Scientia Horticulturae 100 (2004) 153–159

Auxins increase the occurrence of leaf-colourvariants inCaladium regenerated from leaf explants

E.U. Ahmed∗, T. Hayashi, S. YazawaLaboratory of Vegetable and Ornamental Horticulture, Division of Agronomy and Horticultural Science,

Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa,Sakyo-Ku, Kyoto 606-8502, Japan

Accepted 11 August 2003

Abstract

Leaf explants ofCaladium ‘Pink Cloud’ were cultured in vitro on MS medium containing variousauxins (NAA, IBA, IAA, 2,4,5-T and 2,4-D) in combination with cytokinin (BA). NAA gave the mostvigorous in vitro propagation of this plant, and only 15% of the plants were leaf-colour variants onthe medium containing 0.5�mol NAA. Leaf colour variation was observed in all plants regeneratedon the medium containing 2,4-D at 0.5–4.5�mol. In hormone-free medium, only a few leaf-colourvariants (6%) occurred, but the rate of plant regeneration was very low. Application of 0.5�mol NAAtogether with 4.5�mol BA seemed to be the most appropriate for in vitro propagation ofCaladium‘Pink Cloud’ with only a few leaf-colour variants.© 2003 Elsevier B.V. All rights reserved.

Keywords: Caladium; Auxin; Variant; Micropropagation

1. Introduction

Certain plant species propagated by in vitro culture over a long period exhibit variantsin regenerated plants. Tissue culture-derived variation is highly undesirable in microprop-agation. Such changes are disadvantageous for the commercial grower, whose objectiveis to produce a large number of uniform plants.Caladium plants have been propagated invitro by using the young leaf tissue for 20 years (Sahavacharin, 1982). However, cultivardependent variation has been observed during tissue culture, and this occurs frequently in‘Pink Cloud’ regenerated in vitro (Zhu et al., 1993a).

∗ Corresponding author. Tel.:+81-75-753-6048; fax:+81-75-753-6068.E-mail address: eakhlas@yahoo.com (E.U. Ahmed).

0304-4238/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.scienta.2003.08.012

154 E.U. Ahmed et al. / Scientia Horticulturae 100 (2004) 153–159

For several species, the occurrence of variant leaves seems to be associated with highconcentration of auxin in the medium (Vajrabhaya, 1977; George and Sherrington, 1984).However, there is a possibility that the tissue culture procedure itself is mutagenic. Inan assay on colour mutagenesis, in stamen hairs ofTradescantia, medium componentsincluding 2,4-D (2,4-dichlorophenoxyacetic acid) appeared not to be mutagenic (Dolezeland Novak, 1984). Micropropagation ofGerbera jamesonii is enhanced by increasing thelevel of cytokinin in the medium. This may satisfy the demand for rapid propagation, butcould lead to the abnormality called bushiness, resulting in a production loss of up to 30%(Topoonyanont et al., 1999).

Plant growth regulators in the culture medium influence the frequency of karyotypicalterations in cells (Bayliss, 1975; Skirvin, 1978) and subsequent occurrence of variants insome systems. In regenerated plants, variation has been observed in plant morphology, leafshape and leaf colour. The expanding leaves of ‘Pink Cloud’ were cultured on modified MSmedium supplemented with 5�mol/l NAA and 4.5�mol/l BA from which the occurrenceof leaf-colour variants in regenerated plant has been reported byZhu et al. (1993b).

Therefore, the objective of the current study was to compare different auxin types atthree concentrations on the stability of ‘Pink Cloud’Caladium plants regenerated from leafexplants.

2. Materials and methods

2.1. Plant material, growth conditions and preparation of explants

Caladium ‘Pink Cloud’ stock plants were grown in a glasshouse in summer. Air temper-ature ranged between 25 and 35◦C in the daytime. Plants were grown in 15 cm plastic potsand fertilized with N–P–K (10–10–10) at the concentration of 5 g l−1 and insecticide alsoapplied when it was needed. After 3–4 months, fully expanded young leaves were taken forculture.

