Seisuke & Neelima 2008

Emerging Model Organisms
Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop

Seisuke Kimura and Neelima Sinha1
Department of Plant Biology, University of California, Davis, CA 95616, USA
INTRODUCTION
Tomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. It origi-
nated in western South America, and domestication is thought to have occurred in Central America.
Because of its importance as food, tomato has been bred to improve productivity, fruit quality, and
resistance to biotic and abiotic stresses. Tomato has been widely used not only as food, but also as
research material. The tomato plant has many interesting features such as fleshy fruit, a sympodial
shoot, and compound leaves, which other model plants (e.g., rice and Arabidopsis) do not have. Most
of these traits are agronomically important and cannot be studied using other model plant systems.
There are 13 recognized wild tomato species that display a great variety of phenotypes and can be
crossed with the cultivated tomato. These wild tomatoes are important for breeding, as sources of
desirable traits, and for evolutionary studies. Current progress on the tomato genome sequencing
project has generated useful information to help in the study of tomato. In addition, the tomato
belongs to the extremely large family Solanaceae and is closely related to many commercially impor-
tant plants such as potato, eggplant, peppers, tobacco, and petunias. Knowledge obtained from stud-
ies conducted on tomato can be easily applied to these plants, which makes tomato important
research material. Because of these facts, tomato serves as a model organism for the family Solanaceae
and, specifically, for fleshy-fruited plants.
RELATED INFORMATION
For a history of tomato genetics, see Rick (1991a). A review of quantitative trait loci (QTL) analyses in
tomato is provided by Lippman et al. (2007). An outline of the effort to integrate genomic and taxo-
nomic information for the members of the diverse Solanaceae plant family is also available (Knapp et
al. 2005). For an overview of the International Solanaceae Genome Project (SOL) Genomics Network
(SGN) (a comparative resource for Solanaceae biology), see Mueller et al. (2005).
The key traits involved in tomato (and pepper) domestication, as well as efforts to identify the
genes responsible for those traits, are reviewed by Paran and van der Knaap (2007). For additional dis-
cussion of the genes and genetic changes involved in the domestication of crop plants, including
tomato, see Doebley et al. (2006). A review of the genetic control and development of shoot branch-
ing in tomato (and Arabidopsis) is provided by Schmitz and Theres (1999). For a review of the genetic
control and development of compound leaves in tomato (and other plant species), see Sinha (1999).
Several Web-based resources are also available: Tomato expressed sequence tag (EST) clones are
available for purchase through http://ted.bti.cornell.edu/; the BAC/EST Resource Center at the
Clemson University Genomics Institute (http://www.genome.clemson.edu/capabilities/bacCenter.shtml)
has tomato bacterial artificial chromosome (BAC) libraries and clones available for purchase; the C.M.
Rick Tomato Genetics Resource Center (TGRC) (http://tgrc.ucdavis.edu) contains a collection of wild
relatives, monogenic mutants, and miscellaneous genetic stocks of tomato; the Tomato Expression
Database (http://ted.bti.cornell.edu) includes the tomato microarray data warehouse, tomato
microarray expression data, and tomato digital expression data; the Kazusa Full-length Tomato cDNA
1
Corresponding author (nrsinha@ucdavis.edu)
Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.emo105 www.cshprotocols.org
© 2008 Cold Spring Harbor Laboratory Press 1 Vol. 3, Issue 11, November 2008
Database (http://www.pgb.kazusa.or.jp/kaftom) provides information about full-length cDNA clones
derived from the miniature cultivar Micro-Tom; the International Tomato Sequencing Project
(http://www.sgn.cornell.edu/index.pl), part of the SGN, tracks the progress of the tomato genome
sequencing project and includes links to additional tomato- and Solanaceae-related resources;
http://zamir.sgn.cornell.edu/mutants (The Genes That Make Tomatoes) provides information about
the saturated mutation library of tomato; and KOMICS (Kazusa OMICS; http://webs2.kazusa.or.jp/
komics) provides a database for metabolites.
Protocols describing How to Grow Tomatoes (Kimura and Sinha 2008a), Crossing Tomato
Plants (Kimura and Sinha 2008b), Grafting Tomato Plants (Kimura and Sinha 2008c), and Tomato
Transformation (Kimura and Sinha 2008d) are also available.
BACKGROUND INFORMATION
The tomato (Solanum lycopersicum) (Fig. 1) and its wild relatives (genus Solanum, section Lycopersicon)
originated in western South America (Ecuador, Peru, and Chile). Wild tomatoes can still be found
along the western coast of South America, in the Andes, and on the Galapagos Islands. Although the
center of diversity of wild tomatoes is in Peru (Rick 1991b), genetic analysis of primitive cultivars has
shown that the center of diversity of cultivated tomatoes is in Mexico. This indicates that tomato
domestication may have occurred in Central America (Rick 1991b). When the conquistadors from
Europe came to the Americas, there was widespread cultivation of tomato. It is likely that Europeans
distributed the tomato from the Americas to Europe and European colonies during the 16th century.
Interestingly, the tomato was introduced to the United States by European immigrants, not from
Mexico. Today, the tomato is one of the major vegetable crop plants in the world. Currently, more
than 120 million tons of tomatoes are produced annually worldwide.
Carolus Linnaeus (1753) placed the tomato in the genus Solanum as Solanum lycopersicum L. (lyco
means “wolf,” and persicum means “peach”). However, Philip Miller (1754) segregated a new genus,
Lycopersicon, from Solanum to accommodate the tomato and several other species and named the
tomato Lycopersicon esculentum Mill. (esculentum means “edible”). At the time, many people thought
that the tomato fruit was poisonous; Miller may have wanted to emphasize that it was edible. This
name (Lycopersicon esculentum) breaches the International Code of Botanical Nomenclature (ICBN);
technically, the name Lycopersicon lycopersicum should have been used. Nevertheless, Lycopersicon
esculentum was widely used until recently.
FIGURE 1. Tomato plants and parts: (A) seedling; (B) 40-d-old

plant; (C) leaf; (D) flowers; (E) fruit; (F) seeds. Bar, 2 cm.
www.cshprotocols.org 2 Cold Spring Harbor Protocols

