6.

208

Review

The Role of Italy in the Use of

Advanced Plant Genomic
Techniques on Fruit Trees: State of
the Art and Future Perspectives

Luca Nerva, Lorenza Dalla Costa , Angelo Ciacciulli, Silvia Sabbadini, Vera Pavese, Luca Dondini,
Elisa Vendramin, Emilia Caboni, Irene Perrone, Andrea Moglia et al.

Special Issue
State-of-the-Art Molecular Plant Sciences in Italy
Edited by
Dr. Silvia Celletti and Dr. Stefania Astolfi

https://doi.org/10.3390/ijms24020977
 International Journal of
Molecular Sciences

Review
The Role of Italy in the Use of Advanced Plant Genomic
Techniques on Fruit Trees: State of the Art and
Future Perspectives
Luca Nerva 1,2,† , Lorenza Dalla Costa 3,† , Angelo Ciacciulli 4,† , Silvia Sabbadini 5 , Vera Pavese 6 ,
Luca Dondini 7 , Elisa Vendramin 8 , Emilia Caboni 8 , Irene Perrone 2 , Andrea Moglia 6 , Sara Zenoni 9 ,
Vania Michelotti 10 , Sabrina Micali 8 , Stefano La Malfa 11 , Alessandra Gentile 11 , Stefano Tartarini 7 ,
Bruno Mezzetti 5 , Roberto Botta 6 , Ignazio Verde 8 , Riccardo Velasco 1 , Mickael Arnaud Malnoy 3, *
and Concetta Licciardello 4, *

1 Research Center for Viticulture and Enology, Council for Agricultural Research and Economics,
31015 Conegliano, Italy
2 Institute for Sustainable Plant Protection, National Research Council, 10135 Torino, Italy
3 Research and Innovation Centre, Foundation Edmund Mach, 38098 San Michele all’Adige, Italy
4 Research Center for Olive Fruit and Citrus Crops, Council for Agricultural Research and Economics,
95024 Acireale, Italy
5 Department of Agricultural, Food, and Environmental Sciences, Marche Polytechnic University,
60131 Ancona, Italy
6 Department of Agricultural, Forest and Food Sciences, University of Torino, 10095 Torino, Italy
7 Department of Agricultural and Food Sciences, University of Bologna, 40127 Bologna, Italy
8 Research Center for Olive Fruit and Citrus Crops, Council for Agricultural Research and Economics,
00134 Rome, Italy
9 Department of Biotechnology, University of Verona, 37134 Verona, Italy
10 Research Center for Genomics and Bioinformatics, Council for Agricultural Research and Economics,
29017 Fiorenzuola D’Arda, Italy
11 Department of Biotechnology, University of Catania, 95124 Catania, Italy
* Correspondence: mickael.malnoy@fmach.it (M.A.M.); concetta.licciardello@crea.gov.it (C.L.);
Citation: Nerva, L.; Dalla Costa, L.; Tel.: +39-04-6161-5536 (M.A.M.); +39-09-5765-3104 (C.L.)
Ciacciulli, A.; Sabbadini, S.; Pavese, † These authors contributed equally to this work.
V.; Dondini, L.; Vendramin, E.;
Caboni, E.; Perrone, I.; Moglia, A.; Abstract: Climate change is deeply impacting the food chain production, lowering quality and yield.
et al. The Role of Italy in the Use of In this context, the international scientific community has dedicated many efforts to enhancing
Advanced Plant Genomic Techniques resilience and sustainability in agriculture. Italy is among the main European producers of several
on Fruit Trees: State of the Art and
fruit trees; therefore, national research centers and universities undertook several initiatives to
Future Perspectives. Int. J. Mol. Sci.
maintain the specificity of the ‘Made in Italy’ label. Despite their importance, fruit crops are suffering
2023, 24, 977. https://doi.org/
from difficulties associated with the conventional breeding approaches, especially in terms of financial
10.3390/ijms24020977
commitment, land resources availability, and long generation times. The ‘new genomic techniques’
Academic Editor: Samuel De Visser (NGTs), renamed in Italy as ‘technologies for assisted evolution’ (TEAs), reduce the time required
Received: 15 November 2022
to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This
Revised: 28 December 2022 review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific
Accepted: 29 December 2022 focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop,
Published: 4 January 2023 the key genes and traits on which the scientific community is working, as well as the technological
improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus
is placed on the legal aspects in the European and in Italian contexts.

Copyright: © 2023 by the authors.

Keywords: woody plants; qualitative traits; new genomic techniques; climate change resilience;
Licensee MDPI, Basel, Switzerland.
genome editing; cisgenesis; intragenesis
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Int. J. Mol. Sci. 2023, 24, 977. https://doi.org/10.3390/ijms24020977 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 977 2 of 30

1. Application of New Genomic Techniques in Fruit Tree Species

Fruit trees represent a fundamental group of crops for the agri-food sector of Italy. In
2020, Italian fruit farming produced a turnover of more than 10 billion EUR, accounting for
more than 10% of the entire agricultural sector (Faostat data, accessed on 10 October 2022).
Indeed, Italy is among the top countries for the production of citrus, grapevine, pome
(apple and pear) and stone (peach, cherry, plum, apricot and almond) fruits, kiwi, chestnut,
and strawberry. To protect the economic value of agricultural productions from the ongoing
climate change, genetic improvement of fruit trees is needed to enhance resilience and
sustainability, also in light of the ongoing international sustainable development programs
(i.e. New Green Deal, European Green Deal, Farm to Fork, and Paris Agreement on Climate
Change) [1,2]. Furthermore, among the breeding objectives, special attention is dedicated
to the expansion of the flowering and ripening calendar, pomological diversification, the
extension of fruit shelf-life, and the development of resistance/tolerance to biotic and/or
environmental stresses.
It is worth noting that tree fruit species contribute to satisfying the requirements for
vitamins, carotenoids and polyphenolic compounds, and fibers thanks to the consumption
of fresh and derived products [3]. Fruits and vegetables are the basis for a healthy and
sustainable diet, contributing to improve the quality of life for present and future genera-
tions, even though not all people are in the conditions (availability, affordability, or lack of
knowledge and awareness) to consume a minimum of 900 g of fruit and vegetables each
day, the doses suggested by expert bodies [4]. The fruit market costs are influenced by
several factors including the incidence and severity of plant pest and pathogen attacks, as
well as the susceptibility to abiotic stress such as the limited availability of water or extreme
temperatures [5]. Considering the ongoing climate change, susceptibility to both biotic and
abiotic stresses is predicted to increase, with important drawbacks on productivity and,
therefore, an unavoidable impact on market costs [6]. Furthermore, the energy crisis is now
impacting the agricultural sector, highlighting the need for more sustainable and resilient
agronomic practices [7] with a reduced input demand.
Despite their importance, fruit species are suffering from difficulties related to classical
breeding techniques, in terms of financial commitment, land resources availability, and
long generation times. It is important to recall that, in fruit trees, the juvenile phase can
last up to 10 years, and the length is influenced by environmental and genetic factors [8].
This means that, after crossing, breeders need to wait long time to observe the ameliorated
trait, especially if it concerns the fruit. For these reasons, genetic engineering may represent
a valuable strategy to improve fruit crop species [9]. The most recent discoveries in the
field of molecular biology have led to the development of the ‘new genomic techniques’
(NGTs) or ‘technologies for assisted evolution’ (TEAs), as they have been renamed in Italy.
Compared with conventional breeding methods, NGTs allow the time required to obtain
new varieties to be reduced, as well as to precisely target specific DNA sequences without
altering any other trait [10].
Genome editing uses engineered nucleases, including meganucleases (MNs), zinc fin-
ger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR
(clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated)9.
After the first application of ZFNs in Arabidopsis [11], this technology was also used to
target endogenous genes in apples [12] and poplars [13], albeit with a limited diffusion
because of its off-target effects, difficult procedures, and unsustainable costs. On the other
hand, TALENs are more precise and efficient than ZFNs, but the large carriers and high cost
limit their applications in plants [14]. Compared to ZFNs and TALENs, the CRISPR/Cas9
system is undoubtedly simpler, cheaper, and more efficient; this is the reason why its
use was generally most diffused and otherwise adaptable also for woody plants and fruit
trees. Notably, the CRISPR/Cas9 system can be designed for any genomic targets and
multiplexed by adding multiple gRNAs. In fact, CRISPR/Cas9 represents an innovative
technique to modify specific genome sites, as well as to introduce new genetic variants by
DNA double-strand breaks (DSBs) and nonhomologous end joining (NHEJ), or homology-
Int. J. Mol. Sci. 2023, 24, 977 3 of 30

directed recombination (HDR) [15]. Due to the efficiency and simplicity of this system, it
is increasingly being used for modifying the traits of many plants, including important
crops, and for developing improved germplasm resources. The CRISPR/Cas9 machinery
can be delivered to the plant cell in two different ways: as stable integration of the DNA
cassette, under selective pressure for DNA integration, as transient transformation by
direct mRNA delivery avoiding selective pressure [16], as guide RNA plus Cas proteins
as ribonucleoprotein (RNP) complexes [17], or through virus-based vector [18]. Several
issues are associated with stable transformation using the CRISPR/Cas cassette such as the
random integration into the host genome, with the risk of impacting coding or important
regulative regions, and the necessity to perform backcrosses in order to segregate the
exogenous DNA cassette while preserving the editing event(s) [10]. For this reason, the
application of DNA-free approaches exploiting protoplasts is now increasingly reported
in the literature [19]. Overall, the use of NGTs represents a valid instrument to speed up
the long times required by classical breeding, intervening in a surgically precise manner
in target sequences, while maintaining the rest of the genetic background unaltered. In
fact, this approach would allow the value of the main fruit species cultivated in Italy to be
increased, improving several aspects that, to date, penalize the final products disregarding
the requests of national and international sustainability programs, as well as those from
consumers and producers.
At the international level, the application of these technologies is impressive, the
main players being the United States and China (https://publications.jrc.ec.europa.eu/
repository/handle/JRC123830, Accessed on 9 November 2022). Looking at the most recent
worldwide studies in the fruit crops sector (Table 1), in general, most of the studies focused
on defense against biotic stresses. In some cases, the effort was mainly focused on a specific
pathogen, such as the bacterium Xanthomonas axonopodis that causes the Citrus bacterial
canker (CBC) disease in the genus Citrus, or the Plum pox virus affecting Prunus species,
while, in other cases, such as in grapevine, a wider range of pathogens was considered. In
addition, in some species, current research is directed toward the improvement of traits
linked to yields, such as in kiwifruit and strawberries.

Table 1. State of progress in the application of genetic engineering techniques outside Italy. Many
studies report the applications of new genomics techniques and Agrobacterium-mediated transforma-
tion using several types of starting explants that are mainly focused on the introduction of resistance
to biotic and abiotic stresses in Citrus, Vitis, Castanea, and Malus. In other species such as kiwifruit
and strawberries, most recent studies are mainly directed toward the improvement of traits linked to
yields and early flowering.

Genus Species Trait Modified Gene(s) Approach Ref.

Loss of function of CRISPR/Cas9;
C. sinensis CsNPR3 that Protoplast transfection with [20]
represses NPR1 Lipofectamine
Mutation of an EBE CRISPR/Cas9;
C. paradisi in the promoter of A. tumefaciens infection of grapefruit [21]
LOB1 epicotyls
Resistance to Citrus
Citrus CRISPR/Cas9;
canker disease Mutation of an EBE
A. tumefaciens infection of epicotyls
C. sinensis in the promoter of [21]
and protoplast transfection.
LOB1
Improved binary vector
C. paradisi
Loss of function of CRISPR/Cas9;
C. sinensis × Poncirus [22]
DMR6 A. tumefaciens infection of epicotyls
trifoliata
Int. J. Mol. Sci. 2023, 24, 977 4 of 30

Table 1. Cont.

Genus Species Trait Modified Gene(s) Approach Ref.

CRISPR/Cas9;
Resistance to Botrytis Loss of function of
V. vinifera A. tumefaciens infection of [23]
cinerea VvWRKY52
embryogenic callus
Tolerance to downy CRISPR/Cas9;
Loss of function of
V. vinifera mildew caused by A. tumefaciens infection of [24]
PR4
Plasmopara viticola embryogenic callus
Resistance to
Loss of function of CRISPR/Cas9;
powdery mildew
V. vinifera VvMLO3 and A. tumefaciens infection of [25]
caused by Erysiphe
Vitis VvMLO4 embryogenic callus
necator
Tolerance to Pierce’s CRISPR/Cas9;
V. riparia × V. Disruption of the
disease and Red A. tumefaciens infection of [26]
rupestris miRNA gene TAS4a/b
Blotch Disease embryogenic callus
CRISPR/Cas9;
V. vinifera × V. Control of grapevine Loss of function of
A. tumefaciens infection of [27]
berlandieri shoot branching CCD7 and CCD8
embryogenic callus
CRISPR/Cas9;
Response to cold Loss of function of
V. amurensis A. tumefaciens infection of [28]
stress PAT1
embryogenic callus
Overexpression of Agrobacterium-mediated
C. sativa Tolerance to Chestnut [29]
the CsCh3 transformation of somatic embryos
Castanea blight
Overexpression of Agrobacterium-mediated
C. dentata [30]
the wheat OxO transformation of somatic embryos
Tolerance to
Loss of function of CRISPR/Cas9;
Malus × domestica Botryosphaeria [31]
CNGC A. tumefaciens infection of leaf explants
dothidea
Loss of function of CRISPR/Cas9;
Malus × domestica Early flowering [32]
Malus TFL1 A. tumefaciens infection of leaf explants
Proof of concept of Base editing of ALS CRISPR/Cas9;
Malus × domestica [33]
base editing and PDS A. tumefaciens infection of leaf explants
application CRISPR/Cas9;
M. sieversii Knockout of PDS [34]
A. tumefaciens infection of leaf explants
Loss of function of CRISPR/Cas9;
P. communis Early flowering [32]
TFL1 A. tumefaciens infection of leaf explants
Pyrus Proof of concept of
Base editing of ALS CRISPR/Cas9;
P. communis base editing [33]
and PDS A. tumefaciens infection of leaf explants
application
Investigation of the
auxin synthesis sites Loss of function of CRISPR/Cas9;
Fragaria F. vesca [35]
during fruit and root FveYUC10 A. tumefaciens infection of leaf strips
development
Compactness of
CRISPR/Cas9;
growth habit, early Loss of function of
A. chinensis A. tumefaciens-mediated [36]
flowering, and fruit AcCEN4 and AcCEN
transformation of leaf explants
development
Self-pollination and Loss of function of CRISPR/Cas9;
Actinidia
A. chinensis fast-flowering SyGl and CEN-like A. tumefaciens-mediated [37]
offspring. genes transformation of leaf explants
Evergrowing but not CRISPR/Cas9;
Genome editing of
A. chinensis early flowering A. tumefaciens-mediated [38]
AcBFT2
phenotype transformation of leaf explants

In addition to genome editing, cisgenesis and intragenesis techniques also belonging

to the NGTs allow a speed up in comparison to conventional breeding approaches. The
results of cisgenic approaches are plants modified with the addition of gene(s) isolated
exclusively from sexually compatible species, including introns and regulative regions (i.e.
promoters and terminators) in their native sense orientation [39,40]. In the case of intragenic
plants, the inserted sequence is the result of a new arrangement of genetic elements isolated
from the same species or different sexually compatible ones (e.g., a gene promoter from
one species and a coding sequence from another one, both sexually compatible) [41]. In
both cases (cisgenic and intragenic plants), the presence of selectable markers genes is
forbidden. The advantage of these approaches is that the plants obtained resemble those
Int. J. Mol. Sci. 2023, 24, 977 5 of 30

derived from conventional breeding approaches, without the inconvenient integration of

undesired traits [42]. The latter phenomenon, known as linkage drag, is usually solved
by the double pseudo-testcross strategy which can require decades in the case of woody
plants with long juvenile phases. Cisgenesis and intragenesis allow the linkage drag issues
to be bypassed by transferring only the desired gene(s) in a single step, preserving all the
quality traits selected in elite cultivars appreciated by the final consumers.
Notwithstanding the potentiality of NGTs, they suffer from drawbacks that strongly
limit their use. First of all, the knowledge of genes responsible for specific traits of interest
is not always available. While the availability of the genome represents a starting point, it
must be integrated with more accurate information such as gene annotation and knowl-
edge of the protein interactions and cellular functions [43]. In the framework of fruit trees
species, several genomes of important species have already been made available, including
grapevine [44,45], citrus [46,47], peach [48,49], apricot [50,51], cherry [52,53], apple [54,55],
pear [56,57], kiwifruit [58], strawberry [59,60], and chestnut [61,62]. Therefore, the knowl-
edge on genes controlling traits of interest must be elucidated using the most modern
technologies and combining data from multidisciplinary approaches to identify the best
candidates (Figure 1).

Figure 1. Overview of the multidisciplinary approaches to identify candidate gene(s) exploitable for
genome editing or cisgenesis approaches. Starting from the natural occurring phenotypes, including
Int. J. Mol. Sci. 2023, 24, 977 6 of 30

both cultivated and wild genotypes, it is possible to identify genetic sequences involved in the
determination of specific traits. More in detail, combining genomic analysis with gene expression
and metabolomic data, it is possible to identify pathways and key genes controlling the trait(s) of
interest. Once identified, if concerning negative regulator(s), such as stress-related susceptibility
genes, it is possible to apply genome editing to obtain an improved genotype where the expression
of such gene(s) will be knocked-out. On the contrary, the identification of positive regulator(s) can
be exploited in cisgenesis approaches to obtain new genotypes expressing the resistance/tolerance-
related gene(s).

As a second step of complexity, the ability to transform and regenerate elite fruit crop
genotypes represents the most dramatic bottleneck. In fact, the selection of the best starting
tissue for the transformation event, similarly to the selection of the most suitable tissue for
protoplasts isolation, is not easily achievable in all fruit crops (Figure 2).

Figure 2. Representation of the main workflows exploited for the application of the new genomic tech-
niques (NGTs) in some fruit crops selected as an example using somatic embryos. From left to right:
Int. J. Mol. Sci. 2023, 24, 977 7 of 30

in grapevine, the most exploited tissues to produce embryogenic calli belong to flowers (e.g., anthers
and ovaries); in Citrus, hypocotyls are exploited to obtain embryogenic calli; in apple, there is no
need for embryogenic calli since, if cultured in specific conditions, the leaf cells are able to regenerate
the entire plant via organogenesis. Once the morphogenic competent tissue is obtained, there
are two main approaches to apply NGTs: the classical method through Agrobacterium-mediated
transformation or the isolation and transfection of protoplasts (DNA-free approach). Once the
transformation/transfection event is completed, embryos are regenerated, and the genetic and
phenotypic analyses are then performed.

