The state-of-the-art of grapevine biotechnology and new breeding technologies (NBTS) - OENO One
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Received : 23 February 2019 y Accepted y 21 April 2019 y Published y 14 May 2019
DOI : 10.20870/oeno-one.2019.53.2.2405
VINE AND WINE
OPEN ACCESS JOURNAL
The state-of-the-art of grapevine biotechnology
and new breeding technologies (NBTS)
Lorenza Dalla Costa1, Mickael Malnoy1, David Lecourieux2, Laurent Deluc3,
Fatma Ouaked-Lecourieux2, Mark R. Thomas4 and Laurent Torregrosa5*
1 Dept. of Biology and Genomic of Fruit Plants, Foundation E. Mach, 38010 San Michele all’Adige, Italy
2 ISVV-EGFV, CNRS, INRA, Bordeaux University, 33883 Villenave d’Ornon, France
3 Dept. of Horticulture, Oregon State University, OR 97331, Corvallis, USA
4 CSIRO Agriculture and Food, Hartley Grove, Urrbrae SA 5064, Australia
5
AGAP, Montpellier University, CIRAD, INRA, Montpellier SupAgro, Montpellier, France
Corresponding author : laurent.torregrosa@supagro.fr
This article is published in cooperation with the 21th GIESCO International Meeting, June 23-28 2019, Thessaloniki, Greece.
Guests editors : Stefanos Koundouras and Laurent Torregrosa
ABSTRACT
Context of the review: The manipulation of the genetic basis controlling grapevine adaptation and phenotypic
plasticity can be performed either by classical genetics or biotechnologies. In the last 15 years, considerable
knowledge has accumulated about the grapevine genome as well as the mechanisms involved in the interaction of
the vine with the environment, pests and diseases. Despite the difficulties associated with genetic mapping in this
species (allele diversity, chimerism, long generation intervals…), several major QTLs (quantitative trait loci)
controlling important vegetative or reproductive traits have been identified. Considering the huge genotypic and
phenotypic diversities existing in Vitis, breeding offers a substantial range of options to improve the performances of
cultivars. However, even if marker-assisted selection was largely developed to shorten breeding programs, the
selection of improved cultivars, whether for agronomic traits or disease tolerances, is still long and uncertain.
Moreover, breeding by crossing does not preserve cultivar genetic background, when the wine industry and market
are still based on varietal wines.
Significance of the review: In grapevine, pioneering biotechnologies were set up in the 1960s to propagate and/or
clean the material from micro-organisms. In the 1990s, the basis of genetic engineering was primary established
through biolistic or Agrobacterium with several derived technologies refined in the last 10 years. The latest advance
is represented by a group of technologies based on genome editing which allows a much more precise modification
of the genome. These technologies, so-called NBTs (new breeding technologies), which theoretically do not
deconstruct the phenotype of existing cultivars, could be potentially better accepted by the wine industry and
consumers than previous GMO (genetically modified organism) approaches. This paper reviews the current state-of-
the-art of the biotechnologies available for grapevine genome manipulation and future prospects for genetic
improvement.
KEYWORDS
grapevine, biotechnologies, gene transfer, genome editing, genetic improvement
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Lorenza Dalla Costa et al.
INTRODUCTION genome editing), which can be used to modify
WHAT DOES GRAPEVINE gene structure or functioning.
BIOTECHNOLOGY MEAN?
HISTORY OF GRAPEVINE
The term biotechnology refers to any process of BIOTECHNOLOGIES
cultivation, multiplication or genetic The first reports (Table 1) dealing with the
modification that uses techniques or conditions application of biotechnology of the vine are due
of implementation that do not exist in the nature to Georges Morel (1944). These early works
(Torregrosa and Bouquet, 1993). This term can concerned the in vitro culture of tissues under
therefore be applied to a large number of aseptic conditions (Torregrosa et al., 2001;
technologies used to multiply, select elite Bouquet and Torregrosa, 2003). As soon as the
individuals or modify their sanitary or genetic first in vitro culture media were developed, the
status. Biotechnologies can be classified into first applications consisted of defining the
three categories. The first one involves all conditions of tissue or organ development from
techniques used for conservation or pre-existing meristems, for propagation
multiplication (e.g. in vitro micropropagation to purposes. From the 1970s-80s, these techniques
establish collections of varieties under aseptic allowed the development of viral sanitation
conditions). The second category is related to a procedures and in vitro propagation to be used
set of techniques that supports the sanitary (e.g. for the vegetative and genetic improvement of
micrografting) or genetic (e.g. embryo rescue) rootstock and scion varieties.
selection of elite individuals, without causing From the 1980s, thanks to advances in the
genetic modification. The last category includes understanding of the hormonal control of plant
technologies that modify the genome or the organogenesis, regeneration techniques were
epigenome (e.g. somaclonal variation or in vitro developed to induce the differentiation of shoot
mutagenesis, genetic transformation or recently meristems from undifferentiated cells. The first
TABLE 1. Grapevine biotechnologies (in bold major advances).
Achievement Reference
Aseptic tissue culture Morel (1944)
Micropropagation Galzy (1961)
Isolated meristem culture Galzy (1972)
Somatic embryogenesis Mullins and Srinivasan (1976)
Adventitious organogenesis Favre (1977)
Axillary bud proliferation Jona and Webb (1978)
Adventitious caulogenesis Rajasekaran and Mullins (1981)
Engineered hairy roots Guellec et al. (1990)
Transgenic vines Mullins et al. (1990)
Particle bombardment Hébert et al. (1993)
Agronomic trait manipulation Le Gall et al. (1994)
Protoplast technology Reustle et al. (1994)
L1/L2 cell layer dissociation Franks et al. (2002)
Cell suspension expression Torregrosa et al. (2002)
Transgenic organogenesis Mezzetti et al. (2002)
Minimal cassette technology Vidal et al. (2006)
Virus inducing gene silencing Muruganantham et al. (2009)
Microvine transformation Chaib et al. (2010)
CRISPR/Cas9 mutagenesis Ren et al. (2016)
DNA-Free genetic edition Malnoy et al. (2016)
206 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 2, 205-228
somatic regenerations were obtained by grapevine, a first proof-of-concept was provided
adventive organogenesis, i.e. by inducing the by Ren et al. (2016) who modified the
development of neo-buds (Barlass and Skene, metabolism of tartaric acid by mutating the
1978). In parallel, somatic embryogenesis has L-idonate dehydrogenase (IdnDH) enzyme of
also been developed for a large number of Vitis Chardonnay. Since then, several research groups
genotypes (Martinelli and Gribaudo, 2001). The have been attempting to develop the technology
latter technology, which makes it possible to to induce genetic modifications without the
obtain thousands of somatic embryos from a few incorporation of viral or bacterial genetic
hundred mg of calli or embryogenic cell backbones. These new technologies represent
suspensions, forms the basis of genetic interesting prospects for both functional
transformation or genome editing procedures. genomics studies and for genetic improvement
of grapevine (Dalla Costa et al., 2017). These
The first publication mentioning the successful technologies are detailed in the next sections.