Recent studies using morphological characters (Child 1990) and molecular data from both chloro-
plast and nuclear genomes (Spooner et al. 1993; Bohs and Olmstead 1997) have shown that toma-
toes are a sister group of potatoes (Solanum tuberosum L.). Tomatoes, potatoes, and their relatives are
now taxonomically treated as part of the same genus (Solanum) to retain the monophyly of the taxon.
Therefore, tomato is currently placed in the genus Solanum and named Solanum lycopersicum per
Linnaeus (Darwin et al. 2003; Spooner et al. 2005). The word Lycopersicon is now used as one of the
section names of Solanum.
Currently, 13 wild tomato species are known (Peralta et al. 2005; Spooner et al. 2005). All are
diploid (2n = 24), can be crossed with the cultivated tomato, and serve as a breeding source for desir-
able traits such as increased productivity yield, fruit quality, and resistance to pathogens and abiotic
stresses. Wild tomato species are also important for evolutionary studies. Tomato belongs to the
extremely large family Solanaceae, and it is closely related to many commercially important plants
such as potato, eggplant, peppers, tobacco, and petunias. The knowledge obtained from tomato
studies can be easily applied to these plants, making tomatoes important research material. As
described here, genetic research on tomato has resulted in linkage maps, molecular markers, mutants,
and introgression lines. Because of these excellent resources, tomato serves as a model organism for
the family Solanaceae generally and for fleshy-fruited plants specifically.
SOURCES AND HUSBANDRY

The C.M. Rick Tomato Genetics Resource Center (TGRC) at the University of California, Davis main-
tains seed stocks of accessions of wild relatives, monogenic mutants, and miscellaneous genetic stocks
of tomato and distributes the seeds to researchers worldwide. It also stocks introgression lines repre-
senting the genomes of wild tomatoes Solanum habrochaites and Solanum pennellii on the S. lycoper-
sicum background. The TGRC maintains a user-friendly database (http://tgrc.ucdavis.edu) that
includes general information on all accessions, detailed collection notes for wild species, mutant lists,
and images of tomato stocks.
A comprehensive mutant population is a basic resource for studying gene function. An isogenic
mutant library was developed for tomato (Menda et al. 2004) using two mutagens, EMS (ethyl-
methanesulfonate) and fast neutron. A total of 13,000 M2 families were grown in the field, pheno-
typed, and categorized. Currently, 3417 mutations have been cataloged, and all of the information
(phenotypes, images, etc.) is available on a website called “The Genes That Make Tomatoes”
(http://zamir.sgn.cornell.edu/mutants). The mutants can be searched by keyword and phenotype,
and mutant seeds can be requested through this site.
There are more than 5000 different cultivars of tomato. Seeds of many of these cultivars can be
purchased from commercial companies, but most of them are hybrid stocks. For basic research, culti-
vars such as M82, Ailsa Craig, and VF36 are usually used. Recently, Micro-Tom, a dwarf cultivar, was
introduced as a preferred variety to perform molecular research because of its compact habit
(Meissner et al. 1997). Micro-Tom is only 4-7 in. high and is one of the smallest tomato plants in the
world. Its seeds are distributed by the TGRC. Tomatoes can be easily grown in the field, in green-
houses, and in growth cabinets.
RELATED SPECIES
The genus Solanum is one of the largest genera of angiosperms. The number of species in this genus
is estimated to be ~1500. It also includes potato (Solanum tuberosum), eggplant (Solanum melongena),
and wild tomato (Solanum L. section Lycopersicon [Mill.]). Currently, there are 13 recognized wild
tomato species, and these are the most important genetic resource for tomato breeders. Wild toma-
toes can be found in many environments, from high mountains to dry coastal regions, and they
exhibit great differences in morphological characters, mating systems, habitat preferences, disease
susceptibility, and stress resistance. They have been used as a source of desirable traits such as
increased productivity yield, fruit quality, and resistance to pathogens and abiotic stresses (Stevens and
Rick 1986).
Solanum belongs to an extremely large plant family, the Solanaceae, which is one of the most
important to humans. Plants within this family (Fig. 2) are used as food sources (e.g., potato, egg-
plant, bell peppers, chili peppers [Capsicum annuum, Capsicum frutescens, and Capsicum chinense], and
tomatillo [Physalis philadelphica]); as drugs (e.g., tobacco [Nicotiana tabacum], deadly nightshade

FIGURE 2. Many economically important plants belong to the
Solanaceae family: (counterclockwise from top, right) tomato,
potato, eggplant, red and green bell peppers, chili peppers,
tomatillo, and tobacco.
[Atropa belladonna], mandrake [Mandragora officinarum], and jimson weed [Datura stramonium]); and
as ornamentals (e.g., petunia [Petunia hybrida]). The presence of many economically important plants
in the Solanaceae family makes tomato pivotal as a model plant species.
USES OF THE TOMATO MODEL SYSTEM

