2014 BiotechAdv Bergougnoux

Biotechnology Advances 32 (2014) 170189
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
The history of tomato: From domestication to biopharming

Vronique Bergougnoux 1
Department of Molecular Biology, Center of the Region Han for Biotechnological Research, Palack University, lechtitel 11, 783 71 Olomouc, Czech Republic
a r t i c l e
i n f o
Available online 7 November 2013

Keywords:
Tomato
Domestication
Breeding
Genetic engineering
Biopharming
a b s t r a c t
Imported from the Andean region to Europe in the 16th century, today tomato is widespread throughout the
world and represents the most economically important vegetable crop worldwide. Tomato is not only traded
in the fresh market but is also used in the processing industry in soups, as paste, concentrate, juice, and ketchup.
It is an incredible source of important nutrients such as lycopene, -carotene and vitamin C, which all have
positive impacts on human health. Its production and consumption is increasing with population growth. In
this review, we report how tomato was already domesticated by the ancient Incan and Aztec civilizations, and
how it came to Europe, where its breeding history started. The development of genetic, molecular biology and
plant biotechnology have opened the doors towards the modern genetic engineering of tomato. The different
goals of tomato genetic engineering are presented, as well as examples of successfully engineered tomatoes in
terms of resistance to biotic and abiotic stresses, and fruit quality. The development of GM tomato for
biopharming is also described.
2013 Elsevier Inc. All rights reserved.
Contents
1.
Introduction to tomato . . . . . . . . . . . . . . . . . . . . . .
1.1.
Economic importance of tomato and its botanical description .
1.2.
Habitat and diversity of tomato . . . . . . . . . . . . . . .
1.3.
History of domestication . . . . . . . . . . . . . . . . . .
1.4.
The tomato today: a model organism for scientists . . . . . .
2.
Challenges of tomato breeding and genetic bases of important traits . .
2.1.
Tomato yields . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Resistance to biotic and abiotic stresses . . . . . . . . . . .
2.3.
Size and shape of fruit . . . . . . . . . . . . . . . . . . .
2.4.
Ripening and color establishment . . . . . . . . . . . . . .
2.5.
Nutritional value . . . . . . . . . . . . . . . . . . . . . .
2.6.
Limits of classical breeding: the new area of genetic engineering
3.
Genetic engineering of tomato . . . . . . . . . . . . . . . . . . .
3.1.
Agrobacterium-mediated transformation of tomato . . . . . .
3.2.
Selection of transgenic plants . . . . . . . . . . . . . . . .
3.3.
Temporal and/or organ-specic expression: choice of promoter
4.
Transgenic tomatoes with enhanced agronomic traits . . . . . . . .
4.1.
Resistance to biotic stresses . . . . . . . . . . . . . . . . .
4.2.
Resistance to abiotic stresses . . . . . . . . . . . . . . . .
4.3.
Fruit quality . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Biopharming . . . . . . . . . . . . . . . . . . . . . . .
5.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E-mail address: veronique.bergougnoux@upol.cz.

Tel.: +420 585 634 740.
0734-9750/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.11.003
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V. Bergougnoux / Biotechnology Advances 32 (2014) 170189
1. Introduction to tomato
1.1. Economic importance of tomato and its botanical description
Today, tomato is not only sold fresh but also processed as paste,
soup, juice, sauce, powder, concentrate or whole. Tomato is one of the
most consumed vegetables in the world, after potatoes and before onions (FAOSTAT, http://faostat3.fao.org/home/index.html), and probably
the most preferred garden crop. With worldwide production reaching
almost 160 million tons in 2011, tomato is the seventh most important
crop species after maize, rice, wheat, potatoes, soybeans and cassava.
During the last 20 years, tomato production, as well as the area dedicated to its culture, has doubled (Fig. 1A). Surprisingly, whereas 20 years
ago, Europe and the Americas represented the most important producers, today Asia dominates the tomato market with China ranking
rst, followed in decreasing order by India, USA, Turkey, Egypt, Iran,
Italy, Brazil, Spain and Uzbekistan (Fig. 1BC). Interestingly the countries harboring the highest yield are from northern Europe, where the
climatic conditions are not favorable to the culture of tomato and
where the area dedicated to tomato culture is very small (Table 1). It
is noteworthy that these countries produce most of their tomatoes
171
under controlled greenhouse conditions. The recent increase in tomato

production responds to the increased consumption of tomatoes during
the same period (Fig. 1A), reaching an average consumption of
20.5 kg/capita/year in 2009. The three countries where tomato is
consumed the most are Libya, Egypt and Greece, with consumption
exceeding 100 kg/capita/year. From a general point of view, it is in
the Mediterranean and Arabian countries that the consumption of
tomatoes is the highest with averages between 40 and 100 kg/capita/
year (Table 2).
From the botanical point of view, tomato (Solanum lycopersicum L.)
is a fruit berry, and not a vegetable. This misunderstanding was a question of debates during the 19th century in USA, with the special case of
Nix vs. Hedden 149 U.S. 304 (1893). In 1887, Nix contested the decision of the tax collector of the port of New York to recover taxes on
tomatoes imported from the West Indies in the spring of 1886, which
the collector assessed as a vegetable. The court opined: Botanically
speaking, tomatoes are the fruit of a vine, just as are cucumbers,
squashes, beans, and peas. But in the common language of the people,
[] all these are vegetables which are grown in kitchen gardens, and
which, whether eaten cooked or raw, are, like potatoes, carrots, parsnips,
turnips, beets, cauliower, cabbage, celery, and lettuce, usually served at
Fig. 1. Metrics of tomato production in the world. 1A Tomato production and the area dedicated to tomato culture worldwide for the period 19902011 The frame depicts the increase in
tomato consumption for the same period. 1B Weight (in %) of the different continents in tomato production, comparison between 1990 and 2010. 1C Production of the nine leading
producers (source: FAO Statistics; http://faostat3.fao.org/home/index.html).
172
Table 1
Tomato yield in different countries and comparison with production rank and harvested area.
1
2
3
4
5
6
7
8
9
10
19
24
35
40
45
46
48
55
57
92
Country
Yield (Hg/Ha)
Rank of production
Harvested area (Ha)
Netherlands
Belgium
Norway
United Kingdom
Ireland
Iceland
Denmark
Finland
Sweden
Austria
United States of America
Spain
Brazil
Italy
China
Uzbekistan
Turkey
Egypt
Iran
India
4,788,484
4,608,333
4,237,742
4,157,407
4,131,563
4,012,500
3,550,000
3,523,070
2,821,458
2,723,730
848,833
765,630
617,947
572,919
492,714
445,690
408,162
381,521
371,025
194,520
25
55
117
75
116
143
113
93
115
87
3
9
8
7
1
10
4
5
6
2
1,702
474
31
216
32
4
40
114
48
185
148,730
49,913
71,473
103,858
985,903
58,000
269,584
212,446
183,931
865,000
dinner in, with, or after the soup, sh, or meats which constitute the
principal part of the repast, and not, like fruits generally, as dessert.
(http://supreme.justia.com/cases/federal/us/149/304/case.html).
The tomato belongs to the Solanaceae family, containing more than
3000 species including many plants of economic importance including
potatoes, eggplants, petunias, tobacco, peppers (Capsicum) and Physalis.
Solanum is the largest genus in the Solanaceae family, encompassing
1250 to 1700 species. Species of the Solanum genus are present on all
temperate and tropical continents and are remarkable for their morphological and ecological diversity. Solanum is probably the most economically important genus, containing crop species and many other species
producing poisonous or medicinal compounds (Weese and Bohs,
2007). Since its introduction in Europe in the 16th century, it was assumed that the tomato was closely related to the genus Solanum and
was identied as Solanum pomiferum. In 1753, Linnaeus classied for
the rst time tomatoes in the genus Solanum under the specic name
of S. lycopersicum. Nevertheless, the genus and designation of tomato
were for a long time a subject of debate, as reported by several authors
(Foolad, 2007; Peralta and Spooner, 2007). The use of molecular data
allowed revision of the phylogenetic classication of the Solanaceae
and the genus Lycopersicon was re-introduced in the Solanum genus
into the section Lycopersicon. It is interesting to note that 200 years of
debate were necessary to conrm the description of Linnaeus. The
ingroup organization of tomato has also evolved since the rst
Table 2
Food supply quantity (kg/capita/year) in the 15 countries consuming the most tomatoes.
The countries with the highest production are highlighted in pale gray.
Country
Food supply quantity (kg/capita/year)
Rank of production
Libya
Egypt
Greece
Tunisia
Turkey
Armenia
Lebanon
Uzbekistan
Iran
Italy
Spain
Cuba
United Arab Emirates
Portugal
Turkmenistan
150.3
115.9
105.3
94.9
90.5
87.3
75.4
74.4
71.6
60.5
58.9
58.7
57.8
57.7
50.2
54
5
18
101
13
50
45
106
6
7
133
33
3
16
68
description in 1940 done by Mller, who identied six tomato species separated into two sections: subgenus eulycopersicon, including
Lycopersicon esculentum and L. pimpinellifolium; and the subgenus
eriopersicon, regrouping L. peruvianum, L. cheesmaniae, L. hirsutum
and L. glandulosum. Rick (1960, 1979) proposed a tomato classication relying on the ability of the wild species to cross with the cultivated tomato. He recognized nine wild tomato species divided into
two main groups according their crossability: the Esculentum and
Peruvianum complexes. All the species of the Esculentum complex
(L. esculentum, L. pimpinellifolium, L. cheesmaniae, L. pennellii, L. hirsutum,
L. chmielewskii and L. parviorum) can be hybridized with the cultivated
tomato and represent potential sources of resistance to biotic and
abiotic stresses as well as other desirable characters; the species
from the Peruvianum complex (L. chilense and L. peruvianum) are extremely diverse and represent real potential for crop improvement.
The use of these latter species was strongly limited due to their
poor hybridization capacity with the cultivated tomato and the necessity to develop special methods such as embryo rescue (Foolad,
2007). The actual description of the tomato is more complex than
was thought initially and Peralta and Spooner (2001) reported the
nal organization of the Solanum sec. Lycopersicon. Their phylogenetic study, based on the sequence of the granule-bound starch synthase (GBSSI) gene, allowed them to ascertain the outgroup
organization within the Solanum subgenus Potatoe. This study supported Solanum juglandifolium and Solanum ochranthum as the closest
outgroup of tomato (Fig. 2) and divided nally the tomato into three
groups: sect. Lycopersicon subsect. Lycopersicon, ser. Lycopersicon, ser.
Eriopersicon and ser. Neolycopersicon. The nal classication of tomato
recognizes the cultivated tomato (S. lycopersicum) and its twelve wild relatives, the two species S. galapense and S. cheesmaniae being endemic to
the Galapagos Islands. The species S. peruvianum was divided into northern and southern species, and a more precise analysis identied four species: S. arcanum, S. huaylasense, S. peruvianum and S. corneliomulleri
(Peralta et al., 2005, 2008). The different wild tomato species, according
to their relationships into the Solanum genus, are summarized in
Table 3.
1.2. Habitat and diversity of tomato
Wild tomato species are native to western South America along the
coast and high Andes from central Ecuador, through Peru, to northern
Chile, and in the Galapagos Islands. Consequently tomato wild species
grow in a variety of habitats ranging from sea level on the Pacic coast
up to 3300 m asl. in the Andean Highlands, and from arid to rainy
173
Fig. 2. General phylogenetic tree based on the analysis of GBSSI gene sequences from 65 accessions of the 9 tomato species and 14 outgroup taxa. Numbers indicate bootstrap values, and
decay values are indicated between parentheses.
Extracted from Peralta and Spooner (2005).
climates (Table 3). Tomato wild species are often restricted to narrow
and isolated valleys where they adapted to particular climatic and soil
types. It is probable that the Andean geography, the diverse ecological
habitats and the different climates together contributed to wild tomato
diversity. This hypothesis is supported by a very recent study based on
the two closely related wild tomato species S. lycopersicum and S.
pimpinellifolium (Nakazato and Housworth, 2011). This diversity is
expressed through morphological, physiological and sexual characteristics (Peralta and Spooner, 2005; Spooner et al., 2005).
The mating system plays a key role in species diversication and
outcrossing constitutes an important and widespread strategy for maintaining genetic variability. Genetically controlled self-incompatibility
is a very common mechanism in the plant kingdom. In tomato, the
self-incompatibility is gametophytic and varies from allogamous selfincompatible, to facultative allogamous, to autogamous and selfcompatible (Table 3). Self-incompatibility is strongly correlated
with the degree of outcrossing, allelic diversity, oral display and degree of stigma exsertion in wild tomatoes (Peralta et al., 2008). Indeed,
an exserted stigma above the anthers will promote outcrossing by buzz
pollination, whereas a recessed stigma below the anthers will promote
self-fertilization (Chen and Tanksley, 2004). By investigating the bases
of self-compatibility/-incompatibility and ower characteristics, Rick
(1982) came to the conclusion that the mating system evolved from
self-incompatible, as the ancestral condition, to self-compatible. Changes from self-incompatibility to self-compatibility are events which are
expected to happen infrequently and independently. This is the case
of S. habrochaites and S. pennellii, for which both self-compatible and
self-incompatible populations have been identied (Rick, 1982). The
self-incompatible populations have a higher degree of diversity, larger
ower parts, and exserted stigma, as opposed to self-compatible populations characterized by reduced genetic diversity, smaller ower parts
and little or no stigma exsertion. Thus, in tomato, ower stigma exsertion and gametophytic incompatibility contribute to greater
outcrossing and genetic diversity.
1.3. History of domestication
Despite the centuries since tomato was introduced to Europe, the
origin of its domestication is still unclear, and two hypotheses are still
being contended: the Peruvian and Mexican hypotheses. How can the
place of domestication of a crop be determined? In an attempt to
solve this question, DeCandolle (1886) used an approach combining
botany, archeology and paleontology, history and philology. Botany
consists of observing the natural occurrence of the crop and/or its putative relative wild species; archeology and paleontology focus on studying fossils from caves, burial sites or other preserved deposits; history
looks for evidence of the crop in the early reports of people; and nally
philology or linguistic evidence is based on the comparison of native
names to prior languages.
DeCandolle expressed for the rst time the Peruvian origin of tomato
domestication. His conclusions were based on the fact that: i) no unambiguous natural records of tomato were identied out of the Americas
after its European discovery, ii) mala peruviana and pomi del Peru
were used to refer to the tomato, suggesting its initial domestication
and transport from Peru to Europe, iii) the cultivated tomato was
thought to originate from the wild cherry tomato which was known
to be localized from coastal Peru through Mexico to the southwestern
USA (California), iv) the distribution of the cultivated tomato and its
progenitors arose from Peru by garden escapes, v) the domestication occurred before the discovery of America but not very long before that
(reviewed in Peralta and Spooner, 2007). The Mexican origin of domestication was proposed by Jenkins in 1948. It was mainly justied by the
fact that no evidence of pre-Colombian tomato cultivation in South
America was available, compared to good evidence in Mexico. Following
the philology, Jenkins also argued that the name tomato comes from
the Mexican Nahua word tomatl which refers to plants bearing
globous and juicy fruit (Bauchet and Causse, 2012). However to date,
the origin of the domestication of tomato is unsolved, even though it
has been reported that tomatoes from Europe and North America
share similar isozymes and molecular markers with those from Mexico
and Central America, suggesting that the tomato was introduced to
Europe and North America from these regions (Bauchet and Causse,
2012; Peralta and Spooner, 2007). None of the hypotheses on the origin
of tomato domestication is more conclusive than the other. It might be
that domestication occurred independently in both regions. The most
likely ancestor of cultivated tomatoes is the wild cherry tomato, usually
identied as S. lycopersicum var. cerasiforme because of its wide representation in Central America. Nevertheless the genetic investigations made
by Nesbitt and Tanksley in 2002 demonstrated that the plants known as
cerasiforme are a mixture of wild and cultivated tomatoes, rather
than the direct ancestor of the cultivated tomato. A very recent
study based on the analysis of single nucleotide polymorphisms
not only conrms that S. lycopersicum var. cerasiforme is not the ancestor of the cultivated tomato but also reinforces the model that a
pre-domestication of the tomato occurred in the Andean region
(Peruvian hypothesis), with the domestication being completed in
174
Table 3
Comparison of wild tomato species (Solanum L. section Lycopersicon subsection Lycopersicon) adapted from Peralta et al. (2005) and Spooner et al. (2005). SC: self-compatible, SI: self-incompatible, At: autogamous, Al: allogamous.
Lycopersicon equivalent
Distribution and habitat
Fruit color
Reproductive system
Importance for breeding purposes
S. cheesmaniae
L. cheesmaniae
Yellow, orange
SC, exclusively At
Salt tolerance; Lepidoptera and virus resistance