gene transfer in grapevine reported the recovery
of calli and roots transformed by Agrobacterium PROPAGATION
tumefaciens-derived plasmids (Hemstad and AND CONSERVATION
Reisch, 1985). Baribault et al. (1989) obtained OF ORIGINAL RESOURCES
vegetative organs of Cabernet-Sauvignon
ectopically expressing transgenes but could not The reliable propagation of grapevine genotypes,
achieve the regeneration of stable plants. The i.e. without morphogenetic changes, cannot be
first transgenic vines were obtained by Mullins achieved by somatic regeneration because of
et al. (1990) who coupled the transformation high levels of growth regulators that are required
through Agrobacterium-disarmed vectors with to maintain this status also can potentially cause
the regeneration by somatic embryogenesis of somatic variations or mutations. In addition, by
rootstocks. The first attempt to incorporate genes regenerating an individual from a single cell or a
of agronomic interest occurred a few years after small number of cells, the regeneration process
by Le Gall et al. (1994) who incorporated a gene modifies the genetic structure of chimeric
encoding the coat protein of GCMV (Grapevine genotypes (Torregrosa et al., 2011). Thus, in
Chrome Mosaic Virus) into rootstocks and vitro propagation is best achieved by
interspecific Vitis x Muscadinia hybrids. microcutting, a technique that does not need the
use of plant growth regulators (Galzy, 1961).
Over the years, gene transfer technology has Each bud develops into a single vegetative axis
been improved and applied to a range of Vitis producing 2-4 phytomers per month. The annual
species (Bouquet et al., 2006 and 2008; theoretical production can reach 10 3 plants
Torregrosa et al., 2015). Agrobacterium- depending on the varieties (Figure 1). Another
mediated transformation has also been developed technique so-called axillary micropropagation
for the microvine (Chaib et al., 2010), a requires the use of cytokinins to suppress apical
promising model for boosting physiology and dominance and induce axillary shoot
genetics studies in grapevine (Torregrosa et al., proliferations (Silvestroni, 1981). Because the
2016). However, despite many improvements, hormonal balance imposed to obtain the axillary
the regeneration of non-chimeric transgenic proliferation masses inhibits the rhizogenesis,
plants remains a long and complex process in the rooting of the shoots isolated from
many grapevine genotypes. Nevertheless, proliferation masses requires an additional step
various alternative techniques using physical using an auxin-enriched media (IAA, IAB or
vectors or viruses have been developed to obtain NAA). By this technique, it is theoretically
ectopic gene expression in individual cells, cell possible to obtain from a single bud more than
suspension cultures, tissues or non-caulinary 104 plants per year.
organs (Vidal et al., 2010).
The establishment of genetic germplasms
More recently, genetic modification techniques maintained by micropropagation is an alternative
have improved significantly with the to the current repositories maintained in
development of genome editing approaches. greenhouses or outdoors, exposing genotypes to
These technologies, in particular those based on biotic and abiotic risks. However, the major
CRISPR/Cas9, use endonucleases that change weakness of in vitro repositories is the cost of
gene structure and expression in a more specific subcultures. To decrease the frequency of sub-
and targeted way than previous technologies. For culturing by decreasing in vitro plant growth,
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Lorenza Dalla Costa et al.
FIGURE 1. Propagation through microcuttings (top) or axillary proliferations (bottom). On the top right,
a one-month plantlet developed from a microcutting. On the bottom right, a microproliferation mass
before and after shoot separation.
several approaches have been tried, e.g. apex (200-500 μm) on in vitro plant internodes
decreasing temperature conditions, using or on hypocotyl fragments of a zygotic or
sugarless culture media or using plant growth somatic embryo (Bass et al., 1976; Torres-Viñals
inhibitors such as chlorocholine chloride (Galzy, et al., 2004). Thermotherapy can be performed in
1985; Harst-Langenbucher and Alleweldt, 1990). combination with meristem culture, apex
All of these systems can be successfully applied micrografting or somatic embryogenesis to
to a large cultivar (genotypic) range. In contrast, increase the efficiency of viral elimination
cryopreservation of apex or buds which has also (Gribaudo et al., 2006; Maliogka et al., 2015).
been investigated stays only applicable to a Some authors have shown that viral load can be
limited number of genotypes and with a low reduced during cryopreservation steps (Wang et
level of reproducibility (Plessis et al., 1991; al., 2003), but this approach is still under
Marković et al., 2015). investigation. Therefore, there is a range of
techniques that can be implemented in clonal and
VEGETATIVE GENETIC sanitary programs of ancient and/or rare
OR SANITARY SELECTION varieties, as a certain percentage of identified
selections are systematically infected with one or
To deliver genetically homogenous and virus- more virus strains.
free varieties, most countries perform clonal
propagation (Mannini, 2000). For the In vitro embryo rescue techniques (Figure 2) are
elimination of viruses, in vitro thermotherapy also used to support the establishment of
was the first method proposed (Galzy, 1963). genetically segregating populations, especially
Meristem culture and somatic embryogenesis when parents are carrying: i) the seedlessness
have also been reported as effective methods for trait, a maternal fruit character that interferes
the elimination of grapevine viruses (Goussard, with embryo development (Ramming et al.,
1981; Goussard et al., 1991) and have been 1990) or ii) the Vvgai1 mutation, which
shown to be virus strain- and grapevine accentuates seed integument dormancy
genotype-dependent. Today, the most widely (Chatbanyong and Torregrosa, 2015). The
used method consists of micrografting a shoot different technologies to induce the development
208 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 2, 205-228FIGURE 2. Zygotic embryo rescue to recover individuals from crosses between seedless varieties.
of a zygotic embryo extracted from a fertilized generate the final cassette (insert + vector) to be
ovary are known as embryo rescue (Sharma et transferred into a host organism via a
al., 1996; Ramming et al., 2000). The transformation procedure. There are several
effectiveness of embryo rescue can be optimized strategies available to either conduct one
by selecting specific developmental stages of the insertion event or the assembly of several DNA
fruit after anthesis (Pommer et al., 1995), the modules (Table 2).