Because of its importance as a food source, tomato has been bred and studied for a long time. The
tomato has many interesting features that other model plants, such as rice and Arabidopsis, do not
have. For example, tomato plants produce fleshy fruits that are important for the human diet (Fig. 1E);
botanically, tomato fruits are berries. The tomato has sympodial shoots, and it is the only model plant
with compound leaves (Fig. 1C) that has extensive genome sequence data available. Most of these
traits are agronomically important and have been the major targets for domestication and breeding.
Phenotypes of most of these traits are determined by the collective effects of many loci (quanti-
tative trait loci, or QTL). The tomato is an ideal plant for QTL analysis because of the phenotypic diver-
sity of these traits in cultivated and wild tomatoes. In addition, a large number of mutants have been
generated, which facilitates the study of these important traits. In this section, we describe current
research on the genes and the genetic and molecular mechanisms controlling several of the traits that
are well studied in tomatoes.
Fruit Characteristics
Fruit Size
Fruit size is one of the most important traits in agriculture and was a major selection target during
domestication and breeding. The presumed ancestor of cultivated tomato, Solanum pimpinellifolium,
has tiny fruits that weigh only a few grams each. The fruit of the cherry tomato plant (S. lycopersicum
subspecies cerasiforme), which is thought to be the direct ancestor of cultivated tomatoes, is only
slightly larger than that of S. pimpinellifolium. Domestication and breeding resulted in modern culti-
vated tomatoes, with fruits that are more than 10 cm in diameter and weigh more than 500 g.
Fruit size in tomato is a quantitative trait, and studies have identified many QTL controlling vari-
ation in fruit size (Tanksley 2004). So far, only one of the QTL, fw2.2 (the second fruit weight [fw] QTL
on Chromosome 2), has been cloned (Frary et al. 2000). This QTL explains as much as 30% of the dif-
ference in fruit size between wild and cultivated tomatoes. fw2.2 encodes a protein that shares homol-
ogy with the human RAS oncogene and acts as a negative regulator of cell division during fruit
development. Differences in fruit size between wild and cultivated tomatoes are thought to be caused
by differences in the transcriptional regulation of this gene.
Fruit Shape
Fruit shape is also an important agricultural trait, and several loci controlling fruit shape have been
identified and characterized (Tanksley 2004). One of the QTL, SUN, accounts for 58% of the pheno-
typic variation in a cross between S. lycopersicum variety Sun1642 and wild tomato S. pimpinellifolium
(Xiao et al. 2008). S. pimpinellifolium has round fruit, whereas Sun1642 has elongated fruits. Map-
based cloning revealed that the SUN locus includes the IDQ12 gene; the corresponding IDQ12 protein

is a member of the IQ67-domain-containing family. Interestingly, a retrotransposon (Rider)-mediated
gene duplication resulted in the fusion of the DEFL1 gene promoter region with the IDQ12 gene,
which, in turn, resulted in the overexpression of IDQ12. This IDQ12 overexpression is thought to
cause the elongated fruit shape by altering plant hormone and/or secondary metabolite levels (Xiao
et al. 2008).
The OVATE gene, also identified by QTL analysis, is involved in making pear-shaped fruits and
was found in the S. lycopersicum subspecies cerasiforme variety “Yellow Pear” (Liu et al. 2002).
Overexpression of the OVATE gene converts pear-shaped tomatoes to round-shape tomatoes but does
not affect fruit size. OVATE is a novel regulatory gene with a putative nuclear localization signal and
homology with human von Willebrand factor genes.
Fruit Color
is determined by the quantities of carotenoids, such as lycopene and β-carotene, and chlorophylls.
Tomato fruit colors vary from green and yellow to orange and red. In most cases, the color of the fruit
Red and orange colors result from the accumulation of lycopene and β-carotene, respectively. Green
fruits contain chlorophylls, which usually degrade during the ripening process. Multiple genetic loci
control tomato fruit color, and most of the loci encode enzymes of the carotenoid biosynthetic path-
way or their regulators.
Solanum cheesmaniae (previously known as Lycopersicon cheesmanii f. major) and Solanum galapa-
Islands, have orange fruits that contain fivefold to 10-fold more β-carotene than red-colored fruits do
gense (previously known as Lycopersicon cheesmanii f. minor), wild tomatoes endemic to the Galapagos
gene encodes a chromoplast-specific lycopene β-cyclase (CYC-B), an enzyme that converts lycopene
(Ronen et al. 2000). This color change is regulated by a dominant allele (Beta) of the B gene. The B
to β-carotene. Expression of the B gene in ripening fruit is dramatically higher in the Beta mutant than
in wild-type fruit and results in an accumulation of β-carotene in the fruit. The recessive old-gold (og)
B gene (Ronen et al. 2000). The complete absence of β-carotene in og fruits (due to the lack of CYC-
mutation results in deep-red-colored fruits. Molecular analysis has shown that og is a null allele of the
The DELTA locus encodes lycopene ∈-cyclase, which converts lycopene to δ-carotene (Ronen et
B) causes their deep-red color.
mulation of δ-carotene and orange-colored fruit. The recessive tangerine mutant also has orange
al. 1999). The dominant allele Delta exhibits increased gene expression, which results in an accu-
fruit. The TANGERINE gene encodes a carotenoid isomerase (CRTISO) that is required during
carotenoid desaturation. The expression of CRTISO is up-regulated during fruit ripening, but its
expression is abolished in the tangerine mutant. In the tangerine mutant, prolycopene (instead of
all-trans-lycopene) accumulates, thereby making the fruit color orange (Isaacson et al. 2002).
The yellow-fruited tomato mutant yellow flesh has a mutation in the PHYTOENE SYNTHASE 1
(PSY1) gene (Fray and Grierson 1993), which results in the production of a truncated protein. The
yellow color is thought to be caused by lack of carotenoids because the truncated protein is unable
to convert geranylgeranyl diphosphate to phytoene.
Total Soluble Solids

One of the major objectives for tomato breeders is to increase the contents of total soluble solids (Brix,
measured by a refractometer in Brix units) in the fruit (Eshed and Zamir 1995). Brix is a measure of
sugars and acids in fruit, is an important fruit taste trait, and is associated with commercial tomato
yields. Brix values in the wild tomato S. pennellii are significantly higher than those in cultivated toma-
toes, and QTL analyses have identified more than 20 QTL that increase Brix (Eshed and Zamir 1995).
One of the QTL identified in S. pennellii, Brix9-2-5, improves Brix by more than 20% and was cloned
by map-based cloning (Fridman et al. 2004). The Brix9-2-5 locus encompasses a gene for a cell wall
invertase (LIN5). The functional polymorphism of the Brix9-2-5 locus causes an amino acid substitu-
tion near the catalytic site of the invertase, affecting enzyme kinetics and resulting in fruits with high
sugar contents.
Floral System and Plant Architecture