For example, this issue is quite relevant in grapevine, where the most diffuse tissue
type for transformation events and the only one available for totipotent protoplasts isolation
and transfection is the embryogenic callus [63,64]. In grapevine, it can be obtained from
floral tissues (e.g., anthers and ovaries) but the competency in producing embryogenic
callus is genotype-specific, and many varieties are still recalcitrant [65]. Comparable, or
even more challenging, is the case of peach where reports of genetically engineered plants
are limited to few cases [66]. Indeed, for species such as almond and apricot, there are
no studies in the scientific literature reporting the application of NGTs (Table 1). Taking
these difficulties into consideration, despite the efforts made in terms of gene selection and
technology implementation, many challenges are still to be addressed.
The last limiting aspect that grips the European scientific community concerns the
legislative framework. Indeed, while many countries in the world issued or are adopt-
ing a regulatory framework that excludes from genetically modified organisms (GMOs)
legislation those products with small mutations (small insertions or deletions, or base
substitutions) induced by site-directed nucleases (e.g., CRISPR/Cas9 system), in Europe,
genome-edited, cisgenic, and intragenic plants must undergo the same environmental and
food and feed risk assessment as is required for the first generation of GMO plants [67].
While the internal debate is still ongoing and the European Parliament will likely discuss
the possibility to deregulate at least the gene-edited plants free of exogenous DNA (see
Section 6), the scientific community is working to limit as much as possible the exogenous
DNA transferred into the plant genome, by also removing unwanted exogenous sequences
from the final products. The latter purposes can be achieved by using constructs including
site-specific recombination systems such as FLP/Frt [68,69] or Cre-loxP [70,71], which allow
transgene-free genome-edited plants to be established. The elimination of the Cas9 gene
from genome-edited plants would also prevent the occurrence of mutations at untargeted
loci (off-target effect) due to the unspecific recognition of off-target sites by the sgRNA–Cas9
complex [72].
This review aims to illustrate the role of the Italian public research in the application
of NGTs thanks to the support of Italian funding, with specific focus on some of the main
fruit crops, including Citrus, Vitis (as grapevine and table grape), Pomaceae (such as Malus
and Pyrus), Castanea, Fragaria, Prunus (peach, apricot and cherry), and Actinidia. For each
crop, the principal traits on which the scientific community is working and the main active
projects are presented. In particular, most of the results regarding citrus, grape, strawberry,
apple, pear, and kiwifruit have been produced in the framework of BIOTECH, the first
project funded by the Italian Ministry of Agriculture that, from 2018 to 2023, invested
around 6 million EUR to apply NGTs on several crops, including fruit trees (subprojects
named CITRUS for Citrus spp., VITECH for Vitis spp. (rootstocks, wine, and table grapes),
and BioSOSFru for stone fruit, strawberry, apple, pear, and kiwifruit. Lastly, a focus is
placed on the legislative aspects within the European context.

2. Improving Citrus Fruit Quality by NGT Approaches—The CITRUS Contribution

Citrus fruits are cultivated for different purposes, mainly for fruit production (used as
fresh product or processed), and for ornamental use. The production of high-quality citrus
fruits (in terms of size, sugar and acidity balance, juice yield, seedlessness, and presence
of bioactive compounds) represents the main objective of breeding, along with a higher
Int. J. Mol. Sci. 2023, 24, 977 8 of 30

tolerance or resistance toward the main biotic and abiotic stresses. The traditional breeding
strategies that have been developed at CREA and at the University of Catania for lemon,
sweet orange, and grapefruit are focused on clonal selection (bud or nucellar selection). The
selection considers different traits, including pomological and qualitative characteristics
of the fruit, plant productivity (yield and yield precocity), and resistance to biotic and
abiotic stresses, and it has led to the release of different improved citrus cultivars, such
as the ‘Femminello Zagara Bianca’ lemon (a vigorous and productive clone with white
flowers and green young shoots), the ‘Tarocco tdv’ (a clone with a deeper pigmentation
than the old Tarocco lines), and the ‘Tarocco meli’ and ‘Tarocco ippolito’ (late maturing
clones with high firmness) sweet oranges [73]. New mandarin varieties and rootstocks
were obtained mainly by crossing. In particular, the diploid crossing resulted in seedy
new accessions, such as the ‘Sun Red’, a deeply pigmented mandarin-like fruit [74], and
the ‘F6 P12′ (C. latipes × Poncirus trifoliata) rootstock [75]. Variation of ploidy was also
applied allowing the obtainment of triploid seedless varieties, such as the pigmented
mandarin-like ‘Mandared’ and ‘Alkantara’, the early ripening grapefruit-like ‘Bellini’ with
a reduced content of furanocoumarins (responsible for the ‘grapefruit juice effect’), and
the lemon-like ‘Lemox’ that bear fruits with high quality in summer [76]. These varieties
represent the result of a long and hard work that Italian citrus breeders did for more than
40 years. In Citrus, mutation breeding was used more than in other tree crops, and, in
the last few decades, new grapefruit, lemon, mandarin, and orange cultivars have been
obtained thanks to the effort of both public and private breeding programs in different
countries [77]. Huanglongbing (HLB), CBC, and Citrus black spot are the most devasting
quarantine diseases that are threatening the worldwide citriculture, putting at risk the fruits
production and their commercialization. These diseases are licking the European basin,
giving little advantage to finding the right strategies to deal with them. Unfortunately,
for Citrus, as well as for many other fruit tree species, conventional breeding is a long-
term and expensive process, such that molecular breeding approaches and, more recently,
NGTs are expected to play an important role in speeding up breeding programs. To date,
very few studies have been reported about the successful application of the CRISPR/Cas9
technology; generally, they are mainly based on the use of the Golden Gate strategy. This
approach was used to introduce the resistance to Xanthomonas citri to susceptible sweet
orange and grapefruit species [78,79]. In Carrizo citrange, a genome-editing approach
was developed to produce a double thorn phenotype [80]. Very recently, Alquézar and
colleagues [81] induced through base editing resistance against the herbicide selection
agent imazapyr, bypassing the limitations of the presence of the transgene for the selection
and obtaining transgene free plants.
In the framework of BIOTECH project, CITRUS was addressed to take advantage
of the NGTs to improve two important traits for consumer’s needs, so as to combine
anthocyanins and lycopene in a unique fruit, as well as reduce seed content (in size or
in number) in mandarin and mandarin-like varieties. Transformation and regeneration
in Citrus are variety-specific; so far, most of the results have been achieved on Carrizo
citrange, ‘Duncan’ grapefruit, and ‘Valencia’, ‘Pineapple’, and ‘Jincheng’ sweet oranges,
whereas mandarins and clementine and, to a lesser extent, sour orange and lemon species
are considered recalcitrant or less prone to be transformed (reviewed in [82]).

2.1. Two Approaches to Produce Lycopene-Rich Citrus Varieties with Anthocyanins

To date, no reference has reported citrus varieties accumulating both anthocyanins and
lycopene in the pulp; these traits are difficult to be combined due to the long juvenility of
pummelo, grapefruit, and sweet orange, because of the apomictic nature of lycopene-rich
grapefruit and sweet orange varieties, as well as that of anthocyanin-rich sweet orange.
Moreover, very little information on the genetic variants responsible for the accumulation
of lycopene is available, as well as the presence of locus or markers associated with the trait.
The use of cisgenesis and genome editing could contribute to overcoming most of these lim-
its. Ruby, a Myb-like transcription factor, was described to be responsible for the control of
Int. J. Mol. Sci. 2023, 24, 977 9 of 30

anthocyanin production in blood oranges, specifically for the directed association with the
upstream long terminal repeat (LTR) acting as promoter, absent in common nonpigmented
oranges [83,84]. The role of Ruby has furthermore been demonstrated in transgenic experi-
ments, increasing the anthocyanin accumulation in nonpigmented Citrus species [85,86].
For our aim, we used Ruby as cisgene in lycopene-rich accessions (i.e., ‘Vaniglia Sanguigno’,
an acidless red-orange grapefruit, and ‘Star Ruby’, a pink grapefruit). We developed two
constructs [87], one consisting of a cisgenic vector for the Ruby gene, and a second harboring
the Cas9 and sgRNA designed to induce the knockout in two Terminal Flowering Locus 1
(TFL1) genes, Cs8g15080 and Cs6g15000, with the aim to induce the precocious flowering
in Carrizo and in ‘Tarocco tringale’ (an anthocyanin-rich sweet orange variety), as reported
for CENTRORDIALIS in Actinidia [88]. Both vectors contained a FLP/FRT system to excise
the cassette after a heat-shock treatment and obtain marker-free cisgenic plants, similarly
to what has been described in apple [72]. The regenerated plantlets were mini-grafted onto
Carrizo rootstock. For the Ruby construct, the PCR screening identified four ‘Star Ruby’
positive lines obtained from epicotyl transformation, and 32 ‘Vaniglia Sanguigno’ positive
lines (six obtained by the transformation of cotyledons and 26 by the transformation of
epicotyls); the resulting efficiency was 1.7% for ‘Star Ruby’, while that for ‘Vaniglia San-
guigno’ was 6.45% and 6% from epicotyls and seed explants, respectively [87]. For the
TFL1 construct, the mutations induced by Cas9 were investigated by amplicon sequencing
through high-throughput sequencing (HTS); as results, we obtained three Carrizo and
three ‘Tarocco tringale’ edited for both Cs8g15080 and Cs6g15000, and six ‘Tarocco tringale’
edited only for Cs8g15080. The efficiency was 1.5% for Carrizo and a total of 6.77% for
‘Tarocco tringale’, showing for the first time the possibility of using one single guide to edit
two targets in anthocyainin-rich sweet orange varieties. The promising results obtained by
both constructs let us to proceed with the fusion of the two constructs. The fused plasmid
would be able to introduce Ruby and validate the phenotype by early flowering [87].
Few studies used the genome-editing approach to improve fruit quality; for example,
in tomato, the biosynthesis of lycopene was promoted by inhibiting the conversion from
lycopene to β- and α-carotene [89,90]. The genome editing of β-cyclase was also used to
develop the β-carotene-enriched banana variety [91]. In our case, in Citrus, the knockout of
β-LCY2, through a dual-single guide approach, was aimed at producing loss-of-function
mutants to induce lycopene accumulation in anthocyanin-rich sweet oranges [92]. For the
first time, genome editing has been adopted to improve citrus fruit qualitative traits. Several
edited anthocyanin-pigmented sweet oranges have been produced, including nonchimeric
genotypes, as supported by HTS analysis. Among sweet oranges, a few studies described
that anthocyanin-pigmented varieties, such as ‘Tarocco’ and ‘Maltese half-blood’, starting
either from mature or from young tissues, can be regenerated and transformed [93,94]. A
strong effort has also been made to optimize the protocols for those varieties belonging
to sweet orange and grapefruit for which regeneration was needed. In the meantime,
transformation using seed-derived tissues in sweet orange has been reported, mainly on
a series of anthocyanin-rich sweet oranges never tested before [87]. Moreover, the use of
mini-grafting in a nonsterile environment represents a novelty, allowing a faster recovery
of transformed shoots.

2.2. The Production of Seedlessness Mandarin-like Fruits

Seedlessness in Citrus is a complex trait under the control of several biological pro-
cesses. It can be observed in parthenocarpic and stenospermocarpic varieties, such as
seedless mandarin Mukaku Kishu-type [95], as well as in self-incompatible (SI) ones, such
as clementine, grown in isolated blocks, far from cross-pollinators [96]. In other cases,
such as ‘Satsuma’ mandarin and ‘Washington Navel’ cultivars, male or female sterility
contributes to producing fruits with no seeds or a significant reduction in their number [97].
Lastly, triploids produce seedless fruits, due to their sterility [98]. Therefore, the identi-
fication of genes that can be managed to produce seedless varieties is very complicated.
In Arabidopsis it is reported that iku (IKU1 and IKU2) and miniseed3 (MINI3) mutations
Int. J. Mol. Sci. 2023, 24, 977 10 of 30

specifically affected the reduction in seed size through regulating endosperm proliferation
and cellularization [99–101]; the loss-of-function mutations in IKU pathway genes cause a
decrease in seed size. Therefore, we used a dual single-guide approach on the homologous
of IKU1 in Citrus [102], hypothesizing that it could regulate seed size affecting the develop-
ment of the zygotic tissues also in this species, as previously described in Arabidopsis [103].
So far, Carrizo citrange plantlets have been recovered after editing, and then propagated;
different types of mutations (insertions, deletions, and inversions) have been obtained for
both sgRNAs, and the deduced sequence of the edited IKU gene showed in most cases the
introduction of a stop codon responsible for premature termination of the protein. Further
analyses on edited plantlets are ongoing [102].
In 2020, investigating self-incompatibility (SI) systems in many different Citrus species,
Liang et al. [104], discovered that a predominant single-nucleotide mutation called Sm -
RNase, present in self-compatible (SC) accessions, was responsible for the loss of SI in the
SC accessions. In order to recover the SI mechanism in sweet orange, we developed a
genome-editing vector containing a sgRNA that targets the Sm -RNase polymorphism [102].
Transformation experiments using ‘Doppio Sanguigno’ sweet orange seedlings are ongoing.

3. VITECH: Improving Grapevine Genetics Features to Develop a Resilient and

Sustainable Vineyard
Among woody crops, grapevine is one of the most important species in the EU,
accounting for about 50% of the grape cultivated area in the world, with more than
3.4 million hectares in 2020 (Faostat, accessed on 5 October 2022). Furthermore, the value
of this crop amounts to about 30 billion EUR in Europe, of which more than 3 billion
belongs to Italy (Faostat, accessed on 5 October 2022). Conventional breeding approaches
have been employed to face urgent issues related to the modern viticulture [105]. With
the aim of reducing the massive use of chemical compounds in vineyards against fungal
pathogens, in 1998 scientists from the University of Udine and the Institute of Applied
Genomics (IGA) of Udine started the genetic improvement program which led in 2015 and
2020 to the registration in the Italian National Catalog of new fungi-resistant wine varieties
(https://vivairauscedo.com/contributi/download/stato-dell-arte-2022-it.pdf, accessed
on 20 December 2022). Evaluations of their agronomic traits, enological features, and field
resistance to downy and powdery mildews are reported in literature [106–108]. In 2020, the
Foundation Edmund Mach together with Civit (Consorzio Innovazione Vite) also registered
four wine varieties resistant to fungal pathogens (https://www.vinievitiresistenti.it/2020
/10/09/nuovi-nomi-per-le-quattro-varieta-2020-di-fem-e-civit/, accessed on 20 December
2022). Likewise, also at CREA (Arezzo, Conegliano, Turi, and Velletri), breeding activities
are underway to obtain wine varieties resistant to fungi (https://www.crea.gov.it/web/
viticoltura-e-enologia/breeding-vite-da-vino-arezzo-conegliano-turi-e-velletri-, accessed
on 20 December 2022). Regarding table grapes, it is only since the last few decades that
Italy has resumed the activity of genetic improvement by breeding aimed at obtaining table
varieties featuring resistance to disease and abiotic stresses, with naturally large berry size,
preferably seedless, good firmness of the pulp, and good resistance to handling, transport,
and storage (https://vivairauscedo.com/contributi/download/stato-dell-arte-2022-it.pdf,
accessed on 20 December 2022). Although most cultivars in Italy are with seeds (e.g.,
Italia, Victoria, and Red Globe), interest in seedless varieties that better respond to mar-
ket needs has recently grown. Lastly, the modern viticulture requires a renewed inter-
face between grape and soil, able to face abiotic stresses exacerbated by the ongoing
climate change. Recently, scientists from University of Milano developed a breeding pro-
gram focused on rootstocks in order to select new genotypes resistant to abiotic stresses,
such as drought and calcareous soils, through the development and validation of ge-
netic and physiological markers. Four new genotypes have been obtained (‘M’ series)
and registered to the Italian National Catalog of Grape Varieties. Their features are well
described in the literature [109–111]. Moreover, the University of Bologna is carrying
out a breeding program for the development of rootstocks to reduce grapevine vigor
Int. J. Mol. Sci. 2023, 24, 977 11 of 30

(https://agrimpresaonline.it/due-nuovi-portinnesti-per-ridurre-la-vigoria-della-vite/, ac-
cessed on 20 December 2022).
In this context, VITECH is dedicated to improve the resilience and sustainability of
grapevine (Vitis spp.) production by means of NGTs, pursuing the same main objectives of
conventional breeding: (i) enhancing resilience to biotic stresses exploiting both cisgenesis
and genome-editing strategies, (ii) using the genome-editing approach to develop elite
seedless table grape cultivars (e.g., cv. Italia), and (iii) developing improved cultivars and
rootstocks genotypes to cope with climate change, specifically with drought.

3.1. Grapevine Tolerance to Powdery and Downy Mildews

To produce pathogen-tolerant grape plants, which require lower pesticide treatments,
many efforts have been focused on the development of several genome-editing approaches
applied on susceptibility genes [112] and on other important genes involved in the reg-
ulation of the responses to biotrophic pathogens [113]. The loss-of-function mutation,
mediated by CRISPR/Cas9, of susceptibility genes to powdery and downy mildew was the
most used strategy [114,115]. In parallel, a cisgenic approach was developed to introduce
resistance to Plasmopara viticola (RPV3-1 locus) [116] into elite wine grape cultivars (e.g.,
Glera, Sangiovese, Chardonnay, and Pinot noir). Due to the limitations of working with
woody plants with highly heterozygous genomes and, therefore, the impossibility to un-
dergo backcross strategies [10], a DNA-free approach through transfection of embryogenic
callus-derived protoplasts by means of a CRISPR/Cas9 RNP system is highly desired. Very
recently, the possibility to obtain transgene-free edited grapevine plants through the regen-
eration of edited protoplasts has been demonstrated in ‘Thompos seedless’, encouraging the
use of genome editing for the genetic improvement of grapevine [117]. Grapevine-specific
miRNAs 3632 and 3623 (not yet characterized) and miR482 (already known as involved
in disease response in tomato and cotton [118,119]) regulate NBS–LRR disease-resistance
genes. SgRNAs targeting these miRNAs have been used, in order to improve grapevine
responses to pathogens. Regenerated embryos of ‘Chardonnay’ and ‘Brachetto’ cultivars
are now in culture and will be analyzed for editing events.