type and level of growth regulators (Agüero et
al., 2000) and the progenitors (Hewstone et al., Traditional cloning usually refers to the use of
2006). These technologies, which are now restriction endonuclease to generate DNA
commonly used in table grape breeding fragments with complementary end sequences
programs, provide germination percentages of up that can be joined together with a DNA ligase
to 50% (Hewstone et al., 2006). Applied to enzyme. This usually involves preparing both
crosses involving microvine parents, embryo the insert and the vector by cutting with two
rescue results in a very high rate of germination, unique restriction enzymes. The use of two
in particular when embryos are extracted at an restriction enzymes that generates two non-
early stage of berry development (Chatbanyong compatible ends results in a directional cloning
and Torregrosa, 2015). of the insert into the vector, thereby lowering the
transformation background for self-ligation
CELL AND ORGAN TRANSIENT events of the vector. The discovery of the
OR STABLE TRANSFORMATION Polymerase Chain Reaction (PCR) has
1. Cloning strategies and tools expanded the use of restriction cloning to PCR
cloning by the introduction of restriction sites
Molecular cloning is a fundamental step of necessary for the ligation reaction with the
genetic engineering. It refers to the process by vector at the end of PCR-amplified insert.
which DNA/RNA molecules are produced and
transformed into a host organism. It typically In the recent years, the seamless cloning method
consists of the following components: i) the has gained scientific attention because it enables
DNA fragment of interest to be inserted, and ii) sequence-independent insertion of one or more
the accepting vector/plasmid backbone that inserts into the vector. The workflow involves
contains all the components for replication in the PCR to amplify the gene of interest, an
host. Each component (insert and vector) will go exonuclease generating cohesive ends for the
through a series of preparative steps necessary to insert and vector, and either a ligase or a
OENO One 2019, 2, 205-228 © 2019 International Viticulture and Enology Society - IVES 209Lorenza Dalla Costa et al.
recombinase to join the insert to the vector. The that enable transfer of a DNA fragment from one
ability to join 5 to 10 fragments in a vector to another without the use of restriction
predetermined order, without any sequence enzymes and ligases. Multi-round Gateway
restrictions or artifacts, is a very appealing and technologies (recombinase) can produce the
powerful tool in the light of synthetic biology. delivery of multiple transgenes through multiple
The most popular method is the Gibson reactions (Vemanna et al., 2013) but they leave a
assembly method developed by Gibson et al. 21-bp DNA artifact between building blocks.
(2009). This in vitro recombination is a one-step Yet, the combination Golden Gate Entry Vector
isothermal reaction employing a 5’ exonuclease along with a Gateway Recombination system is
(generation of overhang ends), a polymerase to becoming extremely useful as part of
fill in the gaps of the annealed single-stranded multiplexed Plant Genome Editing and
regions, and a DNA ligase to seal the nicks. Transcriptional Regulation approaches
employing CRISPR/Cas9 (Lowder et al., 2015).
The Golden Gate assembly is another approach
of seamless cloning that exploits the use of Type As generally conceived, the Golden Gate
IIS restriction enzymes to cleave DNA outside of technology was not reusable and thereby not
the recognition site (Engler et al., 2008 and useful for multigene engineering (modular
2009). There are several advantages to this cloning). Two new strategies for DNA assembly
methodology. First, the overhang sequence were created to enable the reusability for
created is not dictated by the restriction enzyme, instance of the Golden Gate cloning scheme:
and no additional DNA is introduced. Second, Moclo (Weber et al., 2011) and GoldenBraid
the fragment-specific sequence of the overhangs (Sarrion-Perdigones et al., 2011). Both strategies
enables the assembly of multiple fragments in a use the Golden Gate Property to build
specific sequential order. Third, the restriction transcriptional units (TUs) and design
site is eliminated from the ligated product, so the destination vectors that enable the TUs to be
digestion and ligation reactions can take place assembled among them. Those two strategies are
simultaneously. Both methods (Gibson and advantageous for the development of modular
Golden Gate Assembly) were recently reported assembly systems in plant synthetic biology as
for grapevine as part of a cloning strategy aimed long as common standards are adopted by the
to generate a TAS3-resistant ARF4 gene community so that the building blocks can be
construct and an amiRNA (artificial microRNA) shared by as many users as possible (Vazquez-
targeting the endogenous ARF4 gene (Gouthu et Vilar et al., 2017).
al., 2018).
Finally, Ligation Independent Cloning (LIC) is
For the past decade, recombinational cloning an alternative technique to restriction
has become very popular in genetic engineering enzyme/ligase cloning (Aslanidis and de Jong,
of plants using either integrases or recombinases 1990). Inserts are PCR amplified and vectors are
TABLE 2. Summary of the physical and chemical DNA delivery methods (from Cunningham et al., 2018).
Delivery method Adverse effects Targets Cargo types Limitations
Physical methods
• Effectiveness highly species and cultivar
• Damage to target tissue & cargo
specific
DNA, siRNA,
Biolistic
miRNA, • Targeting leaves limits time to observe
or particle-mediated • Low penetration depth Calli, embryos, leaves
ribonucleoproteins, delivery effects
delivery large size
• Targeting embryos requires laborious
• Random integration
regeneration procedure
• Damage to target tissue • Protoplasts
Nucleic acids
• Non-specific transport of material
Electroporation through pores
• Meristems (DNA, siRNA, Limited cargo-carrying capacity
miRNA)
• May lead to improper cell function • Pollen grains
Chemical methods
• Species amenable to Nucleic acids
PEG-mediated Regeneration is inefficient for most
High density can induce cytotoxicity. protoplast regeneration (DNA, siRNAs,
delivery species in transient studies
• Protoplasts miRNAs)
210 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 2, 205-228made linear either by restriction enzyme Biolistic experiments were conducted in
digestion or by PCR. The use of 3’ → 5’ grapevine in the late 1990s and early 2000s. In
exonuclease activity of T4 DNA Polymerase 1996, Kikkert and colleagues reported on the
creates overhangs with complementarity between first generation of transgenic grapevine plants
the vector and insert. The incorporation of dGTP using the Vitis Hybrid Chancellor. Later on,
in the reaction limits the exonuclease processing Vidal et al. (2003) adapted a reliable and
to the first complementary C residue, which efficient system for stable transformation and
facilitates the generation of sticky ends PCR regeneration of ‘Chardonnay’ plants using
products complementary to the vector. Joined microprojectile bombardment. In grapevine, the
fragments (insert+vector) have four nicks that biolistic method was also found to be extremely
are repaired by E. coli during transformation. popular for transient assays in various plant
This technique allows efficient creation of materials (cell suspension culture, leaf sections,
recombinant plasmids without the introduction and somatic embryos). Promoter analyses in
of DNA artifacts. One particular variation of the Cabernet Sauvignon suspension cells were
ligation independent cloning - the SLIC method successful in studying the effect of anaerobiosis
for Sequence and Ligation independent cloning - on the regulation of VvAdh gene expression
has been adopted by many researchers. As many (Torregrosa et al., 2002). Co-transformation
as five inserts can be assembled in one reaction experiments using GFP (green fluorescent
simultaneously with great efficiency (Li and protein), GUS (beta-glucuronidase) and
Elledge, 2012). More marginal but still efficient Luciferase for the validation of protein-DNA
is the Uracil-Specific Excision Reagent Fusion interactions were routinely conducted to
(Geu-Flores et al., 2007), which consists of the examine the trans-activation of MYB and MYC
use of PCR primers that contain a single transcription factors on promoters of several
deoxyuridine residue near the 5’ end. Subsequent flavonoid-related genes (Bogs et al., 2007;
treatment of the PCR products with deoxyuridine- Walker et al., 2007; Deluc et al., 2008; Czemmel
excision reagents generates long 3’ overhangs et al., 2009; Hichri et al., 2010). More recently,
designed to complement each other and thereby promoter deletion analysis of VitViGIP1 was
facilitating their fusion. The combination of this performed on leaf discs and somatic embryos
approach with improved cloning techniques has and revealed the core promoter of this gene and
led to the development of an efficient toolbox for several other cis-acting regulatory elements
transformation in cereals (Hebelstrup et al., (Joubert et al., 2013).
