Scientia Horticulturae 122 (2009) 501–505

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Scientia Horticulturae
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Short communication

A practical and reliable procedure for in vitro induction of tetraploid tomato

Milene Miranda Praça, Carlos Roberto Carvalho *, Wellington Ronildo Clarindo
Laboratório de Citogenética e Citometria, Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: A relatively rapid, practical and reliable procedure for in vitro production of tetraploid tomato using
Received 19 March 2009 colchicine is described. Using this procedure 11.11% generation of tetraploids was obtained after
Received in revised form 26 May 2009 exposure of seedling shoot meristem explants with 8 mM colchicine for 96 h. Confirmation of tetraploid
Accepted 27 May 2009
production (2n = 48), was determined by flow cytometry and verified by cytogenetic analysis of root tip
preparations. The results indicate that the procedure is adequate for induction and screening of
Keywords: tetraploid tomato plantlets.
Colchicine
ß 2009 Elsevier B.V. All rights reserved.
Chromosome
Flow cytometry
Solanum lycopersicum

1. Introduction S. bulbocastanum, S. chacoense and hybrids as S. tuberosum 

Polyploidy induction has been successfully applied in vege-
tables, ornamental and medicinal plants in order to obtain lines
exhibiting new agronomical characteristics. This procedure has
provided plants without seeds, with significantly larger fruits and
flowers, longer lasting flowers, which overcome hybridization
barriers, show enhanced pest resistance and physical stress
tolerance (Sanford, 1983; Predieri, 2001). These effects can vary
according to species, degree of heterozygosity and ploidy level
(Zhang et al., 2008).
Polyploid tomato plants have been obtained in three different
ex vitro approaches: spontaneously (Winkler, 1916), decapitation
of diploid plants (Jorgensen, 1928) and colchicine treatment (Stair
and Showalter, 1942). Zhang et al. (2008) reported that the ex vitro
procedures render low efficiency for polyploid plant production
and causes high frequency of mixoploid plants. The nuclear DNA
ploidy level stability of mixoploid plants is often disturbed after
successive cell division cycles. As the diploid cells proliferate at
higher rates than the cells with other ploidy levels, mixoploidy
reverts partially or totally to the diploid condition (Mergen and
Lester, 1971).
With the development of tissue culture techniques, in vitro
polyploidization has become the main method for induction of
polyploid plants (Zhang et al., 2008). Some researchers have
applied this technique in species such as: Solanum tuberosum
and S. chacoense (Cardi et al., 1992), Solanum sparsipilum,
* Corresponding author. Tel.: +55 31 38992568; fax: +55 31 38992549.
E-mail address: ccarvalh@ufv.br (C.R. Carvalho).
Solanum sp. (Chauvin et al., 2003).
As in vitro polyploidy induction generates plantlets with
distinct DNA ploidy levels, a practical and efficient method for
DNA ploidy screening is necessary (Roy et al., 2001). Different
studies have showed that flow cytometry is the most practical,
rapid and reliable method for this purpose (Van Duren et al., 1996;
Roy et al., 2001; Clarindo et al., 2008). Additionally, in the flow
cytometry protocols, plant samples can be prepared and analyzed
in one working day without requirement of dividing cells (Dolezel,
1997). Nevertheless, chromosome counting has been traditionally
used (Carvalho et al., 2005; Zhang et al., 2008) and can be used to
confirm flow cytometry data.
Jacobs and Yoder (1989) obtained tomato plantlets showing
different nuclear DNA ploidy levels after in vitro genetic
transformation using the vector pMAC. These authors determined
the germline ploidy of tomato primary transformants by meiotic
chromosome counting and chloroplast number. Polyploid tomato
plantlets were also observed by Ellul et al. (2003) in in vitro genetic
transformation experiments mediated with Agrobacterium. The
nuclear DNA ploidy of these plants was verified by flow cytometry.
Tomato plantlets, exhibiting tetraploid and rare diploid cells, were
regenerated from embryogenic calli obtained from anther culture
by Brasileiro et al. (1999). These authors used mitotic chromosome
counting for nuclear DNA ploidy level determination. In spite of
these similar studies, polyploidy in the tomato plantlets was
obtained as a secondary result. Therefore, the reliability of these
methods for generating polyploid tomato was not determined.
Given that protocols for in vitro polyploidization have not been
applied in tomato, in the present work we describe a rapid,
practical and reliable procedure for in vitro induction of tetraploidy
in tomato.

