedromoters

Tissue-specific promoters activate gene expression only in par-icular types of cells, tissues, or organs, whereas inducible andevelopmentally regulated promoters are activated in response topecific cues and at certain stages of plant development, respec-ively. The development of a novel trait using precision breedingecessitates the availability of plant regulated promoters. For

nstance, engineering a fruit quality trait requires the use of aruit-active promoter such as the V. vinifera thaumatin-like proteinVvTL-1) gene promoter [60]; likewise, improvement of root growthequires root-specific promoters. Thus far, only an extremelyimited number of regulated promoters in grapevine are known.


hey include a seed-specific 2S albumin gene VvAlb1 promoter,wo alcohol dehydrogenase gene promoters; a stilbene synthaseene promoter and a number of PR1 gene promoters [50,52,61–63].he lack of characterized regulated promoters limits the


deployment of grapevine regulatory elements via precision breed-ing and inevitably attracts the use of non-grapevine materialsthat are not biologically consistent with the Vitis lifecycle [64].Therefore, the discovery and functional verification of candidategrapevine promoters is crucial to further progress in precisionbreeding [65–68].

Resistance/tolerance genes to biotic and abiotic stresses

Antifungal genes

The development of highly reproducible genetic engineeringprotocols for a wide array of grapevine cultivars and rootstocks nowallows identification and screening of grapevine-derived genes forintroducing desirable traits, particularly disease resistance. A num-ber of field studies for screening precision-bred grapevines arecurrently in advanced stages of testing prior to commercialization[see [69] for a complete list].

Grapevines are affected by a number of fungal pathogens world-wide and require intensive fungicide spray regimes for sustainablecrop production [70,71]. Major improvement efforts have beendirected toward enhancing fungal-disease resistance in table andwine grape cultivars [69]. A number of pathogenesis-related (PR)proteins were screened for their response to fungal pathogeninfection. Genetically modified ‘Neo Muscat’ and ‘Pusa Seedless’grapevines constitutively expressing rice chitinase genes exhib-ited enhanced resistance to anthracnose and powdery mildew[72,73]. However, no resistance to powdery mildew was observedin ‘Seyval Blanc’ plants expressing barley chitinase genes [74].Enhanced resistance to Eutypa lata was observed in ‘Richter 110′

grapevines that constitutively expressed a Vigna radiata eutypinedetoxyfing gene (Vr-ERE), which converts eutypine toxin producedby the pathogen to non-toxic eutypinol [75]. Stilbene synthasegenes encoding resveratrol were isolated from several Vitis speciesand engineered for constitutive expression to improve fungalresistance [76,77]. A VvTL-1 gene previously reported to inhibitElsinoe ampelina spore germination and hyphal growth was con-stitutively expressed in ‘Thompson Seedless’ grapevines [35,78].Enhanced resistance to foliar fungal diseases and decreased inci-dence of sour bunch rot in berries was observed in greenhouseand field tests. Constitutive expression of a V. vinifera WRKY1(VvWRKY1) transcription factor gene in ‘41B’ rootstock plantletsresulted in activation of regulatory genes in the jasmonic acidpathway and upregulation of several defense related proteins. Areduction in downy mildew symptoms caused by Plasmopora viti-cola was observed on in vitro-derived genetically modified plantleaves compared to the controls, when inoculated under simi-lar conditions [79]. Other non-grapevine derived genes such aslytic peptides encoding magainin and polygalactouranase inhibi-ting proteins (PGIP) were demonstrated to improve fungal diseaseresistance [80,81].

Antibacterial genes

Significant efforts have been directed toward improvinggrapevine resistance to bacterial crown gall disease (Agrobacteriumvitis) and Pierce’s disease (PD) (Xylella fastidiosa). A number of non-plant antimicrobial peptides from various sources were tested fortheir in vitro efficacy against A. vitis and X. fastidiosa prior to func-tional analyses [82,83]. Genetically modified grapevines expressingthe animal-derived lytic peptide, magainin, and truncated Agrobac-


terium virE2 protein, exhibited decreased crown gall symptomscaused by A. vitis [81,84]. Other lytic peptides such as cecropin,mellitin and their hybrid derivatives, LIMA1 and LIMA 2, wereinserted into elite table and wine grape cultivars [85–89]. Lytic


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eptides were detected in such genetically modified grapevinessing ELISA, and enhanced PD resistance was observed in repeatedreenhouse trials and field tests for several years [69]. A chimericeptide to incorporate bacterial resistance was developed by fus-

ng a pear-derived bacterial cell surface recognition domain with aecropin-derived lytic domain [90]. A significant reduction in bac-erial colonization and leaf scorching symptoms associated with PDere observed in modified ‘Thompson Seedless’ and ‘Chardonnay’

rapevines in the greenhouse environment, however resistance inhe field was not reported.