2010).
Electroporation - Initially developed for
4.2. Physical and chemical delivery methods protoplast transformation, this technology was
Biolistic, also called particle bombardment expanded to intact plant cells in rice and wheat
(Table 3), is a physical method developed in the (Shimamoto et al., 1989; He et al., 1994). The
1980s intended to deliver into plant cells principle requires the application of a strong
microparticles (gold or tungsten) coated with a electric field to cells that creates temporary pore
genetic cargo containing the genetic information in cell membranes, thereby facilitating the
of interest (Klein et al., 1987). Unlike uptake of the genetic cargo into the cell. In the
Agrobacterium-mediated transformation, past decade, electroporation protocols for plant
biolistic delivery can result in transformation of transformation have been optimized and
nuclear, plastidial, or mitochondrial genomes. standardized for several plant species (including
However, its effectiveness is highly dependent tobacco, rice, wheat, and maize) using
on the species/cultivars, making its impact very commercially available electroporators
limited. The nature and the size of the genetic (Barampuram and Zhang, 2011). Although the
cargo have evolved along with technology methodology is fast and inexpensive, it has some
advances of genetic engineering. DNA, siRNAs constraints such as the thickness of the cell wall,
(small interfering RNAs), miRNAs, ribonucleo- and the impact of the strong electric field pulse
proteins (RNPs) have been demonstrated to be that can damage the structure of the delivered
efficiently transferred through biolistic methods gene by creating inaccurate translational end
although plasmid DNA remains the most products (Rakoczy-Trojanowska, 2002). To date,
common genetic material transferred through there is no study using this method in grapevine
this mode. genetic studies.
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TABLE 3. Advantages and disadvantages of cloning and assembly methods.
Cloning method Advantage Disadvantage
Low cost
Sequence constraints due to presence and/or translation
Traditional Versatile
of restriction site
Directional cloning
Limited vector choices
High efficiency Higher cost
PCR
Amenable to high throughput Lack of sequence control at junction
Directional cloning may be difficult
The overhang sequence not dictated
by the restriction enzyme Low cloning efficiency may require the use
Seamless
Allows for orderly assembly for ultracompetent chemically competent cells
of multiple fragments simultaneously
Relatively more expensive
Allows high-throughput vector creation
Recombinational compared to traditional methods
Widely available ORF collections Vector sets defined by supplier
Ligation-independent Low cost
Some type of sequence modifications not possible
Cloning Many different vector choices
PEG-mediated transformation uses an inert are described as powerful tools for delivering
hydrophilic polymer of ethylene oxide that can genes of interest into a host plant. Despite being
help the transfer of DNA into protoplasts. In this technically challenging, these Agrobacterium-
approach, DNA molecules are directly incubated mediated approaches are still preferred for
with protoplasts and the transfer is initiated by transgenic plant production, as they present
the addition of divalent cations to the mixture. several advantages: they allow i) the transfer of
The addition of PEG to the protoplast mixture large and intact DNA segments, ii) the insertion
destabilizes the permeability of the plant of simple transgenes with defined ends and low
membrane and allows free DNA to enter the copy number, iii) stable integration and
plant cytoplasm. On the one hand, the lack of an inheritance, and iv) consistent gene expression
efficient technique and methodology to over generations (Barampuram and Zhang,
regenerate whole plants from protoplasts renders 2011).
the use of PEG-mediated delivery system for
Agrobacterium-mediated transformation
mature plant transformations marginal. On the
methods are divided into two categories: stable
other hand, it has been extensively used for
transformation and transient transformation.
transient assays in plants to validate gene
function. In grapevine, PEG-treated protoplasts Stable transformation occurs when the DNA
from Cabernet-Sauvignon cell suspension transferred inside the plant nucleus is inserted
cultures were used to study protein subcellular into the plant genome for inheritance to
localization (Hichri et al., 2010), promoter subsequent generations. Alternatively, transient
analysis (Saumonneau et al., 2012), transformation refers to the situation in which
protein/protein (Saumonneau et al., 2008) and the foreign DNA transiently remains in the
DNA/protein interactions (Marchive et al., nucleus without being integrated into the plant
2013). More recently, PEG-mediated genome but is transcribed to express the genes of
transformation of Chardonnay protoplasts was interest. In grapevine, these Agrobacterium-
exploited as preferential method for the direct mediated gene transfer methods were developed
delivery of purified CRISPR/Cas9 RNPs in the early 1990s (Baribault et al., 1989) and
(Malnoy et al., 2016). underwent continuous improvement to allow the
characterization of the function and regulation of
3. Agrobacterium-derived technologies several genes (Table 4).
Among the different plant genetic transformation The main Agrobacterium strain used for grape
methods, Agrobacterium-mediated technologies transformation is A. tumefaciens, but the use of
212 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 2, 205-228TABLE 4. Examples of Agrobacterium-mediated transformation assays (modified from Jelly et al., 2014).