Salt tolerance; Lepidoptera and virus resistance
S. galapagense
L. cheesmaniae var. minor
Yellow, orange
SC, exclusively At
S. lycopersicum
L. esculentum
Red
SC, facultative Al
Moisture-tolerance, resistance to wilt,

root-rotting, and leaf-spotting fungi
S. pimpinellifolium
L. pimpinellifolium
Red
SC, At, facultative Al
S. chilense
L. chilense
Green, purple stripes
SC, Al
Color and fruit quality; resistance to insect,

nematode and disease
Drought resistance
S. chmielewskii
S. habrochaites
L. chmielewskii
L. hirsutum
Green
Green
SC, facultative Al
SI
High sugar content

Cold and frost tolerance; resistance to insects
due to their glandular hairs
S. pennellii
L. pennellii
Green
SI
Drought resistance; resistance to insects
S. neorickii
L. parviorum
Endemic to the Galapagos Islands, Ecuador; wide variety of

habitat; sea level to 500 m
Endemic to the Galapagos Islands; mostly occurring on
coastal lava to within 1 m of high tide mark within range of
sea spray, but occasionally inland; sea level to 50 m
Known only from cultivation or escapes; world-wide
in a variety of habitats, many escaped plants have smaller
fruits (cerasiforme); sea level to 4000 m
Central Ecuador to central Chile; dry coastal habitats;
0-500 m but exceptionally up to 1400 m
S Peru to N Chile; in hyper-arid rocky plains and coastal
deserts; sea level to 3250 m
S Peru to N Bolivia; high dry Andean valleys; 1600-3200 m
Central Ecuador to Central Peru, on the western slopes of the Andes;
in a variety of forest types from premontane forest to
dry forests; (40)-200-3300 m
N Peru to N Chile; dry rocky hillsides and sandy areas; sea
level to 2300 m
S Ecuador to S Peru; dry inter-Andean valleys, often found
trailing over rocky banks and roadsides; (920)-1950-2600 m
N Peru, coastal and inland Andean valleys; lomas, dry valleys
and dry rocky slopes; 100 to 2800 m
N Peru; rocky slopes of the Callejon de Huaylas along the Rio
Santa and in the adjacent Rio Fortaleza drainage;
(940)1700-3000 m
Central Peru to N Chile; coastal lomas formations and
occasionally in coastal deserts, occasionally as a weed at eld
edges in coastal river valleys; sea level to 600 m
Central to S Peru, W slops of the Andes; landslides and rocky
slopes; (40)200-3300 m
Pale green
SC, At
Green
Typically SI, Al, rare

population
SC, At with a trend to reduce
variability in Northern races
S. peruvianum
north
S. peruvianum
south
S. arcanum
S. huaylasense
L. peruvianum
var. hirsutum
L. peruvianum
S. peruvianum
L. peruvianum
S. corneliomuelleri
L. peruvianum
var. glandulosum
Green
Green
Green
Resistance to virus, bacteria, fungi,

aphid and nematode
Species (Solanum name)
Mesoamerica (Mexican hypothesis), followed by its introduction to

Europe by Spaniards and then spread all over the world (Blanca et al.,
2012).
The history of tomato's use (and probable consequent domestication) was reported by George McCue (1952), who did a remarkable bibliographical investigation. It was probably the Spanish conquistador
Cortes who rst introduced the small yellow tomato to Spain after the
capture in 1521 of Tenochtitlan, the Aztec city known today as Mexico
City. From Spain, the tomato reached Italy through Naples, which was
Spanish property at that time. The rst description of the tomato in
Europe was found in a herbarium written in 1544 by Petrus Matthiolius.
Due to its botanical closeness with the mandrake, Matthiolius described
the tomato thusly: another species [of Mandrake] has been brought to
Italy in our time, attened like the melerose and segmented, green at
rst and when ripe of a golden color, which is eaten in the same manner
[as the eggplant fried in oil with salt and pepper, like mushrooms].
Tomato was probably used for human consumption very early after its
introduction to Europe as cookbooks referred to its use in gazpacho by
the beginning of the 17th century. Nevertheless, due to its resemblance
with toxic Solanum, such as mandrake and belladonna, the tomato was
long used only for ornamental purposes. Thus, in Italy, the fruit was
used solely as decoration and was incorporated into the local cuisine
only late in the 17th or early 18th century. In 1760, Blois reported that
tomato was used in France for its ornamental properties and 18 years
later, he mentioned that the catalog of seeds of the Maison grainiere
Andrieux Vilmorin, which still exists to this day, offered seeds of tomato as a vegetable. Following a south/north axis, tomato consumption
expanded to the north. In England, tomato consumption was very
common by the mid-18th century. From England, tomatoes were
exported to the Middle East/Asia by John Baker, British consul in
Aleppo. Tomatoes migrated then to North America due to English
colonization. The real domestication of the tomato as an edible vegetable started during the 19th century. Thus, in 1820, Sabine referenced that four red tomatoes and two yellow were cultivated in
Europe; he even gave advice on how to cultivate them in the specic
conditions of England. In America, Alexander W. Livingston promoted the tomato and was the rst to be able to improve the wild tomato, developing and stabilizing the plants. This contribution to tomato
development in the USA was so important that in 1937 it was admitted that the majority of the varieties resulted from the ability of
Livington to evaluate and perpetuate superior material in the
tomato.
The numerous cultivars available since the end of the 19th century
were produced by open pollination under the auspices of farms or
small collectives. Development of new cultivars happened by spontaneous mutation, natural outcrossing or recombination of pre-existing
genetic variation (Bauchet and Causse, 2012). Because tomatoes are
mostly autogamous (Table 4), crosses between two different individuals were quite rare and the plants developing from the seeds had a
parental phenotype. This allowed obtaining and maintaining xed
populations called heirlooms which were unique in their size, color
and shape. The best example of tomato breeding is probably the one
of Alexander Livingston, who wanted to obtain tomato fruits smooth
in shape, uniform in size and with better avor. For this purpose, he selected in his eld tomatoes with different traits. He saved the seeds,
grew them in elds, selected repeatedly over 5 years, until he obtained
a eshier and larger fruit (Livingston, 1893, reprint 1998). In 1870, he
introduced on the market the cultivar Paragon which is still grown
today in the USA. With expansion of tomato's use, the 20th century
was marked by the development of private seed industries which developed the principle of the F1 hybrid. Indeed, hybrids combine the
agronomical traits of the two parents, but because these characters segregate in the progeny, farmers are not able to maintain these hybrids by
seed collecting and further eld exploitation, forcing them to buy new
seeds each growing season (Bai and Lindhout, 2007). From an evolutionary point of view, domestication and breeding programs induced
175
Table 4
Non exhaustive list of important pests and diseases of tomato with their causal agents.
Bacterial diseases
Bacterial canker
Bacterial speck
Bacterial spot
Bacterial stem rot and
fruit rot
Syringae leaf spot
Clavibacter michiganensis subsp. michiganensis

Pseudomonas syringae pv. tomato
Xanthomonas campestris pv. vesicatoria
Erwinia carotovora subsp. carotovora
Pseudomonas syringae pv. syringae
Fungal diseases
Alternaria stem canker
Black mold rot
Black shoulder
Early blight
Fusarium crown and
root rot
Fusarium wilt
Gray mold
Late blight
Leaf mold
Powdery mildew
Pythium damping-off
and fruit rot
Verticillium wilt
White mold
Alternaria alternata f.sp. lycopersici