Young leaves were washed with tap water followed by 10 min in a 0.5% active sodiumhypochlorite solution containing 0.1% Tween-20. The leaves were rinsed three times withsterilized distilled water and cut into 0.5 cm×0.5 cm segments. Leaf segments were culturedon MS medium containing Fe-EDTA (Murashige and Skoog, 1962) supplemented withvitamins (Ringe and Nitsch, 1968), 20 g l−1 sucrose and 8 g l−1 agar (Wako Pure ChemicalIndustries).

2.2. Effects of various auxins (Experiment 1)

1-Naphthaleneacetic acid (NAA), 3-indolebutyric acid (IBA), 3-indoleacetic acid (IAA),2,4,5-trichlorophenoxyacetic acid (2,4,5-T) or 2,4-D was added to the culture medium at0.4–5.7�mol together with 4.5�mol 6-benzyladenine (BA). All media were adjusted to pH5.8 before the addition of agar and autoclaved at 121◦C for 1 h. Explants were placed on theagar medium (20 ml) in 100 ml test tube. Cultures were incubated at 25◦C under continuouscool white fluorescent light (25�mol m−2 s−1) for 90 days. Explants with multiple shootsinitiated were sub-cultured on MS basal medium without growth regulators at 4-weekinterval for 3 months.

E.U. Ahmed et al. / Scientia Horticulturae 100 (2004) 153–159 155

The regenerated plantlets, about 1–2 cm in height, were transferred to vermiculite in celltrays (cell size 5.0 cm× 5.0 cm× 7.0 cm; 50 cells/1 tray) and kept in a mist house forhardening for 1 month. When reaching 2–3 cm in height, all the regenerated plantlets weretransferred to vermiculite in 12 cm plastic pots and the liquid fertilizer N–P–K (5–10–5) wereapplied at the concentration of 2 ml l−1 once a week. The regenerated plants were grownin a glasshouse for about 3 months to evaluate leaf colour characteristics. The leaf-colourvariants were determined by visual assessments in comparison with mother plants. Theplants with distinct leaf-colour variants such as vein colour, the percentage of red, greenand grey colour area was considered during data collection.

2.3. Effect of NAA (Experiment 2)

Leaf explants were cultured on media containing 0.005–50�mol NAA together with4.5�mol BA and the occurrence of variants in the regenerated plants was examined.Leaf segments were also cultured on growth regulator-free medium and examined forthe occurrence of variants. All multiple shoots initiated were sub-cultured as describedabove.

Explant sources were excised from 10 stock plants. Ten tubes (replication) were selectedfrom each stock plants. The colour patterns of the leaves from five or six regenerated plantswere examined from one tube.

3. Results and discussion

3.1. Effects of auxins on the occurrence of variants (Experiment 1)

3.1.1. Type of auxinsWe examined the effects of different type of auxins on somaclonal variation in regener-

ated plants.De Klerk (1990)argued the need for an assay to assess the extent of somaclonalvariation in a population of regenerated plants. The effect of leaf colour variation on thelevel of the phenotype is usually determined as the percentage of plants that show vari-ants for one or more defined characteristics. The leaf-colour variants varied with auxintype more than with concentration of specific auxins (Fig. 1). On the medium containing0.5�mol NAA, regenerated plants had only 15% leaf-colour variants. On the other hand,the number of variant plants was high on media containing 2,4,5-T or 2,4-D. Also, darkgreen callus that gave rise to fewer variants were produced more often on media containinglow concentrations of NAA or IBA. Partly green callus produced on IAA-containing mediaregenerated more variants (>70%), and white callus produced on media containing 2,4,5-Tor 2,4-D regenerated leaf-colour variants 80–100% of the time. Low concentration of NAAmay induce organized callus in contrast to the unorganized callus induced by 2,4-D. It hasbeen reported that 2,4-D did not have a direct mutagenic effect but caused abnormalities in-directly by stimulating disorganized growth (Bayliss, 1980). Leaf variegation did not seemto influence development and size of plant leaves during tissue culture. Variegated leavesdeveloped normally but anthocyanin was not produced in variant plants. Auxin may influ-ence the phenotypic response in regenerated plants. It may share its biological effectiveness