Method Tissue Cultivar Strain Application Genes/seq. References
Syringe infiltration Leaves of in vitro plantlets Superior seedless AGL1, GV3101 Gene silencing hpRNA against VvPDS Urso et al. (2013)
Vacuum infiltration Thompson seedless C58C1 (pCH32) Overexpression D4E1 (synthetic AMP) Visser et al. (2012)
Leaves of in vitro plantlets
Cabernet Franc, Syrah, Zinfandel EHA105 Viral vector engineering GLRaV‐2 cDNA Kurth et al. (2012)
Detached leaves Cabernet Franc GV3101 Gene silencing hpRNA against VvPGIP1 Bertazzon et al. (2012)
OENO One 2019, 2, 205-228
of in vitro plantlets Cabernet-Sauvignon, Cinsault, C58C1 (pCH32) Overexpression VvVST1 Santos‐Rosa et al. (2008)
Muscat, Carignane GV3101 Overexpression VpGLOX Guan et al. (2011)
Overexpression VpPR10.2 He et al. (2013)
Promoter analysis VpSTS Xu et al. (2010)
LBA4404 Overexpression VpPR10.1 Xu et al. (2014)
Leaves Grenache GV2260 Viral vector engineering GLRaV‐2 cDNA Liu et al. (2009)
Syrah GV3101 Overexpression VvNPR1 Le Henanff et al. (2009)
Prime, Thompson seedless EHA105 Viral vector engineering GRSPaV cDNA Meng et al. (2013)
Agro‐drenching Roots of in vitro plantlets
Prime EHA105 Viral vector engineering GVA cDNA Muruganantham et al. (2009)
Cell suspension Gamay Red EHA105 Promoter analysis VvDFR Gollop et al. (2002)
Somatic embryos Ami‐RNA validation amiRNAs against Grapevine fanleaf virus Jelly et al. (2012)
Chardonnay GV3101
(cotransformation) and GUS sensor CaMV35S, CsVMV,
Thompson seedless EHA105 promoters testing At ACT2 promoters Li et al. (2001)
promoter testing BDDPs with CaMV35S, CsVMV Li et al. (2004 ; 2011)
Thompson seedless EHA105 promoters and enhancers
31 grapevine Li et al. (2012)
Co-cultivation Freedom EHA105 Overexpression VvCBF4 Tillet et al. (2011)
VvSK1 Lecourieux et al. (2010)
41B EHA105 Overexpression VvCEB1 Nicolas et al. (2013)
VvABF2 Nicolas et al. (2014)
Chardonnay, Thompson seedless EHA101 Overexpression Pear PGIP Agüero et al. (2015)
VpSTS Fan et al. (2008)
Thompson seedless GV3101 Overexpression
VpPUB23 Zhou et al. (2014)
© 2019 International Viticulture and Enology Society - IVES 213Lorenza Dalla Costa et al.
A. rhizogenes strains to transform roots (hairy biolistic bombardment, electroporation or
roots) also provides an interesting system to protoplast transfection. Janssen and Gardner
perform functional studies (Hu and Du, 2006). (1990) showed that a short period of co-
The most frequently A. tumefaciens strains used cultivation with A. tumefaciens led to the active
for grapevine transformation are EHA105 (Hood transcription of many copies of the transgene in
et al., 1993), GV3101 (Koncz and Schell, 1986) the mesophyll cells. In grapevine, this
and C58C1 (Hamilton et al., 1996). technology was successfully used to monitor
elicitor-induced calcium changes by
Stable transformation methods - In grapevine, overexpressing apoaequorin in V. vinifera cv.
stable transformation is a long and sometime Gamay cells (Vandelle et al., 2006; Aziz et al.,
difficult process. Its efficiency strongly depends 2007). Other methods exist: i) A. tumefaciens
on the genotype, the explant source, the medium can be infiltrated into plant leaves using a
composition and the transformation method needleless syringe to inject the Agrobacterium
used. Co-cultivation of somatic embryos with A. suspension through the stomata (Zottini et al.,
tumefaciens is the most commonly used method 2008), or ii) the leaves (or entire plant) can be
to stably transform grapevine. It was largely flooded with the bacterial suspension under
exploited to achieve proof-of-concepts vacuum pressure to facilitate liquid penetration
(Torregrosa et al., 2002; Chaib et al., 2010; Ren into the cells. This second method has the
et al., 2016) and functional characterization of advantage of allowing gene expression in the
putative key genes (Lecourieux et al., 2010; Li et whole leaf (or plant). A number of studies have
al., 2012; Nicolas et al., 2013, 2014; Pessina et reported the successful use of vacuum-
al., 2016; He et al., 2018; Sun et al., 2018). infiltration experiments either with detached
leaves (Santos-Rosa et al., 2008; Zottini et al.,
Because regeneration of transgenic plants from 2008; Le Henanff et al., 2009; Xu et al., 2010;
stably transformed embryos is long, gene Guan et al., 2011; Bertazzon et al., 2012; He et
function can also be addressed using certain al., 2013; Xu et al., 2014) or with entire plants
plant organs. In this regard, transgenic hairy (Kurth et al., 2012; Visser et al., 2012). The
roots induced by A. rhizogenes provide a success of agroinfiltration experiments was
powerful system for functional studies (Hu and shown to be cultivar dependent (Santos-Rosa et
Du, 2006; Gomez et al., 2009; Terrier et al., al., 2008).
2009; Höll et al., 2013) as their production takes
only a few weeks (Torregrosa and Bouquet, For some particular cases, other methods were
1997). developed. An agrodrenching method was
developed using roots of young in vitro
Non-embryonic cell cultures can also be stably grapevine plantlets to deliver an infectious viral
transformed by Agrobacterium. This was shown cDNA clone of GVA that could not be inoculated
to be particularly interesting for the production using leaf agroinfiltration (Muruganantham et
of bioactive compounds (Martínez-Márquez et al., 2009). This method has been validated for
al., 2015). To improve Agrobacterium-mediated Prime and Thompson seedless grapevine
transformation efficiency of dedifferentiated V. cultivars (Muruganantham et al., 2009; Meng et
vinifera cv Monastrell cells, Chu et al. (2016) al., 2013). As the production of transgenic fruits
used the Sonication-Assisted Agrobacterium- after grapevine transformation takes time, berry
mediated Transformation (SAAT) method that agro-injection was developed to shorten the time
was previously developed for soybean needed to study gene function in fruits. This
transformation (Trick and Finer, 1997). This method is derived from the transient
method significantly increases transformation methodology successfully applied to tomato fruit
efficiency (Chialva et al., 2016). (Orzaez et al., 2006) based on Agrobacterium
cultures injection through the fruit stylar apex
Transient transformation methods - To rapidly using a syringe with needle. Using this transient
investigate unknown gene function, several overexpression technique in the “Kyoho” grape
transient transformation systems have been fruit, Gao et al. (2018) showed the involvement
developed. These assays are faster and of the ABA Receptor VlPYL1 in the regulation
convenient alternatives to the time-consuming of anthocyanin accumulation in grape berry skin.
stable transformation method. They mainly Similarly, Sun et al. (2017) showed that
include Agrobacterium-mediated transient VvVHP1;2 (vacuolar H + -PPase 1) over-
transformation and direct gene transfer by expression promoted anthocyanin accumulation
214 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 2, 205-228in berry skins. These reports underline the
possible application of the transient
Lovato et al. (2014)
Kurth et al. (2012)
Meng et al. (2013)
overexpression technique for studying grapevine
(using N. benthamania) (using N. benthamania) Park et al. (2016)
Muruganantham
Liu et al. (2009)
Reference
fruit development.
et al. (2009)
4. Virus-derived technologies
An alternative approach to perform functional
studies in grape is the use of viral vectors
PDS and ChlI silencing
VIGS Transcription
(Table 5). For a long time, modified plant
viruses have been used for several purposes
PDS silencing
including the transient expression of
heterologous proteins (with protein size
limitations according to the viral system used) or
ChlH
the silencing of host genes (known as Virus
nd
Induced Gene Silencing or VIGS).