The shoot systems of flowering plants display great variation in their architecture and growth habits.
The model plant Arabidopsis thaliana has monopodial shoot architecture; the apical meristem is inde-
terminant and active throughout the plant life cycle (Pnueli et al. 1998). In contrast, tomato has

sympodial shoot development: The primary vegetative shoot terminates in a flower after the devel-
opment of eight to 12 leaves. Subsequently, new vegetative shoots arise from the axillary bud just
below the terminating inflorescence. This new shoot, in turn, terminates again after making three
leaves, and the next shoot arises from the newly formed axillary bud. This cycle is repeated continu-
ously to form sympodial shoots. By definition, tomato shoots are considered to be “determinate”
because each shoot terminates in a flower. However, the wild-type growth habit of tomatoes is classi-
fied as “indeterminate” because they continuously produce sympodial units.
The SELF-PRUNING (SP) gene regulates the switch from vegetative to reproductive growth. sp
mutants show a “determinate” shoot habit (a limited number of sympodial shoots), resulting in lim-
ited shoot growth, a reduction in the number of leaves between inflorescences (which, in turn, results
in closely set fruit), and bushy compact plants with nearly homogeneous fruit settings. In contrast, the
overexpression of SP results in an extended vegetative phase of sympodial shoot development. The
tomato SP gene is the ortholog of the A. thaliana TERMINAL FLOWER 1 gene, which maintains the inde-
terminate state of the inflorescence meristem. SP is thought to have a role in preventing early flower-
ing in each of the developing shoot meristems (Pnueli et al. 1998). The efficiency of mechanical fruit
harvesting in field-grown tomatoes has been increased by introducing the sp mutation into cultivated
tomatoes (Atherton and Harris 1986).
Compound Leaf Development

In general, plant leaves are characterized by a striking diversity in shape. The most conspicuous char-
acteristic of leaf shape is the degree to which the leaf is subdivided into smaller segments. Leaves lack-
ing subdivision are termed “simple,” whereas divided leaves are termed “compound.” Leaf complexity
is known to directly affect several processes critical to plant survival, including efficiency of photosyn-
thesis, rate of gas exchange, and resistance to herbivore damage. This indicates that the diversity in
leaf form is the result of adaptation to growth in varied environments.
The tomato has unipinnately compound leaves (Fig. 1C) and is the only model plant with com-
pound leaves that has extensive genomic and genetic data available. Leaf shape varies among wild
and cultivated tomatoes, which makes QTL analyses possible (Holtan and Hake 2003; Frary et al.
2004). A large number of leaf mutants are also known. These facts make tomato ideally suited for the
study of the molecular mechanisms of compound leaf development.
Tomato leaf mutants have varying degrees of leaf developmental defects. Some mutations change
compound leaves into simple or nearly simple leaves. Lanceolate (La) and entire (e) mutants belong to
this category. The LA gene encodes a TCP family transcription factor that contains a microRNA
(miR319)-binding site (Ori et al. 2007). Mutations are in the miR319-binding site, so the mutated tran-
scripts cannot be recognized by miR319, resulting in a partial resistance of the La transcript to miRNA-
directed inhibition. Elevated La transcripts in young leaf primordia are thought to promote the
precocious differentiation of leaf margins, leading to the La phenotype (Ori et al. 2007). The entire
mutant also has nearly simple leaves. An alteration in the coding region of the tomato IAA9 gene, a
member of the Aux/IAA gene family, was shown to result in the entire mutant phenotype (Zhang et al.
2007). The function of IAA9 in compound leaf development is still unclear, but results indicate the
involvement of the auxin pathway in compound leaf development.
Other mutations, such as Mouse ear (Me), Peteroselinum (Pts), Curl (Cu), and bipinnata (bip),
increase leaf complexity. Mouse ear (Me) is a dominant mutation that arose spontaneously in the
tomato cultivar Rutgers and shows excessively proliferated leaves, with three or four orders of leaflets
(Chen et al. 1997; Parnis et al. 1997). The Me phenotype resembles the phenotype of transgenic
plants that overexpress the tomato gene LeT6, a member of the KNOX gene family (L. esculentum [S.
lycopersicum] T6). Molecular analysis showed that the Me phenotype was caused by a spontaneous
fusion of the PFP gene, which encodes a metabolic enzyme (β-subunit of PPi-dependent phospho-
fructokinase), and LeT6, resulting in the altered expression pattern and elevated expression levels of
LeT6 (Chen et al. 1997; Parnis et al. 1997). The Cu is a dominant mutant with ramified compound
leaves that have wrinkled and curled blades (Parnis et al. 1997). The Me and Cu loci are tightly linked
to each other. LeT6 is overexpressed in Cu (as it is in Me), indicating that Cu is also caused by the over-
expression of LeT6 (Parnis et al. 1997).
The most dramatic example of leaf natural variation was found between the closely related wild
tomato species S. cheesmaniae and S. galapagense (Darwin et al. 2003). S. cheesmaniae leaves are sim-
ilar to leaves of cultivated tomatoes, whereas the leaves of S. galapagense are more complex. The
introgression of the leaf complexity trait from S. galapagense into cultivated tomato demonstrated that

this phenotype is conferred by a single semidominant allele named Petroselinum (Pts) (Rick 1980).
Map-based cloning of Pts showed that this locus encompasses a novel KNOX gene that lacks a home-
odomain (Kimura et al. 2008). A mutation in the promoter of this gene up-regulates the gene prod-
uct in leaves and is responsible for increased leaf complexity. The classic tomato mutation bipinnata
(bip) closely resembles the Pts phenotype and has been shown to be a loss-of-function mutant of the
BEL-LIKE HOMEODOMAIN (BELL) gene (Kimura et al. 2008).
GENETICS AND GENOMICS RESOURCES