0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2009.05.032
502 M.M. Praça et al. / Scientia Horticulturae 122 (2009) 501–505
2. Materials and methods
2.1. Plant material and in vitro culture
Solanum lycopersicum L. ‘Stupické’ seeds were supplied by Dr.
Jaroslav Doležel from the Experimental Institute of Botany, Czech
Republic. For in vitro cultures, seeds were collected from fruits of
plants growing in an experimental field at the Universidade
Federal de Viçosa, Minas Gerais, Brazil.
Under a laminar flow hood, seeds were held in 70% (v/v) ethanol
(Merck1) for 20 s, 2.5% (v/v) sodium hypochlorite (Merck1) and
0.1% (v/v) Tween 20 (Merck1) for 20 min and then rinsed five times
with sterile dH2O (deionized water). Seeds were germinated in
60 mm  15 mm Petri dishes (J. Prolab1) containing MS salts
(Murashige and Skoog, 1962, Sigma1) supplemented with B5
vitamins (Gamborg et al., 1968), 3.0% (w/v) sucrose and 0.7% agar
type-A (Sigma1-A4550). The pH of this medium was adjusted to
5.7 prior to autoclaving. Petri dishes were sealed with PVC film
(Goodyear1) and cultures were kept in the dark at 27 8C. After 6–8
days, germinated seeds were transferred to test tubes
(15 mm  25 mm) containing the medium described above.
Cultures were maintained at 27 8C under a 16/8 h light/dark
regime with 36 mmol m 2 s 1 light radiation provided by fluor-
escent lamps (20 W, Osram1) for 20 days.
2.2. In vitro polyploidization induction
Shoot tips explants were excised and placed in Erlenmeyer
flasks containing 10 mL of polyploidization medium. This culture
medium had similar composition to the one previously described,
but was devoid of the gelling agent agar and supplemented with
different concentrations of filter-sterilized colchicine (Sigma1)
(0.0, 5.0, 6.5 and 8.0 mM). The flasks were shaken (40 rpm) in a
growth room with a 96 or 120 h pulse of 0.0, 5.0, 6.5 or 8.0 mM
colchicine. Each flask contained three shoot tips explants,
providing two replicates for each treatment except the control.
The experiment was repeated twice, on different days, totalizing
84 explants (72 treated and 12 controls). The shoot tips were rinsed
three times with dH2O, and then returned to regular tissue culture
media. Cultures were maintained at 27 8C under a 16/8 h light/dark
regime with 36 mmol m 2 s 1 light radiation provided by two
fluorescent lamps (20 W, Osram1).
2.3. Plant screening by flow cytometry
After 1 month of colchicine treatment, leaves were excised
from regenerated S. lycopersicum plantlets (samples). In vitro
germinated and greenhouse seed-raised tomato plants were used
as diploid internal standards. The leaves of the sample and
standard were simultaneously placed in dH2O at 4 8C and cut into
2 cm2 fragments. Nuclei were extracted by chopping (Galbraight
et al., 1983) in 0.5 mL of OTTO-I lysis buffer (Otto, 1990)
supplemented with 2.0 mM dithiothreitol (Sigma1) and
50 mg mL 1 RNAse (Sigma1), pH 2.3. Subsequently, 0.5 mL of
the same buffer was added. The suspension was sieved through a
nylon 30 mm mesh diameter (Partec1), transferred to 2 mL
microcentrifuge tubes (Eppendorf1) and centrifuged at 100  g.
After 5 min, the pellet was incubated for 10 min in a 100 mL
OTTO-I lysis buffer. The nuclei suspension was stained with
1.5 mL of 1:2 (Loureiro et al., 2006) OTTO-I:OTTO-II solution
(Otto, 1990), supplemented with 75 mM propidium iodide (PI,
Sigma1), 50 mg mL 1 RNAse (Sigma1) and 2.0 mM dithiothreitol
(Sigma1), pH 8.6 (Carvalho et al., 2008). The nuclear suspension