Reporter gene
VIGS requires engineered infectious viral clones
that generate double-stranded RNA resulting in
GFP and GUS
post-transcriptional silencing of a target gene as
part of the plant’s own defense response against
GFP
GFP
GFP
invading virus (Baulcombe, 1999). VIGS is one
of the most widely used genomic tools,
Infectivity with Syrah
V. vinifera varieties
displaying several advantages such as allowing
easy and rapid gene silencing, with no need for
Prime Thomson
and Cab. Franc.
stable plant transformation, and enabling both
forward and reverse genetic approaches in many
Brachetto
Nebbiolo
Corvina
Sultana
crop species (Senthil-Kumar and Mysore, 2011).
Prime
Syrah
VIGS has been successfully used to better define
TABLE 5. Potential plant virus-based vectors suitable for VIGS in grapevine.
the role of genes involved in various plant
Recomb. virus
processes including fruit development
Infectivity /
Yes / Leaves
Yes / Leaves
Yes / Leaves
Yes / Roots
(Fernandez-Moreno et al., 2013) or abiotic stress
tolerance (Ramegowda et al., 2014).
The majority of the available plant virus-based
Agroinoc. of viral vector
vectors displays a host range mainly restricted to
Agro-drenching of roots
Vacuum agroinfiltration
Vacuum agroinfiltration
annual or herbaceous plants. In this context,
of the whole plantlets
several grapevine infectious viral vectors were
of plantlet’s roots
Agro-drenching
engineered, including those based on the
of plantlets
Vitivirus Grapevine Virus A (GVA)
(Muruganantham et al., 2009), the Closterovirus
Grapevine leafroll-associated virus-2 (GLRaV-
2) (Kurth et al., 2012), and the Foveavirus
RNA / Closterovirus
Virus type / Genus
RNA / Tombusvirus
Grapevine rupestris stem pitting-associated
RNA / Foveavirus
virus (GRSPaV) (Meng et al., 2013). Generation
Grapevine virus A (GVA) RNA / Vitivirus
of recombinant GFP-expressing viral cDNA
clones inserted into binary plasmids allowed the
agroinoculation of different grapevine cultivars
and highlighted the infectivity of these viral
Grapevine Algerian latent
constructs by GFP visualization. Four weeks
Grapevine rupestris stem
after agroinoculation, the virus was detected in
Grapevine leaf rool-
pitting-assoc. virus
the V. vinifera infected plantlets regardless of the
associated virus-2
Virus
viral-derived vector used. GVA-GFP and
virus (GALV)
(GLRa-V2)
GRSPaV-GFP fluorescent signals were detected
(GRSPaV)
in plantlet leaves and roots, respectively
(Muruganantham et al., 2009; Meng et al.,
OENO One 2019, 2, 205-228 © 2019 International Viticulture and Enology Society - IVES 215Lorenza Dalla Costa et al. 2013). In the case of GLRaV-2, infected cells This challenge may explain, at least in part, the appeared first in the stem phloem and then lack of functional studies using these grape colonized all plant organs including berries, thus infectious viral vectors. Other viral vectors like revealing a systemic virus expression Tobacco rattle virus (TRV) and Apple latent propagating through the phloem. Interestingly, spherical virus (ASLV) vectors can provoke the GLRaV-2-derived vector infection was VIGS in many plant species (Senthil-Kumar and shown to be transmitted by grafting to a wide Mysore, 2011) but not in grapevine, as recently range of grape varieties and to be genetically confirmed by Gao et al. (2018) who failed to highly stable within infected plants that express silence VlPYL1 (pyrabactin resistance/PYL the exogenous transmitted sequence up to 3- (PYR-like)) using the TRV-mediated silencing in years post-inoculation (Dolja and Koonin, 2013). “Kyoho” grapes. To the best of our knowledge, GVA- and GLRaV-2-mediated transcriptional only one viral-derived vector was successfully gene silencing efficiency was demonstrated used to perform functional studies in grapevine. using derived vectors harboring fragments of In 2007, Peretz and co-workers engineered the targeted host genes. Thus, virus-induced IL-60 system derived from the geminivirus silencing of endogenous PDS (phytoene Tomato yellow leaf curl virus (TYLCV). IL-60 desaturase) or ChlI (subunit I of magnesium- was defined as a universal DNA plant vector protoporphyrin IX chelatase) was observed in system providing efficient expression or infected grapevines, displaying leaf bleaching silencing in numerous plant species (Peretz et symptoms resulting from the loss of chlorophyll al., 2007). Ten years later, Sun et al. (2017) (Muruganantham et al., 2009; Kurth et al., successfully exploited the IL-60 viral transient 2012). The capacity of such grapevine specific expression system to overexpress VvVHP1 virus vectors to silence genes in particular (vacuolar H + -PPase 1) in “Kyoho” berries. organs (i.e. roots and berries), or in specific However, the efficiency of IL-60 system to tissues (i.e. fruit skin) remains an open question. silence grape genes remains to be demonstrated. FIGURE 3. Scheme showing the functioning of CRISPR-Cas9 nuclease. PAM=protospacer adjacent motif; NHEJ=non-homologous end joining; HDR=homology directed repair. 216 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 2, 205-228
Recently, viral-based vectors have been interspersed short palindromic repeats (CRISPR)
developed as promising tools for plant genome with the associated nuclease Cas9. The
engineering including the use of genome editing CRISPR/Cas9 system is the most powerful gene
(Zaidi and Mansoor, 2017). Gene targeting (GT) editing technique now available due to its
efficiency relies on the delivery method of GT simplicity of application and effectiveness
reagents (sequence specific nucleases and repair (Puchta and Fauser, 2014). Originally discovered
templates) to plant cells. In this way, as a part of adaptive immunity in Streptococcus
autonomously replicating virus-based vectors pyogenes, CRISPR/Cas9 technology has been
have been demonstrated as efficient means of exploited to introduce desired mutations in genes
delivering GT reagents into plants. Plant RNA of interest in medical, plant and animal studies.