Alternaria alternata, Stemphylium botryosum, Pleospora
tarda, Stemphylium herbarum, Pleospora herbarum,
Ulocladium consortiale
Alternaria alternata
Alternaria solani
Fusarium oxysporum f.sp. radicis-lycopersici
Fusarium oxysporum f.sp. lycopersici
Botrytis cinerea, Botryotinia fuckeliana
Phytophthora infestans
Fulvia fulva
Oidiopsis sicula, Leveillula taurica
Pythium aphanidermatum, Pythium arrhenomanes, Pythium
debaryanum, Pythium myriotylum, Pythium ultimum
Verticillium albo-atrum, Verticillium dahliae
Sclerotinia sclerotiorum, Sclerotinia minor
Nematodes, parasitic
Root-knot
Sting
Stubby-root
Meloidogyne spp.
Belonolaimus longicaudatus
Paratrichodorus spp., Trichodorus spp.
Viral, viroid and mycoplasmalike organisms [MLO] diseases

Common mosaic of
tomato
Curly top
Potato virus Y
Tomato bushy stunt
Tomato mosaic
Tomato mottle
Tomato spotted wilt
Tomato yellow leaf curl
Tomato yellow top
Tomato bunchy top
Tomato planto macho
Aster yellows
Tomato big bud
Tobacco mosaic virus (TMV)

Curly top virus
Potato virus Y
Tomato bushy stunt virus
Tomato mosaic virus (ToMV)
Tomato mottle gemini virus
Tomato spotted wilt virus
Tomato yellow leaf curl virus
Tomato yellow top virus
Tomato bunchy top viroid
Tomato planto macho viroid
MLO
MLO
drastic physiological and morphological changes, but this articial selection reduced the genetic diversity of cultivated tomato.
1.4. The tomato today: a model organism for scientists
The popularity of the tomato for scientists has increased over the
years, until it has become a model organism for research programs,
both for applied and theoretical purposes. This is probably due to
1) the possibility of growing tomato in different conditions, allowing
an understanding of the adaptability of tomato to different abiotic
stresses (cold, drought, etc.), 2) its relative short life cycle, 3) its photoperiod insensitivity, i.e. the ability to ower, and subsequently produce
seeds in any condition of day length, 4) its high self-fertility and homozygosity, characteristics leading to the breeding of heirlooms by the end of
19th century, 5) the ease of controlled pollination and hybridization,
6) the simplicity of its genetics with a relatively small genome (estimated
to be approximately 900 Mb for the inbred tomato cultivar Heinz 1706,
which was used for the recent sequencing of the tomato genome; The
Tomato Genome Consortium, 2012) and lack of gene duplication, and
7) its ability to be propagated asexually by grafting, or to regenerate
whole plants from different parts of the plant.
176
Other plants are already model organisms for scientists, such as

Arabidopsis, maize, rice, or poplar. But rst, the tomato is phylogenetically distant from these plants, and second, it possesses specic morphological traits which are not shared with other model plants. For
example, it has an indeterminate growth habit due to reiterative
switches from vegetative to reproductive phases. A large set of mutants,
spontaneous or induced by chemicals or irradiation, is available, and
represents an important pool of resources for breeders as well as for scientists to isolate and understand the function of genes which regulate
development and growth of tomato (Lozano et al., 2009).
2. Challenges of tomato breeding and genetic bases of important
traits
Domestication is characterized by the modication of a wide range
of morphological and physiological traits of the crop compared to its
wild ancestor. This process is called the domestication syndrome
(Hammer 1984 in Doebley et al, 2006). The domestication syndrome
varies from crop to crop but generally focuses on growth habit which
becomes more compact, increased earliness of the crop, reduction/loss
of seed dispersal and dormancy, gigantism and increased morphological
diversity in the consumed part of the crop. In tomato, the breeding objectives are to produce and distribute new tomato cultivars with improved agronomical traits, depending on which market the cultivar is
dedicated to: the fresh or the processed market. Processing tomatoes
are mainly cultivated in elds, whereas fresh tomatoes are grown either
in elds or in greenhouses with or without temperature control. Breeding objectives have evolved over time with the cultivars released and
modications of growing systems. Despite the fact that three main objectives are recurrent (adaptability to the environment, resistance to
pests and diseases and fruit yield and quality), the breeding history
has passed through four phases: breeding for yield in the 1970, for
shelf-life in the 1980, for taste in the 1990 and since then for nutritional
value (Bai and Lindhout, 2007; Bauchet and Causse, 2012; Causse et al.,
2007; Foolad, 2007). The molecular basis of the domestication syndrome has been studied for growth habit (self-pruning, plant height
and earliness) and fruit traits (set, size, shape, color, morphology).
This has led to the identication of qualitative genes and quantitative
trait loci (QTLs).
2.1. Tomato yields
Despite its potentially improved agronomical traits, a cultivar which
does not have higher or at least equal yield than the already available
cultivars will not be considered in breeding programs. Yield takes into
account both fruit number and fruit weight. Over time, the improvement of cultural practices (notably the use of fertilizers) has contributed
to the same extent as tomato breeding to increase yield. It is obvious
that tomato yield is not an isolated trait, as it is strongly correlated
with factors inuencing the overall growth of the plant. Temperature
is one of these factors inuencing plant growth and indirectly yield. By
breeding tomatoes for resistance to high temperatures, Scott was able
to increase yield under hot and humid condition (Scott et al., 1997).
2.2. Resistance to biotic and abiotic stresses
One of the most prominent issues in tomato breeding is resistance to
biotic stresses represented by destructive pests and diseases which can
cause signicant economic losses (Bai and Lindhout, 2007). Tomato is
the target of more than 200 pests and diseases. A non-exhaustive list
is given in Table 4. These pests and diseases are controlled via chemical
treatments, which produce several negative effects: development of resistance to the chemicals and the subsequent necessity to develop new
chemicals, and harm to the environment, the farmer and the consumer.
Moreover, their use increases the costs of crop production and is subjected to chemical-use laws. In order to limit the use of chemicals in
agricultural practices, breeders turned to the wild species. Indeed the

rst introgression of interesting agronomical traits from wild species
to the cultivated tomato was reported by Walter (1967). He reported
the development of a cultivated tomato resistant to Cladosporium fulvum,
a fungus responsible for leaf mold, by crosses with S. pimpinellifolium.
2.3. Size and shape of fruit
In order to understand the modication of tomato fruit that occurred
during domestication, it is necessary to review what a tomato fruit is.
The eshy-fruit corresponds to the ovary of the plant, and is composed
of an epidermis, a thick pericarp and the placental tissues surrounding
the seeds (Fig. 3A). The pericarp is the outer wall of the gynoecium,
which is composed of at least two carpels, determining the number of
locules of the fruit. There are essentially four stages in the development
of the fruit: 1) 2- to 3-weeks of ower development, 2) a period of
intensive mitotic division activity which is initiated by fertilization and
lasts for approximately 2 weeks, 3) a period of cell expansion (up to
20-fold), characterized by intense endoreduplication and the establishment of highly polyploid cells, and 4) a ripening or maturation
phase which arises after growth stops and is characterized by chemical,
biochemical and structural changes (Fig. 3B; Tanksley, 2004).
The most prominent changes observed during domestication and
breeding of tomato are the intrinsic qualities of the fruit such as size,
shape, color, fruit rmness and shelf-life. If one considers wild tomato
species, the fruits are very small, intended to propagate the species
and not to feed humans. But the modern cultivated tomatoes offer a
large variation in fruit size, ranging from the cherry tomato (less than
20 g) to the beef tomato (up to 500 g). The potential size of the fruit depends on the cell number which is established at the pre-anthesis stage
but the nal fruit size depends on the rate and duration of cell enlargement. Endoreduplication plays a major role in the cell enlargement observed during fruit development (Chevalier et al., 2011). Six QTLs seem
to be responsible for the enlargement of the fruit during domestication.
One of them is fruit weight 2.2 which can increase the size of the fruit by
30%. Its further description indicated that it encodes a negative repressor of cell division, and large fruits are characterized by a higher mitotic
activity during the cell division phase of fruit development (Cong et al.,
2002). Two loci, fasciated and locule-number, are responsible for fruit
size changes via the modication of the number of carpels in the ower.
The fasciated locus has a stronger effect on fruit morphology than the
locule-number locus. Plants bearing the fasciated mutation can develop
more than 15 locules. Nevertheless, it is noteworthy that plants developing fruits whose weight exceeds 500 g are the result of the cumulative
effect of both mutations (Lippman and Tanksley, 2001). The attempt to
identify the gene responsible for this phenotype revealed that the fasciated gene regulates oral meristem size and is expressed very early during
oral organogenesis. Nevertheless, none of the tomato homologs of
Arabidopsis genes known to regulate this process were identied as responsible for the fasciated phenotype in tomato (Barrero et al., 2006).
Large choices of fruit shape are also available: round, oblate, pear-,
torpedo- or bell-shaped. Today it is considered that fewer than ten
QTLs are responsible for the majority of the size and shape modications
associated with the history of cultivated tomato (Tanksley, 2004). Only
few of them are described here. At the beginning of the 20th century,
segregation of a locus conditioning the pear-shape of tomato fruit was
described in the same time as oblate- to oval-shape fruits; 75 more
years were necessary before researchers demonstrated that these two
phenotypes are mediated through the same gene, ovate (Ku et al.,
1999). This gene is expressed during early ower development and
the two rst weeks following anthesis. Ovate results in a more or less
pronounced asymmetric elongation, giving rise to a more or less pronounced pear shape. Interestingly, the mutation leads to different morphology depending on the genetic background in which it is expressed,
suggesting that ovate interacts with an unknown locus (Tanksley, 2004;
Van der Knaap and Tanksley, 2001). The ovate gene encodes a nuclear
177
Fig. 3. Schematic representation of development of the ower into a fruit after pollination/fertilization (A) and of the physiological processes occurring during fruit development (B, from
Tanksley, 2004).
protein (Liu et al., 2002). Two other loci are important in the control of
fruit shape: sun and fs8.1. The locus fs8.1 is responsible for the square
tomato which is the result of adaptation of the tomato for mechanical
harvest (Grandillo et al., 1996). Like ovate, fs8.1 is expressed during
ovary development. As with the ovate locus, the sun mutation induces
an increase in the length of fruit; but in the case of the sun mutant, the
elongation occurs in both longitudinal directions, conferring a bilateral
symmetry (Van der Knaap and Tanksley, 2001). Moreover, whereas
ovate is expressed early during ower development, sun is expressed
only during the phase of cell division. All together demonstrate that,
although the two mutations confer similar phenotypes, they surely
have different genetic base (Tanksley, 2004).
2.4. Ripening and color establishment
Ripening, or fruit maturation, is the physiological process giving rise
to red, fully developed mature fruit. During ripening, important biochemical reactions occur. Some are benecial to the fruit, such as acquisition of color, accumulation of sugars and volatile compounds. Others
are detrimental to long storage, such as loosening of the cell wall,
which leads to loss of fruit rmness and reduction of shelf-life. In
the climacteric fruit of tomato, the onset of ripening is preceded by
the increase of respiration and the biosynthesis of ethylene (Lelievre
et al., 1997). Ethylene results from the methionine metabolism
(Yang, 1985). The S-adenosylmethionine is converted via ACC synthase
(ACS) to 1-amino-cyclopropane-1-carboxylic acid (ACC), which is subsequently converted to ethylene via ACC oxidase (ACO). In addition to
ACC, ACS produces methylthioadenosine, which is used to synthesize