156 E.U. Ahmed et al. / Scientia Horticulturae 100 (2004) 153–159

Fig. 1. Effect of auxin given together with BA on the percentage of variants ofCaladium plants. Asterisk (∗)represents the calli produced on media containing various auxins at 5�mol/l together with 4.5�mol/l BA. Valuesare means (n = 5) ± S.E.

with such diverse synthetic chemicals as NAA, IBA, IAA, 2,4,5-T and 2,4-D containingnarrow to wide range (0.4–5.7�mol) concentration of compounds.

3.1.2. Mode of variationThe occurrence of leaf-colour variants was influenced by the kind of auxin and its con-

centration. Plants regenerated in this study were either the same as mother plant (Fig. 2A) orproduced lamina-colour variants, which included green or grey leaves with reduced antho-cyanin production (Fig. 2B and C), and leaf vein alteration from white to green were observed(Fig. 2D). The significant effects of concentrations of plant growth regulators on variationwere demonstrated in ornamental plants byJain (1993)andBouman and De Klerk (1996).Green leaf vein variants are commonly observed in regenerated plants but grey leaf-colourvariants are rarely observed during micropropagation in ‘Pink Cloud’. In this experiment,many green leaf vein variants (42–60%) were observed on media supplemented with IBA orIAA. Green lamina variants appeared at a high frequency on media containing two, 4-5-T or2,4-D (73–100%) (Table 1). In the present study, variability decreased as the concentration

E.U. Ahmed et al. / Scientia Horticulturae 100 (2004) 153–159 157

Fig. 2. Types of variation observed in plants regenerated fromCaladium leaf explants. (A) Normal leaf, (B) variantwith green lamina, (C) variant with grey lamina and (D) green leaf vein variant.

Table 1Caladium plants regenerated from leaf explants cultured on media containing different auxina

Auxin type Concentration(�mol)

Total evaluatedplants

Normal plants Leaf colour variants

Lamina Vein green

Green Grey

NAA 0.5 54 46 2 (4) 0 6 (11)2.7 52 36 5 (10) 3 (6) 8 (15)5.3 58 33 6 (10) 5 (9) 14 (24)

IBA 0.5 50 20 9 (18) 0 21 (42)2.3 52 19 7 (13) 0 26 (50)4.5 53 13 10 (19) 1 (2) 29 (55)

IAA 0.6 55 16 13 (24) 2 (4) 23 (42)2.8 58 11 17 (29) 1 (1) 29 (50)5.7 57 5 14 (25) 4 (7) 34 (60)

2,4,5-T 0.4 51 10 37 (73) 0 4 (8)2 54 10 34 (63) 0 7 (13)4 56 8 43 (77) 0 5 (9)

2,4-D 0.5 56 0 56 (100) 0 02.3 54 0 54 (100) 0 04.5 54 0 54 (100) 0 0

a 50–58 plants per treatment. Values in parentheses indicate the percentage of variant plants.

158 E.U. Ahmed et al. / Scientia Horticulturae 100 (2004) 153–159

= 44.4 x 0.25

R2 = 0.9

01 0.1 1 10 100

NAA concentration ( µmol/l )

y

0

20

40

60

80

100

0.001 0.