viruses like the potato virus X (PVX) and the It operates through guide RNAs composed of a
tobacco rattle virus (TRV) were used for spacer, complementary to a desired DNA
delivering ZFN nucleases (Marton et al., 2010) sequence, and a scaffold forming complex with
or the CRISPR guide RNAs (gRNA) (Ali et al., Cas9 (van der Oost et al., 2014). The Cas9/guide
2015). Using the CRISPR-Cas system combined RNA complex scans the genome searching for
with the bean yellow dwarf virus (BEyDV) complementarity by unwinding double stranded
rolling circle replicon, Dahan-Meir et al. (2018) DNA. The unwinding process, prerequisite for
optimized a method to target mutagenesis and to checking and finding the complementary target
replace genes in tomato, without any selection site, is allowed by the protospacer-adjacent motif
marker or reporter genes. The single viral- (PAM) recognized by the nuclease. Once the
derived construct described in this study contains right target has been found, the nuclease can
both the CRISPR-Cas9 and a gemini viral generate a DSB with the effect of introducing
replicon system, as well as a guide RNA and a insertion/deletion (INDEL) mutations in the
donor fragment that can be exchanged in a specific gene sequence. In a recent paper, Jones
modular manner. This makes the present viral- and colleagues (2017) evaluated the kinetics of
derived vector an interesting tool that could Cas9 nuclease by measuring the timeframes
potentially be adapted to grapevine. needed for unwinding double stranded DNA and
for checking complementarity to the guide RNA
5. Genome editing in living E. coli. They found that each potential
The term ‘New Breeding Technologies (NBTs)’ target - each region with a PAM site (for SpCas9
comprises several techniques having different is the triplet NGG) - is bound for less than 30
purposes and methodologies. The best known milliseconds allowing to find a specific single
and the one which can allow precise genetic target sequence in about six hours.
changes is genome editing. The application of The application of these new technologies may
programmable nucleases for genome editing has be particularly useful in grapevine since they
been one of the greatest scientific breakthroughs produce minimal and precise modifications in
in the field of plant genetic engineering (Kim selected genotypes of interest, such as elite
and Kim, 2014). These nucleases are enzymes cultivars sought-after by the wine market,
which cut the DNA at specific desired sites in the without altering the genetic background as
genome. The induced double stranded breaks happens in traditional breeding. CRISPR/Cas9
(DSBs) are then repaired by the cell natural technology has been successfully applied to
repair mechanism, either non-homologous end generate edited grapevine plants (Osakabe et al.,
joining (NHEJ), which may introduce nucleotide 2018). Two different delivery systems have been
variation, or homologous recombination (HDR) used: one based on the stable integration of the
when a donor DNA with homologous arms is genetic components in the genome by means of
present. The main achievable outcomes are gene A. tumefaciens gene transfer and the second one
knockout or gene replacement (Figure 3). based on the direct delivery of purified Cas9
5.1. State-of-the art of the NBTs protein and gRNAs (Figure 4). Ren et al. (2016)
transformed ‘Chardonnay’ embryogenic cell
Three major classes of programmable nucleases masses to gain point mutations in the L-idonate
for precision genome editing have been used dehydrogenase gene. They were able to
over the past 15 years: zinc finger nucleases regenerate plants with an altered production of
(ZFNs), transcription activator-like effector tartaric acid and vitamin C. In 2017, ‘Neo
nucleases (TALENs), and clustered regularly Muscat’ somatic embryos were transformed with
OENO One 2019, 2, 205-228 © 2019 International Viticulture and Enology Society - IVES 217Lorenza Dalla Costa et al.
FIGURE 4. Current options to perform genome editing with grapevine using CRISPR-Cas9 nuclease.
a CRISPR/Cas9 editing construct targeting the editing machinery via endonuclease complexes
phytoene desaturase gene and plants with albino or by viral particles. Accordingly, technological
leaves were produced (Nakajima et al., 2017). improvements of SpCas9 have been focused on
Transgenic ‘Thompson Seedless’ plants have three main goals: increasing accuracy and
also recently been produced with mutated specificity of targeting, expanding the set of
versions of the WRKY52 transcription factor PAM sites recognized by the nuclease, and
gene under both mono- and bi-allelic conditions reducing the molecular size. At present, many
(Wang et al., 2018). In an attempt to produce Cas variants more specific, accurate and able to
non-transgenic edited grapevines, Malnoy and recognize different PAM sequences have been
colleagues (2016) directly delivered the purified obtained by mutagenizing key amino acids of
Cas9 and gRNAs into ‘Chardonnay’ protoplasts SpCas9. As well, several SpCas9 orthologues
generating edited protoplasts, but not whole were found in other bacterial species which show
plants. However, the rate of protoplasts different PAM site specificities and smaller size
presenting a mutation was relatively low, around (Cebrian-Serrano and Davies, 2017).
0.5%.
Another line of research is the methodological
5.2. Technical challenges for NBTs improvement for the delivery of CRISPR/Cas
machinery in plant cells. This is an important
The main issues limiting the potential of the line of research since the availability of methods
commonly used S. pyogenes Cas9 are: i) the which avoid the incorporation of foreign DNA in
occurrence of off-target editing which are highly the host genome could be crucial in paving the
undesired, ii) the requirement of a specific PAM way for the exemption of genome editing
site (NGG) adjacent to the target site which products from GMO legislation. Recent
limits the number of potential targets and iii) the developments and techniques in the field of
large size of the nuclease (approximately DNA-free genome editing are summarized in
160 KDa) which may hamper the delivery of the Metje-Sprink et al. (2019) and they rely mainly
218 © 2019 International Viticulture and Enology Society - IVES OENO One 2019, 2, 205-228on the delivery of protein-RNA complex (RNP) cultivars of multiple disease resistance genes or
to protoplasts by electroporation, polyethylene polygenic quality traits as well as the modulation
glycol (PEG) vesicles, biolistics (see paragraph of important metabolic pathways by using
4.2). The main pitfalls of these approaches in multiple guide RNAs and multiple donor DNA
comparison to classical transformation system simultaneously but this ambitious goal will
based on A. tumefaciens, are the low editing require a long time and strong technological
efficiency, the lack of a selection phase to enrich progress. Finally, CRISPR/Cas technology may
for positively edited plants and the need of also be used to produce genetic variability for
sequencing for screening. On the other hand, studying plant biology and for obtaining new
new approaches are being evaluated for the allelic variants with agronomic/qualitative
elimination of exogenous DNA when classical advantages.