3.2. Seedlessness Table Grape

To reach the second objective, many efforts have been focused on the development of
embryogenic calli from important table grape varieties (e.g., cultivars Italia, Victoria, and
Red Globe) allowing the setup of an improved protocol [120]. This objective is linked to
the fact that seedlessness is one of the most desired traits by consumers of table grapes.
Several studies already defined the central role of an MADS-type transcription factor,
VviAGL11, in the process of stenospermocarpy [121], which consists of an embryo abortion
with consequent seed development regression after full fertilization. However, no working
mechanisms have been reported yet. Additionally, the existence of specific promoter-coding
sequence (CDS) combinations, that directly affect the VviAGL11 expression level, has al-
ready been demonstrated. Transcriptomic analyses on ovule and developing seeds in seedy
and seedless varieties have highlighted the role of VviAGL11 in hormone signaling and
phenylpropanoid metabolism, identifying a methyl jasmonate esterase, an indole-3-acetate
beta-glucosyltransferase, and an isoflavone reductase, as direct targets of VviAGL11. The
dominant negative effect of the mutated VviAGL11 CDS [122] on target gene activation was
molecularly validated, and a new regulatory mechanism correlating VviAGL11 haplotype
assortment and seedlessness class in grapevine was proposed [123]. The next step will be
the functional characterization of VviAGL11 through the generation of edited microvine
plants, a model system which makes it possible to obtain fruits in a few months accelerating
the long generation cycles of common cultivars [124].

3.3. Resistance to Drought

One of the projects dedicated to improve resilience to the ongoing climate change (i.e.,
drought), a rapid and innovative method consists of the application of spray-induced gene
Int. J. Mol. Sci. 2023, 24, 977 12 of 30

silencing (SIGS), to functionally validate genes in rootstocks [10]. Thanks to this approach,
a gene belonging to the glutathione S-transferase family, namely, VvGST40, was charac-
terized as a potential candidate for the application of gene editing to counteract drought
effects. Once downregulated using the SIGS approach, the resulting plants displayed an
increased abscisic acid (ABA) accumulation and an improved antioxidant arsenal limiting
the negative effects of drought stress [125]. In parallel, the study of the genetic basis of
stomata formation in the leaf, which is a key trait for plant response to drought and heat
stress, has been initiated. The two grapevine isoforms of epidermal patterning factor-like
9, known to be a key regulator of stomata formation in model plants and cereals, were
chosen as target for knockout (KO) and overexpression experiments. Epfl9-1 KO ‘Sugraone’
mutants showed reduced stomatal density [126], and further research is ongoing to depict
the role and peculiarities of VvEPFL9-2.

4. Chestnut: Genome Editing to Introduce the Tolerance to Biotic Stress

The European chestnut (Castanea sativa Mill.) is a woody species of high economic
interest, widely cultivated in Italy, especially in Campania, Calabria, Tuscany, Latium, and
Piedmont Regions. It is considered a multipurpose species for the nutritional value of
its nuts, the timber quality, the role in modeling the landscape of mountain areas, and
the ecological importance of its forests and orchards. At the moment, in most areas of
Italy, the chestnut industry is still built on ancient orchards, often over 100 years old,
since the renewal of plantings is very poor, in spite of the high nut demand from the
confectionery industry and the fresh market. This is due to several causes, one being the
presence of pathogens that are particularly aggressive against young plants and require
intensive care in the first years after planting. Chestnut breeding has been focused on
obtaining cultivars with higher yield, nut and timber quality (large nut size, easy peeling
and good wood quality, fast growth), and tolerance to the major pests and pathogens of the
species. Among chestnut species, C. mollissima and C. crenata are tolerant to Phytophthora
spp. [127] and have been used to obtain hybrids bearing this trait [128]. Breeding programs
carried out at INRAE, crossing C. sativa and C. crenata, produced a set of hybrid selections
with a higher tolerance to Phytophthora that are currently used as rootstocks and as
direct producers, despite the lower quality of nuts [129,130]. Concerning the canker blight
disease, caused by C. parasitica, no breeding programs for obtaining improved varieties
are currently underway in Europe. On the contrary, a large breeding program has been
carried out in the USA to restore the species C. dentata by introgressing resistance genes
from C. mollissima. Breeding programs aimed at selecting resistant genotypes to the Asian
gall wasp Dryocosmus kuriphilus and introducing resistance in cultivars were active in Japan
and Italy [131], before biological control was established. C. sativa is highly susceptible
to two severe diseases that undermine its survival: ink disease induced by the oomycete
Phytophthora spp., and chestnut blight caused by the fungus Cryphonectria parasitica [132].
Moreover, over the past 20 years, chestnut has been affected by Dryocosmus kuriphilus
Yasumatsu, the Asian gall wasp, an insect that produces galls on the shoots of the host
plant, and by the fungus Gnomoniopsis castaneae G. Tamietti, the nut rot agent representing
a new plant health emergency [133,134]. For these reasons, currently, the chestnut breeding
programs are mainly focused on increasing tolerance to pathogens using genotypes with
high yield and nut quality. In [132], two candidate genes Powdery mildew resistance 4 (pmr4)
and Downy mildew resistance 6 (dmr6) were found involved in the chestnut susceptibility
response to P. cinnamomi and C. parasitica. The ongoing research is currently focused on the
knockout of pmr4 and dmr6 genes using the CRISPR/Cas9 technology in order to obtain
tolerant plants, an important goal for the success of the future chestnut industry in a highly
reduced time.

4.1. The Genome Editing of PDS Gene as Proof of Concept

Very recently, the use of CRISPR/Cas9 technology in the Castanea genus was used to
target the phytoene desaturase (pds) marker gene, involved in chlorophyll biosynthesis [135];
Int. J. Mol. Sci. 2023, 24, 977 13 of 30

the silencing of this gene causes the appearance of an albino phenotype [136,137] and is a
visual system commonly used to test the editing technique efficiency for the first time in
a new species [32,138,139]. The pds vector was designed using the GoldenBraid assembly
system, targeting two gRNAs located in two conserved regions of the chestnut pds gene.
Somatic embryos were chosen as starting material due to their high transformation rate
compared to other explants; moreover, the possibility of obtaining regenerated chimeric
plants is reduced using somatic embryos as target material [29]. Nonpigmented ‘albino’
shoots obtained from in vitro cultures were associated with the successful editing of pds
gene with an average gene efficiency of 61% for gRNA1 and 56% for gRNA2. This work
opens the way for the use of the CRISPR/Cas9 system in European chestnut for breeding
improvement and the valorization of ecotypes, acting on genes responsible for tolerance
to biotic/abiotic stresses and quality. The innovative achievements will also provide the
acquisition of new knowledge that will make it possible to extend this technique to other
woody species.

4.2. A Successful Use of Chestnut Protoplasts to Produce Trangene-Free Edited Plants

To avoid the stable integration of recombinant DNA, the CRISPR/Cas9 RNP approach
can be an innovative way to obtain transgene-free plants leading to a better acceptance of
these improved plants by consumers as compared to classic GMOs [140,141]. The proto-
plast represents a valuable material for the transfection process due to its high permeability
to exogenous DNA [142]. The first experiment conducted in chestnut reported the setup of
a protoplast isolation protocol and a transfection event using the pds gene [143]. A high
number of protoplasts/mL was obtained (4,500,000), 91% viable. The number of isolated
protoplasts was close to the number of protoplasts extracted from grape and apple tis-
sues [144,145], and higher compared to Quercus ilex and Populus alba [146]. Protoplasts were
transfected with the RNP complex targeting the pds gene; an editing efficiency ranging from
15 to 20% was obtained, in agreement with data observed in rice (8.4–19%) and Arabidopsis
(16%), and higher than in grapevine and apple (0.1% and 0.5–6.9%, respectively) [140,144].
Compared to the CRISPR/Cas9 traditional approach [135], the gRNA editing efficiency
estimated using the same gRNA with RNPs was lower. Different regeneration media were
tested to optimize the regeneration process from protoplasts, a step that represents a major
bottleneck in the application of transformation techniques in chestnut. Future research
will focus on obtaining protoplasts directly from somatic tissues (leaves), to preserve the
genotype of the mother plant, and on improving the regeneration technique. This CRISPR
RNP-based genome editing protocol represents a powerful breeding tool for chestnut and
can be transferred to other woody species that present a high genome complexity and a
long juvenile phase.

5. BioSOSFru: A Large Project to Improve the Main Fruit Species Qualitative Features
and Response to Biotic Stresses
Italy is among the first countries in the world for cultivated area of pome fruits (apple
and pear), stone fruits (peach, cherry, plum, apricot, and almond), kiwi, and strawberry.
The BioSOSFru aims at developing superior varieties for agronomically relevant traits and
resistance to biotic stresses in stone fruits, pome fruits, strawberry, and kiwifruits through
cisgenesis and genome editing (i.e., CRISPR/Cas9).

5.1. Apple and Pear

Genetic improvement of pome fruit trees species can be conducted through intra-
or interspecific crosses, but this approach is very lengthy and not able to guarantee the
maintenance of all the traits typical of a well-known cultivar. As an example, it took over
70 years to introduce scab resistance into commercial apple cultivars starting from the
wild species Malus floribunda 821 [147]. Pyramidations for cumulating scab and fire blight
resistance [148], or scab and rosy apple aphid resistance [149] have also been described
in apple. Fire blight-resistant apple and pear cultivars have also been obtained through
Int. J. Mol. Sci. 2023, 24, 977 14 of 30

conventional breeding [150,151]; generally, it takes several years, too much if we want to
try to protect these species from pests. As an alternative to crossbreeding, the conventional
fruit tree genetic improvement has made extensive use of clonal selection or artificial
mutagenesis, using chemical or physical mutagenic agents (i.e., γ-rays [152]). Several pome
fruit varieties were obtained by clonal selection or mutagenesis with a good commercial
success. No adverse effects on humans or the environment have been shown due to the use
of varieties obtained by mutagenesis; however, this is a random process and, therefore, it is
not possible to direct it and predict which and how many genes will be modified.
Intense research work is being carried out at the international and national level to
apply TEAs to pome crops. This is not surprising, considering the versatility of these
techniques, which are potentially applicable to even minor but locally important genotypes,
and the speed with which they enable the development of new market-ready varieties.

5.1.1. A Cisgenic Approach to Introduce Scab and Fire Blight Resistances

The first cisgenic apple was developed by transferring the HcrVf2 gene for scab
resistance [153], but various studies on apple were mainly devoted to the optimization of
the cisgenic approach [154]. After the first successful application, other cisgenic apple plants
were also obtained for inducing fire blight resistance with a gene identified in M. × robusta
5 (FB_MR5 gene [155]). To date, there are two cisgenic apple clones under evaluation in
Switzerland and in the Netherlands, obtained by transferring resistance to fire blight (from
M. × robusta 5) and scab (from M. floribunda) into the ‘Gala’ variety. The gene pool available
for pome fruits improvement by cisgenesis is wide since there is a lot of experience in the
use of interspecific crosses in apple and pear breeding. Furthermore, the availability of
Malus/Pyrus hybrids was reported, and this is opening the way for gene transfer between
the two species [156]. To this extent, the successful transfer of a scab resistance gene from
apple to pear was recently demonstrated and the resulting lines were resistant to the pear
scab caused by Venturia pyrina [157].
In Italy, some cisgenic apple lines have been produced by inserting the putative apple
scab gene Rvi12 and putative fire blight resistance gene from M. fusca [158]. Then, some
European pear lines putatively resistant to fire blight have been produced [159] by inserting
the already characterized FB_MR5 apple fire blight resistance gene from M. × robusta 5.
Furthermore, the insertion of another putative fire blight resistance gene from M. fusca
(FB_Mfu10 [160,161]) into pear is also under testing.

5.1.2. The Genome Editing to Produce Apple and Pear Resistant to Fire Blight and
Self-Compatible
Most of the DNA-editing experiments in apple have been conducted to optimize the
protocol by knocking down genes inducing albinism (PDS [162]) or by promoting early
flowering (TFL1 or terminal flowering gene [32]). Analogously, successful DNA editing in
pear was also recently reported by using the same genes reported in apple [32]. A precise
nucleotide substitution without double-stranded breaks (base editing) was also recently
demonstrated in pear; by co-editing the acetolactate synthase (ALS, conferring resistance to
chlorsulfuron) and PDS, chlorsulfuron-resistant and albin lines have been obtained [33].
In Italy, the CRISPR/Cas9 system has already been successfully applied in the apple
varieties ‘Golden Delicious’ and ‘Gala’ to reduce susceptibility to fire blight via editing of
the DIPM4 gene [72]. Remarkably, the CRISPR/Cas9 transgene in the fire blight improved
‘Golden Delicious’ and ‘Gala’ plants was nearly completely removed by a programmed
transgene self-elimination strategy, leaving behind a minimal trace of foreign DNA. This
residual DNA does not contain any protein-coding sequence, which makes CRISPR/Cas9-
based gene editing in apple a promising approach for disease control. The joint silencing of
three susceptibility factors (an HIPM and a DIPM gene for fire blight, and the mlo gene for
powdery mildew) is also under testing on an apple selection that is genetically resistant to
scab and rosy apple aphid in order to obtain a multi-resistant genotype [159].
Int. J. Mol. Sci. 2023, 24, 977 15 of 30

Regarding European pear, research is currently carried out to knock-down the RNase
gene involved in SI, or the fire blight and powdery mildew susceptibility genes described
above for apple [159].
Despite recent progress, the application of CRISPR/Cas9 in fruit tree species needs
to overcome some existing problems. A crucial point is to define a GMO-free system for
Cas9/gRNA delivery to the plant cells. A purified Cas9 protein was used for DNA editing
in isolated apple protoplasts, without involving any foreign DNA [72,144]. Unfortunately,
a large-scale application of this technique is strongly hampered by the difficulties in the
regeneration of apple plants from protoplasts. Modern technologies for drug delivery al-
ready available in animal model systems [163] should be considered in the future, knowing
that the plant cell walls could represent an additional problem to be solved.

5.2. Strawberry
Strawberry is one of the most popular and appreciated fruits by consumers for its
aroma and nutraceutical properties. During the last 10 years, strawberry world production
increased by 40% of global production according to the Food and Agriculture Organization
of the United Nations.
Currently, strawberry genetic improvement programs are focused on the identification
of varieties sustainable for the farmer and appreciated by the consumer, with increased
sensorial and qualitative aspects of the fruit. The huge amount of data acquired during
the last years through strawberry genotyping and mapping has increased the use of DNA
information among breeding programs to identify lineage of parentals used in programmed
crosses or to select seedlings. The advances in the field of genome sequencing and bioin-
formatics, together with the optimization of molecular strategies, such as QTL mapping
and genome-wide prediction, have also led to the discovery of important agronomic loci
and complex traits in the cultivated strawberry, such as those related to flowering, dis-
ease resistance, fruit quality, and yield (reviewed by Whitaker et al. [164]). However, the
genetic improvement of the octoploid strawberry is often limited by the fact that some
traits are controlled by multiple loci, distributed in different subgenomes, and many of
the genes and molecular markers related to important agronomic characteristics are still
unrevealed. These limitations could be overcome through the complementary use of NGTs,
especially for the obtainment of genetically modified plants with sequences belonging to
the same species or to sexually compatible ones (cisgenesis/intragenesis), as happens with
traditional breeding techniques. However, one of the aspects that remains problematic in
many cases in obtaining cisgenic/intragenic lines is represented by the absence of suitable
cisgenic constitutive promoters, as well as of an optimized system for the selection of
transformed lines by only using selectable marker or reporter genes of vegetable origin.
Few examples of plant gene sequences can confer resistance to usually toxic reagents, such
as herbicides represented by the 3-phosphoscichimate 1-carboxyvinyltransferase (EPSPS),
which confers resistance to glyphosate, a total nonselective herbicide, when overexpressed
in plants. Recently, this isolated strawberry sequence was studied in the work of Carvalho
and Folta [165], as a possible nontransgenic selectable marker gene, to be used for obtaining
cisgenic/intragenic strawberry plants. In this same work, several strawberry promoters
were also studied to be used as constitutive promoters to replace the use of the 35S promoter
of the cauliflower mosaic virus, normally used in transgenic systems. In particular, the
FanAPA1-related promoters 1 and 2 and FanUBCE2 isolated from F. × ananassa were found
to be candidate constitutive promoters for the expression of sequences of interest in all
plant tissues. These results served as a starting point for the design and preparation of
intragenic constructs aimed at constitutively expressing the recently characterized straw-
berry FveFT2 gene, in both diploid and octoploid strawberries, as a non-photoperiodic
florigene, capable of conferring a remontant character in seasonal flowering strawberry
varieties when overexpressed [166].
Int. J. Mol. Sci. 2023, 24, 977 16 of 30

5.2.1. An Example of Cisgenic/Intragenic Strawberry System

In the framework of BioSOSFru, one of the objectives is the introduction of the remon-
tant trait in a seasonal flowering strawberry variety with the inclusion of only sequences
of strawberry or of vegetable origin, such as MdMYB10 of apple, which has been used
as reporter gene in apple transformation trials [154]. The FveFT2 gene was placed under
the control of the FaAPA1-R2 promoter, while the MdMYB10 and FvEPSPS genes were
controlled by the FaUBCE2 promoter [167]. The FveFT2 gene was also expressed without
the flanking of any reporter or marker gene, because it is able to induce a re-flowering phe-
notype even in vitro when overexpressed in the plant, giving the possibility of identifying
any transformation events without the use of marker genes [166].