Tomato is one of the most well-studied organisms for genetics research and, because of its agricultural
importance, has a long history of breeding. The cultivated tomato is diploid and self-pollinating and
has a relatively short generation time. Wild tomatoes are a valuable resource for desirable traits
because they show great phenotypic variation (e.g., morphology and resistance against diseases and
abiotic stresses) and can be crossed with cultivated tomato. These features make tomato ideal mate-
rial for genetic research.
Classical genetics research has created more than 1000 tomato mutants that include spontaneous
and induced (via radiation and chemicals) mutations. Loci responsible for many of these mutants have
been genetically mapped (Stevens and Rick 1986). Recently, an isogenic tomato mutant library was
developed on the genetic background of the inbred variety M82 (Menda et al. 2004). Currently, 3417
mutations have been cataloged in this mutant library. These collections of mutants are used to inves-
tigate the functions of genes in tomato. Nomenclature guidelines for tomato genetics were published
in 1955 (Barton et al. 1955).
Names and symbols of mutant genes are designated by letters and are italicized. The mutant
name should indicate the main character of the phenotype. The initial letter of a dominant allele is
uppercased, whereas a recessive allele is written with all letters in lowercase. The normal (wild-type)
allele is indicated by adding the superscript “+” after the name of the gene, and additional alleles at
the same locus, except for the first allele, are designated by numbered superscripts (e.g., bip+, bip, bip2,
and bip3). Mutations at different loci whose effects are indistinguishable by phenotype are called
“mimics.” Mimics are preferably designated by different names, but a numbered series can be applied,
too. In the latter case, mimics are designated by the same name, followed by a hyphen and a num-
ber (e.g., wiry-1 and wiry-3).
The first-generation linkage map of the tomato contained more than 200 morphological and
isozyme loci (Stevens and Rick 1986). Subsequently, high-density molecular linkage maps were con-
structed by mapping restriction fragment length polymorphism (RFLP) and amplified fragment
length polymorphism (AFLP) markers to the tomato genome using S. lycopersicum × S. pennellii F2
populations (Haanstra et al. 1999). Some of the morphological and isozyme markers were mapped
to the RFLP map to integrate the linkage and molecular maps. Linkage maps have also been gener-
ated using S. lycopersicum × S. pimpinellifolium (Doganlar et al. 2002) and S. lycopersicum × S.
habrochaites (Bernacchi and Tanksley 1997). These linkage maps have made map-based cloning, QTL
analysis, marker-based breeding, and genome sequence assembly feasible in tomato. Information
about these maps is available on the website for the International Tomato Genome Sequencing
Project (http://www.sgn.cornell.edu/about/tomato_sequencing.pl).
The ongoing International Tomato Genome Sequencing Project is the cornerstone of a larger proj-
ect, the International Solanaceae Genome Project (SOL), that aims to develop the family Solanaceae
as a model for understanding plant adaptation and diversification through systems biology
(http://sgn.cornell.edu/solanaceae-project). Macrosynteny and microsynteny conservation among the
genomes of tomato, potato, pepper, and eggplant is very high because there were no large-scale
duplication events such as polyploidization early in the radiation of the Solanaceae family. This
allows one to easily apply the knowledge obtained from tomato genome sequence data to other
Solananceae species.
The tomato genome contains 950 Mb of DNA (n = 12) (Arumuganathan and Earle 1991); of that,
only 220 Mb (25%) is gene-rich euchromatin (Wang et al. 2006). The remaining 730 Mb (75%) is
heterochromatin and largely devoid of genes. In tomato, heterochromatin is concentrated around
centromeres, and euchromatin is located in the distal portion of each chromosome. In contrast, in the
chromosomes of maize and rice, heterochromatin and euchromatin are interspersed. This interesting
feature of the tomato genome allowed the International Tomato Genome Sequencing Project to focus
on sequencing only the euchromatic regions. BAC clones that mapped to euchromatic regions were

selected and used as “seed BACs” to identify BAC clones that covered entire euchromatic regions.
These BACs are currently being sequenced by shotgun sequencing. This strategy is expected to give
us more useful data than would be obtained from the whole-genome shotgun approach. Currently,
~25% of euchromatic DNA has been sequenced, and the sequencing project is expected to be
finished in 2008. The sequence data, other useful information (annotations, maps, markers, etc.), and
tools are available on the website for the International Tomato Genome Sequencing Project
(http://www.sgn.cornell.edu/index.pl).
EST, unigene, and BAC information is also available on the website for the International Tomato
Genome Sequencing Project. The entire tomato BAC library, BAC filters, or individual BAC clones can
be purchased from the Clemson University Genomics Institute (CUGI; http://www.genome.
clemson.edu/capabilities/bacCenter.shtml). Tomato EST clones can also be ordered online
(http://ted.bti.cornell.edu/). Information on full-length cDNAs from Micro-Tom is available from the
Kazusa Full-length Tomato cDNA Dababase (KaFTom; http://www.pgb.kazusa.or.jp/kaftom).
Microarray expression data are available from the Tomato Expression Database (http://ted.bti.
cornell.edu). A database for metabolites detected in tomato fruit is available from KOMICS
(http://webs2.kazusa.or.jp/komics).
TECHNICAL APPROACHES
Tomato is excellent research material. Protocols that are useful for tomato research are described in
How to Grow Tomatoes (Kimura and Sinha 2008a), Crossing Tomato Plants (Kimura and Sinha
2008b), Grafting Tomato Plants (Kimura and Sinha 2008c), and Tomato Transformation (Kimura
and Sinha 2008d). Most basic techniques, such as genomic DNA isolation, RNA isolation, and pro-
tein extraction, are easy, and protocols for the model plant Arabidopsis can be applied to tomato.
Tomatoes can be stably transformed using tissue culture methods (see Tomato Transformation
[Kimura and Sinha 2008d]), so investigators can subsequently perform overexpression, RNA interfer-
ence (RNAi), promoter-β-glucuronidase (GUS) analysis, and other assays to functionally characterize
a gene of interest.
REFERENCES
Arumuganathan, K. and Earle, E. 1991. Estimation of nuclear DNA Lycopersicon pennellii in the cultivated tomato enables the identi-
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Protocol
How to Grow Tomatoes