Agrobacterium-mediated transformation is used.
In grapevine, if the cultivar genome is to be 6. Regulatory framework and prospects for
preserved then backcrossing and screening of the NBTs
progeny is not a feasible strategy and methods
for site-specific DNA cassette elimination should Development in genome editing technologies
be considered and tested (Dalla Costa et al., can decrease the cost and time required to
2019). improve agronomic as well as horticultural crops
in the future, but the broad adoption of NBT
Future prospects for grapevine genetic requires government support in setting up an
improvement by means of the CRISPR/Cas updated regulatory framework (Ma et al., 2018).
system may concern the optimization of the National policies concerning NBT products for
targeted insertion of donor DNA at the cleavage feed and food will probably play a key role in
site driven by homology (knock-in strategy). At determining their success in the foreseeable
the moment the knock-in approach remains very future. Two opposite attitudes towards green
challenging compared to gene knock-out biotechnologies may be observed: one is focused
(nucleotide mutations at the cleavage target site) on the final product while the other pays more
but would be very useful for targeted gene attention to the process through which a specific
insertion or allele replacement. The donor DNA product has been generated. The first approach is
can contain an allelic variant with a loss or gain followed by USA, Argentina, Australia and
of function compared to the targeted allele or Brazil which have established that if no foreign
could be a gene deriving from wild relatives genes or genetic material is present in a genome-
which can confer specific pathogen resistance. A edited variety, then it will not be subject to
visionary objective is the introgression in elite additional regulatory oversight and risk
TABLE 6. Main principles underpinning the European legislation on GMO.
Principle Legislative act Requirement
The applicant must present a request for GMO authorization to the Member
State which will transmit it to EFSA (European Food Safety Authority) for
Dir. 2001/18/EC
the risk assessment. EFSA’s opinion will be submitted to European
Reg. (EC) No 1829/2003
Authorization Commission for the final decision. Authorizations are valid for a maximum
Dir. (EU) 2015/412
of 10 years (renewable)
Member States have the possibility to restrict or prohibit GMO cultivation on
their territory
GMOs and products produced from GMOs must be traced at all stages of
their placing on the market. The information “the product/ingredient consists
Traceability Reg. (EC) No 1830/2003 of or contains or is produced from GMOs” must be transmitted to all the
operators receiving the product along the production and distribution chains
GMOs information must be recorded in a public available register
Specific product labelling is required when GM material > 0.9% of the food
Reg. (EC) No 1829/2003 ingredient/feed material while labelling is not compulsory when GM
Labelling
Reg. (EC) No 1830/2003 material ! 0.9%, provided that this presence is adventitious or technically
unavoidable
OENO One 2019, 2, 205-228 © 2019 International Viticulture and Enology Society - IVES 219Lorenza Dalla Costa et al.
assessment as in the case of GMO (Eriksson et 0CRISPR%2012%20Nov%202018.pdf). In this
al., 2019). The second approach has been document, scientists claim that regulating
historically adopted by Europe, where, on 25th genome editing as GMOs will have negative
July 2018, the Court of Justice of the European consequences for agriculture, society and
Union ruled that organisms obtained by economy in Europe and hope that organisms
mutagenesis (including genome editing) are containing small genetic alterations and which
GMO within the scope of the GMO European do not contain foreign genes will not be subject
Directive 2001/18/EC (http://curia.europa.eu to the provisions of the EU GMO Directive but
/juris/document/document.jsf?text=&docid=204 instead will fall under the regulatory regime that
387&pageIndex=0&doclang=EN&mode=req&d applies to classically bred varieties. As observed
ir=&occ=first&part=1&cid=747443). This by Tyczewska et al. (2018) the EU has incurred
decision strongly reaffirms the precautionary large investment costs in research as well as in
principle and forces gene-edited plants with NBTs that should promote competitiveness of
small and targeted nucleotide modifications to European agriculture. The huge development of
go through the very long and expensive analytical tools over the last years could likely
regulatory process intended for classical help to get out of the Manichaean diatribe
transgenic plants and defined by the Regulations “product vs. process”. At present, we have the
(EC) 1829/2003 and (EC) 1830/2003. This legal technology and the knowledge to deeply evaluate
framework relies mainly on the safety the plant that has been endowed with new trait,
assessment of the highest possible standards, full at both phenotypic and genomic level. Regarding
traceability throughout the production/ this last aspect, next generation sequencing
distribution chain and labelling requirement (NGS) platforms and bioinformatics can make
(Table 6). whole genome sequencing possible in short
times and with low costs, allowing to select the
A crucial point concerns the requirement of an proper and safer plant material to be propagated
analytical method for the unambiguous and commercialized. Such scientific data should
identification of the specific genetically guide the actions of politicians and regulators to
modified organism. In the case of genome edited valorize innovations for the benefit of society.
organisms, nucleotide mutations produced by a
gene-editing mechanism might be CONCLUSION
indistinguishable from the naturally occurring WHAT PERSPECTIVES
ones (or from those induced chemically or by FOR GRAPEVINE
irradiation) by means of the current analytical BIOTECHNOLOGIES AND NBTS ?
techniques available (Grohmann et al., 2019). It
follows that a method allowing to unequivocally Biotechnologies cover a very large number of
trace the origin of this mutation cannot be applications to multiply or improve sanitary or
provided as it cannot be excluded that the genetic of grapevine varieties. Some of these
identical DNA alterations occurred already technologies are now so integrated in the
spontaneously, were introduced by random selection of rootstock and scion varieties that
mutagenesis or were/will be created in an they are no longer debatable. Thus, without the
independent editing experiment. This uncertainty use of apex culture or micrografting, that makes
will have consequences for enforcement of the possible the elimination of pathogenic viruses or
GMO legislation (ENGL, 2019). bacteria, it would not be possible to provide the
industry with healthy clonal material. As a
The scientific community (upon the initiative of reminder, 100% of the rootstocks distributed in
VIB, a life science research institute based in France are certified clones, the majority of which
Belgium) expressed its opposition to the ruling have been sanitized by thermotherapy and
of the Court of Justice, to safeguard genome micrografting. This is also true for a significant
editing from GMO legislation. A hundred of proportion of scion clones. In terms of varietal
European research centers (University and innovation, the use of embryo rescue is
public and private institutes) have signed an widespread in the breeding of seedless table
important letter which has been submitted to the grapes, leading to a very innovative range of
President of the European Commission, Jean- varieties.
Claude Juncker on 24th January 2019
(www.vib.be/en/news/Documents/Position%20p The latest biotechnologies, genetic transfor-
aper%20on%20the%20ECJ%20ruling%20on%2 mation developed in the 1990s, and genome
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