5.2.2. The Genome-Editing Approach Applied to Strawberry

Mutation breeding in strawberry has been carried out through conventional mutagene-
sis techniques in the past, by exploiting both gamma radiation and chemical mutagens [168].
Currently, point-mutagenized strawberry plants can be obtained in a more precise man-
ner, by both stable transformation (mediated by Agrobacterium or direct) and transient
expression to produce CRISPR/Cas9-edited plants [138,169]. However, the production
of a CRISPR/Cas9-mutagenized plant excluding other genome modification by stable
transformation can be time-consuming and not easily practicable in clonally propagated
heterozygote crops, with the risk of losing most of the important traits of the cultivar.
Protoplasts are the most widely used plant material for genome editing by instantaneous
transient expression [142,144]. This method can be used to test the on-target efficiency of
gRNA and the successful construction of the CRISPR vector [170]. Despite its importance
as a horticultural model plant; however, there are currently very few reports of genome
editing in strawberry protoplasts [171], demonstrating the potential of a highly efficient
mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9-
mediated genome editing. However, the regeneration efficiency of protoplast-derived calli
remains strongly genotype-dependent, thus limiting the application of an easy production
of protoplast-derived genome edited strawberry plants of the main commercial cultivars.
A first application of the CRISPR/Cas9 genome-editing system in an octoploid species
to characterize the function of TM6 in strawberry flower development was reported [172].
This result was achieved by using both transient expression of the sgRNA1-2/Cas9 binary
vector by infiltration of a suspension of Agrobacterium tumefaciens into fruits, and by the
stable transformation of F. × ananassa cv. Camarosa. Gaston and colleagues [166] compared
the gene-editing approach with gene-silencing and gene-overexpression strategies to in-
duce perpetual flowering in strawberry, evidencing the most efficient result in transforming
a short-day cultivar into a perpetual flowering one. Recently, Gou and colleagues [171]
applied CRISPR/Cas9 to knockout the Reduced anthocyanins in petioles (RAP) gene in the cul-
tivated strawberry F. × ananassa, obtaining both T0 generation and T1 progeny (segregating
the CRISPR/Cas9 transgene) with a green stem trait, demonstrating that the RAP gene can
be a promising candidate in fruit color breeding of strawberry. However, future application
of gene editing in strawberry remains limited to the availability of high-efficiency regen-
eration protocols from protoplasts; otherwise, the only possibility is the stable expression
mediated by Agrobacterium-mediated transformation of somatic tissues [173].

5.3. Prunus
The genus Prunus encompasses about 250 species including important temperate fruit
and nut crops such as peach, almond, apricot, plums, and cherry with a small nondupli-
cated genome (<300 mb) and extensive synteny and collinearity [174]. Prunus crops are
characterized by a long juvenile phase (3–8 years) that hampers breeding procedures. Basi-
cally, Prunus species have been genetically improved using classical breeding methods such
as parental crossing, introgression, and untargeted mutations with physical and chemical
mutagens. The advent of the genomic era allowed the design of important genetic tools
such as peach and cherry SNP arrays [175,176], enabling the application of marker-assisted
Int. J. Mol. Sci. 2023, 24, 977 17 of 30

breeding (MAB), marker-assisted introgression (MAI), and haplotype analysis [177]. The
main traits addressed are related to fruit quality (such as firmness, solid soluble content,
aroma, skin cracking, and split pit), flowering and maturity date, and biotic (e.g., Sharka
and Powdery Mildew) and abiotic resistance in rootstocks. For auto-incompatible species
such as apricot, almond, plum and cherry self-compatible cultivars were also obtained
in breeding programs. The availability of genetic and genomic tools is crucial for the
application of NGTs as it facilitates causal gene discovery. Since the public release of the
peach genome in 2010 and its subsequent publication [48,49], several genes controlling
traits of interest have been identified, mainly regarding fruit, habitus and disease resistance
characteristics [177,178]. The availability of a genomic sequence also allows genome scan-
ning for the identification of off-target regions to avoid in the choice of a sgRNA. A major
challenge in Prunus is represented by the in vitro steps since these species are recalcitrant
to regeneration and Agrobacterium-mediated transformation.

5.3.1. Resistance to Biotic Stress

Resistance to major pests and diseases is of strategic importance in Prunus breeding.
NGTs offer the possibility for an elite cultivar to maintain its superior genotype while
introducing only the resistance by gene transfer from a donor or by de novo creating
alleles, through genome editing. In the framework of BioSOSFru, a cisgene approach based
on the Ma gene belonging to TNL receptors [179,180], identified in Prunus cerasifera, is
being applied to obtain rootstocks resistant to root-knot nematodes. Ma confers broad-
spectrum dominant resistance to the most aggressive Meloidogyne species, including M.
floridensis, which has overcome the resistance induced by R genes from P. persica and
P. dulcis [179,181,182].
Due to the lack of R genes to Sharka disease in peach [183], CRISPR/Cas9 is being
applied to address recessive resistance to this detrimental virosis, targeting susceptibility
genes [184,185]. Silencing of eIFiso4E in P. domestica and eIFiso4G11 in P. salicina, coding for
members of the eukaryotic initiation complex eIFiso4F involved in Plum pox virus infection,
resulted in resistant plants [186]. Different regions of eIFiso4E are being targeted with a
different single sgRNA with the intent of obtaining an allelic series of mutations [187,188].

5.3.2. Pillar Habitus

In peach, the columnar or Pillar habitus, a homozygous recessive phenotype (br/br)
characterized by narrow branches and a reduced canopy diameter, could potentially boost
productivity by intercepting light in a more efficient way and improving dry matter parti-
tioning [189]. The peach Pillar phenotype is due to the knockout of the PpeTAC1 [178,190],
an ortholog of the Tiller Angle Control 1 (TAC1) that promotes horizontal branch growth in
rice and in different plant species [191–194]. Mutations on this gene lead to compact habitus
and vertical branch growth. PpeTAC1 occurs as a single-copy gene in most plant genomes
and is highly conserved. This makes it the perfect candidate to apply a genome-editing
approach in peach, cherry, and apricot. Actually, a double-sgRNA CRISPR/Cas9 vector
was obtained to silence the gene. As a result of the high level of conservation, the peach
sgRNAs were constructed and are being used in the three species [187,188].

5.3.3. Early Flowering

Stone fruit breeding is a slow process due to the unproductive juvenile phase ranging
from 3 to 8 years. Shortening it and achieving fast introgression of desirable traits is a great
challenge [195]. Two genes have a central position in mediating the onset of flowering: the
TFL, a floral repressor, and the Flowering locus T (FT), a floral inducer [196,197]. FT over-
expression and TFL silencing both lead to accelerated flowering [32]. The overexpression
of a heterologous FT gene in plum led to early flowering [198]. In the peach genome, TFL
was initially annotated as a gene with unknown function (Prupe7.G112600), and, in the
framework of BioSOSFru, a protein sequence phylogenetic analysis clustered it with other
Int. J. Mol. Sci. 2023, 24, 977 18 of 30

TFLs, allowing its function to be inferred. To overcome the production of GMOs, a genome
editing construct has been produced and used to silence TFL [187,188].

5.4. Kiwifruit
Actinidia spp. are deciduous and dioecious woody climbing perennial species charac-
terized by a relatively large genome with the basic chromosome number x = 29 and ploidy
variation [199]. Kiwifruit domestication started in the early 20th century, and the available
cultivars of the two commercially most important species (Actinidia deliciosa and A. chinensis)
are the result of more recent, with respect to other fruit species, breeding programs [200,201].
The intensive cultivation of clonally propagated kiwifruit provided the occasion for the
appearance, first in 1984 and then from 2008 to date, of the pandemic pathogen Pseudomonas
syringae pv. actinidiae (Psa), the Gram-negative bacterium responsible for bacterial canker
of kiwifruit, which has in the last years caused huge economic losses for the major global
kiwifruit producers, such as China, Italy, and New Zealand [202,203]. Psa is by far the
most destructive disease of cultivated kiwifruit in the world, causing leaf spotting, leaf loss,
bud browning and drop, fruit desiccation and shriveling, and plant death, and infected
orchards can be destroyed within 2–3 years [204]. The bacterium can asymptomatically
survive for long periods in the phyllosphere and host invasion occurs through the lesions or
open stomata, which are considered a major entry point [205]. The host response is partly
dictated by Psa, which reduces the defense capacity of the plant. Currently, disease control
is based on application of a copper foliar spray for prevention, and of acibenzolar-S-methyl
as inductor of natural plant resistance [206–208]. The application of genome editing with
CRISPR/Cas9 has emerged as an effective tool for highly efficient target shooting [209].
Biotechnology offers the possibility to apply genome-editing methods and to manipulate
plant response through overexpression or downregulation of endogenous genes, but ef-
ficient regeneration systems are necessary to develop these approaches; in Actinidia spp.
they are already available for some commercial cultivars [210,211].

An Attempt of Application of CRISPR/Cas9 to Produce Kiwifruit Resistant to

Bacterial Canker
Recently, in the framework of the BioSOSFru, some CRISPR/Cas9 vectors were de-
veloped and used for genome editing of some A. chinensis var. chinensis selections [212].
A gene belonging to AP2/ERF transcription factors was selected as a target sequence. The
Cas-Designer tool was performed on the genomic sequence of the transcription factor
belonging to the AP2/ERF family to reach all possible targets. Furthermore, for the selection
of the CRISPR/Cas9 target sequence, the cDNA sequences of AP2/ERF genes of A. chinensis
var. chinensis were alienated to identify the homologous region from them. These regions,
as well as the conserved domain AP2, were excluded from the pool of the target sequence.
In the end, four possible sgRNAs were obtained. To develop the CRISPR/Cas9 vectors with
multiplex sgRNA, two intermediate expression cassettes containing the sgRNA sequence
were previously constructed [213]. Leaf explants prepared as previously reported [210]
were used for infection with A. tumefaciens carrying these vectors, and regenerated shoots
were obtained. Molecular analyses were performed using specific primers designed to am-
plify the insert carrying the sgRNA transcriptional cassette within the CRISPR/cas9 vectors
and the presence of the insertion was observed. Further molecular analyses are in progress
to estimate the type and the severity of the mutations. The above-described preliminary
study represents an encouraging indication on the feasibility of application of CRISPR/Cas9
vectors in A. chinensis in future approaches for inducing Psa resistance/tolerance. Evalua-
tion of edited regenerated plants is in progress, either in vitro or in vivo, to characterize
the response to Psa infection also through gene expression studies. This study represents a
step toward the confirmation that genome editing with CRISPR/Cas9 could be a useful
tool to accelerate the development of new improved varieties in fruit trees.
Int. J. Mol. Sci. 2023, 24, 977 19 of 30

6. Regulatory Issues in Europe, with a Focus on Italy

In 2019, the European Council commissioned a study from the European Commission
(EC) regarding the status of NGTs under Union law. The council aimed to investigate
whether the current regulatory framework that applies to GMOs, which in 2018 was also
extended to NGT products (by the European Court of Justice with the judgment in Case C-
528/16), fits for such technologies that have particularly evolved over the last decade. The
EC study, published in April 2021, took into consideration views on NGTs from Member
States and relevant EU-level stakeholders, scientifically supported by the European Food
Safety Authority and by the Commission’s Joint Research Center. The study has recorded a
very high number of NGT applications underway in Europe and around the world, which
could be very useful for the sustainability of the agro-food sector in the future. However,
while most of the extra-European countries have adopted or are adopting legislations that
differentiate many NGT products from GMOs, making their authorization for the market
more streamlined and faster, the current European regulatory framework would constitute
a strong obstacle to the development of NGTs in Europe [214]. On a more technical level, the
EC identified substantial issues concerning traceability of some products obtained through
NGTs because it is possible to analytically detect single-point mutations in a target sequence
but not their origin, whether natural or induced in the laboratory [215]. Therefore, to keep
pace with the scientific and technological development and favor European economic and
social progress, the EC has decided to test whether the conditions exist in Europe for a
change in the legislative framework on NGTs in plants.

6.1. Public Consultation Promoted by the European Commission

In September 2021, the EC initiated a policy action to assess the impact of different
legislative options. Alongside this impact assessment, an online public consultation was
launched through the internet portal of the EC. The consultation included two phases to
collect the plurality of voices of European stakeholders and citizens. In the first phase it was
possible to release a free comment over 1 month (24 September 2021–22 October 2021), while
the second phase (29 April 2022–22 July 2022) involved the compilation of a questionnaire
concerning the issues of risk assessment, sustainability, traceability, and labeling.
The outcome of this second phase of the consultation was published in September
2022 (https://ec.europa.eu/info/law/better-regulation/have-your-say/initiatives/13119-
Legislation-for-plants-produced-by-certain-new-genomic-techniques/public-consultation_
en, accessed 5 October 2022). Regarding the current rules of GMO legislation, 79% of re-
spondents considered them inadequate for plants obtained by targeted mutagenesis or
cisgenesis. Moreover, 61% of participants are in favor of a different risk assessment ap-
proach than the one applied to GMOs. In terms of sustainability, 51% were in favor of
specific regulatory provisions such as regulatory incentives or actual requirements. As
regards traceability, according to most of the participants, the current requirements of the
GMO legislation should be revised.
The last step of this political action, expected by mid-2023, affects the definition of a
legislative proposal to be submitted to the European Institutions holding legislative power,
the European Parliament, and the Council. Through its representatives who sit in the
European Institutions, Italy will take part in the discussion which will lead to the final
adoption or rejection of the EC legislative proposal.

6.2. The Position of Italy

Compared to the first phase of consultation in which Italy was the sixth European
country in terms of feedback, with 2% of responses (Germany resulted the most participa-
tory country, with 46% of responses, followed by France), in the second phase, Italy was
second in terms of number of answers given (23%), behind Germany (26%) (France, with
15%, was third).
Locally, on 12 July 2022 the Italian joint commissions on Social Affairs and Agriculture
argued favorably on the possibility of conducting out field trials on plants produced with
Int. J. Mol. Sci. 2023, 24, 977 20 of 30

genome editing and cisgenesis techniques for experimental and scientific purposes, under
the aegis of public research [216]. Indeed, Italy invested substantial effort in this sector,
and in-field experimentation could strengthen its leading role in crop genetic improvement
through new genomic techniques.

7. Concluding Remarks and Future Perspectives

In the last few years, Italy has invested millions of euros through national and inter-
national funding to apply NGTs on several crops, woody plants and fruit trees included.
In this way, it was possible to acquire know-how on the most advanced technologies and
train a new generation of scientists. Recent sources guaranteed to develop genome editing
and intra/cisgenic constructs on traits the genes of which were already known, as well as
on new candidate genes; moreover, new optimized protocols were successfully adapted
to important varieties for the ‘Made in Italy’, mainly focused on improving regeneration
capabilities from different explants (Figure 3).

Figure 3. Summary of the main achievements reached by the Italian scientific community over the
last years exploiting new genomic techniques (NGTs). From left to right, the species selected for
genetic improvement, the gene(s) identified as target (for chestnut the selected gene was chosen as
proof of concept), the technique exploited for the selected gene(s) (i.e., genome editing or cisgene-
sis/intragenesis), and the selected approach (i.e., Agrobacterium-mediated marker free transformation
or DNA-free through protoplasts transfection). In the last column, we highlight if improved plants
were already regenerated, and which cultivars were regenerated (published data reported in the
previous sections). The hourglass represents that work is in progress.

The possibility to test in field the genotypes already evaluated in the laboratory
represents an opportunity to conjugate basic and applied research, encountering the needs
of the national primary sector through biotechnologies. One of the most urgent scopes
of application consists of protection from biotic emergencies that can compromise the
Int. J. Mol. Sci. 2023, 24, 977 21 of 30

survival of the local species/variety, lowering the biodiversity. In grapevine, for example,
the susceptibility to powdery and downy mildew threatens the production of wine grape
cultivars such as Glera, Sangiovese, Chardonnay, and Pinot noir. In this context, the use
of conventional breeding cannot be sufficient to produce resistant varieties due to the
admixture of the genomes. The use of genome editing allows resistance to be introduced
while maintaining and protecting the quality of the elite accessions. Similarly, in Citrus,
local lemon clones suffer, for more than a century, from Mal secco disease, thus heavily
limiting cultivation. Throughout years, breeding programs were not able to introduce
the resistance to the main lemon varieties, putting at risk the production of those fruits
so appreciated for their nutritional and antioxidant properties. We are sure that the use
NGTs can contribute to speed up the recovery of lemon growing, which has recently been
suffering. Moreover, the close income of HLB in Europe would contribute to reducing, until
the disappearance, the worldwide citriculture; all Citrus species commonly used for fruits
consumption and rootstocks are susceptible. The urgency to find solutions is due to the
detection of vectors transmitting the bacterium Candidatus liberibacter (causal agent of the
disease) in Portugal and Spain (2014) and in Israel (2022). The introduction of the resistance
genes in the most appreciated varieties of all the main Citrus species (e.g., sweet oranges,
lemons, mandarins, and grapefruits) and the main rootstocks will contribute to recovering
and protecting the entire citrus production. Lastly, from a qualitative point of view, the use
of NGTs will contribute to protecting the mandarin ‘Tardivo of Ciaculli’, as an example,
unique for its flavor and aroma, but naturally seedy and, therefore, little appreciated by
consumers. If proven effective, the silencing of susceptibility genes in chestnut would
greatly improve the possibility of establishing new orchards of C. sativa in many areas,
escaping the risk of infections by canker blight and ink disease, as well as hindering the
current trend toward the planting of hybrid cultivars, more tolerant to pathogens but of
lower nut quality.
Generally, this path would allow for more sustainable crops from an environmental
point of view; the new varieties would allow the reduction in agrochemicals and fertilizers,
lowering the needs of chemical inputs. In parallel, genetic improvement will relieve the
effects of climate change, considering both biotic and abiotic stresses, with huge benefits for
the agricultural sector, as well as the final consumers, while also preserving the peculiarity
of the wide agrobiodiversity in Italian agriculture.
The efforts made by the Italian research community, from both the economic and
the advanced know-how in research perspectives, are now freely available worldwide,
allowing the exploitation of such results in many countries. This will lead to the possibility
of extending the effectiveness of NGTs to agroecosystems of low- and middle-income
countries, improving sustainability and resilience not only from an economic perspective
but also from a social point of view. On the contrary, due to the restrictive EU and Italian
regulations on the use of plants resulting from NGTs, the exploitation of the results in
Italy, as well as in Europe, can suffer from huge limitations, with severe drawbacks on
important currently ongoing national and international programs dealing with mitigation
of climate change effects (e.g., Farm to Fork, New Green Deal, and Paris Agreement on
Climate Change).