was sieved through a nylon filter with 20 mm mesh diameter
(Partec1) and maintained in the dark for 30 min (Clarindo and
Carvalho, 2009).
The suspension was analyzed with a Partec PAS1 flow
cytometer (Partec1). The nuclear DNA ploidy level of the samples
was determined by using channel values corresponding to the
average G0/G1 peaks of sample and standard plants. The peak
relative to the standard nuclei was set to channel 100. Three
independent repetitions were performed, with over 10,000 nuclei
being analyzed in each.
The polyploid plantlets selected by flow cytometry were
maintained in tissue culture medium. Nuclear DNA ploidy level
stability of these plantlets was reassessed after 4 weeks.
2.4. Chromosome counting
The nuclear DNA ploidy level of the plantlets analyzed by flow
cytometry was also confirmed by cytogenetic preparations. Root
tips (0.5–1.0 cm long) from standards and plantlets selected by
flow cytometry were pre-treated with the microtubule inhibiting
agent amiprophos-methyl (APM-Nihon Bayer Agrochem K. K.1) in
a final concentration of 4.0 mM, for 19 h, at 4 8C. The root tips were
washed with dH2O for 15 min, and then fixed in fresh cold
methanol:acetic acid solution (3:1). The fixative was changed
three times and the root tips were stored at 20 8C (Praça et al.,
2008).
After 24 h, root tips were washed three times with dH2O and
macerated in a pectinase solution (Sigma1), diluted 1:10 in dH2O,
for 2 h at 34 8C. Roots were washed for 20 min with dH2O and fixed
again at 20 8C. Slides were prepared by meristematic cellular
dissociation, air-dried and placed on a hot-plate (50 8C) for 20 min.
The slides were immediately stained with a 5% Giemsa (Merck1)
solution in phosphate buffer (pH 6.8) for 4 min, washed twice in
dH2O and air-dried (Praça et al., 2008).
Images of the chromosomes were captured with a Photometrics
CoolSNAP Pro1 cf (Roper Scientific1) monochromatic CCD video
camera of 12 bits gray, attached to an OlympusTM BX-60
microscope. The frame was digitized using the Kit Cool-SNAP
Pro1, and image analysis was performed using the Image Pro-
Plus1 6.1 analysis system (Media Cybernetics).
2.5. Statistical analysis
The mean number of diploid, tetraploid and mixoploidy
tomato plantlets was obtained from flow cytometry screening.
The mean values, and consequently the efficiency of treatments,
were statistically analyzed by the F-test and t-test methods.
This analysis was performed on statistical Genes Program
(Cruz, 1997).
3. Results and discussion
The tissue culture MS medium, as recommended for in vitro
tomato culture by Park et al. (2001), was considered adequate for
germination, propagation, polyploidization-treatment and rooting
of the S. lycopersicum explants. All tomato seeds germinated in this
medium.
The optimal colchicine treatments (5.0, 6.5 and 8.0 mM pulse of
96 and 120 h) were selected from previous experiments in our
laboratory. Other concentrations and/or times for colchicine
application, as well as others chromosome doubling agent as
oryzalin and APM, did not induce polyploids or caused plant death
(data not shown). The results reinforce the principle that colchicine
concentration is crucial for the success of in vitro polyploidy
induction (Zhang et al., 2008).
In potato (S. tuberosum), chromosome doubling agent such as
oryzalin and APM are more efficient for polyploidization than
colchicine. Ramulu et al. (1991) showed, by flow cytometry and
chromosome counting, that oryzalin was the most efficient in
 M.M. Praça et al. / Scientia Horticulturae 122 (2009) 501–505 503