The possibility of imparting PD resistance to unmodified scionsrafted on genetically modified rootstocks expressing lytic peptideenes also was studied [91]. Non-modified ‘Cabernet Sauvignon’cions grafted onto modified ‘Thompson Seedless’ plants express-ng an animal-based synthetic lytic peptide were used as anxperimental rootstock [92]. Lytic peptide could be detected inylem sap of unmodified scions at levels similar to that of genet-cally modified rootstocks. In other studies, a non-grapevine PGIProtein could be detected in xylem sap of unmodified scions graftednto modified ‘Thompson Seedless’ and ‘Chardonnay’ vines consti-utively expressing PGIP [80]. Factors including protein size that cane transmitted from a modified rootstock to a grafted unmodifiedcion via the xylem and its threshold concentration necessary tompart disease resistance in grafted vines are being evaluated [93].onferring PD resistance using a mobile rootstock-derived anti-acterial peptide would eliminate the need for creating geneticallyodified versions of multiple scion cultivars.

ntiviral genes

Virus resistance was obtained using the strategies of para-ite derived resistance and RNA interference [94,95]. The coatrotein gene of several viral pathogens including Arabis mosaicirus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine chromeosaic virus (GCMV), Grapevine virus A and B (GVA and GVB)ere expressed in modified grapevines [96–98]. Unmodified scions

rafted onto modified rootstocks expressing GFLV CP exhibited noisual disease symptoms after 3 years of natural infection in twoineyard sites [99]. No viral recombination between modified andative GFLV isolates was observed in field tests, thus demonstrat-

ng the safety of this approach. In other studies, an inverted repeatonstruct and artificial microRNAs (amiRNA) were inserted intorapevine plants to evaluate GFLV resistance using RNA interfer-nce [100,101].

biotic stress tolerance genes

Phenotypic plasticity may cause the same genotype to exhibitignificant variability in fruit yield and quality in different envi-onmental conditions [102]. The availability of the sequencedrapevine genome combined with high-throughput expressionrofiling technologies such as microarrays, differential display andNA sequencing now makes it possible to analyze gene expression

n several Vitis species under varying environmental conditions.ranscriptome analysis was successfully applied to identify genesnvolved in vegetative and reproductive growth and to study vineesponse to abiotic and biotic stress factors. It has also was pro-osed to be an alternative to reference genome sequencing fortudying variability that might exist among cultivars with respecto enological characteristics [103].

Transcriptome analysis of grapevine tissues at various develop-ental stages revealed significant differences in gene expression


etween actively growing, green/vegetative tissues and matureoody tissues [104]. Differential gene expression was observeduring tissue maturation as a result of coactivation of pathways thatere not expressed in actively growing tissues. Such transcriptome


reprogramming during maturation has been specifically observedin woody plants. Transcriptome analysis of berry tissues during var-ious stages of development and ripening has provided a significantamount of information on the expression of transcription factorsand genes involved in the production of organic acids, tannins andother secondary metabolites [105].

Drought, salinity and temperature fluctuations adversely affectgrapevine production worldwide. These stresses reduce crop yieldand quality, although irrigation management strategies may beused to concentrate pigments and flavor of wine grapes andimprove enological qualities [106]. Response to abiotic stressprimarily occurs through altered physiological processes, whichultimately affects vegetative and reproductive growth patterns[107]. Grapevine species exhibit significant differences in transcrip-tome and protein patterns under conditions of abiotic stress; suchinformation may be useful to elucidate underlying differences inphenotypic response and ultimately improve performance underadverse growing environments.