INTRODUCTION
Tomatoes can be easily grown in a field, in a greenhouse, or in a growth cabinet. They need acidic
soil (pH 6.0-6.8), a lot of light, and water. The optimum temperature for growing tomato plants and
fruit is 18°C-24°C. This protocol describes how to germinate tomato seeds, cultivate adult plants, and
harvest seeds from fruit.
RELATED INFORMATION
For an overview of tomato as a model organism, see Tomato (Solanum lycopersicum): A Model Fruit-
Bearing Crop (Kimura and Sinha 2008a). Protocols describing Crossing Tomato Plants (Kimura and
Sinha 2008b), Grafting Tomato Plants (Kimura and Sinha 2008c), and Tomato Transformation
(Kimura and Sinha 2008d) are also available.
MATERIALS
CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, and
recipes for reagents marked with <R>.
Reagents
<!>Bleach (sodium hypochlorite, 2.7%) (optional; see Step 1)
Tomato seeds
Equipment
Container for seed collection (see Step 9)
Flats with plastic covers, or Petri dishes (100 mm diameter × 15 mm high) or other transparent
containers lined with wet filter paper (see Steps 1 and 2)
Greenhouse or growth cabinet, set at 18°C-24°C (optional; see Steps 2 and 4)
Knife
Paper towels
Pots (15 cm diameter × 15 cm high) or field (see Step 3)
Scissors (optional; see Step 6)
Sieve (optional; see Step 10)
Soil, gardening (pH 6.0-6.8)
Stakes or tomato cages (optional; see Step 5)
1
Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5081 www.cshprotocols.org
METHOD
Germination
1. Sow the seeds in flats containing soil (0.5-2 cm from the surface of the soil) or on wet filter paper
in a Petri dish. Keep the seeds 2.5-5 cm apart.
Seeds of some tomato accessions, such as wild tomato and introgression lines, are resistant to germination. To
improve the germination rate, completely immerse the seeds in 2.7% bleach (sodium hypochlorite) for 30 min at
room temperature. Then wash off the bleach completely by rinsing the seeds in H2O before sowing.
2. Allow seeds to germinate as follows:
If using soil:
i. Place the flats in a greenhouse or growth cabinet. Cover them with a plastic cover to keep
the soil moist while the seeds are germinating.
If using wet filter paper:
ii. Keep the seeds in the dark at room temperature until germination.
The seeds will germinate in 7-14 d.
3. After germination, transplant the seedlings from the flats or Petri dishes to a field (30-60 cm apart)
or into pots (15 cm diameter × 15 cm high).
4. Keep the soil moist by watering the plants once a day. If maintaining the plants in a greenhouse
or growth cabinet, keep the temperature between 18°C and 24°C and the light intensity relatively
strong (50,000-60,000 lx).
5. If necessary, use stakes or a tomato cage to support the tomato vine after it grows in height.
6. If necessary, prune the plants using scissors.

Determinate tomatoes need no pruning. For indeterminate tomatoes, pruning “suckers” (lateral shoots that form
between the main stem and the leaf) makes the main stem stronger.
7. Once flowering begins, shake the tomato plants gently once or twice each week to promote
pollination.
This increases fruit production. Usually, fruit ripens within 90-120 d after germination.
Seed Collection
8. After ripening, harvest the fruit.
9. Cut the fruit in half with a knife and squeeze the seeds into a container.
10. Add tap water to the container so that it is ~70%-80% full. Keep it for ~3 d at room temperature.
This ferments the seeds, which allows the removal of the gelatinous coating that covers them. Once fermented,
mold will form on the surface of the water. The coating can also be mechanically removed without fermentation
by smashing the seeds in a sieve with a lot of water.
11. Repeatedly wash the seeds with tap water at room temperature (~5-10 washes, for 5-10 sec each
wash) to remove the coating.
12. Dry the seeds by leaving them on a paper towel overnight at room temperature.
REFERENCES
Kimura, S. and Sinha, N. 2008a. Tomato (Solanum lycopersicum): A Kimura, S. and Sinha, N. 2008c. Grafting tomato plants. Cold Spring
model fruit-bearing crop. Cold Spring Harb. Protoc. (this issue). Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5083.
doi: 10.1101/pdb.emo105. Kimura, S. and Sinha, N. 2008d. Tomato transformation. Cold Spring
Kimura, S. and Sinha, N. 2008b. Crossing tomato plants. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5084.
Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5082.

Protocol
Crossing Tomato Plants

INTRODUCTION
This protocol describes how to cross tomato plants. Crossing is important for the genetic analysis and
breeding of tomatoes. Tomatoes are self-pollinating plants; thus, emasculation (removal of the anthers
from the female parent) is essential. All wild tomato species can be crossed with cultivated tomatoes
(although it may be difficult); this is useful because wild tomatoes are a great source of desirable traits.
Most commercial tomatoes are F1 hybrids, and the seeds for them were produced by crossing two
parent tomatoes.
RELATED INFORMATION
For an overview of tomato as a model organism, see Tomato (Solanum lycopersicum): A Model Fruit-
Bearing Crop (Kimura and Sinha 2008a). Protocols describing How to Grow Tomatoes (Kimura and
Sinha 2008b), Grafting Tomato Plants (Kimura and Sinha 2008c), and Tomato Transformation
MATERIALS
Reagents
Tomato plants, adult (see How to Grow Tomatoes [Kimura and Sinha 2008b])
Equipment
Forceps
Paint brush, small (optional; see Step 3)
Pencil
Razor blade, sharp
Tags for labeling crosses
Tubes (1.5-mL microcentrifuge)
Alternatively, use empty gelatin capsules (available at most drug stores).
METHOD
1. Select a female parent. Choose flowers that have not yet opened but are about to turn yellow in
color. Emasculate the flowers by carefully removing the sepals, petals, and anther cone with
forceps to expose the stigma and style.
1
2. Select a male parent (pollen source). Choose flowers that are opened or partially opened. Use a
sharp razor blade to cut a slit in the anther cone to facilitate the release of pollen. Collect the pollen
in a 1.5-mL microcentrifuge tube by holding the anther with a forceps and tapping the forceps
with a pencil. Do not tap the anther directly.
3. Transfer pollen to the stigma by one of the following methods:
i. Directly dip the stigma into the collected pollen.