Table 1
Generation of diploid, tetraploid and mixoploid S. lycopersicum plantlets observed after treatment of shoot tips with three concentrations of colchicine and two exposure
times.

Colchicine concentration (mM) Treatment duration (h) Number of individuals treated Number of individuals (%) by ploidy level

Diploid (2) Tetraploid (4) Mixoploid (2/4)

0 96 6 6 (100) – –
120 6 6 (100) – –

5 96 12 – 2 (2.78)b 10 (13.88)
120 12 6 (8.33) 3 (4.17)b 3 (4.17)

6.5 96 12 6 (8.33) 4 (5.55)ab 2 (2.78)

120 12 4 (5.55) 6 (8.33)ab 2 (2.78)

8 96 12 1 (1.39) 8 (11.11)a 3 (4.17)

120 12 5 (6.94) 3 (4.17)b 4 (5.55)

Total 84 34 26 24

Means followed by the same letter are not significantly different at the p = 0.05 by t-test.

Fig. 1. (a and c) Representative flow cytometry histograms of nuclei isolated from leaves of a diploid and tetraploid Solanum lycopersicum. (a) Histogram showing G1/G0 peak of
the standard and diploid samples (channel 100, 2C = 2). (c) Histogram showing G1/G0 peak of the standard (channel 100, 2C = 2) and tetraploid samples (channel 198,
2C = 4). (b and d) Metaphasic chromosomes of Solanum lycopersicum obtained of root tip treated with APM 4.0 mM for a period of 19 h at 4 8C and stained with Giemsa 5%. (b)
Chromosome number of the diploid plantlets (2n = 2 = 24). (d) Chromosome number of the tetraploid plantlets (2n = 4 = 48) (Bar = 5 mm).
504 M.M. Praça et al. / Scientia Horticulturae 122 (2009) 501–505
vitro chromosome doubling agent, followed by APM and
colchicine.
The DNA ploidy level of the tomato plantlets recovered was
verified by flow cytometry 1 month after treatment. Plantlets
cultured in MS medium without colchicine as well as internal
standards exhibited G0/G1 peaks at the same channel. Therefore,
these plantlets show the same nuclear DNA ploidy level
(2C = 2) (Table 1 and Fig. 1a). Flow cytometry histograms also
showed that heteroploid (mixoploid and/or tetraploid) tomato
plantlets were produced in the medium containing colchicine,
corresponding to 69.44% of the regenerants (Table 1). Consider-
ing the 72 explants treated, 30.54% of the tomato plantlets
displayed the same nuclear DNA ploidy level of the standard
(2C = 2, diploids), 33.33% showed G0/G1 cells 2C = 2 and
2C = 4 (mixoploids), and 36.11% presented G0/G1 cells 2C = 4
(tetraploids) (Table 1 and Fig. 1c). Exposure to 8 mM colchicine
for 96 h provided the best percentage of tetraploid tomato
plantlets (11.11%), followed by treatment with 6 mM at 120 h
(8.33%) and 6 mM at 96 h (5.55%) (Table 1), in accordance to
statistical t-test (p = 0.05). In other Solanum species, the
percentage of tetraploids, related in in vitro experiments with
the oryzalin doubling technique, varied from 6% for hybrid S.
tuberosum  S. spegazzinii to 29% for S. tuberosum  S. sparsipilum
(Chauvin et al., 2003).
Subsequent flow cytometry analyses were applied to verify the
stability of the nuclear DNA ploidy level of the plantlets. The
results showed that DNA ploidy remained stable in all cloned
polyploids, including the mixoploids. According to Mergen and
Lester (1971), diploid cells of the mixoploids proliferate at higher
rates than the cells with other ploidy levels. As a result, nuclear
DNA ploidy level stability of these plants is often disturbed after
successive cell division cycles. As the nuclear DNA ploidy of
mixoploids is unstable (Carvalho et al., 2005), we discarded these
plantlets.
Flow cytometry was an important, practical, reliable and rapid
methodology for nuclear DNA ploidy screening of the regenerated
tomato plantlets. Owing to these advantages, Dolezel (1997) and
Clarindo et al. (2008) recommended flow cytometry for nuclear