Microarray analyses of salt and water stressed ‘Cabernet Sauvi-gnon’ grapevines demonstrated that more than 2000 genes weredifferentially expressed, and expression was influenced by bothdrought and ABA [108]. Significant differences as well as com-mon attributes were observed in gene response to varying stressfactors. These included transcription factors, genes involved insignal transduction and carbohydrate metabolism, which may ulti-mately contribute to enhanced abiotic stress tolerance [109]. Waterdeficit imposed by drought appeared to have a more severe effectthan that imposed from salinity. Differences in protein expressionwere observed between V. vinifera cultivars Chardonnay and Caber-net Sauvignon exposed to drought and salinity stress. Decrease ingrowth parameters were correlated with corresponding reductionsin the amounts of proteins involved in photosynthesis [110].

A number of cold-inducible transcription factors recently wereisolated from grapevines and shown to act as master switches forstress-induced activation of several genes [67,111,112]. Transcrip-tome analysis of V. amurensis and V. vinifera ‘Hamburg Muscat’grapevines indicated large differences when vines were exposedto cold-stress [113]. A higher number of unidentified sequenceswere obtained in cold-hardy V. amurensis when compared with thereference genome, possibly due to the large phylogenetic distancebetween the two species.

Results of genetic engineering of grapevine to improve abi-otic stress tolerance and qualitative traits are just emerging. Forinstance, modified grapevines expressing a Medicago sativa fer-ritin gene and Arabidopsis CBF1 transcription factor were shownto exhibit improved abiotic stress tolerance [114,115]. Enhancedfreezing tolerance was observed in Vitis interspecific hybrid ‘Free-dom’ that constitutively expressed a V. vinifera CBF4 transcriptionfactor [116]. Genetically engineered grapevines with improvedfecundity and reduced berry browning currently are being eval-uated [117,118]. Nevertheless, with the tremendous amount ofinformation being accumulated through genome mining andexpression analysis, more critical genes/promoters will be revealedand become available for use in grapevine improvement.

Precision bred plants under field testing

The majority of previous attempts to increase stress resistancein grapevine employed non-grape genes or promoters. However,in ongoing research, a number of grapevine-derived genetic ele-ments, including several pathogenesis-related genes were isolatedfrom disease resistant, sexually-compatible V. vinifera hybrids and


plants overexpressing these genes were placed under field condi-tions [1,119]. These plants contain genes encoding the PR1 variants[119], VvTL1 (PR5) [35], VvAlb1 [52], homologues of VvAMP1 andVvAMP2/defensin [120] and an orthologue of Snakin-1 [121]. It


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s expected that results from the long-term, real world tests willnhance understanding of gene function and expand our abilityo engineer resistant grapevine using only genetic materials fromexually-compatible grapevines.

onclusion

Significant advancements in cell culture, gene discoverynd gene insertion technologies were only recently merged toully enable precision breeding for the genetic improvement ofrapevine. It should be noted that the results of precision breed-ng are fully in sync with the developmental biology of grapevine,ut provide a significantly more efficient and predictable methodf genetic improvement compared to that of conventional breed-ng. With precision breeding, only genetic elements from grapevinere used, but certain key obstacles, including inbreeding depres-ion (resulting in the inability to “self” desirable lines), that stiflemprovement via conventional breeding are completely overcome.he results obtained by precision breeding are more predictablehan that of conventional breeding, lowering the risk of unintendedutcomes. With precision breeding, a major goal is to create newersions of elite cultivars that not only maintain their desirableraits, but are able to be managed with less-to-no chemical inter-ention. Such precise modification cannot be obtained throughonventional breeding within any practical timeframe. Currently,rototype plants harboring putative grapevine resistance genesnd promoters are in the field to test for trait expression. Plantsontaining only genetic elements from grapevine will begin to belanted for testing within the next year. However, more widespreadnd robust evaluations, as is the norm for conventional breeding,ust occur to confirm the utility of cultivars produced by precision

reeding.

cknowledgements

This research was supported in part by the Florida Agriculturalxperiment Station and grants from the Florida Department of Agri-ulture and Consumer Services’ Viticulture Trust Fund (grant no.0079907) and the USDA/NIFA Specialty Crops Research Initiativegrant no. 2011-51181-30668).

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