new methionine via a modied cycle, called Yang cycle (Fig. 4). This
alternative methionine pathway ensures that high rates of ethylene
can be maintained even when the pool of methionine is limited
(Alexander and Grierson, 2002; Bapat et al., 2010). Ethylene can be
produced by two distinct systems. The system 1 is responsible for
the low production of ethylene in all tissues, is autoinhibited by ethylene
and functions during normal vegetative development. The system 2,
characterized as autocatalytic, is responsible for an auto-stimulated massive ethylene production, requires the induction of new ACS and ACO
isoforms, and is specic of climacteric fruits and petal senescence
(Bapat et al., 2010; Lin et al., 2009). The control of ripening can be done
at several points: ethylene synthesis, ethylene perception, ethylene signaling pathway. Several tomato germplasms with altered ripening were
identied and a non-exhaustive list can be found in Moore et al. (2002).
Among them, one can cite: ripening-inhibitor (rin), never-ripe (Nr),
non-ripening (nor), high-pigment 2 (hp-2) or colorless non-ripening
(Cnr). The Nr mutant is an ethylene receptor mutant which results in
non-ripening, ethylene insensitive fruit (Wilkinson et al., 1995). The
analysis of the tomato germplasms revealed that climacteric ripening
represents a combination of ethylene mediated and non-ethylene mediated regulation. Indeed the initial evidence of non-ethylene mediated
regulation came from the analysis of the two mutants, rin and nor, that
do not produce autocatalytic ethylene, do not ripen, and more importantly do not ripen in response to exogenous ethylene. The molecular
characterization identied rin as encoding a transcription factor belonging the MADS-box family whereas nor encodes a transcription factor of
178
Fig. 4. Ethylene biosynthetic pathway and ripening regulated processes. 1: adomet synthetase, 2: kinase, 3: aminotransferase, 4: ACC synthase (ACS), 5: ACC oxydase (ACO).
the NAC domain family (Martel et al., 2011; Vrebalov et al., 2002). If
these mutations are homozygous, the process of ripening is inhibited,
the fruits remain yellow or light orange and can be stored for months
at room temperature. When the mutations are heterozygous, the fruit
ripens slowly and has an extended shelf-life. Interestingly, the rin mutation in its form rin/Rin constitutes the basis for most of the tomato hybrids with slow ripening and long shelf-life (Giovannoni, 2007). In a
recent study, Martel et al. (2011) demonstrated that RIN interacts
Fig. 5. Carotenoids biosynthetic pathway and tomato mutants. Pictures are from the Tomato Genetic Resource Centre. ABA: abscissic acid; AO, adldehyde oxidase; CrtR-b: carotenehydroxylase; Cyc-B: chromoplast specic lycopene synthase; DMAPP: dimethylallyl diphosphate; GPP: genaryl diphosphate; GGPP: geranylgeranyl diphosphate; Ggps: GGPPsynthase; IPP: isopentenyl diphosphate; Lcy-b: lycopene -cyclase; Lcy-e: lycopene -cyclase; MEP: methylerythritolphosphate; Nxs: neoxanthin synthase; Pds: phytoene desaturase;
Psy: phytoene synthase; Vde1: violaxanthin deepoxidase; VNCED: 9-cis-epoxycarotenoid dioxygenase; Zds: -carotene desaturase; Zep1: zeaxanthin epoxidase.
with the promoter of genes involved in the major processes observed

during ripening: ethylene biosynthesis, perception and signaling, cell
wall metabolism, and carotenoid.
Color change is the most obvious trait of tomato ripening. The color
of fruit depends on its content of carotenoid pigments, mainly lycopene
and to a lesser extent -carotene. The rst step in carotenoid synthesis
is the condensation of two molecules of GGPP (geranylgeranyl diphosphate, synthesized from isopentenyl diphosphate/IPP and dimethylallyl
diphosphate/DMAPP) to produce phytoene by phytoene synthase
(PSY). Phytoene is converted into lycopene via the production of the intermediate -carotene; this reaction involves two enzymes: phytoene
desaturase (PDS) and -carotene desaturase (ZDS). The cyclization of
lycopene is an important branching point in the pathway as it can give
rise to -carotene and xanthophylls on one side, or to -carotene/carotene and lutein on the other side. -carotene is synthesized by a
two-step reaction mediated by lycopene -cyclase (LCY-B/CRTL-B)
with the production of -carotene as intermediate. Lycopene -cyclase
(LCY-E/CRTL-E) allows the synthesis of -carotene from lycopene. The
production of other compounds results from hydroxylation of the cyclic
carotenes by two carotenoid hydroxylases (CHYs), one specic for rings (BCH type) and the other one catalyzing hydroxylation of both
- and -rings (CYP97 type) (Ruiz-Sola and Rodrguez-Concepcin,
2012). The next steps are epoxydation by means of ZEP1 (Fig. 5;
Hirschberg, 2001). In tomato, several mutants affected in color are
available: r (yellow esh, yellow color of the ripe fruit esh), sh (sherry,
yellow fruit fresh with reddish tinge), hp-1 (high pigment1, higher content
in chlorophyll, carotenoids and ascorbic acid), tg (tangerine, fruit esh
and stamens are orange colored), B (beta-carotene, high -carotene
and lycopene content in ripe fruit), at (apricot, yellow-pinkish color of
the fruit esh), og (old gold, increased lycopene content), DEL (delta,
reddish-orange mature fruit color). These mutants contributed to the
elucidation of the carotenoid synthesis pathway (Causse et al., 2007)
and their molecular characterization contributed to the identication
of the genes responsible for the mutations/phenotypes. The mutant r
encodes the phytoene synthase, PSY1, the rst specic step of the carotenoid biosynthesis pathway (Hirschberg, 2001). The orange color of the
mutant tg is due to the accumulation of prolycopene and encodes a
Table 5
Nutritional value of 100 g of red fresh tomato (source: USDA, http://www.usda.gov/wps/
portal/usda/usdahome).
Proximates
Water
Energy
Protein
Total lipid
Fibers
Sugars
g
kcal
g
g
g
g
94.52
18
0.88
0.2
1.2
2.63
Minerals
Calcium
Magnesium
Phosphorus
Potassium
Sodium
Fluoride
mg
mg
mg
mg
mg
g
10
11
24
237
5
2.3
mg
mg
g
g
g
g
g
g
13.7
6.7
42
449
101
2573
123
7.9
Vitamins
Vitamin C
Choline
Vitamin A
-Carotene
-Carotene
Lycopene
Lutein + zeaxanthin
Vitamin K
179
carotenoid isomerase. The Del mutant accumulates high levels of carotene and encodes a lycopene -cyclase, like the mutation B, which
is characterized by the accumulation of -carotene (Lewinsohn et al.,
2005).
2.5. Nutritional value
If one takes into consideration only proteins/lipids/sugars content to
describe the nutritional value, it appears clearly that tomato does not
have a high nutritional value. Nevertheless, tomatoes represent an important source of nutrients which are important for human health
such as antioxidants, represented by the content in lycopene, vitamin
A (-carotene) and ascorbic acid (vitamin C) (Table 5). Thus, tomatoes
represent the main source of lycopene, which has antioxidant properties
and is considered to protect against cancer or cardiovascular diseases
(Rao and Agarwal, 2000). The cross between S. lycopersicum cv. Floradade
and the wild relative S. galapense (L. cheesmanii f. minor C.H. Mull), bearing the Beta (B) gene, gave rise to three lines with enhanced fruit carotene content, and consequently higher nutritional value (Stommel,
2001). Tomatoes are also an important and remarkable source of ascorbic
acid. The primary route of ascorbic acid synthesis is the L-galactose
WheelerSmirnoff pathway in which ascorbic acid is synthesized from
mannose-6-phosphate via GDP-mannose and GDP-L-galactose. More
pathways have been described notably the alternatice pathway with an
L -galactonic acid intermediate, deriving from cell wall polymers
(Di Matteo et al., 2010; Ioannidi et al., 2009; Stevens et al., 2007).
Compared to the modern cultivated tomato, wild tomato varieties
are rich in ascorbic acid and can contain up to 5 times more ascorbic
acid than the cultivated counterpart (Stevens, 1986). Stevens et al.
(2007) investigated the QTLs and candidate genes affecting fruit
ascorbic acid. The recent work of Di Matteo et al. (2010) demonstrated that the accumulation of ascorbic acid is achieved by increasing
pectin degradation and may be triggered by ethylene. Some cultivars
with enhanced nutritional value were thus successfully developed,
but reduction of the yield in these new cultivars hindered their commercial success (Causse et al., 2007).
Soluble and total solids are important traits for processing tomatoes
and contribute to the denition of the concentrated tomato product.
Soluble solids represent sugars and organic acids whose ratio, together
with the composition in volatile aroma, characterizes the avor of the
fruit. The organic acids, alone, determine the pH of the nal product. A
pH above 4.5 will allow the development of microorganisms, spoiling
the nal product. Increased temperatures and extended processing
time are the only ways to get rid of this problem, but they also increase
the costs linked to processing. Insoluble solids, represented by components of the cell wall and proteins, dene the rmness of the fruit but
also the viscosity of the nal products, such as tomato juice, ketchup,
soups and paste. Two other criteria are of high importance for the
breeding of new cultivars for processing tomatoes: growth habit and
ease of harvest. The spontaneous self-pruning (sp) mutation appeared
in 1914, allowing the development of cultivars with bushy growth
habit. In addition, sp induces the concentration of owers and consequently of fruits, and contributes to fruit rmness and resistance to
over-ripening. All these characteristics made cultivars bearing this mutation material of choice for mechanical harvest. The jointless mutations (j and j2) are characterized by no abscission zone in fruit pedicel,
enabling harvest without calyx and pedicel, i.e. the fruit free from any
green parts.
2.6. Limits of classical breeding: the new area of genetic engineering
In order to obtain new cultivars with improved agronomical traits,
three objectives have to be taken into account: environmental conditions, mode of culture and mode of harvest. Efcient breeding relies
on the availability of genetic diversity and the heritability of the traits
of interest. Very often, many traits must be simultaneously improved
180
and introduced into the new cultivar and most of them are controlled by
several genes and/or inuenced by the environment. Finally, and importantly, the organoleptic signature, considered often as a side criterion
for selection, is evaluated by sensorial analyses which cannot be developed on the scale of mass screening (Causse et al., 2007). Traditional
breeding usually starts from a cross between elite lines of adapted cultivars, or between an elite line and either wild species or close outgroup
related species (S. juglandifolium and S. ochranthum). It must be noted
that the production of a new cultivar from a cross between two cultivars
takes 5 to 7 years, and the incorporation of new genes from wild relatives takes about 20 years, the complexity of the breeding program increasing with the complexity between parents (Causse et al., 2007).
The choice of parental lines is thus crucial and demands good knowledge of the available germplasm. In the case of tomato, more than
83,000 accessions are stored in seed banks throughout the world, placing tomato as the number one collected and conserved vegetable species (Bauchet and Causse, 2012). The main collections are: the Tomato
Genetic Resource Center in California (USA TGRC, http://tgrc.
ucdavis.edu/), the USDA collection (USA www.ars.usda.gov), the
World Vegetable Center in Taiwan (www.avrdc.org) and other collections in Europe. In the frame of the European Solanaceae project
(EU-SOL, www.eu-sol.wur.nl), approximately 7000 domesticated
tomato accessions, alongside representative wild species, were enumerated. These collections represent a precious source of information
for subsequent breeding programs. All wild species can be crossed
with the cultivated tomato (S. lycopersicum) with more or less high
efciency if cultivated tomato is used as the female (Bedinger et al.,
2011). When two species are non-crossable, in vitro techniques are required, such as cell fusion and regeneration from tissue or single cells, or
embryo-rescue (Bai and Lindhout, 2007; Rick, 1974); this is particularly
necessary in the case of crosses with S. peruvianum. Table 6 gives a nonexhaustive list of traits which have a potential for breeding and their
germplasm origin. A few examples of the improvement of cultivated tomato by introgression of wild characters can be mentioned here: the
use of L. hirsutum for improving cold tolerance, of L. chilense for drought
tolerance, of L. cheesmanii for soluble solids and salt tolerance (Hobson
and Grierson, 1993), or of L. pennellii for sugar yield (Fridman et al.,
2000; Ikeda et al., 2013) or yield and tness (Semel et al., 2006). Recently, in an attempt to increase the endogenous content of avonoids,
compounds with a positive impact on human health, S. pennellii var.
puberulum was crossed with cultivated tomato (Willits et al., 2005).
The plants accumulated high levels of quercetin in the fruit. Some accessions accumulated lycopene or ascorbic acid at levels twice those of traditional cultivars. Thus carotene content can be signicantly increased
in cultivated tomato by using S. hirsutum in crosses. Analogously, a
strategy based on S. pimpinellifolium, S. cheesmanii and S. chmielewskii
has increased the sugar content (Stevens, 1986). The method of Gas
chromatographymass spectrometry was used to characterize the metabolic prole of leaves and fruits of wild tomato species in comparison
to cultivated tomato. The method allowed identifying more than 90
metabolites constituting the metabolic signature of each species. This
is of importance as it can constitute a way of selecting parental lines
used in crosses (Schauer et al., 2005).
By 2050, the world population is estimated to reach 9.07 billion,
with 62% of people living in Africa, Southern Eastern Asia. These same
regions are where a huge proportion of the population suffers from
hunger (FAO, data collected for the period 20102012). Traditional agriculture and breeding cannot sustain the increasing food demand for
several reasons: 1) decreasing arable land giving way to living space,
2) increasing problem of drought and salt stresses due to environmental
and climate changes, and 3) slow and laborious empirical selection
(Ahmad et al., 2012). The development of molecular biology offers
breeders a new prospect for genetic improvement based on molecular
markers and genetic engineering. Molecular markers used in markerassisted selection (MAS) are not reviewed in this work as they have
been extensively reported in various recent reviews (Bauchet and
Table 6
Non-exhaustive list of agronomic traits of interest available from wild tomato species
(extracted from Foolad, 2007, for more details see the references therein).
Phenotype
Biotic stress
Resistance to bacteria
Resistance to fungi
Resistance to virus
Resistance to insects
Germplasm source (Lycopersicum name)