DNA ploidy level determination, especially in analyses of many
individuals.
The chromosomic complement of the diploid and tetraploid
tomato plantlets was also verified by chromosome counting. All
root tips of the diploid and standard plantlets showed 2n = 24
(Fig. 1b), as previously described by Banks (1984). On the order
hand, all root tip cells of the tetraploid plantlets presented 2n = 48
chromosomes (Fig. 1d). Seeing that all mitotic cells of the diploids
and tetraploids showed 2n = 24 and 2n = 48 chromosomes,
respectively, cytogenetic APM pre-treatment did not induce
polyploidization in the root apical meristem cells.
After flow cytometry for screening and chromosome counting
for ploidy confirmation, the tetraploid plantlets were acclimatized.
A standard plant (2) and all tetraploid plants were transferred to
plastic pots containing coconut substrate.
Our results indicate that the procedure was adequate for
induction, propagation and recovery of tetraploid tomato plantlets
in vitro. Screening of nuclear DNA ploidy by flow cytometry was a
practical and rapid strategy for selection of polyploids. Chromo-
some counting was also essential for attesting the relationship
between chromosome complement and ploidy level in the nuclear
DNA.
Other in vitro procedures have also produced tomato polyploidy
plantlets. Jacobs and Yoder (1989) observed 22% of polyploids in
the genetic transformation experiments. Ellul et al. (2003) found
24.5–80% tetraploids in Agrobacterium-mediated transformation
approaches from cotyledonary explants. Polyploids were also
recovered from embryogenic calli derived from anther culture
(Brasileiro et al., 1999), using different combinations of growth
regulators. In these three works, the polyploid tomato plants were
recovered from embryogenic cultures as a sub-product and,
therefore, may not be obtained in further repetitions of the same
experiments. In our work, an efficient 80–90-day procedure for
tetraploid generation in vitro was demonstrated. The method used
is reliable for routine in vitro production of polyploid tomatoes and
it is expected to be applicable with minor modifications to other
plant species.
Acknowledgements
We are grateful to the Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (FAPEMIG, Brazil), Conselho Nacional de
Pesquisa (CNPq, Brazil) and Coordenação de Aperfeiçoamento de
Pessoal de Nı́vel Superior (CAPES, Brazil), for financial support.
References
Banks, P., 1984. A new diploid chromosome number for tomato (Lycopersicon
esculentum). Can. J. Genet. Cytol. 26, 636–639.
Brasileiro, A.C.R., Willadino, L., Carvalheira, G.G., Guerra, M., 1999. Callus induction
and plant regeneration of tomato (Lycopersicon esculentum CV, IPA 5) via anther
culture. Ciência Rural 29, 619–623.
Cardi, T., Carputo, D., Frusciante, L., 1992. In vitro shoot regeneration and chromo-
some doubling in 2 and 3 potato clones. Am. J. Potato Res. 69, 1–12.
Carvalho, C.R., Clarindo, W.R., Praça, M.M., Araújo, F.S., Carels, N., 2008. Genome size,
base composition and karyotype of Jatropha curcas L., an important biofuel
plant. Plant Sci. 174, 613–617.
Carvalho, J.F.R., Carvalho, C.R., Otoni, W.C., 2005. In vitro induction of polyploidy in
annatto (Bixa orellana). Plant Cell Tissue Org. Cult. 80, 69–75.
Chauvin, J.E., Souchet, C., Dantec, J.P., Ellisseche, D., 2003. Chromosome doubling
of 2 Solanum species by oryzalin: method development and comparison
with spontaneous chromosome doubling in vitro. Plant Cell Tissue Org. Cult.
73, 65–73.
Clarindo, W.R., Carvalho, C.R., 2009. Comparison of the Coffea canephora and C.
arabica karyotypes based on chromosomal DNA content. Plant Cell Rep. 28,
73–81.