Author Contributions: Conceptualization, C.L. and M.A.M.; A.C., C.L., S.L.M. and A.G. contributed
to writing the section on Citrus; L.N., R.V., S.Z., I.P. and L.D.C. contributed to writing the section
on grape; V.P., A.M. and R.B. contributed to writing the section on chestnut; L.D., S.T. and M.A.M.
contributed to writing the section on apple and pear; S.S. and B.M. contributed to writing the
section on strawberry; E.V., S.M. and I.V. contributed to writing the section on Prunus; E.C. and V.M.
contributed to writing the session on kiwifruit; L.N., L.D.C. and A.C. prepared the original draft; C.L.
was responsible for review and editing. All authors have read and agreed to the published version of
the manuscript.
Int. J. Mol. Sci. 2023, 24, 977 22 of 30

Funding: This research was funded by the Italian Ministry of Agriculture Food and Forestry through
the project “BIOTECH—Biotecnologie sostenibili per l’agricoltura italiana” (DM 15930/7305/2018),
by the Horizon 2020 MSCA RISE project TESS—Targeted engineering of stone fruit tree genomes
for resistance to Sharka (GA n. 777794), and by the Fondazione Cassa di Risparmio di Torino (CRT)
through the project “EditGrape: Biotecnologie sostenibili al servizio della viticoltura” (Fondazione
CRT prot n. 2017.AI1909.U2180 del 14 December 2017). Research at FEM was funded by the
Autonomous Province of Trento. Research on chestnut was funded by Fondazione CRT.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this review are openly available in the
cited literature.
Acknowledgments: S.Z. and R.V. thank Alessandra Amato for her contribution to the VvAGL11
promoter study and trascriptomic data interpretation. S.S. and B.M. thank Amelia Gaston, Beatrice
Denoyes (INRAE, Bordeaux, France), and José L. Caballero (Departamento Bioquímica y Biología
Molecular, Universidad de Córdoba, Cordoba, Spain) for helping in designing the intragenic con-
structs for strawberry transformation. S.M., E.V., and I.V. thank Veronique Decroocq and colleagues
from INRA, as well as Humberto Prieto and colleagues from INIA, for their invaluable contributions
to the genome editing for Sharka resistance and in vitro transformation of peach, in the framework of
the TESS project. V.M. and E.C. thank Gianni Tacconi for the helpful discussion during all phases
of the project and for the maintenance of kiwi regenerated plants at CREA-Genomics and Bioinfor-
matics of Fiorenzuola D’Arda (Italy). The authors thank Alison Garside for the manuscript English
editing service.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Arabska, E. From Farm to Fork: Human Health and Well-Being through Sustainable Agri-Food Systems. J. Life Econ. 2021, 8,
11–27.
2. Eckert, E.; Kovalevska, O. Sustainability in the European Union: Analyzing the Discourse of the European Green Deal. J. Risk
Financ. Manag. 2021, 14, 80. [CrossRef]
3. Bazzano, L.A.; Serdula, M.K.; Liu, S. Dietary Intake of Fruits and Vegetables and Risk of Cardiovascular Disease. Curr. Atheroscler.
Rep. 2003, 5, 492–499. [CrossRef] [PubMed]
4. Rejman, K.; Górska-Warsewicz, H.; Kaczorowska, J.; Laskowski, W. Nutritional Significance of Fruit and Fruit Products in the
Average Polish Diet. Nutrients 2021, 13, 2079. [CrossRef] [PubMed]
5. Anderson, R.; Bayer, P.E.; Edwards, D. Climate Change and the Need for Agricultural Adaptation. Curr. Opin. Plant Biol. 2020, 56,
197–202. [CrossRef] [PubMed]
6. Kompas, T.; Pham, V.H.; Che, T.N. The Effects of Climate Change on GDP by Country and the Global Economic Gains from
Complying with the Paris Climate Accord. Earths Future 2018, 6, 1153–1173. [CrossRef]
7. Rahman, M.M.; Khan, I.; Field, D.L.; Techato, K.; Alameh, K. Powering Agriculture: Present Status, Future Potential, and
Challenges of Renewable Energy Applications. Renew. Energy 2022, 188, 731–749. [CrossRef]
8. Hackett, W. Juvenility and Maturity. In Cell and Tissue Culture in Forestry; Springer: Dordrecht, The Netherlands, 1987;
Volume 24–26, pp. 216–231.
9. Limera, C.; Sabbadini, S.; Sweet, J.B.; Mezzetti, B. New Biotechnological Tools for the Genetic Improvement of Major Woody Fruit
Species. Front. Plant Sci. 2017, 8, 1418. [CrossRef]
10. Giudice, G.; Moffa, L.; Varotto, S.; Cardone, M.F.; Bergamini, C.; De Lorenzis, G.; Velasco, R.; Nerva, L.; Chitarra, W. Novel and
Emerging Biotechnological Crop Protection Approaches. Plant Biotechnol. J. 2021, 19, 1495–1510. [CrossRef]
11. Lloyd, A.; Plaisier, C.L.; Carroll, D.; Drews, G.N. Targeted Mutagenesis Using Zinc-Finger Nucleases in Arabidopsis. Proc. Natl.
Acad. Sci. USA 2005, 102, 2232–2237. [CrossRef]
12. Peer, R.; Rivlin, G.; Golobovitch, S.; Lapidot, M.; Gal-On, A.; Vainstein, A.; Tzfira, T.; Flaishman, M.A. Targeted Mutagenesis
Using Zinc-Finger Nucleases in Perennial Fruit Trees. Planta 2015, 241, 941–951. [CrossRef] [PubMed]
13. Lu, H.; Klocko, A.L.; Dow, M.; Ma, C.; Amarasinghe, V.; Strauss, S.H. Low Frequency of Zinc-Finger Nuclease-Induced
Mutagenesis in Populus. Mol. Breed. 2016, 36, 121. [CrossRef]
14. Min, T.; Hwarari, D.; Li, D.; Movahedi, A.; Yang, L. CRISPR-Based Genome Editing and Its Applications in Woody Plants. Int. J.
Mol. Sci. 2022, 23, 10175. [CrossRef]
15. Zaboikin, M.; Zaboikina, T.; Freter, C.; Srinivasakumar, N. Non-Homologous End Joining and Homology Directed DNA Repair
Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents. PLoS ONE 2017, 12, e0169931. [CrossRef]
[PubMed]
Int. J. Mol. Sci. 2023, 24, 977 23 of 30

16. Zhang, Y.; Liang, Z.; Zong, Y.; Wang, Y.; Liu, J.; Chen, K.; Qiu, J.-L.; Gao, C. Efficient and Transgene-Free Genome Editing in
Wheat through Transient Expression of CRISPR/Cas9 DNA or RNA. Nat. Commun. 2016, 7, 12617. [CrossRef] [PubMed]
17. Shan, Q.; Wang, Y.; Li, J.; Zhang, Y.; Chen, K.; Liang, Z.; Zhang, K.; Liu, J.; Xi, J.J.; Qiu, J.-L. Targeted Genome Modification of Crop
Plants Using a CRISPR-Cas System. Nat. Biotechnol. 2013, 31, 686–688. [CrossRef]
18. Wang, M.; Lu, Y.; Botella, J.R.; Mao, Y.; Hua, K.; Zhu, J. Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-
Based CRISPR/Cas9 System. Mol. Plant 2017, 10, 1007–1010. [CrossRef]
19. Yue, J.-J.; Yuan, J.-L.; Wu, F.-H.; Yuan, Y.-H.; Cheng, Q.-W.; Hsu, C.-T.; Lin, C.-S. Protoplasts: From Isolation to CRISPR/Cas
Genome Editing Application. Front. Genome Ed. 2021, 3, 717017. [CrossRef]
20. Mahmoud, L.M.; Kaur, P.; Stanton, D.; Grosser, J.W.; Dutt, M. A Cationic Lipid Mediated CRISPR/Cas9 Technique for the
Production of Stable Genome Edited Citrus Plants. Plant Methods 2022, 18, 33. [CrossRef]
21. Huang, X.; Wang, Y.; Wang, N. Highly Efficient Generation of Canker-Resistant Sweet Orange Enabled by an Improved
CRISPR/Cas9 System. Front. Plant Sci. 2021, 12, 769907. [CrossRef]
22. Parajuli, S.; Huo, H.; Gmitter, F.G.; Duan, Y.; Luo, F.; Deng, Z. Editing the CsDMR6 Gene in Citrus Results in Resistance to the
Bacterial Disease Citrus Canker. Hortic. Res. 2022, 9, uhac082. [CrossRef] [PubMed]
23. Wang, X.; Tu, M.; Wang, D.; Liu, J.; Li, Y.; Li, Z.; Wang, Y.; Wang, X. CRISPR/Cas9-mediated Efficient Targeted Mutagenesis in
Grape in the First Generation. Plant Biotechnol. J. 2018, 16, 844–855. [CrossRef] [PubMed]
24. Li, M.-Y.; Jiao, Y.-T.; Wang, Y.-T.; Zhang, N.; Wang, B.-B.; Liu, R.-Q.; Yin, X.; Xu, Y.; Liu, G.-T. CRISPR/Cas9-Mediated VvPR4b
Editing Decreases Downy Mildew Resistance in Grapevine (Vitis vinifera L.). Hortic. Res. 2020, 7, 149. [CrossRef] [PubMed]
25. Wan, D.-Y.; Guo, Y.; Cheng, Y.; Hu, Y.; Xiao, S.; Wang, Y.; Wen, Y.-Q. CRISPR/Cas9-Mediated Mutagenesis of VvMLO3 Results in
Enhanced Resistance to Powdery Mildew in Grapevine (Vitis vinifera). Hortic. Res. 2020, 7, 116. [CrossRef]
26. Sunitha, S.; Rock, C.D. CRISPR/Cas9-Mediated Targeted Mutagenesis of TAS4 and MYBA7 Loci in Grapevine Rootstock 101-14.
Transgenic Res. 2020, 29, 355–367. [CrossRef]
27. Ren, C.; Guo, Y.; Kong, J.; Lecourieux, F.; Dai, Z.; Li, S.; Liang, Z. Knockout of VvCCD8 Gene in Grapevine Affects Shoot Branching.
BMC Plant Biol. 2020, 20, 47. [CrossRef]
28. Wang, Z.; Wong, D.C.J.; Wang, Y.; Xu, G.; Ren, C.; Liu, Y.; Kuang, Y.; Fan, P.; Li, S.; Xin, H. GRAS-Domain Transcription Factor
PAT1 Regulates Jasmonic Acid Biosynthesis in Grape Cold Stress Response. Plant Physiol. 2021, 186, 1660–1678. [CrossRef]
29. Corredoira, E.; San José, M.C.; Vieitez, A.; Allona, I.; Aragoncillo, C.; Ballester, A. Agrobacterium-Mediated Transformation of
European Chestnut Somatic Embryos with a Castanea sativa (Mill.) Endochitinase Gene. New For. 2016, 47, 669–684. [CrossRef]
30. Carlson, E.; Stewart, K.; Baier, K.; McGuigan, L.; Culpepper, T.; Powell, W. Pathogen-induced Expression of a Blight Tolerance
Transgene in American Chestnut. Mol. Plant Pathol. 2022, 23, 370–382. [CrossRef]
31. Zhou, H.; Bai, S.; Wang, N.; Sun, X.; Zhang, Y.; Zhu, J.; Dong, C. CRISPR/Cas9-Mediated Mutagenesis of MdCNGC2 in Apple
Callus and VIGS-Mediated Silencing of MdCNGC2 in Fruits Improve Resistance to Botryosphaeria dothidea. Front. Plant Sci. 2020,
11, 575477. [CrossRef]
32. Charrier, A.; Vergne, E.; Dousset, N.; Richer, A.; Petiteau, A.; Chevreau, E. Efficient Targeted Mutagenesis in Apple and First Time
Edition of Pear Using the CRISPR-Cas9 System. Front. Plant Sci. 2019, 10, 40. [CrossRef] [PubMed]
33. Malabarba, J.; Chevreau, E.; Dousset, N.; Veillet, F.; Moizan, J.; Vergne, E. New Strategies to Overcome Present CRISPR/Cas9
Limitations in Apple and Pear: Efficient Dechimerization and Base Editing. Int. J. Mol. Sci. 2020, 22, 319. [CrossRef] [PubMed]
34. Zhang, Y.; Zhou, P.; Bozorov, T.A.; Zhang, D. Application of CRISPR/Cas9 Technology in Wild Apple (Malus sieverii) for Paired
Sites Gene Editing. Plant Methods 2021, 17, 79. [CrossRef] [PubMed]
35. Feng, J.; Dai, C.; Luo, H.; Han, Y.; Liu, Z.; Kang, C. Reporter Gene Expression Reveals Precise Auxin Synthesis Sites during Fruit
and Root Development in Wild Strawberry. J. Exp. Bot. 2019, 70, 563–574. [CrossRef]
36. Varkonyi-Gasic, E.; Wang, T.; Voogd, C.; Jeon, S.; Drummond, R.S.; Gleave, A.P.; Allan, A.C. Mutagenesis of Kiwifruit
CENTRORADIALIS-like Genes Transforms a Climbing Woody Perennial with Long Juvenility and Axillary Flowering into
a Compact Plant with Rapid Terminal Flowering. Plant Biotechnol. J. 2019, 17, 869–880. [CrossRef]
37. Varkonyi-Gasic, E.; Wang, T.; Cooney, J.; Jeon, S.; Voogd, C.; Douglas, M.J.; Pilkington, S.M.; Akagi, T.; Allan, A.C. Shy Girl, a
Kiwifruit Suppressor of Feminization, Restricts Gynoecium Development via Regulation of Cytokinin Metabolism and Signalling.
New Phytol. 2021, 230, 1461–1475. [CrossRef]
38. Herath, D.; Voogd, C.; Mayo-Smith, M.; Yang, B.; Allan, A.C.; Putterill, J.; Varkonyi-Gasic, E. CRISPR-Cas9-mediated Mutagenesis
of Kiwifruit BFT Genes Results in an Evergrowing but Not Early Flowering Phenotype. Plant Biotechnol. J. 2022, 20, 2064–2076.
[CrossRef]
39. Schouten, H.J.; Krens, F.A.; Jacobsen, E. Do Cisgenic Plants Warrant Less Stringent Oversight? Nat. Biotechnol. 2006, 24, 753.
[CrossRef]
40. Schaart, J.G. Towards Consumer-Friendly Cisgenic Strawberries Which Are Less Susceptible to Botrytis cinerea; Wageningen University
and Research: Wageningen, The Netherlands, 2004; ISBN 9798516028632.
41. Holme, I.B.; Wendt, T.; Holm, P.B. Intragenesis and Cisgenesis as Alternatives to Transgenic Crop Development. Plant Biotechnol.
J. 2013, 11, 395–407. [CrossRef]
42. Hospital, F. Selection in Backcross Programmes. Philos. Trans. R. Soc. B Biol. Sci. 2005, 360, 1503–1511. [CrossRef]
43. Wang, M.; Wang, S.; Liang, Z.; Shi, W.; Gao, C.; Xia, G. From Genetic Stock to Genome Editing: Gene Exploitation in Wheat.
Trends Biotechnol. 2018, 36, 160–172. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 977 24 of 30