L. pimpinellifolium, L. hirsutum, L. pennellii, L. peruvianum
L. cheesmaniae, L. esculentum, L. pimpinellifolium,
L. chilense, L. hirsutum, L. pennellii, L. parviorum,
L. peruvianum
L. esculentum, L. pimpinellifolium, L. chilense,
L. hirsutum, L. peruvianum
L. pimpinellifolium, L. peruvianum
Abiotic stress
Cold (low temperature) L. pimpinellifolium, L. hirsutum
Drought
L. pimpinellifolium, L. pennellii
Salt
L. pimpinellifolium, L. pennellii
Fruit characteristics
Antioxidant capacity
Ascorbic acid
Citric acid
Color
Jointless
L. pennellii
L. pennellii
L. pennelli
L. pimpinellifolium, L. peruvianum, L. hirsutum,
L. parviorum, L. chmielewski, L. pennellii
L. cheesmanii, L. hirsutum, L. pennellii, L. parviorum
L. pimpinellifolium, L. parviorum, L. pennellii
L. pennellii
L. parviorum
L. pennellii, L. pimpinellifolium
L. pimpinellifolium
L. parviorum, L. pennellii
L. parviorum, L. chmielewskii, L. pennellii
L. pennellii, L. hirsutum
L. pimpinellifolium
L. pimpinellifolium
L. pimpinellifolium, L. peruvianum, L. hirsutum, L. parviorum
L. pennellii, L. pimpinellifolium, L. peruvianum, L. cheesmanii
L. chmielewskii, L. cheesmanii, L. pennelli,
L. pimpinellifolium, L. hirsutum
L. parviorum, L. pennellii
L. pennellii, L. chmielewskii, L. cheesmanii,
L. pimpinellifolium, L. peruvianum, L. hirsutum
L. chmielewskii, L. pennelli, L. pimpinellifolium,
L. peruvianum, L. hirsutum, L. parviorum
L. cheesmanii
Plant characteristics
Branch number
Male sterility
Growth habit
Height
Self-pruning
L. cheesmanii
L. pimpinellifolium
L. peruvianum, L. pimpinellifolium
L. pennelli, L. pimpinellifolium, L. hirsutum, L. cheesmanii
L. chmielewskii, L. pimpinellifolium
-carotene
Lycopene
Orange
Yellow
Cracking
Diameter
Shape
Firmness
Sugars
Length
Locule number
Maturity
Ripening
Soluble solids
Viscosity
Weight
Yield
Causse, 2012; Causse et al., 2007). Attempts to understand the plant genome, i.e. how the plant functions and how a gene can make a plant, led
in 2003 to the development of the international Solanaceae Genomics
Network (SOL) project. This consortium encompasses several databases resulting from gene sequencing, gene expression proling,
metabolites proling, genome sequencing and annotation. The fullysequenced tomato genome was released in 2012, and its annotation is
still underway (The Tomato Genome Consortium, 2012). All this information allows a better understanding of the plant growth and development; today a specic character can be easily introduced in a cultivar of
interest by means of plant transformation and regeneration in a shorter
time than traditional breeding. Moreover, genetic engineering surmounts the barriers intra-/inter-crossability. Indeed, a gene from a
non-related species can be introduced into the crop of interest which
would not have been possible by classical breeding, due to interspecic
incompatibility.
3. Genetic engineering of tomato

Genetic engineering, i.e. the introduction of a gene or its silencing,
provides the tools for fast and specic improvement of tomato
agronomical traits. Except for the report on chloroplast transformation
using particle bombardment (Ruf et al., 2001), the most widely used
method for transferring genes into tomato is Agrobacterium-mediated
transformation. The FLAVR-SAVR tomato variety was the rst genetically engineered crop to be commercialized (Kramer and Redenbaugh,
1994). Despite the fact that this new tomato also contained a bacterial
gene encoding resistance for kanamycin (used for selection), the new
cultivar was introduced on the fresh tomato market on May 21, 1994.
The FLAVR-SAVR tomato found an important albeit brief success,
due to the costs linked to engineering and the newly growing negative
consumer concerns about GMO (genetically modied organisms;
Bruening and Lyons, 2000).
3.1. Agrobacterium-mediated transformation of tomato
For 30 years now, Agrobacterium has been used for research purposes, and it has also been used to produce genetically engineered
crops for commercial purposes. The Gram negative soil bacterium
Agrobacterium tumefaciens is a phytopathogen responsible for crown
gall disease in a wide range of plants (Alimohammadi and BagheriehNajjar, 2009). The molecular basis of plant cell reprogramming by the
bacterium and the principles of Agrobacterium-mediated transformation have been already extensively reviewed (Binns and Thomashaw,
1988; Shantha et al., 2012). The traditional protocol for Agrobacteriummediated transformation is based on the co-culture of explant, with a
bacterial culture containing the cloning vector and the property of a
plant cell to regenerate a full plantlet. Frary and Van Eck (2005) describe
a standard protocol, based on the initial work of McCormick et al.
(1986) and Fillatti et al. (1987). In brief, after an overnight
preculture step, the cotyledon explants are cocultivated with
Agrobacterium for two days. Afterward the explants are transferred
to a selective regeneration medium containing zeatin. The rooting
is ensured on a separate selective medium.
The rst tomato transformation by Agrobacterium was done in 1986
(McCormick et al., 1986) in order to avoid the need to develop a system
of plant regeneration from protoplasts. The leaf disk system was
adapted for tomato transformation. This rst Agrobacterium-mediated
transformation of tomato opened the door to the functional characterization of tomato genes and/or promoters as well as the expression of
heterologous genes. The explants used were as diverse as cotyledons,
hypocotyls, stems and leaves of the tomato (Bird et al., 1988; Davis
and Miller, 1991; McCormick et al., 1986; Ohki et al., 1978). The different conditions relative to efcient transformation have been studied
and reported (Park et al., 2003).
The generation of transgenic plants is routinely developed in most
plant molecular biology research laboratories; nevertheless the classical
method of shoot regeneration from leaf disk/cotyledons tissues cocultivated with disarmed Agrobacterium is a long and expensive process
which can result in undesirable modications, such as somaclonal variation, i.e. modication of the genotype independent from the character introduced. In order to avoid the use of tissue culture, methods called in
planta transformation were developed (Yasmeen et al., 2009). The rst
in planta method developed was the Agrobacterium-mediated oral
dip transformation in Arabidopsis. For this purpose, the plant was grown
until owering, uprooted, fully immersed in a solution of Agrobacterium
with application of vacuum in order to facilitate the penetration of bacteria into the plant tissue, and then planted back into soil. The plants were
then cultivated until the production of seeds. The transgenic progenies
were identied by cultivation of the seeds on a medium supplemented
with the appropriate selective agent (Bechtold et al., 1993). In an
upgraded version of this protocol, Clough and Bent (1998) suppressed
the phase of uprooting/vacuum/replanting, making this method even
181
simpler. In order to study the regulation of ower initiation and development in tomato, Yasmeen et al. (2009) initiated the oral dip transformation of tomato. They observed that owers which did not experience
pollination gave a higher percentage of transformation, similar to what
was observed for Arabidopsis. It is supposed that after anthesis the locule
becomes sealed, enabling the proper penetration of the bacteria to the
ovule (Desfeux et al., 2000). Inltration of tissues for transient gene
expression and localization was developed for several species. The
Agrobacterium-mediated transient expression by leaf inltration is today
the favorite tool in many gene functional analyses. While tobacco leaf is
the material of choice, the efciency of the method has been described
not only for other species, such as alfafa, lettuce, tomato, and Arabidopsis
(Wroblewski et al., 2005), but also for rose petals (Scalliet et al., 2006). In
order to shorten the time in functional studies in fruit development,
Agrobacterium-mediated transformation by inltration of tissues was applied to tomato fruit (Orzaez et al., 2006). In practice, the Agrobacterium
cultures were injected into the mature green fruits through the fruit stylar
apex, which resulted in complete fruit inltration. This new method,
called fruit agroinjection, was found to be a powerful tool for fast transgene expression in fruits and to facilitate functional studies in that organ.
A few years later, Hasan et al. (2008) reported fruit agroinjection as a tool
to stably transform tomato. The efciency of transformation was higher
when the fruit was kept on the plant compared to detached fruit, and mature fruits at the early stage of ripening gave more transformants than
immature, green fruits (Yasmeen et al., 2009).
3.2. Selection of transgenic plants
Because during the process of Agrobacterium-mediated transformation not all cells of the explant are efciently transformed, the use of selection is required to avoid long, costly and laborious screening of the
transgenic progeny. Thus a gene encoding an enzyme conferring resistance to antibiotics or herbicides is introduced concurrently with the
gene of interest. The presence of such genes within the environment
or in the food supply might provoke unpredictable damage to the ecosystem and to human health. The possibility of transmission of the
marker gene through pollen is the major environmental concern
(Tuteja et al., 2012). In the last decade, methodologies have been developed to eliminate marker genes from the process of genetic engineering. Several possibilities were considered. The rst one consisted of
avoiding the use of the marker gene in transgenic plants. This was applied in several crops, but the efciency of transformation rarely
reached 5% (Bai et al., 2009; De Vetten et al., 2003; Jia et al., 2006). A second method is co-transformation, which is a simple and highly effective
method. In this case, the gene of interest and the marker gene are present on two different vectors. The rst generation of the progeny will
bear the two genes in its genome. Because they are not introduced in
the genome at the same loci, they will segregate during sexual reproduction. The marker gene will be eliminated from the genome by further selecting transgenic lines bearing only the gene of interest
(Komari et al, 1996). Transposon-based marker excision is a third strategy which has also been used to remove marker genes from the gene of
interest. This strategy is based on the maize Ac/Ds transposable element
(Upadhyaya et al., 2010). Finally, marker gene removal can be achieved
by a strategy based on the recombination which takes place between
two homologous DNA molecules. Three systems were developed
based on recombination to eliminate the marker gene: the Cre/lox site
specic recombination system (Dale and Ow, 1991), the FLP/FRT recombination system from Saccharomyces cerevisiae (Lyznik et al., 1996) and
the R/RS recombination system from Zygosaccharomyces rouxii (Sugita
et al., 2000). Because all these strategies have been described and reported in detail by several reviewers, they are not explained here
(Darbani et al., 2007; Puchta, 2003; Upadhyaya et al., 2010).
In tomato, the rst report of generation of marker-free transgenic
plants came from Goldsbrough et al. (1993). By using the maize Ac/Ds
transposable element, they were able to obtain NPTII- or GUS-free
182
plants from transgenic plants which originally contained one of these