Clarindo, W.R., Carvalho, C.R., Araújo, F.S., Abreu, I.S., Otoni, W.C., 2008. Recovering
polyploid papaya in vitro regenerants as screened by flow cytometry. Plant Cell
Tissue Org. Cult. 92, 207–214.
Cruz, C.D., 1997. Programa GENES—Aplicativo Computacional em Genética e Esta-
tı́stica. Editora UFV, Viçosa, MG, pp. 442.
Dolezel, J., 1997. Applications of flow cytometry for the study of plant genomes. J.
Appl. Genet. 38, 285–302.
Ellul, P., Garcia-Sogo, B., Pineda, B., Rı́os, G., Roig, L.A., Moreno, V., 2003. The ploidy
level of transgenic plants in Agrobacterium-mediated transformation of tomato
cotyledons (Lycopersicon esculentum L. Mill.) is genotype and procedure depen-
dent. Theor. Appl. Genet. 106, 231–238.
Galbraight, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., Firooza-
bady, E., 1983. Rapid flow cytometric analysis of the cell cycle in intact plant
tissues. Science 220, 1049–1051.
Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Nutrient requirements of suspension
cultures of soybean root cells. Exp. Cell Res. 50, 151–158.
Jacobs, J.P., Yoder, J.I., 1989. Ploidy levels in transgenic tomato plants determined by
chloroplast number. Plant Cell Rep. 7, 662–664.
Jorgensen, C.A., 1928. The experimental formation of heteroploid plants in the
genus Solanum. Genetics 19, 133–211.
Loureiro, J., Rodriguez, E., Dolezel, J., Santos, C., 2006. Comparison of four nuclear
isolation buffers for plant DNA flow cytometry. Ann. Bot. 98, 679–689.
Mergen, F., Lester, D.T., 1971. Colchicine induced polyploidy in Abies. Forest Sci. 7,
314–319.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays
with tobacco tissue culture. Physiol. Plant. 15, 473–497.
Otto, F.J., 1990. DAPI staining of fixed cells for high-resolution flow cytometry of
nuclear DNA. In: Darzynkiewiez, Z., Crissman, H.A., Robinson, J.P. (Eds.),
Methods in Cell Biology, vol. 33. Academic Press, San Diego, pp. 105–110.
Park, J., Yi, B., Lee, C., 2001. Effects of plant growth regulators, bud length, donor
plant age, low temperature treatment and glucose concentration on callus
induction and plant regeneration in anther culture of cherry tomato ‘Mini-
carol’. J. Korean Soc. Hortic. Sci. 42, 32–37.
Praça, M.M., Carvalho, C.R., Marcelino, F.C., Mendonça, M.A.C., 2008. Morphological
aspects of Passiflora edulis f. flavicarpa chromosome using acridine orange
banding and rDNA-FISH tools. Caryologia 61, 154–159.
Predieri, S., 2001. Mutation induction and tissue culture in improving fruits. Plant
Cell Tissue Org. Cult. 64, 185–210.
Ramulu, K.S., Verhoeven, H.A., Dijkhuis, P., 1991. Mitotic blocking, micronucleation,
and chromosome doubling by oryzalin, amiprophos-methyl, and colchicine in
potato. Protoplasma 160, 65–71.
 M.M. Praça et al. / Scientia Horticulturae 122 (2009) 501–505 505

Roy, A.T., Leggett, G., Koutoulis, A., 2001. In vitro tetraploid induction and genera-
tion of tetraploids from mixoploids in hop (Humulus lupulus L.). Plant Cell Rep.
20, 489–495.
Sanford, J.C., 1983. Ploidy manipulations. In: Moore, J.N., Janick, J. (Eds.), Methods in
Fruit Breeding. Purdue Univ. Press, West Lafayette, pp. 100–123.
Stair, E.C., Showalter, R.K., 1942. Tetraploidy in tomatoes induced by the use of
colchicine. P. Am. Soc. Hortic. Sci. 40, 383–386.
Van Duren, M., Mopurgo, R., Dolezel, J., Afza, R., 1996. Induction and verification of
autotetraploids in diploid banana (Musa acuminata) by in vitro techniques.
Euphytica 88, 25–34.
Winkler, H., 1916. Uber die experimentelle Erzeugung von Pflanzen mit abwei-
chenden chromosemenzahlen. Euphytica 8, 417.
Zhang, Z., Dai, H., Xiao, M., Liu, X., 2008. In vitro induction of tetraploids in Phlox
subulata L. Euphytica 159, 59–65.