44. Jaillon, O.; Aury, J.-M.; Noel, B.; Policriti, A.; Clepet, C.; Casagrande, A.; Choisne, N.; Aubourg, S.; Vitulo, N.; Jubin, C. The
Grapevine Genome Sequence Suggests Ancestral Hexaploidization in Major Angiosperm Phyla. Nature 2007, 449, 463. [PubMed]
45. Velasco, R.; Zharkikh, A.; Troggio, M.; Cartwright, D.A.; Cestaro, A.; Pruss, D.; Pindo, M.; FitzGerald, L.M.; Vezzulli, S.; Reid, J.
A High Quality Draft Consensus Sequence of the Genome of a Heterozygous Grapevine Variety. PLoS ONE 2007, 2, e1326.
[CrossRef]
46. Xu, Q.; Chen, L.-L.; Ruan, X.; Chen, D.; Zhu, A.; Chen, C.; Bertrand, D.; Jiao, W.-B.; Hao, B.-H.; Lyon, M.P. The Draft Genome of
Sweet Orange (Citrus sinensis). Nat. Genet. 2013, 45, 59–66. [CrossRef] [PubMed]
47. Di Guardo, M.; Moretto, M.; Moser, M.; Catalano, C.; Troggio, M.; Deng, Z.; Cestaro, A.; Caruso, M.; Distefano, G.; La Malfa, S.
The Haplotype-Resolved Reference Genome of Lemon (Citrus limon L. Burm f.). Tree Genet. Genomes 2021, 17, 46. [CrossRef]
48. Verde, I.; Abbott, A.G.; Scalabrin, S.; Jung, S.; Shu, S.; Marroni, F.; Zhebentyayeva, T.; Dettori, M.T.; Grimwood, J.; Cattonaro, F.
The High-Quality Draft Genome of Peach (Prunus persica) Identifies Unique Patterns of Genetic Diversity, Domestication and
Genome Evolution. Nat. Genet. 2013, 45, 487–494. [CrossRef] [PubMed]
49. Verde, I.; Jenkins, J.; Dondini, L.; Micali, S.; Pagliarani, G.; Vendramin, E.; Paris, R.; Aramini, V.; Gazza, L.; Rossini, L. The Peach
v2. 0 Release: High-Resolution Linkage Mapping and Deep Resequencing Improve Chromosome-Scale Assembly and Contiguity.
BMC Genom. 2017, 18, 225. [CrossRef]
50. Groppi, A.; Liu, S.; Cornille, A.; Decroocq, S.; Bui, Q.T.; Tricon, D.; Cruaud, C.; Arribat, S.; Belser, C.; Marande, W. Population
Genomics of Apricots Unravels Domestication History and Adaptive Events. Nat. Commun. 2021, 12, 3956. [CrossRef]
51. Jiang, F.; Zhang, J.; Wang, S.; Yang, L.; Luo, Y.; Gao, S.; Zhang, M.; Wu, S.; Hu, S.; Sun, H. The Apricot (Prunus armeniaca L.)
Genome Elucidates Rosaceae Evolution and Beta-Carotenoid Synthesis. Hortic. Res. 2019, 6, 128. [CrossRef]
52. Shirasawa, K.; Isuzugawa, K.; Ikenaga, M.; Saito, Y.; Yamamoto, T.; Hirakawa, H.; Isobe, S. The Genome Sequence of Sweet
Cherry (Prunus avium) for Use in Genomics-Assisted Breeding. DNA Res. 2017, 24, 499–508. [CrossRef]
53. Pinosio, S.; Marroni, F.; Zuccolo, A.; Vitulo, N.; Mariette, S.; Sonnante, G.; Aravanopoulos, F.A.; Ganopoulos, I.; Palasciano, M.;
Vidotto, M. A Draft Genome of Sweet Cherry (Prunus avium L.) Reveals Genome-wide and Local Effects of Domestication. Plant J.
2020, 103, 1420–1432. [CrossRef] [PubMed]
54. Kalyanaraman, A.; Fontana, P.; Bhatnagar, S.; Troggio, M.; Pruss, D.; Salvi, S.; Pindo, M.; Baldi, P.; Castelletti, S.; Cavaiuolo, M.
The Genome of the Domesticated Apple (Malus x domestica Borkh.). Nat. Genet. 2010, 42, 833–839.
55. Daccord, N.; Celton, J.-M.; Linsmith, G.; Becker, C.; Choisne, N.; Schijlen, E.; Van de Geest, H.; Bianco, L.; Micheletti, D.; Velasco, R.
High-Quality de Novo Assembly of the Apple Genome and Methylome Dynamics of Early Fruit Development. Nat. Genet. 2017,
49, 1099–1106. [CrossRef] [PubMed]
56. Chagné, D.; Crowhurst, R.N.; Pindo, M.; Thrimawithana, A.; Deng, C.; Ireland, H.; Fiers, M.; Dzierzon, H.; Cestaro, A.; Fontana, P.
The Draft Genome Sequence of European Pear (Pyrus communis L. ‘Bartlett’). PLoS ONE 2014, 9, e92644. [CrossRef]
57. Linsmith, G.; Rombauts, S.; Montanari, S.; Deng, C.H.; Celton, J.-M.; Guérif, P.; Liu, C.; Lohaus, R.; Zurn, J.D.; Cestaro, A.
Pseudo-Chromosome–Length Genome Assembly of a Double Haploid “Bartlett” Pear (Pyrus communis L.). Gigascience 2019,
8, giz138. [CrossRef]
58. Wu, H.; Ma, T.; Kang, M.; Ai, F.; Zhang, J.; Dong, G.; Liu, J. A High-Quality Actinidia chinensis (Kiwifruit) Genome. Hortic. Res.
2019, 6, 117. [CrossRef]
59. Shulaev, V.; Sargent, D.J.; Crowhurst, R.N.; Mockler, T.C.; Folkerts, O.; Delcher, A.L.; Jaiswal, P.; Mockaitis, K.; Liston, A.;
Mane, S.P. The Genome of Woodland Strawberry (Fragaria vesca). Nat. Genet. 2011, 43, 109–116. [CrossRef]
60. Edger, P.P.; Poorten, T.J.; VanBuren, R.; Hardigan, M.A.; Colle, M.; McKain, M.R.; Smith, R.D.; Teresi, S.J.; Nelson, A.D.; Wai, C.M.
Origin and Evolution of the Octoploid Strawberry Genome. Nat. Genet. 2019, 51, 541–547. [CrossRef]
61. Staton, M.; Addo-Quaye, C.; Cannon, N.; Sun, Y.; Zhebentyayeva, T.; Huff, M.; Fan, S.; Bellis, E.; Islam-Faridi, N.; Yu, J. The
Chinese Chestnut Genome: A Reference for Species Restoration. BioRxiv 2019, 615047. [CrossRef]
62. Shirasawa, K.; Nishio, S.; Terakami, S.; Botta, R.; Marinoni, D.T.; Isobe, S. Chromosome-Level Genome Assembly of Japanese
Chestnut (Castanea crenata Sieb. et Zucc.) Reveals Conserved Chromosomal Segments in Woody Rosids. DNA Res. 2021,
28, dsab016. [CrossRef]
63. Gambino, G.; Ruffa, P.; Vallania, R.; Gribaudo, I. Somatic Embryogenesis from Whole Flowers, Anthers and Ovaries of Grapevine
(Vitis spp.). Plant Cell Tissue Organ. Cult. 2007, 90, 79–83. [CrossRef]
64. Capriotti, L.; Limera, C.; Mezzetti, B.; Ricci, A.; Sabbadini, S. From Induction to Embryo Proliferation: Improved Somatic
Embryogenesis Protocol in Grapevine for Italian Cultivars and Hybrid Vitis Rootstocks. Plant Cell Tissue Organ. Cult. PCTOC
2022, 151, 221–233. [CrossRef]
65. Gribaudo, I.; Gambino, G.; Boccacci, P.; Perrone, I.; Cuozzo, D. A Multi-Year Study on the Regenerative Potential of Several Vitis
Genotypes. Acta Hortic. 2017, 1155, 45–50. [CrossRef]
66. Ricci, A.; Sabbadini, S.; Prieto, H.; Padilla, I.M.; Dardick, C.; Li, Z.; Scorza, R.; Limera, C.; Mezzetti, B.; Perez-Jimenez, M. Genetic
Transformation in Peach (Prunus persica L.): Challenges and Ways Forward. Plants 2020, 9, 971. [CrossRef] [PubMed]
67. Court of Justice of the European Union. Judgment of the Court (Grand Chamber) of 25 July 2018 (ECLI:EU:C:2018:583) © Curia,
15/12/2018, curia.europa.eu. Available online: https://curia.europa.eu/juris/document/document.jsf?docid=204387&doclang=
EN (accessed on 20 December 2022).
68. Nandy, S.; Srivastava, V. Site-specific Gene Integration in Rice Genome Mediated by the FLP–FRT Recombination System. Plant
Biotechnol. J. 2011, 9, 713–721. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 977 25 of 30

69. Woo, H.-J.; Cho, H.-S.; Lim, S.-H.; Shin, K.-S.; Lee, S.-M.; Lee, K.-J.; Kim, D.-H.; Cho, Y.-G. Auto-Excision of Selectable Marker
Genes from Transgenic Tobacco via a Stress Inducible FLP/FRT Site-Specific Recombination System. Transgenic Res. 2009, 18,
455–465. [CrossRef]
70. Sreekala, C.; Wu, L.; Gu, K.; Wang, D.; Tian, D.; Yin, Z. Excision of a Selectable Marker in Transgenic Rice (Oryza sativa L.) Using a
Chemically Regulated Cre/LoxP System. Plant Cell Rep. 2005, 24, 86–94. [CrossRef]
71. Cuellar, W.; Gaudin, A.; Solorzano, D.; Casas, A.; Nopo, L.; Chudalayandi, P.; Medrano, G.; Kreuze, J.; Ghislain, M. Self-Excision
of the Antibiotic Resistance Gene NptII Using a Heat Inducible Cre-LoxP System from Transgenic Potato. Plant Mol. Biol. 2006,
62, 71–82. [CrossRef]
72. Pompili, V.; Dalla Costa, L.; Piazza, S.; Pindo, M.; Malnoy, M. Reduced Fire Blight Susceptibility in Apple Cultivars Using a
High-efficiency CRISPR/Cas9-FLP/FRT-based Gene Editing System. Plant Biotechnol. J. 2020, 18, 845–858. [CrossRef]
73. Caruso, M.; Ferlito, F.; Licciardello, C.; Allegra, M.; Strano, M.C.; Di Silvestro, S.; Russo, M.P.; Paolo, D.P.; Caruso, P.; Las Casas, G.
Pomological Diversity of the Italian Blood Orange Germplasm. Sci. Hortic. 2016, 213, 331–339. [CrossRef]
74. Rapisarda, P.; Fabroni, S.; Peterek, S.; Russo, G.; Mock, H.-P. Juice of New Citrus Hybrids (Citrus clementina Hort. Ex Tan.× C.
sinensis L. Osbeck) as a Source of Natural Antioxidants. Food Chem. 2009, 117, 212–218. [CrossRef]
75. Recupero, G.R.; Russo, G.; Recupero, S.; Zurru, R.; Deidda, B.; Mulas, M. Horticultural Evaluation of New Citrus latipes Hybrids
as Rootstocks for Citrus. HortScience 2009, 44, 595–598. [CrossRef]
76. Recupero, G.R.; Russo, G.; Recupero, S. New Promising Citrus Triploid Hybrids Selected from Crosses between Monoembryonic
Diploid Female and Tetraploid Male Parents. HortScience 2005, 40, 516–520. [CrossRef]
77. Talon, M.; Caruso, M.; Gmitter, F.G., Jr. The Genus Citrus; Woodhead Publishing: Sawston, UK, 2020; ISBN 0-12-812217-X.
[CrossRef]
78. Jia, H.; Wang, C.; Zhang, C.; Haider, M.S.; Zhao, P.; Liu, Z.; Shangguan, L.; Pervaiz, T.; Fang, J. Functional Analysis of VvBG1
during Fruit Development and Ripening of Grape. J. Plant Growth Regul. 2016, 35, 987–999. [CrossRef]
79. Jia, H.; Xu, J.; Orbović, V.; Zhang, Y.; Wang, N. Editing Citrus Genome via SaCas9/SgRNA System. Front. Plant Sci. 2017, 8, 2135.
[CrossRef]
80. Zhang, F.; Wang, Y.; Irish, V.F. CENTRORADIALIS Maintains Shoot Meristem Indeterminacy by Antagonizing THORN IDENTITY1
in Citrus. Curr. Biol. 2021, 31, 2237–2242. [CrossRef]
81. Alquézar, B.; Bennici, S.; Carmona, L.; Gentile, A.; Peña, L. Generation of Transfer-DNA-Free Base-Edited Citrus Plants. Front.
Plant Sci. 2022, 13, 835282. [CrossRef]
82. Poles, L.; Licciardello, C.; Distefano, G.; Nicolosi, E.; Gentile, A.; La Malfa, S. Recent Advances of in Vitro Culture for the
Application of New Breeding Techniques in Citrus. Plants 2020, 9, 938. [CrossRef]
83. Butelli, E.; Garcia-Lor, A.; Licciardello, C.; Las Casas, G.; Hill, L. Reforgiato Recupero, G. Changes in anthocyanin production
during domestication of Citrus. Plant Physiol. 2017, 173, 2225–2242. [CrossRef]
84. Butelli, E.; Licciardello, C.; Ramadugu, C.; Durand-Hulak, M.; Celant, A.; Recupero, G.R.; Froelicher, Y.; Martin, C. Noemi
Controls Production of Flavonoid Pigments and Fruit Acidity and Illustrates the Domestication Routes of Modern Citrus Varieties.
Curr. Biol. 2019, 29, 158–164. [CrossRef]
85. Dasgupta, K.; Thilmony, R.; Stover, E.; Oliveira, M.L.; Thomson, J. Novel R2R3-MYB Transcription Factors from Prunus Americana
Regulate Differential Patterns of Anthocyanin Accumulation in Tobacco and Citrus. GM Crop. Food 2017, 8, 85–105. [CrossRef]
[PubMed]
86. Dutt, M.; Stanton, D.; Grosser, J.W. Ornacitrus: Development of Genetically Modified Anthocyanin-Expressing Citrus with Both
Ornamental and Fresh Fruit Potential. J. Am. Soc. Hortic. Sci. 2016, 141, 54–61. [CrossRef]
87. Ciacciulli, A.; Pappalardo, H.D.; Caruso, M.; Pindo, M.; Piazza, S.; Malnoy, M.; Licciardello, C. A Marker-Free Cisgenesis/Genome
Editing System, a New Tool to Produce Fortified Citrus Fruits. Acta Hortic. 2023; in press.
88. Endo, T.; Shimada, T.; Fujii, H.; Kobayashi, Y.; Araki, T.; Omura, M. Ectopic Expression of an FT Homolog from Citrus Confers an
Early Flowering Phenotype on Trifoliate Orange (Poncirus trifoliata L. Raf.). Transgenic Res. 2005, 14, 703–712. [CrossRef]
89. Li, R.; Li, R.; Li, X.; Fu, D.; Zhu, B.; Tian, H.; Luo, Y.; Zhu, H. Multiplexed CRISPR/Cas9-mediated Metabolic Engineering of
Γ-aminobutyric Acid Levels in Solanum lycopersicum. Plant Biotechnol. J. 2018, 16, 415–427. [CrossRef]
90. Natalini, A.; Acciarri, N.; Cardi, T. Breeding for Nutritional and Organoleptic Quality in Vegetable Crops: The Case of Tomato
and Cauliflower. Agriculture 2021, 11, 606. [CrossRef]
91. Kaur, N.; Alok, A.; Kumar, P.; Kaur, N.; Awasthi, P.; Chaturvedi, S.; Pandey, P.; Pandey, A.; Pandey, A.K.; Tiwari, S. CRISPR/Cas9
Directed Editing of Lycopene Epsilon-Cyclase Modulates Metabolic Flux for β-Carotene Biosynthesis in Banana Fruit. Metab. Eng.
2020, 59, 76–86. [CrossRef]
92. Salonia, F.; Ciacciulli, A.; Pappalardo, H.D.; Poles, L.; Pindo, M.; Larger, S.; Caruso, P.; Caruso, M.; Licciardello, C. A Dual
SgRNA-Directed CRISPR/Cas9 Construct Designed for Editing the Fruit-Specific β-Cyclase 2 Gene in Pigmented Citrus Fruits.
Front. Plant Sci. 2022, 13, 975917. [CrossRef]
93. Peng, A.; Zou, X.; Xu, L.; He, Y.; Lei, T.; Yao, L.; Li, Q.; Chen, S. Improved Protocol for the Transformation of Adult Citrus sinensis
Osbeck ‘Tarocco’ Blood Orange Tissues. In Vitro Cell. Dev. Biol.-Plant 2019, 55, 659–667. [CrossRef]
94. Jardak, R.; Boubakri, H.; Zemni, H.; Gandoura, S.; Mejri, S.; Mliki, A.; Ghorbel, A. Establishment of an in Vitro Regeneration
System and Genetic Transformation of the Tunisian ‘Maltese Half-Blood’ (Citrus sinensis): An Agro-Economically Important
Variety. 3 Biotech 2020, 10, 99. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 977 26 of 30

95. Yamasaki, A.; Kitajima, A.; Ohara, N.; Tanaka, M.; Hasegawa, K. Histological Study of Expression of Seedlessness in Citrus
kinokuni ‘Mukaku Kishu’ and Its Progenies. J. Am. Soc. Hortic. Sci. 2007, 132, 869–875. [CrossRef]
96. Caruso, M.; Merelo, P.; Distefano, G.; La Malfa, S.; Lo Piero, A.R.; Tadeo, F.R.; Talon, M.; Gentile, A. Comparative Transcriptome
Analysis of Stylar Canal Cells Identifies Novel Candidate Genes Implicated in the Self-Incompatibility Response of Citrus
clementina. BMC Plant Biol. 2012, 12, 20. [CrossRef] [PubMed]
97. Varoquaux, F.; Blanvillain, R.; Delseny, M.; Gallois, P. Less Is Better: New Approaches for Seedless Fruit Production. Trends
Biotechnol. 2000, 18, 233–242. [CrossRef] [PubMed]
98. Vardi, A.; Levin, I.; Carmi, N. Induction of Seedlessness in Citrus: From Classical Techniques to Emerging Biotechnological
Approaches. J. Am. Soc. Hortic. Sci. 2008, 133, 117–126. [CrossRef]
99. Luo, M.; Dennis, E.S.; Berger, F.; Peacock, W.J.; Chaudhury, A. MINISEED3 (MINI3), a WRKY Family Gene, and HAIKU2 (IKU2),
a Leucine-Rich Repeat (LRR) KINASE Gene, Are Regulators of Seed Size in Arabidopsis. Proc. Natl. Acad. Sci. USA 2005, 102,
17531–17536. [CrossRef]
100. Wang, A.; Garcia, D.; Zhang, H.; Feng, K.; Chaudhury, A.; Berger, F.; Peacock, W.J.; Dennis, E.S.; Luo, M. The VQ Motif Protein
IKU1 Regulates Endosperm Growth and Seed Size in Arabidopsis. Plant J. 2010, 63, 670–679. [CrossRef]
101. Zhou, Y.; Zhang, X.; Kang, X.; Zhao, X.; Zhang, X.; Ni, M. SHORT HYPOCOTYL UNDER BLUE1 Associates with MINISEED3 and
HAIKU2 Promoters in Vivo to Regulate Arabidopsis Seed Development. Plant Cell 2009, 21, 106–117. [CrossRef]
102. Poles, L.; Ciacciulli, A.; Pappalardo, D.H.; Salonia, F.; Distefano, G.; Gentile, A.; Caruso, M.; Larger, S.; Pindo, M.; La Malfa, S.; et al.
Genome Editing of IKU1 to Obtain Citrus Seedless Fruits. Acta Hortic. 2023; in press.
103. Li, N.; Xu, R.; Li, Y. Molecular Networks of Seed Size Control in Plants. Annu. Rev. Plant Biol. 2019, 70, 435–463. [CrossRef]
104. Liang, M.; Cao, Z.; Zhu, A.; Liu, Y.; Tao, M.; Yang, H.; Xu, Q.; Wang, S.; Liu, J.; Li, Y. Evolution of Self-Compatibility by a Mutant
Sm-RNase in Citrus. Nat. Plants 2020, 6, 131–142. [CrossRef]
105. Vezzulli, S.; Gramaje, D.; Tello, J.; Gambino, G.; Bettinelli, P.; Pirrello, C.; Schwandner, A.; Barba, P.; Angelini, E.; Anfora, G.
Genomic Designing for Biotic Stress Resistant Grapevine. In Genomic Designing for Biotic Stress Resistant Fruit Crops; Springer:
Berlin/Heidelberg, Germany, 2022; pp. 87–255. [CrossRef]
106. Foria, S.; Magris, G.; Jurman, I.; Schwope, R.; De Candido, M.; De Luca, E.; Ivanišević, D.; Morgante, M.; Di Gaspero, G. Extent of
Wild–to–Crop Interspecific Introgression in Grapevine (Vitis vinifera) as a Consequence of Resistance Breeding and Implications
for the Crop Species Definition. Hortic. Res. 2022, 9, uhab010. [CrossRef] [PubMed]
107. Cesco, S.; Tolotti, A.; Nadalini, S.; Rizzi, S.; Valentinuzzi, F.; Mimmo, T.; Porfido, C.; Allegretta, I.; Giovannini, O.; Perazzolli, M.
Plasmopara viticola Infection Affects Mineral Elements Allocation and Distribution in Vitis vinifera Leaves. Sci. Rep. 2020, 10, 18759.
[CrossRef] [PubMed]
108. Poni, S.; Chiari, G.; Caffi, T.; Bove, F.; Tombesi, S.; Moncalvo, A.; Gatti, M. Canopy Physiology, Vine Performance and Host-
Pathogen Interaction in a Fungi Resistant Cv. Sangiovese x Bianca Accession vs. a Susceptible Clone. Sci. Rep. 2017, 7, 6092.
[CrossRef] [PubMed]
109. Pagliarani, C.; Vitali, M.; Ferrero, M.; Vitulo, N.; Incarbone, M.; Lovisolo, C.; Valle, G.; Schubert, A. The Accumulation of miRNAs
Differentially Modulated by Drought Stress Is Affected by Grafting in Grapevine. Plant Physiol. 2017, 173, 2180–2195. [CrossRef]
110. Corso, M.; Vannozzi, A.; Maza, E.; Vitulo, N.; Meggio, F.; Pitacco, A.; Telatin, A.; D’angelo, M.; Feltrin, E.; Negri, A.S. Comprehen-
sive Transcript Profiling of Two Grapevine Rootstock Genotypes Contrasting in Drought Susceptibility Links the Phenylpropanoid
Pathway to Enhanced Tolerance. J. Exp. Bot. 2015, 66, 5739–5752. [CrossRef]
111. Bianchi, D.; Caramanico, L.; Grossi, D.; Brancadoro, L.; Lorenzis, G.D. How Do Novel M-Rootstock (Vitis spp.) Genotypes Cope
with Drought? Plants 2020, 9, 1385. [CrossRef]
112. Pessina, S.; Lenzi, L.; Perazzolli, M.; Campa, M.; Dalla Costa, L.; Urso, S.; Valè, G.; Salamini, F.; Velasco, R.; Malnoy, M. Knockdown
of MLO Genes Reduces Susceptibility to Powdery Mildew in Grapevine. Hortic. Res. 2016, 3, 16016. [CrossRef]
113. Liu, X.; Ao, K.; Yao, J.; Zhang, Y.; Li, X. Engineering Plant Disease Resistance against Biotrophic Pathogens. Curr. Opin. Plant Biol.
2021, 60, 101987. [CrossRef]
114. Pirrello, C.; Malacarne, G.; Moretto, M.; Lenzi, L.; Perazzolli, M.; Zeilmaker, T.; Van den Ackerveken, G.; Pilati, S.; Moser, C.;
Giacomelli, L. Grapevine DMR6-1 Is a Candidate Gene for Susceptibility to Downy Mildew. Biomolecules 2022, 12, 182. [CrossRef]
115. Giacomelli, L.; Zeilmaker, T.; Scintilla, S.; Salvagnin, U.; van der Voort, J.R.; Moser, C. Vitis vinifera Plants Edited in DMR6 Genes
Show Improved Resistance to Downy Mildew. bioRxiv 2022. [CrossRef]
116. Foria, S.; Copetti, D.; Eisenmann, B.; Magris, G.; Vidotto, M.; Scalabrin, S.; Testolin, R.; Cipriani, G.; Wiedemann-Merdinoglu, S.;
Bogs, J. Gene Duplication and Transposition of Mobile Elements Drive Evolution of the Rpv3 Resistance Locus in Grapevine.
Plant J. 2020, 101, 529–542. [CrossRef] [PubMed]
117. Najafi, S.; Bertini, E.; D’Incà, E.; Fasoli, M.; Zenoni, S. DNA-Free Genome Editing in Grapevine Using CRISPR/Cas9 Ribonucleo-
protein Complexes Followed by Protoplast Regeneration. Hortic. Res. 2022. [CrossRef]
118. Jiang, N.; Meng, J.; Cui, J.; Sun, G.; Luan, Y. Function Identification of MIR482b, a Negative Regulator during Tomato Resistance
to Phytophthora Infestans. Hortic. Res. 2018, 5, uhac240. [CrossRef] [PubMed]
119. Zhu, Q.; Jin, S.; Yuan, Y.; Liu, Q.; Zhang, X.; Wilson, I. CRISPR/Cas9-mediated Saturated Mutagenesis of the Cotton MIR482
Family for Dissecting the Functionality of Individual Members in Disease Response. Plant Direct 2022, 6, e410. [CrossRef]
[PubMed]
Int. J. Mol. Sci. 2023, 24, 977 27 of 30