ii. Use a small paint brush or finger to transfer the pollen to the stigma.
It is important to ensure that the stigma gets plenty of pollen but suffers no damage.
Leftover pollen can be stored at 0°C and in dry conditions for up to a few months.
4. Mark the crossed flowers with tags.
REFERENCES
Kimura, S. and Sinha, N. 2008a. Tomato (Solanum lycopersicum): A Kimura, S. and Sinha, N. 2008c. Grafting tomato plants. Cold Spring
doi: 10.1101/pdb.emo105. Kimura, S. and Sinha, N. 2008d. Tomato transformation. Cold Spring
Kimura, S. and Sinha, N. 2008b. How to grow tomatoes. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5084.

Protocol
Grafting Tomato Plants

INTRODUCTION
Grafting is agronomically important because one can combine desirable aboveground characteristics
(such as fruit size) and underground characteristics (such as resistance to soil-borne diseases). This protocol
describes the simplest way of grafting tomato plants using “top wedge grafting” or “cleft grafting.”
Potatoes, eggplants, and tobacco plants are closely related to tomatoes, and they can be grafted onto
each other as well. Although the grafting of vegetable crops is still rare, this technique has been useful in
reducing infections caused by pathogens, increasing resistance to drought, and enhancing nutrient uptake.
RELATED INFORMATION
Xoconostle-Cázares et al. (1999) describe the use of grafting techniques to investigate the long-
distance movement of signals through vascular tissue.
For an overview of tomato as a model organism, see Tomato (Solanum lycopersicum): A Model
Fruit-Bearing Crop (Kimura and Sinha 2008a). Protocols describing How to Grow Tomatoes (Kimura
and Sinha 2008b), Crossing Tomato Plants (Kimura and Sinha 2008c), and Tomato Transformation
MATERIALS
Reagents
Tomato plants, adult (see How to Grow Tomatoes [Kimura and Sinha 2008b])
The top portion, which contains the shoot, is called the “scion,” and the bottom part, which contains the roots,
is called the “stock.” It is best to use scion and stock stems with the same (or similar) diameter to increase the
success of the grafting.
Equipment
Razor blades, sharp
Shears, pruning
Surgical tape (Micropore, 3M), plastic tube (~5 mm in diameter), or grafting clip (see Step 4)
METHOD
1. Use pruning shears to cut off the stock ~100 mm above the soil. Use a sharp razor blade to make
a 5- to 15-mm-long slit in the center of the stem. Leave a few leaves on the stock.
1
2. Cut off the scion from the plant with pruning shears. Use a sharp razor blade to cut the lower end
into a V shape (wedge).
3. Insert the scion into the slit of the stock.
4. Tie the junction with tape or use a tiny plastic tube or grafting clip to hold the scion and stock
together and keep moisture in until they heal.
A graft junction takes ~1 wk to heal.
REFERENCES
Kimura, S. and Sinha, N. 2008a. Tomato (Solanum lycopersicum): A Kimura, S. and Sinha, N. 2008d. Tomato transformation. Cold Spring
doi: 10.1101/pdb.emo105. Xoconostle-Cázares, B., Xiang, Y., Ruiz-Medrano, R., Wang, H.L.,
Kimura, S. and Sinha, N. 2008b. How to grow tomatoes. Cold Spring Monzer, J., Yoo, B.C., McFarland, K.C., Franceschi, V.R., and Lucas,
Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5081. W.J. 1999. Plant paralog to viral movement protein that potenti-
Kimura, S. and Sinha, N. 2008c. Crossing tomato plants. Cold Spring ates transport of mRNA into the phloem. Science 283: 94–98.

Protocol
Tomato Transformation
INTRODUCTION
Transformation is an essential technique to analyze the function of genes. Tomato can be stably trans-
formed using Agrobacterium tumefaciens-mediated transfer of T-DNA. This protocol describes an
Agrobacterium-mediated transformation method for tomato.
RELATED INFORMATION
The method described here is a modified version of the protocol by McCormick et al. (1986), the first
report of tomato transformation. For an overview of tomato as a model organism, see Tomato
(Solanum lycopersicum): A Model Fruit-Bearing Crop (Kimura and Sinha 2008a). Protocols describ-
ing How to Grow Tomatoes (Kimura and Sinha 2008b), Crossing Tomato Plants (Kimura and Sinha
2008c), and Grafting Tomato Plants (Kimura and Sinha 2008d) are also available.
MATERIALS
Reagents
Agrobacterium (containing gene of interest)
<!>Bleach (50%)
Ethanol (70%)
<R>Germination medium for tomato (in Magenta boxes)
<R>LB medium for tomato (solid [in Petri dishes] and liquid)
<R>MSO liquid medium
<R>Rooting medium (in Magenta boxes)
<R>Selection medium for tomato (in Petri dishes)
<R>Temporary medium (in Petri dishes)
Tomato seeds
Equipment
Centrifuge
Forceps (sterilized)
Greenhouse
Growth chamber preset to 26°C (with 16-h photoperiod)
Hood (sterile)
1
Incubator preset to 28°C
Inoculating loop
Magenta boxes
Petri dishes
Pipette (transfer)
Pots, moist soil, and clear plastic covers
Razor blade (single-edged, sterilized)
Scissors (sterilized)
Shaking platform
Spectrophotometer
Tape (surgical; Micropore, 3M)
Tubes (culture)
METHOD
Preparing the Plant Material
1. Surface-sterilize ~30 tomato seeds by immersing them in 70% ethanol and swirling them for 2 min
at room temperature.
2. Immerse the seeds in 50% bleach for 15-20 min at room temperature with gentle swirling.
3. Wash off the bleach completely by rinsing the seeds five to 10 times with H2O under a sterile hood.
4. Add 5 mL of H2O to the seeds. Pour the seeds into a Magenta box containing germination
medium for tomato. Place the cover on the Magenta box and put it in a growth chamber (26°C,
16-h photoperiod).
5. Use sterilized forceps and scissors to harvest a cotyledon from an 8- to 10-d-old plant and place it
in a Petri dish. Keep the cotyledons moist by adding ~20 mL of MSO liquid medium to the dish.
6. Cut the tip and base of each cotyledon with a razor blade (see Fig. 1). Wound them with one to
three shallow transverse cuts across the main vein on the adaxial side to facilitate Agrobacterium
infection. Place the explants onto a Petri dish containing 20 mL of temporary medium.
The explants are now ready for cocultivation (Step 10).
Preparing the Agrobacterium Suspension
7. Streak the Agrobacterium onto an LB medium for tomato plate using an inoculating loop and
incubate the plate at 28°C until colonies form.
It typically takes 2-3 d for colonies to form.
FIGURE 1. Tomato cotyledon. The cotyledon is the primary leaf of the embryo of seed plants and emerges first after ger-
mination. The tomato has two cotyledons. After germination, the size of each cotyledon is ~3 cm. For transformation,
cut the tip and base of each cotyledon and wound them with up to three shallow transverse cuts on the adaxial side.