genes in their genome. To our knowledge, Zhang and coworkers obtained the rst genetically engineered marker-free tomato with improved
tolerance to drought, cold and oxidative stresses. These authors obtained transgenic tomatoes by overexpression of the Arabidopsis Ipk2 gene,
in which the marker of selection was removed by the Cre/loxP DNA recombination system (Zhang et al., 2009).
3.3. Temporal and/or organ-specic expression: choice of promoter
The Cauliower mosaic virus 35S (CaMV35S) promoter was, and still
is, routinely used to trigger gene expression in both dicots and monocots. Despite the fact that it is assumed to be a constitutive promoter
(Odell et al., 1985), some studies report that it is not expressed in all
cell and tissue types (reviewed in Sunilkumar et al., 2002). A study
using GFP fusion as the reporter of promoter activity in cotton demonstrated that genes driven by CaMV35S promoter are expressed in all
cells and tissues albeit at different levels (Sunilkumar et al., 2002). The
discrepancy between the study of Sunilkumar et al. (2002) and older
studies is probably due to the GFP reporter assay used which is more
sensitive than classical histological methods using GUS staining. Due
to its viral origin, there are some limitations in its use for genetic engineering of crops. First, despite the lack of scientic support, some fear
its risk to human health; second, the plant can recognize the sequence
of foreign origin and inactivate it (Potenza et al., 2004). Thus other promoters, of plant origin, were developed to drive constitutive expression.
These promoters are derived from actin and ubiquitin genes and induce
strong expression of the gene of interest. One can cite here the promoter
of the Arabidopsis Act2 gene, rice actin 1, maize ubiquitin 1 or Ubi.U4 from
Nicotiana sylvestris (reviewed in Potenza et al., 2004). Constitutive expression can be problematic. Indeed, the gene of interest will be
expressed in all tissues, independently from growth and development,
leading to often undesirable pleiotropic effects which do not reect
the in planta function of the gene. Thus, new promoters driving the expression of the gene of interest in a specic manner were developed.
Fruit and owers are the main targets for genetic engineering of fruit
crops but they are the last to develop, rending their manipulation difcult. Several toolkits have been obtained in order to specically address gene expression in fruit (Fernandez et al., 2009). Ethylene is part
of the process leading to fruit ripening. The E8 gene of tomato encodes
a polypeptide belonging to the family of dioxigenases and participates
in the regulation of ethylene production during ripening. The promoter
analysis of E8 led to the identication of different regions responsible for
both fruit-specic expression and ethylene-regulated expression
(Deikman et al., 1992). This promoter has been successfully used in tomato genetic engineering (Sun et al., 2012). Another promoter used to
specically drive the expression of a gene of interest in the fruit during
ripening is the promoter of the polygalacturonase (PG) gene, encoding
the major cell-wall degrading enzyme (Lau et al., 2009).
When considering genetic engineering for stress tolerance, it is important to develop tools which will respond to a specic stimulus. Indeed, the mechanisms of resistance represent a certain energetic cost
for the plant that remobilizes its metabolism to face the stress. Modern
genetic engineering has a duty to develop plants whose mechanisms of
resistance will be stimulated only if and when the plant will be exposed
to stress. By studying the molecular basis of plant stress responses, researchers have been able to identify genes specically regulated by
stress and use their promoters as tools for bioengineering. As it will be
detailed later, plants respond to stress by producing and accumulating
molecules of low molecular weight, such as glycinebetaine. In higher
plants, glycinebetaine is synthesized in the chloroplast from betaine in
a two-step reaction. The rst reaction is catalyzed by a ferredoxindependent choline mono-oxygenase (CMO) and produces betaine aldehyde as an intermediate. Betaine aldehyde is further transformed into
betaine by the NAD+-dependent betaine aldehyde dehydrogenase
(BADH) (Su et al., 2006). BADH gene expression is stimulated by several
stresses, such as salt, drought, cold and ABA treatment. Analysis of

the promoter sequence of BADH led to the identication of a region
(P5: 300 to + 62 bp) which triggers salt-induced gene expression.
In order to validate that the P5 region of the BADH gene could be used
as a new promoter to drive gene expression specically in response to
saline stress, Wang et al. (2013) transformed tomato plants with the
BADH gene, as reporter gene, under the control of the P5 promoter to
drive salt-inducible expression. The authors could observe that BADH
expression and accumulation of betaine was correlated with the concentration of salt applied. Consequently, the salt-inducible P5 promoter
appears to be an essential tool for further genetic engineering in regard
to salt stress tolerance.
4. Transgenic tomatoes with enhanced agronomic traits
The development and spread of genetically engineered crops encounters negative opinions of consumers and institutions, mainly in
Europe. Thus, the World Health Organization (WHO) has reported
three main concerns to the use of genetically engineered crops: generation of allergenic foods, incorporation of modied food genes into the
human body and the transfer of this information to other wild species.
Despite these barriers, the development of transgenic crops is expanding
all over the world; indeed the surface dedicated to the cultivation of genetically engineered crops has greatly expanded, with 125 million ha in
2008 vs. 1.7 million ha 12 years ago (Ahmad et al., 2012). The leading
countries are without contest the USA with 69.5 million ha dedicated to
genetically engineered crops (maize, soybeans, cotton, canola, sugarbeets,
alfalfa, papayas and squash), followed by Brazil, Argentina, India and
Canada (1040 million ha). Today, China is probably the only country
growing transgenic tomatoes (source: www.isaaa.org, 2012). Indeed, culture of transgenic tomatoes in the USA, which represented 200000 ha in
1998, has been suspended since 2002. The objectives of tomato improvement by genetic engineering focus on: fruit quality, resistance to pests
and diseases, overcoming adverse environments (cold, drought, salinity)
and production of therapeutics. In the scientic literature since 2000,
more than 60 records report on genetic engineering of tomato. Because
it is not possible to describe all of them in the present review, Table 7
gives a non-exhaustive list of successful genetic engineering of tomato;
some of them are discussed in more detail below.
4.1. Resistance to biotic stresses
Economic losses due to pests and diseases are signicant all around
the world. Up to now, pesticides are widely used to control the development of pests and diseases, but several problems have appeared due to
this cultural practice: development of resistance to the chemicals, appearance of new diseases, health disorders of farmers, and detrimental
effects on the environment. Genetic engineering offers a great opportunity to limit the use of such chemicals. To respond to pathogen attack,
plants have evolved a variety of active and passive defense mechanisms:
hypersensitive programmed cell death, expression of defense-related
genes, reinforcement of cell walls, biosynthesis of phytoallexins or phenolic compounds, production of reactive oxygen species (ROS), and initiation of systemic acquired resistance (Li and Steffens, 2002). These
responses are induced upon attack by bacteria, fungi, virus or insects.
Moreover, three hormones, salicylic acid (SA), ad jasmonic acid
(JA) and ethylene, were described to play key roles in the establishment of susceptibility/resistance. It is commonly accepted that SA
and JA/ethylene have an antagonistic role, with SA being involved
in resistance to biotrophic pathogens and JA/ethylene being involved
in resistance to necrotrophic pathogens (Kunkel and Brooks, 2002).
Studies of the molecular basis of resistance have allowed researchers
to identify genes whose manipulation by overexpression could lead
to improved resistance to pathogens. On the other hand, the analysis
of plants with a high susceptibility to pathogens has led to the
183
Table 7
Non-exhaustive list of successful tomato engineering. Reference can be found in Di Matteo et al. (2011). References in bold are discussed in the text.
Trait
Fruit
quality
Inserted target
Reference
Parthenocarpy
Firmness
Arf8, IAA9, iaaM

PG (antisense), Rab11GTPase, TBG4, PME,
ACCO, EXP1A, PLD (antisense), -galactosidase
Size
Flavor
fw2.2
Thaumatin, gdhA, GES, LIS, LeCCD1, LeACCDC1A,
LeACCDC2
Soluble solids
content
Carotenoid content
Lin5, PME
Ficcadenti et al. (1999), Goetz et al. (2007), Wang et al. (2005a)

Behboodian et al. (2012), Brummell et al. (1999),
Langley et al. (1994), Lu et al. (2001); Pinhero et al. (2003),
Smith et al. (1990), Tieman and Handa (1994), Watson et al. (1994),
Xiong et al. (2005)
Frary et al. (2000)
Bartoszewski et al., 2003; Davidovich-Rikanati et al. (2007),
Kisaka and Kida (2003), Lewinsohn et al. (2001);
Simkin et al. (2004), Tieman et al. (2006)
Tieman et al. (1992), Zanor et al. (2009)
Flavonoid content
Dxs, CrtB, CrtI, CrtY, PSY-1, CRY-2, CYC-B, LCY-B, LCY-B,

CHY-B, DET-1, COP1LIKE, CUL4, FIBRILLIN, Spermidine synthase,
PG
CHI, CHS, CHI, F3H, FLS, MYB12, STS, CHR, FNS-II, Del, Ros1
Ascorbic acid content GaLDH, GME, GCHA and/or ADCS

Nutritional value
Abiotic stresses
Crt1, Samdc
aroA, HAL2, HAL1, BADH, AtNHX1, ectoine, GlyI and GlyII,
ACC deaminase, TPS1, APX, Osmotin, CBF1, SAMDC, cAXP
Biotic stresses
Vst1 and Vst2, Cry1Ac, Cry1Ab, Ep5c,

Nucleoprotein gene, CP, p35, PI-II and PCI, Ep5C, PPO
Pharmaceutics and other

compounds
TB1-HBS, CtrB, Spike, miraculin
identication of genes which are involved in this process, and potential targets of genetic engineering by gene silencing.
One of the most rapid responses is the production of superoxide (O2)
and hydrogen peroxide (H2O2), characterizing the oxidative burst
shortly after pathogen recognition. Because H2O2 is highly toxic for
the cell, plants have developed mechanisms of detoxication mediated
by peroxidases. The Ep5c gene, encoding a secreted peroxidase, was
found to accumulate to signicant levels in tomato plants susceptible
to Pseudomonas syringae pv. tomato, leading to the hypothesis that it is
required for disease susceptibility toward this pathogen. Whereas the
inhibition of Ep5C protein accumulation by RNA antisense strategy led
to obtaining transgenic plants resistant to P. syringae pv. tomato, the
mechanisms downstream of Ep5C are still unclear (Coego et al., 2005).
The production of quinones which results from the oxidation of phenols
by the action of polyphenol oxidases (PPO) is also part of the processes
leading to pathogens resistance. Indeed, the active quinones have a direct antibiotic and cytotoxic effect on pathogens. Transgenic tomato
plants constitutively overexpressing the potato PPO gene were characterized by increased PPO activity, leading to a higher rate of phenolic
compound oxidation. These plants were found to be more resistant to
P. syringae pv. tomato. Indeed in these transgenic plants, bacterial
growth was strongly inhibited (100-fold reduction of bacterial population in infected leaves compared to the non-transgenic plants) and the
number of lesions was also signicantly reduced (Li and Steffens, 2002).
The mitochondrial alternative oxidases (AOXs) are components of
the alternative respiratory pathway of plants. They can be induced by
wounding, ethylene, ROS or salicylic acid. The LeAOX gene, encoding a
tomato AOX, was isolated and overexpressed by Agrobacteriummediated transformation in tomato. Multiplication of Tomato spotted
wilt virus (TSWV) was signicantly limited in the transgenic tomato
Davuluri et al. (2005), Dharmapuri et al. (2002),