120. Forleo, L.R.; D’Amico, M.; Basile, T.; Marsico, A.D.; Cardone, M.F.; Maggiolini, F.A.M.; Velasco, R.; Bergamini, C. Somatic
Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes. Horticulturae 2021, 7, 511.
[CrossRef]
121. Mejia, N.; Soto, B.; Guerrero, M.; Casanueva, X.; Houel, C.; de los Angeles Miccono, M.; Ramos, R.; Le Cunff, L.; Boursiquot, J.-M.;
Hinrichsen, P. Molecular, Genetic and Transcriptional Evidence for a Role of VvAGL11 in Stenospermocarpic Seedlessness in
Grapevine. BMC Plant Biol. 2011, 11, 57. [CrossRef]
122. Royo, C.; Torres-Pérez, R.; Mauri, N.; Diestro, N.; Cabezas, J.A.; Marchal, C.; Lacombe, T.; Ibáñez, J.; Tornel, M.; Carreño, J. The
Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box Gene VviAGL11. Plant Physiol. 2018,
177, 1234–1253. [CrossRef]
123. Amato, A.; Cardone, M.F.; Ocarez, N.; Alagna, F.; Ruperti, B.; Fattorini, C.; Velasco, R.; Mejía, N.; Zenoni, S.; Bergamini, C.
VviAGL11 Self-Regulates and Targets Hormone- and Secondary Metabolism-Related Genes during Seed Development. Hortic.
Res. 2022, 9, uhac133. [CrossRef]
124. Torregrosa, L.; Rienth, M.; Romieu, C.; Pellegrino, A. The Microvine, a Model for Studies in Grapevine Physiology and Genetics.
OENO One 2019, 53, 373–391. [CrossRef]
125. Nerva, L.; Guaschino, M.; Pagliarani, C.; De Rosso, M.; Lovisolo, C.; Chitarra, W. Spray-induced Gene Silencing Targeting a
Glutathione S-transferase Gene Improves Resilience to Drought in Grapevine. Plant Cell Environ. 2022, 45, 347–361. [CrossRef]
126. Clemens, M.; Faralli, M.; Lagreze, J.; Bontempo, L.; Piazza, S.; Varotto, C.; Malnoy, M.; Oechel, W.; Rizzoli, A.; Dalla Costa, L.
VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine. Front. Plant Sci. 2022, 13, 878001. [CrossRef]
[PubMed]
127. Fernandes, P.; Machado, H.; Silva, M.C.; Costa, R.L. A Histopathological Study Reveals New Insights Into Responses of Chestnut
(Castanea spp.) to Root Infection by Phytophthora cinnamomi. Phytopathology 2021, 111, 345–355. [CrossRef]
128. Beccaro, G.; Alma, A.; Bounous, G.; Gomes-Laranjo, J. The Chestnut Handbook: Crop & Forest Management; CRC Press: Boca Raton,
FL, USA, 2019; ISBN 0-429-81919-6.
129. Serrazina, S.; Santos, C.; Machado, H.; Pesquita, C.; Vicentini, R.; Pais, M.S.; Sebastiana, M.; Costa, R. Castanea Root Transcriptome
in Response to Phytophthora cinnamomi Challenge. Tree Genet. Genomes 2015, 11, 6. [CrossRef]
130. Santos, C.; Machado, H.; Correia, I.; Gomes, F.; Gomes-Laranjo, J.; Costa, R. Phenotyping Castanea Hybrids for Phytophthora
cinnamomi Resistance. Plant Pathol. 2015, 64, 901–910. [CrossRef]
131. Acquadro, A.; Torello Marinoni, D.; Sartor, C.; Dini, F.; Macchio, M.; Botta, R. Transcriptome Characterization and Expression
Profiling in Chestnut Cultivars Resistant or Susceptible to the Gall Wasp Dryocosmus kuriphilus. Mol. Genet. Genom. 2020, 295,
107–120. [CrossRef]
132. Pavese, V.; Moglia, A.; Gonthier, P.; Torello Marinoni, D.; Cavalet-Giorsa, E.; Botta, R. Identification of Susceptibility Genes in
Castanea Sativa and Their Transcription Dynamics Following Pathogen Infection. Plants 2021, 10, 913. [CrossRef]
133. Aebi, A.; Schönrogge, K.; Melika, G.; Alma, A.; Bosio, G.; Quacchia, A.; Picciau, L.; Abe, Y.; Moriya, S.; Yara, K. Parasitoid
Recruitment to the Globally Invasive Chestnut Gall Wasp Dryocosmus kuriphilus. In Galling Arthropods and Their Associates;
Springer: Tokyo, Japan, 2006; pp. 103–121.
134. Lione, G.; Giordano, L.; Turina, M.; Gonthier, P. Hail-Induced Infections of the Chestnut Blight Pathogen Cryphonectria parasitica
Depend on Wound Size and May Lead to Severe Diebacks. Phytopathology 2020, 110, 1280–1293. [CrossRef]
135. Pavese, V.; Moglia, A.; Corredoira, E.; Martínez, M.T.; Torello Marinoni, D.; Botta, R. First Report of CRISPR/Cas9 Gene Editing
in Castanea sativa Mill. Front. Plant Sci. 2021, 12, 728516. [CrossRef]
136. Pan, C.; Ye, L.; Qin, L.; Liu, X.; He, Y.; Wang, J.; Chen, L.; Lu, G. CRISPR/Cas9-mediated Efficient and Heritable Targeted
Mutagenesis in Tomato Plants in the First and Later Generations. Sci. Rep. 2016, 6, 24765. [CrossRef]
137. Qin, G.; Gu, H.; Ma, L.; Peng, Y.; Deng, X.W.; Chen, Z.; Qu, L.-J. Disruption of Phytoene Desaturase Gene Results in Albino
and Dwarf Phenotypes in Arabidopsis by Impairing Chlorophyll, Carotenoid, and Gibberellin Biosynthesis. Cell Res. 2007, 17,
471–482. [CrossRef]
138. Wilson, F.M.; Harrison, K.; Armitage, A.D.; Simkin, A.J.; Harrison, R.J. CRISPR/Cas9-Mediated Mutagenesis of Phytoene
Desaturase in Diploid and Octoploid Strawberry. Plant Methods 2019, 15, 45. [CrossRef] [PubMed]
139. Walawage, S.L.; Zaini, P.A.; Mubarik, M.S.; Martinelli, F.; Balan, B.; Caruso, T.; Leslie, C.A.; Dandekar, A.M. Deploying Genome
Editing Tools for Dissecting the Biology of Nut Trees. Front. Sustain. Food Syst. 2019, 3, 100. [CrossRef]
140. Woo, J.W.; Kim, J.; Kwon, S.I.; Corvalán, C.; Cho, S.W.; Kim, H.; Kim, S.-G.; Kim, S.-T.; Choe, S.; Kim, J.-S. DNA-Free Genome
Editing in Plants with Preassembled CRISPR-Cas9 Ribonucleoproteins. Nat. Biotechnol. 2015, 33, 1162–1164. [CrossRef] [PubMed]
141. Chen, G.; Abdeen, A.A.; Wang, Y.; Shahi, P.K.; Robertson, S.; Xie, R.; Suzuki, M.; Pattnaik, B.R.; Saha, K.; Gong, S. A Biodegradable
Nanocapsule Delivers a Cas9 Ribonucleoprotein Complex for in Vivo Genome Editing. Nat. Nanotechnol. 2019, 14, 974–980.
[CrossRef]
142. Osakabe, Y.; Liang, Z.; Ren, C.; Nishitani, C.; Osakabe, K.; Wada, M.; Komori, S.; Malnoy, M.; Velasco, R.; Poli, M. CRISPR–Cas9-
Mediated Genome Editing in Apple and Grapevine. Nat. Protoc. 2018, 13, 2844–2863. [CrossRef]
143. Pavese, V.; Moglia, A.; Abbà, S.; Milani, A.M.; Torello Marinoni, D.; Corredoira, E.; Martínez, M.T.; Botta, R. First Report on
Genome Editing via Ribonucleoprotein (RNP) in Castanea Sativa Mill. Int. J. Mol. Sci. 2022, 23, 5762. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 977 28 of 30

144. Malnoy, M.; Viola, R.; Jung, M.-H.; Koo, O.-J.; Kim, S.; Kim, J.-S.; Velasco, R.; Nagamangala Kanchiswamy, C. DNA-Free
Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins. Front. Plant Sci. 2016, 7, 1904.
[CrossRef]
145. Bertini, E.; Tornielli, G.B.; Pezzotti, M.; Zenoni, S. Regeneration of Plants from Embryogenic Callus-Derived Protoplasts of
Garganega and Sangiovese Grapevine (Vitis vinifera L.) Cultivars. Plant Cell Tissue Organ. Cult. PCTOC 2019, 138, 239–246.
[CrossRef]
146. Kuzminsky, E.; Meschini, R.; Terzoli, S.; Pavani, L.; Silvestri, C.; Choury, Z.; Scarascia-Mugnozza, G. Isolation of Mesophyll
Protoplasts from Mediterranean Woody Plants for the Study of DNA Integrity under Abiotic Stress. Front. Plant Sci. 2016, 7, 1168.
[CrossRef]
147. Gessler, C.; Patocchi, A.; Sansavini, S.; Tartarini, S.; Gianfranceschi, L. Venturia Inaequalis Resistance in Apple. Crit. Rev. Plant Sci.
2006, 25, 473–503. [CrossRef]
148. Kellerhals, M.; Baumgartner, I.; Leumann, L.; Lussi, L.; Schütz, S.; Patocchi, A. Breeding High Quality Apples with Fire Blight
Resistance. Acta Hortic. 2013, 1056, 225–230. [CrossRef]
149. Dall’Agata, M.; Pagliarani, G.; Padmarasu, S.; Troggio, M.; Bianco, L.; Dapena, E.; Miñarro, M.; Aubourg, S.; Lespinasse, Y.;
Durel, C.-E. Identification of Candidate Genes at the Dp-Fl Locus Conferring Resistance against the Rosy Apple Aphid Dysaphis
Plantaginea. Tree Genet. Genomes 2018, 14, 12. [CrossRef]
150. De Franceschi, P.; Dondini, L. Molecular Mapping of Major Genes and QTLs in Pear. In The Pear Genome; Springer: Cham,
Switzerland, 2019; pp. 113–131.
151. Peil, A.; Emeriewen, O.F.; Khan, A.; Kostick, S.; Malnoy, M. Status of Fire Blight Resistance Breeding in Malus. J. Plant Pathol.
2021, 103, 3–12. [CrossRef]
152. Predieri, S. Mutation Induction and Tissue Culture in Improving Fruits. Plant Cell Tissue Organ. Cult. 2001, 64, 185–210. [CrossRef]
153. Vanblaere, T.; Szankowski, I.; Schaart, J.; Schouten, H.; Flachowsky, H.; Broggini, G.A.; Gessler, C. The Development of a Cisgenic
Apple Plant. J. Biotechnol. 2011, 154, 304–311. [CrossRef] [PubMed]
154. Krens, F.A.; Schaart, J.G.; Van der Burgh, A.M.; Tinnenbroek-Capel, I.E.; Groenwold, R.; Kodde, L.P.; Broggini, G.A.; Gessler, C.;
Schouten, H.J. Cisgenic Apple Trees; Development, Characterization, and Performance. Front. Plant Sci. 2015, 6, 286. [CrossRef]
155. Kost, T.D.; Gessler, C.; Jänsch, M.; Flachowsky, H.; Patocchi, A.; Broggini, G.A. Development of the First Cisgenic Apple with
Increased Resistance to Fire Blight. PLoS ONE 2015, 10, e0143980. [CrossRef]
156. Fischer, T.; Malnoy, M.; Hofmann, T.; Schwab, W.; Palmieri, L.; Wehrens, R.; Schuch, L.; Müller, M.; Schimmelpfeng, H.; Velasco, R.
F1 Hybrid of Cultivated Apple (Malus × domestica) and European Pear (Pyrus communis) with Fertile F2 Offspring. Mol. Breed.
2014, 34, 817–828. [CrossRef]
157. Perchepied, L.; Chevreau, E.; Ravon, E.; Gaillard, S.; Pelletier, S.; Bahut, M.; Berthelot, P.; Cournol, R.; Schouten, H.; Vergne, E.
Successful Intergeneric Transfer of a Major Apple Scab Resistance Gene (Rvi6) from Apple to Pear and Precise Comparison of the
Downstream Molecular Mechanisms of This Resistance in Both Species. BMC Genom. 2021, 22, 843. [CrossRef]
158. Yousaf, A.; Baldi, P.; Piazza, S.; Patocch, A.; Malnoy, M. A Perspective to Durable Apple Scab Resistance by Attributing Functions
of Rvi12 Gene. In Proceedings of the 10th Rosaceae Genomics Conference, Barcelona, Spain, 1–30 July 2020.
159. Domenichini, C.; Negri, P.; Defrancesco, M.; Alessandri, S.; Bergonzoni, L.; Verde, I.; Malnoy, M.; Broggini, G.; Patocchi, A.;
Peil, A.; et al. New Breeding Technology Approaches to Improve Apple and Pear Varieties. Acta Hortic. 2023; in press.
160. Emeriewen, O.F.; Richter, K.; Piazza, S.; Micheletti, D.; Broggini, G.A.; Berner, T.; Keilwagen, J.; Hanke, M.-V.; Malnoy, M.; Peil, A.
Towards Map-Based Cloning of FB_Mfu10: Identification of a Receptor-like Kinase Candidate Gene Underlying the Malus Fusca
Fire Blight Resistance Locus on Linkage Group 10. Mol. Breed. 2018, 38, 106. [CrossRef]
161. Emeriewen, O.F.; Flachowsky, H.; Peil, A. Characterization of Genomic DNA Sequence of the Candidate Gene for FB_Mfu10
Associated with Fire Blight Resistance in Malus Species. BMC Res. Notes 2021, 14, 291. [CrossRef] [PubMed]
162. Nishitani, C.; Hirai, N.; Komori, S.; Wada, M.; Okada, K.; Osakabe, K.; Yamamoto, T.; Osakabe, Y. Efficient Genome Editing in
Apple Using a CRISPR/Cas9 System. Sci. Rep. 2016, 6, 31481. [CrossRef] [PubMed]
163. Sarker, N.C.; Ray, P.; Pfau, C.; Kalavacharla, V.; Hossain, K.; Quadir, M. Development of Functional Nanomaterials from Wheat
Bran Derived Arabinoxylan for Nucleic Acid Delivery. J. Agric. Food Chem. 2020, 68, 4367–4373. [CrossRef] [PubMed]
164. Whitaker, V.M.; Knapp, S.J.; Hardigan, M.A.; Edger, P.P.; Slovin, J.P.; Bassil, N.V.; Hytönen, T.; Mackenzie, K.K.; Lee, S.; Jung, S. A
Roadmap for Research in Octoploid Strawberry. Hortic. Res. 2020, 7, 33. [CrossRef] [PubMed]
165. Carvalho, R.F.; Carvalho, S.D.; O’Grady, K.; Folta, K.M. Agroinfiltration of Strawberry Fruit—A Powerful Transient Expression
System for Gene Validation. Curr. Plant Biol. 2016, 6, 19–37. [CrossRef]
166. Gaston, A.; Potier, A.; Alonso, M.; Sabbadini, S.; Delmas, F.; Tenreira, T.; Cochetel, N.; Labadie, M.; Prévost, P.; Folta, K.M. The
FveFT2 Florigen/FveTFL1 Antiflorigen Balance Is Critical for the Control of Seasonal Flowering in Strawberry While FveFT3
Modulates Axillary Meristem Fate and Yield. New Phytol. 2021, 232, 372–387. [CrossRef]
167. Carvalho, R.F.; Folta, K.M. Assessment of Promoters and a Selectable Marker for Development of Strawberry Intragenic Vectors.
Plant Cell Tissue Organ. Cult. PCTOC 2017, 128, 259–271. [CrossRef]
168. Kashtwari, M.; Mansoor, S.; Wani, A.A.; Najar, M.A.; Deshmukh, R.K.; Baloch, F.S.; Abidi, I.; Zargar, S.M. Random Mutagenesis in
Vegetatively Propagated Crops: Opportunities, Challenges and Genome Editing Prospects. Mol. Biol. Rep. 2022, 49, 5729–5749.
[CrossRef]
Int. J. Mol. Sci. 2023, 24, 977 29 of 30