Per

cen

tag

e o

f va

rian

ts

Fig. 3. Effect of the concentration of NAA on the percentage of variants inCaladium plants.

of auxin in the medium was lowered. NAA at a concentration of (0.5–5.3�mol) caused(15–43%) leaf-colour variants inCaladium cultured in vitro. It was reported that lowerlevel of auxin (2,4-D) greatly reduced variability in regeneratedHordeum plants, comparedwith a high concentration (Deambrogio and Dale, 1980).

3.2. Effect of NAA concentration (Experiment 2)

In hormone-free medium, only a few leaf-colour variants (6%) occurred, but it was ob-served that the production rate for regeneration was very low (data not shown). NAA at aconcentration of 50�mol induced variants at a high frequency, but that at a concentrationof 0.005–0.5�mol induced variants at a low frequency (Fig. 3). Thus, the medium con-taining 0.5�mol NAA together with 4.5�mol BA was found to be the most appropriatefor propagation of plantlets with high regeneration rate. In this study, we achieved the effi-cient combination of suitable concentration of auxin and cytokinin for the propagation ofCaladium plants with lower variants.

References

Bayliss, M.W., 1975. The effects of growth in vitro on the chromosome complement ofDaucus carota (L.)suspension cultures. Chromosoma 51, 401–411.

Bayliss, M.W., 1980. Chromosomal variation in plant tissues in culture. Int. Rev. Cytol. A 11, 113–144.Bouman, H., De Klerk, G.J., 1996. Somaclonal variation in biotechnology of ornamental plants. In: Geneve,

R., Preece, J., Merkle, S. (Eds.), Biotechnology of Ornamental Plants. CAB International, Wallingford,pp. 165–183.

De Klerk, G.-J., 1990. How to measure somaclonal variation. Acta Bot. Neerl. 39, 129–144.Deambrogio, E., Dale, P.J., 1980. Effect of 2,4-D on the frequency of regenerated plants in barley and on genetic

variability between them. Cereal Res. Comm. 8, 417–423.Dolezel, J., Novak, F.J., 1984. Effect of plant tissue culture media on the frequency of somatic mutations in

Tradescantia stamen hairs. Zeitschrift für pflanzenphysiol. 114, 48–51.

E.U. Ahmed et al. / Scientia Horticulturae 100 (2004) 153–159 159

George, E.F., Sherrington, P.D., 1984. Plant Propagation by Tissue Culture. Exegetics, Eversley.Jain, S.M., 1993. Growth hormonal influence on somaclonal variation in ornamental plants. In: Proceedings of

the XVII Eucarpia Symposium on Creating Genomic Variation in Ornamentals, pp. 93–103.Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and tobacco tissue cultures. Physiol. Plant.

15, 473–497.Ringe, F., Nitsch, J.P., 1968. Conditions leading to flower formation on excised begonia fragments cultured in

vitro. Plant Cell Physiol. 9, 639–652.Sahavacharin, O., 1982. Rapid propagation ofCaladium through tissue culture. In: Plant Tissue Culture.

Proceedings of the Fifth International Congress on Plant Tissue and Cell Culture, pp. 699–700.Skirvin, R.M., 1978. Natural and induced variation in tissue culture. Euphytica 27, 241–266.Topoonyanont, N., Ampawan, R., Debergh, P.C., 1999. Bushiness inGerbera jamesonii: abnormal shoot

development during micropropagation. J. Hort. Sci. Biotech. 74, 675–679.Vajrabhaya, T., 1977. Variation in clonal propagation. In: Orditti, J. (Ed.), Orchid Biology. Cornell University

Press, Ithaca, pp. 179–201.Zhu, Y., Yazawa, S., Asahira, T., 1993a. Varietal differences in leaf color variation of plants regenerated from in

vitro culture of leaf blade inCaladium cultivars. Jpn. Soc. Hort. Sci. 62, 431–435.Zhu, Y., Takemoto, T., Yazawa, S., 1993b. Leaf color of plants regenerated through in vitro culture from variegated

leaf segments ofCaladium. Jpn. Soc. Hort. Sci. 62, 619–624.