Enssi et al. (2005), Fraser et al. (2002), Fray et al. (1995),
Giliberto et al. (2005), Liu et al. (2004), Neily et al. (2011),
Rmer et al. (2000), Ronen et al. (2000), Rosati et al. (2000),
Simkin et al. (2007), Wang et al. (2008), Watson et al. (1994),
Wurbs et al. (2007)
Adato et al. (2009), Butelli et al. (2008), Colliver et al. (2002),
Muir et al. (2001), Schijlen et al. (2006)
de la Garza et al. (2004, 2007), Garcia et al. (2009), Gilbert et al.
(2009), Zhang et al. (2011)
Mehta et al. (2002), Rmer et al. (2000)
lvarez-Viveros et al. (2013), Arillaga et al. (1998), Cheng et al., 2009,
Cortina and Culianez-Macia . (2005), Fillatti et al. (1987), Ghanem et al.
(2011),
Gisbert et al. (2000), Grichko and Glick (2001), Hsieh et al. (2002a,b),
Jia et al. (2002), Moghaieb et al. (2011), Park et al. (2005),
Patade et al. (2013), Wang et al. 2005b,
Wang et al. (2006), Zhang and Blumwald (2001)
Abdeen et al. (2005), Alberto et al. (2005), Coego et al. (2005),
Kumar and Kumar (2004), Li and Steffens (2002), Lin et al. (2004),
Lincoln et al. (2002), Ma et al. (2011), Mandaokar et al. (2000),
Nervo et al. (2003), Raj et al. (2005), Thomzik et al. (1997),
Chen et al. (2009), Hirai et al. (2010), Pogrebnyak et al. (2005),
Shchelkunov et al. (2004), Youm et al. (2008)
compared to what was observed in non-transformed plants (Ma

et al., 2011).
In an incompatible plant-pathogen interaction, the attack by a pathogen not only induces the resistance mechanism at the point of infection, but also induces a systemic acquired resistance (SAR) response in
the tissues which are not infected. The NPR1 gene, encoding a nuclear
protein, is a key regulator of the SA and JA/(ethylene) signals leading
to acquired resistance. One step in genetic engineering for disease resistance would be to stimulate SAR. The Arabidopsis NPR1 gene was introduced into a tomato cultivar possessing resistance to heat-stress and to
tomato mosaic virus (TMV). Characterization of the resulting transgenic
tomato plants revealed that in addition to the innate resistance to
TMV, they were resistant to two diseases conferred by fungi (fusarium
wilt and gray leaf spot) and to two bacterial diseases (bacterial wilt
and bacterial spot) (Lin et al., 2004).
The last example presented in the frame of this review is focused on
resistance to insects. In response to insect attack, plants accumulate an
array of defense proteins, including proteinase inhibitors and secondary
metabolites with harmful activity to the insects. Plant proteinase inhibitors accumulate in plants in response to insect wounding and are part
of the JA-mediated resistance response. The plant proteinase inhibitors
inhibit the activity of insect proteases which are secreted in the digestive tract of larvae in order to assimilate amino acids. The type of proteases is specic to the type of insect. Thus, whereas serine proteinases are
mainly represented in lepidopteran larvae, cysteine and aspartic proteinases are specically found in coleopteran species. Thanks to this
specicity, engineering programs can be initiated to focus on different
groups of tomato pests. In tomato, the leaf-specic expression of two
potato protease inhibitors, belonging to different classes (serineproteinase inhibitor and carboxypeptidase inhibitor), conferred
184
signicantly stronger resistance to damages caused by Heliothis obsoleta

and Liriomyza trifolii larvae (Abdeen et al., 2005). In any genetic engineering strategy, it is recommended that genes involved in different
mechanisms of resistance are used to avoid the risk of pathogenacquired resistance.
4.2. Resistance to abiotic stresses
Drought, salt, cold and heat stresses share common molecular mechanisms: detoxication of ROS, osmoprotection, protection of enzymatic
systems, and water and ion uxes (Zhu, 2002). In northern countries,
tomatoes are grown in greenhouse conditions where temperature has
to be controlled, increasing the expense of culture. The development
of cultivars more tolerant to cold temperature or chilling is an opportunity to decrease this expense. The study of species from temperate
regions, more tolerant to cold, led to the identication of several genes
regulated by cold and grouped under the name of COR genes
(Tomashow, 1998). All the COR genes have two specic sequences in
the promoter: the C-repeat (CRT) and dehydration responsive element
(DRE)-related motifs which interact with the CRT/DRE binding factor 1
(CBF1; Stockinger et al., 1997). When the Arabidopsis CBF1 gene was introduced into tomato, the transgenic plants harbored higher chilling tolerance. This response was associated with a decrease of ROS and
pronounced tolerance to oxidative damages (Hsieh et al., 2002a). Cold
stress also induces osmotic disorders. Thus tolerance to cold induces
the transcription of genes encoding enzymes responsible for the synthesis of osmoprotectants and the antioxidant defense (Goyary, 2009).
Osmotin and osmotin-like proteins were found to accumulate in plants
under a wide range of biotic and abiotic stresses. Patade et al. (2013) reported that transgenic tomato plants accumulating osmotin and proline
during cold treatment had better protection of cell structures and higher
ROS detoxication. As opposed to cold or freezing conditions, the production of plants tolerant to high temperature is of primary interest in
regards to global warming and climate changes. The response of plants
to high temperatures is characterized by metabolic changes which aim
to protect the essential structures and functions of cells. The accumulation of polyamines, such as betaine, putrescine, spermidine or spermine,
is one of these mechanisms. Indeed, betaine was found to protect enzymes, such as photosystem II of the photosynthetic machinery, against
heat-induced inactivation (Allakhverdiev et al., 1996). S-adenosyl-Lmethionine decarboxylase (SAMDC) is one of the key regulators of polyamines synthesis. Tomatoes expressing the SAMDC gene of S. cerevisiae
accumulated substantially higher amounts of spermine and spermidine
and were found to be more tolerant to high temperature. Moreover, the
antioxidant enzymatic activity and protection of lipid membranes
against peroxidation was signicantly enhanced in the transgenic
plants, showing that the accumulation of polyamines is a good strategy
for protection against high temperatures (Cheng et al., 2009). As already
mentioned, heat stress, and all abiotic stresses in general, generates the
production of ROS, possessing highly oxidant properties. In order to protect themselves against ROS-induced damage, plants evolved antioxidant enzymes, such as superoxide dismutase (SOD) and ascorbate
peroxidase (APX). Overexpression of cytosolic APX in tomato allowed
the plant not only to protect itself from damage caused by high temperatures, but also to protect itself against damage caused by exposure to
UV-B (Wang et al., 2006).
Today, more than one-fth of the world's arable land suffers from
salt stress. Salt stress is a major problem in agriculture worldwide, affecting crop growth, development, production, quality and yield. Increased salinization of arable land is expected to have devastating
global effects, resulting in loss of 30% of arable land within the next
25 years, and up to 50% by the year 2050 (Wang et al., 2003). Saline
stress is detrimental to plants because it brings water stress and inhibits
key biochemical reactions due to the excess of sodium (Li et al., 2011;
Moghaieb et al., 2011). To withstand saline stress, plants have developed several strategies: sequestration of solutes, limitation of lipid
peroxidation and/or production of osmoprotectants. The large, acidic

vacuole of plants serves for sequestration of such deleterious solutes.
A huge variety of ion transporters are localized on the vacuolar membrane, allowing the allocation of different ions and molecules into the
vacuole (Martinoia et al., 2000). In tomato, expression of the Arabidopsis
vacuolar Na+/H+ antiport (AtNHX1), which drives the export of salts
from cytoplasm into the vacuole, resulted in the production of transgenic tomato plants able to grow in the presence of 200 mM NaCl. Interestingly, whereas leaves accumulated huge amount of salt, no changes in
salt concentration were observed in fruit (Zhang and Blumwald,
2001). Another consequence of salt stress is lipid peroxidation with
the production of methylglyoxal, a highly mutagenic and cytotoxic compound. The detoxication of methyglyoxal is triggered by specic enzymes, glyoxalase I and II. Recently, the GlyI gene from Brassica juncea
and the GlyII gene from Pennisetum glaucum were simultaneously introduced into tomato. In the transgenic tomatoes expressing both genes,
lipid peroxidation and peroxide production were signicantly reduced.
Moreover degradation of chlorophyll due to saline stress was limited in
transgenic tomato plants. Altogether, this led to the conclusion that engineering the glyoxalase detoxication system is a way to enhance tolerance to high salt concentration in tomato (lvarez-Viveros et al.,
2013). Moghaied et al. (2011) demonstrated that improving the
osmoprotective system is also a good strategy to induce salt stress tolerance in tomato. Ectoine, a common solute of halophilic bacteria, is a
good example of an osmoprotectant. Ectoine is synthesized from Laspartate -semialdehyde in a three-step reaction catalyzed by
enzymes encoded by the ectB, ectA and ectC genes. When the three
genes were introduced into tomato by Agrobacterium-mediated transformation, the resulting transgenic plants accumulated high amounts
of ectoine in a dose-dependent response, i.e. the higher the concentration of salt applied, the more the ectoine accumulated. Water uptake
and transport to leaves was higher in transgenic lines, suggesting that
ectoine contributes to establish a proper water status upon stress. As already mentioned, salt stress negatively inuences the overall growth of
the plant, inhibiting leaf growth and inducing premature senescence.
Plant growth and development require proper hormonal status. In
salt-stressed plants, the general hormonal status is modied, and
more specically the endogenous cytokinin (CK) content is decreased.
Overcoming the salt-induced reduction of CK could be means of engineering tolerance to saline stress. This can be achieved by increasing
CK synthesis via overexpression of genes encoding enzymes for CK biosynthesis. The constitutive expression of the IPT gene, encoding the enzyme responsible for the rst step in CK biosynthesis, led to more than
150-fold increase of the CK content in tobacco and cucumber but had a
deleterious effect inhibiting root growth and inducing water decit syndromes (Smigocki and Owens, 1989). The role of CK in salt stress tolerance in tomato was assessed by two nice experiments (Ghanem et al.,
2011). In the rst experiment, overexpression of the IPT gene was driven by a heat shock inducible promoter. Transient root induction of the
IPT gene resulted in a slight decrease in root biomass but when the saline stress was applied, the higher endogenous CK content delayed stomatal closure and leaf senescence, and induced a 2-fold increase in
shoot growth. In the second experiment, the same authors grafted
non-transgenic tomato plants onto the root systems of transgenic tomato plants (WT/35S::IPT) constitutively expressing the IPT gene. When
the WT/35S::IPT plants were cultivated with moderate salt stress, the
number and size of the fruit produced were signicantly enhanced compared to the non-transgenic, non-grafted tomato plants. These last results, taken together, show how a precise regulation of the hormonal
status can be genetically engineered in order to produce tomato with
enhanced tolerance to salt stress.
Drought or water decit is an important environmental factor greatly affecting agriculture, making the management of water an important
task in agriculture. Genetic engineering for drought tolerance/resistance
is based on the knowledge gained from plants developing in arid and
semi-arid regions. Plants growing in water-limited conditions have
evolved two mechanisms which can be genetically engineered: 1) delay

of drought stress by developing a deep root system, reducing transpiration or increasing wax layers on the leaf surface, 2) tolerance to drought
by reducing the need for water for efcient growth (Kramer and Boyer,
1995). As previously mentioned, mechanisms conferring tolerance to
one stress can also confer tolerance to another one. This is the case of
the CBF1 gene, which was initially found to improved salt tolerance in
tomato (Hsieh et al., 2002a). The same authors demonstrated that the
ectopic expression of Arabidopsis CBF1 in tomato also confers resistance
to drought stress (Hseih et al., 2002b). In order to withstand drought
stress, plants accumulate solutes into their vacuole, modifying the overall osmotic status of the cell favoring water uptake from the soil. This
can be done either by increasing the activity of the vacuolar sodium/
proton antiporter or by increasing the uptake of protons into the vacuole, energizing secondary transporters (Pasapula et al., 2011). Vacuolar
proton uptake is ensured by proton pumps (VP1). Overexpression of the
Arabidopsis vacuolar H+-pyrophosphate (AVP1) in tomato conferred
tolerance to both salt and drought stress. This was marked by greater
import of cations into root vacuoles and higher root biomass (Park
et al., 2005).
4.3. Fruit quality
Tomato cultivars developed by traditional methods soften during
ripening, and require harvesting and transporting while fruits are still
green. Since 1962, it has been clearly dened that ethylene is the hormone driving ripening, as a rise in ethylene occurs before the onset of
ripening (Burg and Burg, 1962) and ethylene production is maintained
throughout ripening. The injection of ethylene allows the fruit to turn
red. The disadvantage of this practice is that the processes which lead
to development of natural avor are not induced, resulting in a tasteless
fresh tomato (Paduchuri et al., 2010). Inhibition of fruit ripening, and
consequently the extension of shelf-life and storage, by genetic engineering can be obtained by reducing or inhibiting ethylene production.
This is reached by the down-regulation or silencing of genes encoding
proteins involved in the ethylene biosynthesis pathway, such as
aminocyclopropane-1-carboxilic acid (ACC) synthase or AAC oxidase
(Hamilton et al., 1990; Oeller et al., 1991). Another possibility is to stimulate degradation of ethylene precursors by overexpressing genes
encoding proteins responsible for this reaction, such as ACC deaminase
or S-adenosyl methionine hydrolase (Good et al., 1994; Klee et al.,
1991). In tomato, the method of hpRNA-mediated silencing was used
to inhibit expression of the ACO1 gene, encoding for an ACC oxidase.
The lower ethylene content in transgenic tomato plants was associated
with a decrease in pectin methylesterase and polygalacturonase (PG)
activities, and consequently a reduced loss of rmness during ripening.
Nevertheless, the transgenic fruits developed their color later than the
non-transformed fruits (Behboodian et al., 2012). This result conrmed
what has been known for a long time: alteration of the ethylene pathway affects color development. This is a negative aspect of the selection
and points out the fact that alteration of the ethylene pathway is probably not the best target of genetic engineering for delayed ripening and/
or rmness. As already mentioned, PG is one of the targets of ethylene.
This enzyme is responsible for the cell wall degradation which occurs
during ripening and leads to fruit softening. In tomato, its expression
was inhibited by the RNA antisense strategy. The resulting transgenic
tomato plants harbored only 1% of residual PG activity and were characterized by rmer fruits. Ethylene production and lycopene accumulation were not modied in these transgenic fruits (Smith et al., 1990).
The color of tomato fruit is due to the presence of -carotene and
lycopene, which both have been found to have a benecial effect on
human health. Several attempts to produce a tomato with higher carotenoid content have been made. The PSY-1 enzyme catalyzes the rst
committed step of the carotenoid biosynthesis pathway by producing
phytoene from GGPP. In order to increase the carotenoid content of
fruit, the Psy-1 gene was constitutively expressed in tomato. When the
185
carotenoid content was signicantly increased in the fruit of transgenic