169. Zhou, J.; Wang, G.; Liu, Z. Efficient Genome Editing of Wild Strawberry Genes, Vector Development and Validation. Plant
Biotechnol. J. 2018, 16, 1868–1877. [CrossRef]
170. Shan, Q.; Wang, Y.; Li, J.; Gao, C. Genome Editing in Rice and Wheat Using the CRISPR/Cas System. Nat. Protoc. 2014, 9,
2395–2410. [CrossRef] [PubMed]
171. Gou, Y.-J.; Li, Y.-L.; Bi, P.-P.; Wang, D.-J.; Ma, Y.-Y.; Hu, Y.; Zhou, H.-C.; Wen, Y.-Q.; Feng, J.-Y. Optimization of the Protoplast
Transient Expression System for Gene Functional Studies in Strawberry (Fragaria vesca). Plant Cell Tissue Organ. Cult. PCTOC
2020, 141, 41–53. [CrossRef]
172. Martín-Pizarro, C.; Triviño, J.C.; Posé, D. Functional Analysis of the TM6 MADS-Box Gene in the Octoploid Strawberry by
CRISPR/Cas9-Directed Mutagenesis. J. Exp. Bot. 2019, 70, 885–895. [CrossRef]
173. Cappelletti, R.; Sabbadini, S.; Mezzetti, B. Strawberry (Fragaria × ananassa). In Agrobacterium Protocols; Springer: New York, NY,
USA, 2015; pp. 217–227.
174. Dirlewanger, E.; Kleinhentz, M.; Voisin, R.; Claverie, M.; Lecouls, A.; Esmenjaud, D.; Poessel, J.; Faurobert, M.; Arús, P.;
Gómez-Aparisi, J.; et al. Breeding for a New Generation of Prunus Rootstocks: An Example of MAS. Acta Hortic. 2004, 658,
581–590. [CrossRef]
175. Verde, I.; Bassil, N.; Scalabrin, S.; Gilmore, B.; Lawley, C.T.; Gasic, K.; Micheletti, D.; Rosyara, U.R.; Cattonaro, F.;
Vendramin, E.; et al. Development and Evaluation of a 9K SNP Array for Peach by Internationally Coordinated SNP De-
tection and Validation in Breeding Germplasm. PLoS ONE 2012, 7, e35668. [CrossRef]
176. Peace, C.; Bassil, N.; Main, D.; Ficklin, S.; Rosyara, U.R.; Stegmeir, T.; Sebolt, A.; Gilmore, B.; Lawley, C.; Mockler, T.C. Development
and Evaluation of a Genome-Wide 6K SNP Array for Diploid Sweet Cherry and Tetraploid Sour Cherry. PLoS ONE 2012, 7, e48305.
[CrossRef]
177. Aranzana, M.J.; Decroocq, V.; Dirlewanger, E.; Eduardo, I.; Gao, Z.S.; Gasic, K.; Iezzoni, A.; Jung, S.; Peace, C.; Prieto, H. Prunus
Genetics and Applications after de Novo Genome Sequencing: Achievements and Prospects. Hortic. Res. 2019, 6, 58. [CrossRef]
178. Dardick, C.; Callahan, A.; Horn, R.; Ruiz, K.B.; Zhebentyayeva, T.; Hollender, C.; Whitaker, M.; Abbott, A.; Scorza, R. P Pe TAC 1
Promotes the Horizontal Growth of Branches in Peach Trees and Is a Member of a Functionally Conserved Gene Family Found in
Diverse Plants Species. Plant J. 2013, 75, 618–630. [CrossRef]
179. Claverie, M.; Dirlewanger, E.; Bosselut, N.; Van Ghelder, C.; Voisin, R.; Kleinhentz, M.; Lafargue, B.; Abad, P.; Rosso, M.-N.;
Chalhoub, B.; et al. The Ma Gene for Complete-Spectrum Resistance to Meloidogyne Species in Prunus Is a TNL with a Huge
Repeated C-Terminal Post-LRR Region. Plant Physiol. 2011, 156, 779–792. [CrossRef]
180. Van Ghelder, C.; Esmenjaud, D.; Callot, C.; Dubois, E.; Mazier, M.; Duval, H. Ma Orthologous Genes in Prunus spp. Shed Light
on a Noteworthy NBS-LRR Cluster Conferring Differential Resistance to Root-Knot Nematodes. Front. Plant Sci. 2018, 9, 1269.
[CrossRef]
181. Claverie, M.; Bosselut, N.; Lecouls, A.; Voisin, R.; Lafargue, B.; Poizat, C.; Kleinhentz, M.; Laigret, F.; Dirlewanger, E.;
Esmenjaud, D. Location of Independent Root-Knot Nematode Resistance Genes in Plum and Peach. Theor. Appl. Genet. 2004, 108,
765–773. [CrossRef] [PubMed]
182. Handoo, Z.; Nyczepir, A.; Esmenjaud, D.; Van der Beek, J.; Castagnone-Sereno, P.; Carta, L.; Skantar, A.; Higgins, J. Morphological,
Molecular, and Differential-Host Characterization of Meloidogyne floridensis n. Sp. (Nematoda: Meloidogynidae), a Root-Knot
Nematode Parasitizing Peach in Florida. J. Nematol. 2004, 36, 20. [PubMed]
183. Clemente-Moreno, M.J.; Hernández, J.A.; Diaz-Vivancos, P. Sharka: How Do Plants Respond to Plum pox virus Infection? J. Exp.
Bot. 2015, 66, 25–35. [CrossRef] [PubMed]
184. Robaglia, C.; Caranta, C. Translation Initiation Factors: A Weak Link in Plant RNA Virus Infection. Trends Plant Sci. 2006, 11,
40–45. [CrossRef]
185. Rodamilans, B.; Valli, A.; García, J.A. Molecular Plant-Plum pox virus Interactions. Mol. Plant. Microbe Interact. 2020, 33, 6–17.
[CrossRef]
186. Rubio, J.; Sánchez, E.; Tricon, D.; Montes, C.; Eyquard, J.-P.; Chague, A.; Aguirre, C.; Prieto, H.; Decroocq, V. Silencing of One
Copy of the Translation Initiation Factor EIFiso4G in Japanese Plum (Prunus salicina) Impacts Susceptibility to Plum pox virus
(PPV) and Small RNA Production. BMC Plant Biol. 2019, 19, 440. [CrossRef]
187. Miccoli, C.; Gambacorta, G.; Urbinati, G.; Santiago-Reyes, M.; Gentile, A.; Monticelli, S.; Caboni, E.; Prieto, H.; Verde, I.;
Decroocq, V.; et al. Novel Breeding Strategies for Tackling Present and Future Challenges in Prunus Species. Acta Hortic. 2022,
1352, 419–426. [CrossRef]
188. Miccoli, C.; Gambacorta, G.; Urbinati, G.; Santiago Reyes, M.; Gentile, A.; Vona, S.; Monticelli, S.; Caboni, E.; Verde, I.;
Vendramin, E.; et al. Application of New Breeding Techniques to Improve Important Agronomical Traits in Prunus Species. In
LXIV SIGA Annual Congress “Plant Genetic Innovation for Food Security in a Climate Change Scenario”; Italian Society of Agricultural
Genetics: Portici, Italy, 2021; ISBN 978-88-944843-2-8. Available online: http://www.geneticagraria.it/congress_abstract.asp?a_
pag=4&id=68#key_M (accessed on 20 December 2022).
189. Glenn, D.; Bassett, C.; Tworkoski, T.; Scorza, R.; Miller, S. Tree Architecture of Pillar and Standard Peach Affect Canopy
Transpiration and Water Use Efficiency. Sci. Hortic. 2015, 187, 30–34. [CrossRef]
190. Hollender, C.A.; Pascal, T.; Tabb, A.; Hadiarto, T.; Srinivasan, C.; Wang, W.; Liu, Z.; Scorza, R.; Dardick, C. Loss of a Highly
Conserved Sterile Alpha Motif Domain Gene (WEEP) Results in Pendulous Branch Growth in Peach Trees. Proc. Natl. Acad. Sci.
USA 2018, 115, E4690–E4699. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 977 30 of 30

191. Ku, L.; Wei, X.; Zhang, S.; Zhang, J.; Guo, S.; Chen, Y. Cloning and Characterization of a Putative TAC1 Ortholog Associated with
Leaf Angle in Maize (Zea mays L.). PLoS ONE 2011, 6, e20621. [CrossRef]
192. Li, H.; Zhang, L.; Hu, J.; Zhang, F.; Chen, B.; Xu, K.; Gao, G.; Li, H.; Zhang, T.; Li, Z. Genome-Wide Association Mapping Reveals
the Genetic Control Underlying Branch Angle in Rapeseed (Brassica napus L.). Front. Plant Sci. 2017, 8, 1054. [CrossRef] [PubMed]
193. Zhao, H.; Huai, Z.; Xiao, Y.; Wang, X.; Yu, J.; Ding, G.; Peng, J. Natural Variation and Genetic Analysis of the Tiller Angle Gene
MsTAC1 in Miscanthus sinensis. Planta 2014, 240, 161–175. [CrossRef] [PubMed]
194. Xu, D.; Qi, X.; Li, J.; Han, X.; Wang, J.; Jiang, Y.; Tian, Y.; Wang, Y. PzTAC and PzLAZY from a Narrow-Crown Poplar Contribute
to Regulation of Branch Angles. Plant Physiol. Biochem. 2017, 118, 571–578. [CrossRef] [PubMed]
195. Flachowsky, H.; Le Roux, P.; Peil, A.; Patocchi, A.; Richter, K.; Hanke, M. Application of a High-speed Breeding Technology to
Apple (Malus × domestica) Based on Transgenic Early Flowering Plants and Marker-assisted Selection. New Phytol. 2011, 192,
364–377. [CrossRef]
196. Zhu, Y.; Klasfeld, S.; Jeong, C.W.; Jin, R.; Goto, K.; Yamaguchi, N.; Wagner, D. TERMINAL FLOWER 1-FD Complex Target Genes
and Competition with FLOWERING LOCUS T. Nat. Commun. 2020, 11, 5118. [CrossRef] [PubMed]
197. Jin, S.; Nasim, Z.; Susila, H.; Ahn, J.H. Evolution and Functional Diversification of FLOWERING LOCUS T/TERMINAL FLOWER
1 Family Genes in Plants. Semin. Cell Dev. Biol. 2020, 109, 20–30. [CrossRef] [PubMed]
198. Srinivasan, C.; Dardick, C.; Callahan, A.; Scorza, R. Plum (Prunus domestica) Trees Transformed with Poplar FT1 Result in Altered
Architecture, Dormancy Requirement, and Continuous Flowering. PLoS ONE 2012, 7, e40715. [CrossRef]
199. Ferguson, A.R. Botanical Description. In The Kiwifruit Genome; Springer: Cham, Vietnam, 2016; pp. 1–13. [CrossRef]
200. Datson, P.; Ferguson, A. Actinidia. In Wild Crop Relatives: Genomic and Breeding Resources; Springer: Berlin/Heidelberg, Germany,
2011; pp. 1–20.
201. Ferguson, A.R.; Huang, H. Genetic Resources of Kiwifruit: Domestication and Breeding. Hortic. Rev. 2007, 33, 1–121.
202. Ferrante, P.; Scortichini, M. Identification of Pseudomonas Syringae Pv. Actinidiae as Causal Agent of Bacterial Canker of Yellow
Kiwifruit (Actinidia chinensis Planchon) in Central Italy. J. Phytopathol. 2009, 157, 768–770. [CrossRef]
203. Donati, I.; Cellini, A.; Sangiorgio, D.; Vanneste, J.L.; Scortichini, M.; Balestra, G.M.; Spinelli, F. Pseudomonas syringae Pv. Actinidiae:
Ecology, Infection Dynamics and Disease Epidemiology. Microb. Ecol. 2020, 80, 81–102. [CrossRef]
204. Vanneste, J.L. The Scientific, Economic, and Social Impacts of the New Zealand Outbreak of Bacterial Canker of Kiwifruit
(Pseudomonas syringae Pv. Actinidiae). Annu. Rev. Phytopathol. 2017, 55, 377–399. [CrossRef] [PubMed]
205. Cellini, A.; Donati, I.; Farneti, B.; Khomenko, I.; Buriani, G.; Biasioli, F.; Cristescu, S.M.; Spinelli, F. A Breach in Plant Defences:
Pseudomonas syringae Pv. Actinidiae Targets Ethylene Signalling to Overcome Actinidia chinensis Pathogen Responses. Int. J. Mol.
Sci. 2021, 22, 4375. [CrossRef] [PubMed]
206. Michelotti, V.; Lamontanara, A.; Buriani, G.; Orrù, L.; Cellini, A.; Donati, I.; Vanneste, J.L.; Cattivelli, L.; Tacconi, G.; Spinelli, F.
Comparative Transcriptome Analysis of the Interaction between Actinidia chinensis Var. Chinensis and Pseudomonas syringae Pv.
Actinidiae in Absence and Presence of Acibenzolar-S-Methyl. BMC Genom. 2018, 19, 585. [CrossRef] [PubMed]
207. Cellini, A.; Fiorentini, L.; Buriani, G.; Yu, J.; Donati, I.; Cornish, D.; Novak, B.; Costa, G.; Vanneste, J.; Spinelli, F. Elicitors of the
Salicylic Acid Pathway Reduce Incidence of Bacterial Canker of Kiwifruit Caused by Pseudomonas syringae Pv. Actinidae. Ann.
Appl. Biol. 2014, 165, 441–453. [CrossRef]
208. Collina, M.; Donati, I.; Bertacchini, E.; Brunelli, A.; Spinelli, F. Greenhouse Assays on the Control of the Bacterial Canker of
Kiwifruit (Pseudomonas syringae Pv. Actinidiae). J. Berry Res. 2016, 6, 407–415. [CrossRef]
209. Wu, X.; Kriz, A.J.; Sharp, P.A. Target Specificity of the CRISPR-Cas9 System. Quant. Biol. 2014, 2, 59–70. [CrossRef]
210. Caboni, E.; Biasi, R.; Delia, G.; Tonelli, M. Effect of CPPU on in Vitro Axillary Shoot Proliferation and Adventitious Shoot
Regeneration in Kiwifruit. Plant Biosyst. 2009, 143, 456–461. [CrossRef]
211. Prado, M.; Gonzalez, M.; Romo, S.; Herrera, M. Adventitious Plant Regeneration on Leaf Explants from Adult Male Kiwifruit and
AFLP Analysis of Genetic Variation. Plant Cell Tissue Organ. Cult. 2007, 88, 1–10. [CrossRef]
212. Michelotti, V.; Urbinati, G.; Gentile, A.; Lucioli, S.; Caboni, E.; Tacconi, G. Preliminary Results on the Development of a Genome
Editing Protocol in Actinidia chinensis Var. Chinensis as Psa Resistance Approach. Acta Hortic. 2022, 1332, 111–116. [CrossRef]
213. Ma, X.; Zhang, Q.; Zhu, Q.; Liu, W.; Chen, Y.; Qiu, R.; Wang, B.; Yang, Z.; Li, H.; Lin, Y. A Robust CRISPR/Cas9 System for
Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol. Plant 2015, 8, 1274–1284. [CrossRef]
214. Mbaya, H.; Lillico, S.; Kemp, S.; Simm, G.; Raybould, A. Regulatory Frameworks Can Facilitate or Hinder the Potential for
Genome Editing to Contribute to Sustainable Agricultural Development. Front. Bioeng. Biotechnol. 2022, 10, 959236. [CrossRef]
[PubMed]
215. Weidner, C.; Edelmann, S.; Moor, D.; Lieske, K.; Savini, C.; Jacchia, S.; Sacco, M.G.; Mazzara, M.; Lämke, J.; Eckermann, K.N.
Assessment of the Real-Time PCR Method Claiming to Be Specific for Detection and Quantification of the First Commercialised
Genome-Edited Plant. Food Anal. Methods 2022, 15, 2107–2125. [CrossRef]
216. Gallinella, C.; Cillis, G.; L’abbate, M.; Manca, A.; Pignatone, D.C.G. AC 3393. Deputy Chamber of Italy. 2021. Available online:
https://documenti.camera.it/leg18/pdl/pdf/leg.18.pdl.camera.3310.18PDL0162050.pdf (accessed on 20 December 2022).

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