Seisuke & Neelima 2008
Seisuke & Neelima 2008
Seisuke & Neelima 2008
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 ([email protected])
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.
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
[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.
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
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.
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-
content of plants by flow cytometry. Plant Mol. Biol. Rep. 9: fication and fine mapping of yield-associated QTL. Genetics 141:
208–218. 1147–1162.
Atherton, J.G. and Harris, G.P. 1986. Flowering. In The tomato crop: A Frary, A., Nesbitt, T.C., Grandillo, S., Knaap, E., Cong, B., Liu, J.,
scientific basis for improvement (eds. J.G. Atherton and J. Rudich), Meller, J., Elber, R., Alpert, K.B., and Tanksley, S.D. 2000. fw2.2: A
pp. 167–200. Chapman and Hall, New York. quantitative trait locus key to the evolution of tomato fruit size.
Barton, D.W., Butler, L., Jenkins, J.A., Rick, C.M., and Young, P.A. Science 289: 85–88.
1955. Rules for nomenclature in tomato genetics. J. Hered. 46: Frary, A., Fritz, L.A., and Tanksley, S.D. 2004. A comparative study of
22–26. the genetic bases of natural variation in tomato leaf, sepal, and
Bernacchi, D. and Tanksley, S.D. 1997. An interspecific backcross of petal morphology. Theor. Appl. Genet. 109: 523–533.
Lycopersicon esculentum × L. hirsutum: Linkage analysis and a QTL Fray, R.G. and Grierson, D. 1993. Identification and genetic analysis
study of sexual compatibility factors and floral traits. Genetics 147: of normal and mutant phytoene synthase genes of tomato by
861–877. sequencing, complementation and co-suppression. Plant Mol.
Bohs, L. and Olmstead, R.G. 1997. Phylogenetic relationships in Biol. 22: 589–602.
Solanum (Solanaceae) based on ndhF sequences. Syst. Bot. 22: Fridman, E., Carrari, F., Liu, Y.S., Fernie, A.R., and Zamir, D. 2004.
5–17. Zooming in on a quantitative trait for tomato yield using inter-
Chen, J.J., Janssen, B.J., Williams, A., and Sinha, N. 1997. A gene specific introgressions. Science 305: 1786–1789.
fusion at a homeobox locus: Alterations in leaf shape and impli- Haanstra, J.P.W., Wye, C., Verbakel, H., Meijer-Dekens, F., van, den
cations for morphological evolution. Plant Cell 9: 1289–1304. Berg P., Odinot, P., van Heusden, A.W., Tanksley, S.D., Lindhout,
Child, A. 1990. A synopsis of Solanum subgenus Potatoe (G. Don) P., and Peleman, J. 1999. An integrated high-density RFLP-AFLP
(D’Arcy) (Tuberarium (Dun.) Bitter (s.l)). Feddes Repert. 101: 209– map of tomato based on two Lycopersicon esculentum × L. pennellii
235. populations. Theor. Appl. Genet. 99: 254–271.
Darwin, S., Knapp, S., and Peralta, I. 2003. Taxonomy of tomatoes in Holtan, H.E. and Hake, S. 2003. Quantitative trait locus analysis of
the Galapagos Islands: Native and introduced species of Solanum leaf dissection in tomato using Lycopersicon pennellii segmental
section Lycopersicon (Solanaceae). Syst. Biodivers. 1: 29–53. introgression lines. Genetics 165: 1541–1550.
Doebley, J.F., Gaut, B.S., and Smith, B.D. 2006. The molecular genet- Isaacson, T., Ronen, G., Zamir, D., and Hirschberg, J. 2002. Cloning
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
Corresponding author ([email protected])
Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5081 www.cshprotocols.org
© 2008 Cold Spring Harbor Laboratory Press 1 Vol. 3, Issue 11, November 2008
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.
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.
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
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.
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
(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
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
Corresponding author ([email protected])
Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5082 www.cshprotocols.org
© 2008 Cold Spring Harbor Laboratory Press 1 Vol. 3, Issue 11, November 2008
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:
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. How to grow tomatoes. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5084.
Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5081.
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
(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
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
Corresponding author ([email protected])
Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5083 www.cshprotocols.org
© 2008 Cold Spring Harbor Laboratory Press 1047 Vol. 3, Issue 11, November 2008
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
model fruit-bearing crop. Cold Spring Harb. Protoc. (this issue). Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5084.
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.
Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5082.
Tomato Transformation
Seisuke Kimura and Neelima Sinha1
Department of Plant Biology, University of California, Davis, CA 95616, USA
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
CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, and
recipes for reagents marked with <R>.
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
Corresponding author ([email protected])
Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5084 www.cshprotocols.org
© 2008 Cold Spring Harbor Laboratory Press 1 Vol. 3, Issue 11, November 2008
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).
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.
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
model fruit-bearing crop. Cold Spring Harb. Protoc. (this issue). Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5083.
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.
Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5082.