plants, some undesirable effects were observed, notably dwarsm of the
transgenic plant, probably due to the lack of gibberellins. Indeed, both
gibberellins and carotenoids are synthesized from GGPP and the probable consequence of accumulation of PSY-1 in the transgenic tomato was
stimulation of carotenoid synthesis in detriment to gibberellins biosynthesis (Fray et al., 1995). The use of the PG promoter drove the expression of Psy-1 specically in the fruit. While high accumulation of
carotenoid could be measured in the fruit, the overall size of the plant
was not affected (Fraser et al., 2002), highlighting again the advantages
of using organ-specic promoters.
The spontaneous tomato high-pigment mutant is characterized by a
high accumulation of carotenoids and the accumulation of other molecules such as avonoids, vitamins C and E, thus enhancing the nutritional quality of the fruit (Azari et al., 2010). Surprisingly the corresponding
gene does not encode an enzyme involved in the carotenoid biosynthesis pathway. Molecular characterization of the mutants revealed that
the mutation affects the tomato homolog of Arabidopsis DEETIOLATED
(DET1), a negative regulator of photomorphogenesis (Mustilli et al.,
1999). The suppression of DET1 in tomatoes by RNAi technology resulted in signicant increases of both carotenoids and avonoids without
deleterious effects on other parameters (Davuluri et al., 2005). It appears that modeling the expression of genes which regulated the biosynthesis pathway but not directly the genes of the biosynthesis
pathway is more efcient in genetic engineering of the tomato and
should be further considered in modern genetic engineering strategies.
Together with color, the taste and avor of fruit are very important
criteria for tomato selection today. Taste is determined by an equilibrium between sugars (sweet) and organic acids (acid). Because it is important to maintain low acidity of the fruit for processing tomatoes
(discussed previously), it is not of interest to modify the acid content
of the fruit. Just the opposite, it is probably preferable to modify the
sweetness of the fruit. This can be achieved by increasing the sugar content, but in a society where people ght obesity, this would not be a
good strategy. The African plant katemfe (Thaumatococcus daniellii
Benth.) produces a sweet-tasting protein called thaumatin (Van der
Wel and Loeve, 1972). In addition to its sweet taste, thaumatin has the
property of intensifying some avors while attenuating others. The
mechanisms of thaumatin action are still unknown. Thaumatin extracted from fruit or synthesized by microorganism was, and still is, widely
used in the food industry. In order to modify the taste of tomato, the sequence encoding thaumatin was introduced by Agrobaterium-mediated
transformation. The transgenic plants produced fruits with enhanced
sweetness (Bartoszewski et al., 2003). This emphasizes the fact that in
order to overcome the lack of taste characteristic of late ripening varieties thaumatin could be introduced into tomato cultivars with rin or nor
background.
The enrichment of tomato avor can be achieved by synthesizing
molecules which are known to participate in the avor and aroma signature of different aromatic plants, such as monoterpenes. Because
monoterpenes, like carotenoids, are synthesized from IPP and DMAPP,
their synthesis can be initiated in tomato fruits by introgression
of the proper gene. Geraniol is the main aromatic compound of
basil (Ocinum basilicum) and confers a characteristic sweet oral
aroma. Thus, Davidovich-Rikanati et al. (2007) expressed the
Ocimum basilicum geraniol synthase gene under the control of the
ripening-specic PG promoter. The avor signature of the tomato fruit
was modied by the accumulation of geraniol in the fruit and greatly
appreciated by the tasters. The negative point of the new transgenic
lines is that the modication of the avor/taste was made in detriment
to lycopene accumulation, resulting in a depreciation of the nutritional
value of the fruit.
-carotene is the precursor of vitamin A, which has a high antioxidant property, making it of interest in human health. As mentioned earlier, the early step of -carotene synthesis is the production of lycopene
from phytoene by the action of the phytoene desaturase. The carotene
186
desaturase (Crtl) gene from Erwinia uredovora was introduced into tomato in order to increase -carotene content. Because the production
of phytoene mediated by phytoene synthase is the limiting step of
carotenoid synthesis, no difference in the total amount of carotene
could be measured in transgenic tomato fruits. Instead, the quality of
the carotenoids produced was modied with a three-fold increase of
-carotene content, representing up to 45% of the total carotenoid content (Rmer et al., 2000).
4.4. Biopharming
In the past few years, a new concept, called biopharming, has been
developing. Biopharming consists of using plants to produce therapeutic molecules which will be further puried or introduced directly into
the human or animal diet. Despite the fact that bacterial fermentation,
yeast systems and mammalian cell cultures are still widely used to produce molecules important in treating cancers or different human diseases in huge amounts, the costs of development linked to purication
are very signicant. Up to now, more than 100 therapeutic and diagnostic recombinant proteins and vaccines have been produced in various
plants, including tobacco, cereals, legumes, fruit and vegetable crops.
Plants have several advantages over the classical modes of production
in term of economy, production scale and safety (Ahmad et al., 2012;
Chen et al., 2009).
Alzheimer's disease (AD) is a neurodegenerative disease leading to
neuron destruction. The toxic brain protein responsible for AD, betaamyloid (A), has been identied and is accumulated in patients developing AD. One strategy to prevent and cure AD would be the use of
vaccines directed against A. Attempts to produce and purify A as an
antigen in Escherichia coli or yeast systems have been laborious because
of the toxicity of the protein itself. Thus Youm et al. (2008) initiated the
production of A in tomato fruit. They were able to produce the protein
in a satisfactory amount and to use it in an immunization assay on mice.
Thymosin 1, a booster of the immune system, is widely used in treatment against viral infections (hepatitis B and C) and cancers. Until now
it has been derived from animal thymus or produced chemically; nevertheless, the amount of T1 produced is not sufcient faced with increasing demand for the medicine. Using Agrobacterium-mediated
transformation, T1 was specically expressed in the fruit of tomato.
The protein could be easily extracted from the fruit and the yield was
up to 6 g/g fresh weight. Moreover, the activity of the T1 produced
by tomato was higher than that produced by E. coli (Chen et al., 2009). Examples of tomato genetic engineering for the purposes of biopharming
are becoming more and more numerous, and tomato has already been
used for the production of different vaccines (reviewed in Ahmad et al.,
2012).
Beside its use to produce vaccines and therapeutic molecules, the tomato is also used as a bioreactor to produce molecules which are more
exotic. This is the case of miraculin. Miraculin is the active compound
of the miracle fruit, Synsepalum dulcum. It has the property to change
any sour taste into a sweet and pleasant savor (Kurihara and Beidler,
1968). The production and purication of miraculin in E. coli systems
was not sufcient enough and it is very recently that researchers have
used tomatoes as bioreactors for widerproduction of the compound.
The amount of miraculin in transgenic tomato fruit was up to 90 g
per g of fresh weight (Hirai et al., 2010).
5. Conclusion
Since its discovery in the 16th century and its rst steps into domestication and breeding more than 600 years ago, tomato has become one
of, if not the most important vegetable crop worldwide. Interest in tomato is increasing with the population. With current predictions of population increase and climatic changes, it is assumed that traditional
breeding of tomato is not sufcient to satisfy future demand and climatic changes. Development of the plant's molecular biology coupled to its
genome sequencing and the study of wild relative species, have allowed
researchers to go beyond the limits of classical breeding. The culture of
genetically modied (GM) tomatoes encounters problems in most of
the leading producer countries. Indeed since the arrest of GM tomato
culture in the USA in 2002, only China remains a producer of GM tomatoes. This is mainly due to the negative opinion of the population that
GM crops are deleterious or, even worse, dangerous for human health
and the environment; this is the consequence of misinformation. As
scientists, our duty is not only to produce crops with engineered
agronomical traits, but also to educate and inform the people around
us. The use of marker-free transgenic plants i.e. devoid of resistance
of antibiotic or herbicides should provide a good argument in favor
of the use of such plants. Moreover, we also have to take advantage of
knowledge from wild relative species. Indeed introgression of genetic
information from wild or related species into the cultivated tomato
and limitation of the transfer of alien information would limit the risk
of deleterious effects on the environment and on human or animal
health. Finally, while GM tomatoes are promising, their potential has
rarely been validated in eld trials. Such trials, as well as study of impact
on health and the environment, have to be developed and the results
have to be brought to the awareness of society. If we do not invest
part of our time in doing this, why should we continue developing
crops useful to humanity?
Acknowledgments
The author would like to thank J. Humplik and P. Galuszka for their
criticism during the preparation of the present manuscript and M.
Sweney for English correction of the text. Finally, the author thanks K.
Janokov for the design of Fig. 3. This work was supported by the OP
RD&I grant ED0007/01/01 Centre of the Region Han for Biotechnological and Agricultural Research from the Ministry of Education Youth and
Sports, Czech Republic and P501/10/0785 from the National Science
Foundation, Czech Republic.
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Biotechnology Advances 32 (2014) 170189

Contents lists available at ScienceDirect

Research review paper

The history of tomato: From domestication to biopharming

Available online 7 November 2013

E-mail address: veronique.bergougnoux@upol.cz.

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

under controlled greenhouse conditions. The recent increase in tomato

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

Harvested area (Ha)

Food supply quantity (kg/capita/year)

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

Distribution and habitat

Importance for breeding purposes

Salt tolerance; Lepidoptera and virus resistance

L. cheesmaniae var. minor

Moisture-tolerance, resistance to wilt,

SC, At, facultative Al

Green, purple stripes

Color and fruit quality; resistance to insect,

High sugar content

Drought resistance; resistance to insects

Endemic to the Galapagos Islands, Ecuador; wide variety of

Typically SI, Al, rare

Resistance to virus, bacteria, fungi,

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

Species (Solanum name)

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

Mesoamerica (Mexican hypothesis), followed by its introduction to

Clavibacter michiganensis subsp. michiganensis

Alternaria alternata f.sp. lycopersici

Viral, viroid and mycoplasmalike organisms [MLO] diseases

Tobacco mosaic virus (TMV)

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

Other plants are already model organisms for scientists, such as

agricultural practices, breeders turned to the wild species. Indeed the

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

ACC, ACS produces methylthioadenosine, which is used to synthesize

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

with the promoter of genes involved in the major processes observed

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

Germplasm source (Lycopersicum name)

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

3. Genetic engineering of tomato

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

plants from transgenic plants which originally contained one of these

stresses, such as salt, drought, cold and ABA treatment. Analysis of

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

Arf8, IAA9, iaaM

Ficcadenti et al. (1999), Goetz et al. (2007), Wang et al. (2005a)

Dxs, CrtB, CrtI, CrtY, PSY-1, CRY-2, CYC-B, LCY-B, LCY-B,

Ascorbic acid content GaLDH, GME, GCHA and/or ADCS

Vst1 and Vst2, Cry1Ac, Cry1Ab, Ep5c,

Pharmaceutics and other

TB1-HBS, CtrB, Spike, miraculin

Davuluri et al. (2005), Dharmapuri et al. (2002),

compared to what was observed in non-transformed plants (Ma

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

signicantly stronger resistance to damages caused by Heliothis obsoleta

peroxidation and/or production of osmoprotectants. The large, acidic

V. Bergougnoux / Biotechnology Advances 32 (2014) 170189

evolved two mechanisms which can be genetically engineered: 1) delay