8. Inoculate 10 mL of liquid LB medium for tomato with a single Agrobacterium colony in a culture
tube. Incubate the culture with shaking overnight at 28°C and then measure the OD600 of the
culture using a spectrophotometer.
The incubation time may be increased to get enough Agrobacterium. The optimum OD600 is 0.6-0.7.
9. Harvest the Agrobacterium by centrifugation at 3000g for 15 min at room temperature and
resuspend the cells with an appropriate amount of MSO liquid medium to make the OD600 of
the suspension ~0.5.
The suspension is now ready for cocultivation (Step 10).
Cocultivation
10. Pour ~5 mL of the Agrobacterium suspension (from Step 9) onto the temporary medium with the
explants (from Step 6).
11. Incubate the explants on the temporary medium for 2 h at room temperature.
12. Remove excess Agrobacterium suspension with a transfer pipette. Seal the plate with surgical tape.
13. Cocultivate explants in the growth chamber (26°C, 16-h photoperiod) for 48 h.
Selection of Transformants
14. Transfer the explants onto a Petri dish containing selection medium for tomato. (Keep the adaxial
side up.) Seal the dish with surgical tape.
15. Keep the explants in the growth chamber (26°C, 16-h photoperiod) until a callus forms. Transfer
the explants to new selection medium for tomato every 2 wk or when Agrobacterium is growing
in the medium.
After a few weeks, the first shoot should form.
Rooting
16. When shoots get to ~2-4 cm, excise them from the explants and transfer them to a Magenta box
that contains rooting medium.
17. Keep the Magenta box in a growth chamber (26°C, 16-h photoperiod).
Roots should form 2-3 wk after transferring to the rooting medium.
Transplanting
18. When the shoots are ~5 cm and the roots are established, take the transformants out of the
Magenta box and gently wash off the agar.
19. Transplant the transformants to a pot with wet soil.
20. Place a clear plastic cover on the pot to keep moisture in and put it in the growth chamber (26°C,
16-h photoperiod) for ~1 wk.
21. After 1 wk, transfer the pot to the greenhouse and let the plants grow.
For general tomato cultivation practices, see How to Grow Tomatoes (Kimura and Sinha 2008b).
REFERENCES
Kimura, S. and Sinha, N. 2008a. Tomato (Solanum lycopersicum): A Kimura, S. and Sinha, N. 2008d. Grafting tomato plants. Cold Spring
doi: 10.1101/pdb.emo105. McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., and
Kimura, S. and Sinha, N. 2008b. How to grow tomatoes. Cold Spring Fraley, R. 1986. Leaf disc transformation of cultivated tomato (L.
Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5081. esculentum) using Agrobacterium tumefaciens. Plant Cell Reports 5:
Kimura, S. and Sinha, N. 2008c. Crossing tomato plants. Cold Spring 81–84.

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Emerging Model Organisms

Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop

FIGURE 1. Tomato plants and parts: (A) seedling; (B) 40-d-old

www.cshprotocols.org 2 Cold Spring Harbor Protocols

SOURCES AND HUSBANDRY

www.cshprotocols.org 3 Cold Spring Harbor Protocols

USES OF THE TOMATO MODEL SYSTEM

www.cshprotocols.org 4 Cold Spring Harbor Protocols

Total Soluble Solids

Floral System and Plant Architecture

www.cshprotocols.org 5 Cold Spring Harbor Protocols

Compound Leaf Development

www.cshprotocols.org 6 Cold Spring Harbor Protocols

GENETICS AND GENOMICS RESOURCES

www.cshprotocols.org 7 Cold Spring Harbor Protocols

for the production of β-carotene and xanthophylls in plants. Plant

www.cshprotocols.org 8 Cold Spring Harbor Protocols

alternative pathway to β-carotene formation in plant chromo-

www.cshprotocols.org 9 Cold Spring Harbor Protocols

How to Grow Tomatoes

2. Allow seeds to germinate as follows:

6. If necessary, prune the plants using scissors.

8. After ripening, harvest the fruit.

www.cshprotocols.org 2 Cold Spring Harbor Protocols

Crossing Tomato Plants

i. Directly dip the stigma into the collected pollen.

4. Mark the crossed flowers with tags.

www.cshprotocols.org 2 Cold Spring Harbor Protocols

Grafting Tomato Plants

www.cshprotocols.org 1048 Cold Spring Harbor Protocols

Preparing the Agrobacterium Suspension

www.cshprotocols.org 2 Cold Spring Harbor Protocols

www.cshprotocols.org 3 Cold Spring Harbor Protocols

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