Plant Regeneration and Chromosome Doubling of Wild Gladiolus Species

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1 Plant Regeneration and Chromosome Doubling of Wild Gladiolus Species K. Suzuki, Y. Takatsu, T. Gonai and M. Kasumi Plant Biotechnology Institute Ibaraki Agricultural Center, Ibaraki Japan Keywords: gladiolus, wild species, callus, plant regeneration, chromosome doubling, colchicine Abstract A plant regeneration system from callus and chromosome doubling by colchicine treatment was investigated in wild Gladiolus species. Corm slices of 3 species, G. priorii, G. tristis and G. virescens, were used and callus was induced in liquid MS medium supplemented with BA or 2,4-D. The resulting callus was classified as two types - a friable type induced by BA and a compact type induced by 2,4-D. The former callus easily regenerated shoots with further cultivation. Chromosome doubling was induced by treating callus initiating shoots with colchicine. The highest efficiency of chromosome doubling was observed in the treatment with colchicine at 0.05% for 72 hours. The regenerants of the wild species having doubled chromosomes will be powerful materials for gladiolus breeding. INTRODUCTION The genus Gladiolus is a member of the family Iridaceae and is distributed in southern Africa, Madagascar and Eurasia. The current number of the species is 255 (Goldblatt and Manning, 1998), however, modern cultivars are considered to be derived from crosses among only a few species (Barnard, 1972; Imanishi, 1989). Therefore, most of the wild species have not been included in breeding programs of cultivars and so they constitute a potentially powerful genetic resource. Some of the wild species have unique traits such as short time to flowering, blue tepals, excellent fragrance, and short plant height (Takatsu et al., 2002). Most of the wild species have diploid chromosome numbers based on x = 15, in contrast with modern cultivars of G. x grandiflora which are tetraploid (Imanishi, 1989). Doubling the chromosome number of wild species (diploid, 2n = 2x = 30) to match modern cultivars (tetraploid, 2n = 4x = 60) is an important procedure for interspecific hybridization breeding. Chromosome doubling has been reported in ornamental plants regenerated through tissue culture (Lindsay et al., 1994; Winkelmann and Grunewaldt, 1995; Oh et al., 1995; Aida and Shibata, 2002) and with plants treated with colchicine (Chen and Goeden-Kallemeyn, 1979; Ishizaka and Uematsu, 1995; Chavadej and Becker, 1984). In this study we investigated induction of callus, regeneration of plants from callus and demonstrated chromosome doubling through colchicine treatment. MATERIALS AND METHODS Plant Materials Three diploid Gladiolus species, G. priorii, G. tristis and G. virescens, were purchased from Silverhill Seed Co. (Capetown, South Africa). The first was an early flowering genotype and the second and third had excellent fragrance. Colchicine Treatment of Corms In Vivo Cotton wool soaked in an aqueous solution of 0.01, 0.05, 0.1 or 0.5% (v/v) colchicine was put on apical buds of G. tristis corms for 12 or 72 hours. To prevent drying, plastic bags were put on the cottons and the colchicine solution was added dropwise every Proc. IX th Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS

2 12 hours. The treated corms were grown in a greenhouse and the success of chromosome doubling was judged by flower morphology. Callus Induction Apical buds of in vitro grown corms 5 to 8 mm in diameter of the Gladiolus species were removed and cut horizontally into 3 slices of 2 to 3 mm thickness. Each three slices was transferred to a 100 ml flask containing 50 ml of liquid medium. The media consisted of the basal medium of MS (Murashige and Skoog, 1962) supplemented with: - NAA (α-naphthaleneacetic acid) - NAA medium: 2,4-D (2,4-dichlorophenoxy- acetic acid) - 2,4-D medium or BA (6-benzyladenine) - BA medium at different concentrations and 3% (w/v) sucrose adjusted to ph 5.8. Cultures were carried out on an orbital shaker at 110 rpm under a 16 hr light photoperiod (50 µmol m -2 sec -1 ) at 20 C. After 2 to 4 months of culture the sliced explants were evaluated visually for induction of callus and regeneration of shoots. Shoot Regeneration After 3 to 4 months of culture, regeneration of shoots from callus was observed in all the species in the BA medium. These shoots were transferred onto the MS medium solidified with 3 g L -1 gellun gum. The compact callus induced by 2,4-D did not show evidence of regeneration and those weighing 100 to 200 mg were placed onto the solid MS medium supplemented with 1 mg L -1 BA. After 2 months this callus was evaluated for the regeneration of shoots and the regenerated shoots were transferred onto the solid MS medium. Treatment of Callus with Colchicine The callus initiating shoot regeneration derived from the tissue culture in BA liquid medium was treated with colchicine at 0.01 or 0.05% (v/v) for 24, 48 and 72 hours and then cultured on solid MS medium. To check the occurrence of chromosome doubling flow cytometric analysis was carried out. Leaf segments about 1 cm long were chopped up well with a razor blade in 0.2 ml of Partec UV solution A from a High Resolution DNA Staining Kit (Partec GmbH, Germany). The resulting suspension with nuclei was filtered through a 50 µm Partec CellTrics TM filter. 1.6 ml of Partec UV solution B from the kit was added and the intensity of fluorescence of nuclei was measured with a Partec Ploidy Analyzer (PA). RESULTS AND DISCUSSION To double the chromosome number of the wild species G. tristis, apical buds of corms were treated with colchicine at different concentrations for 12 and 72 hours (Table 1). However, even at the highest colchicine concentration (0.5%) and the longest treatment time (72 hours), there was no change in subsequent flower morphology indicating no chromosome doubling. This might be because either the colchicine did not reach the growing points through the thicker apical buds of the corms or chimeras might have occurred and not changed flower morphology. A high frequency of chimeras is normally associated with colchicine treatment in vivo (Ackerman and Dermen, 1972; Schifino and Fernandes, 1987). Cohen and Yao (1996) proposed that colchicine treatment in vitro was an excellent technique for chromosome doubling because of a high efficiency of polyploid production and few chimeras. Some researchers have succeeded in chromosome doubling by colchicine treatments in vitro using different types of plant tissue, such as callus (Chen and Goeden-Kallemeyn, 1979; Orton and Steid, 1980; Gmitter et al., 1991), cell suspensions (Chavadej and Becker, 1984; Dolezel and Binarova, 1989) and ovules (Ishizaka and Uematsu, 1995). We therefore tried to establish a plant regeneration system using tissue culture and to double chromosome number in vitro using colchicine treatment. Tissue cultures using corm slices of 3 species, G. priorii, G. tristis and G. virescens, were established in liquid MS medium supplemented with plant growth regulators (Table 176

3 2). Within 2 to 3 months callus was induced by supplementing with 1 or 2 mg L -1 BA or with 2 or 4 mg L -1 2,4-D in all the 3 species. However, corm slices did not show any change and were dead after 4 months in the NAA medium. Two types of callus were distinguishable - BA medium induced friable callus, and 2,4-D medium induced compact callus (Fig. 1). Regeneration of shoots was observed in callus induced by BA in all 3 species within 3 to 4 months. The compact callus induced in the 2,4-D medium remained unchanged during further incubation but regenerated shoots in 2 months after transferring onto the solid MS medium supplemented with 1 mg L -1 BA. Productivity of shoots in the BA liquid medium was superior to that in the 2,4-D liquid medium followed by the BA solid medium. Another trial of in vitro production of G. dalenii and G. tristis revealed that the best production of shoots per corm explant was achieved on a medium supplemented with 2 mg L -1 and 0.5 mg L -1 BA, respectively (De Bruyn and Ferreira, 1992). Therefore we selected BA at 1 mg L -1 as the plant growth regulator for the plant regeneration system of G. priorii, G. tristis and G. virescens. The calluses initiating regeneration of shoots in the BA liquid medium were treated with colchicine and flow cytometric analysis revealed the ploidy levels of regenerants (Fig. 2). In G. tristis 13 (35.1%) and 1 regenerants (2.7%) were obtained as tetraploids (4x) and 2x + 4x chimeras, respectively, by treating with 0.05% colchicine for 72 hours. No change of ploidy level was observed in treating with 0.01 or 0.05% colchicine for 24 and 48 hours (Table 3). In G. virescens tetraploid (4x) regenerants were obtained efficiently (18.2 to 22.2%) from treating with 0.05% colchicine for 24 and 72 hours, and only 1 chimera (9.1%) was observed in 0.05% for 24 hours treatment (Table 4). These results indicated that treating the callus with colchicine at 0.05% for 72 hours would be the best for chromosome doubling of wild Gladiolus species. We then applied these conditions to another species, G. priorii. As expected colchicine treatment at 0.05% for 72 hours produced a high efficiency (42.9%) of chromosome doubling of G. priorii (Table 5). Chromosome doubling has been widely used in plant breeding programs for the production of new cultivars (Griesbach, 1990) and for the restoration the fertility of interspecific or intergeneric hybrids (Ackerman and Dermen, 1972; Orton and Steid, 1980; Anderson et al., 1991; Ishizaka and Uematsu, 1995). In the present study, we have established a plant regeneration system from corm slices in vitro and demonstrated that colchicine treatment of callus at 0.05% for 72 hours was the best for chromosome doubling in the 3 wild Gladiolus species studied. This in vitro method was superior to the in vivo treatment and could be applicable to other species. G. priorii has the desirable agricultural trait of early flowering and G. tristis and G. virescens have excellent fragrance. The tetraploid regenerants (Fig. 3) of the 3 species produced in this study will be powerful materials for interspecific hybridization breeding to produce modern cultivars. Literature Cited Ackerman, W.L. and Dermen, H A fertile colchiploid from a sterile interspecific camellia hybrid. J. Heredity 63: Aida, R. and Shibata, M High frequency of polyploidization in regenerated plants of Kalanchoe blossfeldiana cultivar Tetra Vulcan. Plant Biotech. 19(5): Anderson, J.A., Mousset-Declas, C., Williams, E.G. and Taylor, N.L An in vitro chromosome doubling method for clovers (Trifolium spp.). Genome 34:1-5. Barnard, T.T On hybrids and hybridization. p In: G.J. Lewis, A.A. Obermeyer and T.T. Barnard (eds.), Gladiolus, a revision of the South African species, Suppl. 10, J. S. Afr. Bot. Chavadej, S. and Becker, H Influence of colchicine treatment on chromosome number and growth rate of tissue cultures of Valeriana wallichii DC. Plant Cell, Tissue and Organ Cult. 3: Chen, C.H. and Goeden-Kallemeyn, Y In vitro induction of tetraploid plants from colchicine-treated diploid daylily callus. Euphytica 28: Cohen, D. and Yao, J.L In vitro chromosome doubling of nine Zantedeschia cultivars. Plant Cell, Tissue and Organ Cult. 47:

4 De Bruyn, M.H. and Ferreira, D.I In vitro corm production of Gladiolus dalenii and G. tristis. Plant Cell, Tissue and Organ Cult. 31: Dolezel, J. and Binarova, P The effects of colchicine on ploidy level, morphology and embryogenic capacity of alfalfa suspension cultures. Plant Sci. 64: Gmitter Jr., F.G., Ling, X., Cai, C. and Grosser, J.W Colchicine-induced polyploidy in Citrus. embryogenic cultures, somatic embryos, and regenerated plantlets. Plant Sci. 74: Goldblatt, P. and Manning, J Gladiolus in Southern Africa. Fernwood Press, Vleaberg. Griesbach, R.J Colchicine-induced polyploidy in Eustoma grandiflorum. HortSci. 25: Imanishi, H Gladiolus. p In: T. Matsuo (eds.), Collected data of plant genetic resources, Kodansya Scientific, Tokyo (in Japanese). Ishizaka, H. and Uematsu, J Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses. Euphytica 86: Lindsay, G.C., Hopping, M.E. and O Brien, I.E.W Detection of protoplast-derived DNA tetraploid lisianthus (Eustoma grandiflorum) plants by leaf and flower characteristics and by flow cytometry. Plant Cell, Tissue and Organ Cult. 38: Murashige, T. and Skoog, F A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: Oh, M.-H., Lee, H.S., Song, J.Y., Choi, D.-W., Kwon, Y.M., Lee, J.S. and Kim, S.-G Origin of tetraploidization in protoplast cultures of petunia (Petunia hybrida). J. Heredity 86: Orton, T.J. and Steidl, R.P Cytogenetic analysis of plants regenerated from colchicine-treated callus cultures of an interspecific Hordeum hybrid. Theor. Appl. Genet. 57: Schifino, M.T. and Fernandes, M.I.M Induction of polyploidy and cytological characterization of autotetraploids of Trifolium riograndense Burkart (Leguminosae). Euphytica 36: Takatsu, Y., Manabe, T., Kasumi, M., Yamada, T., Aoki, R., Inoue, E., Morinaka, Y., Marubashi, W. and Hayashi, M Evaluation of germplasm collection in wild gladiolus species of southern Africa. Breeding Res. 4:87-94 (in Japanese). Winkelmann, T. and Grunewaldt, J Analysis of protoplast-derived plants of Saintpaulia ionantha H. Wendl. Plant Breed. 114: Tables Table 1. Colchicine treatment of apical buds in corms of Gladiolus tristis in vivo. Concentration of colchicine (%) treatment (hr) No. of treated plants No. of plants changing flower phenotype 178

5 Table 2. Callus induction and shoot regeneration of wild Gladiolus species on MS medium supplemented with different plant growth regulators. Species Plant growth regulators (mg L -1 ) No. of flasks 1 No. of flasks inducing callus 2 No. of flasks regenerating shoots 3 G. priorii NAA NAA ,4-D ,4-D BA BA G. tristis NAA NAA ,4-D ,4-D BA BA G. virescens NAA NAA ,4-D ,4-D BA BA Each flask containing 3 slices of corm was cultured at 20 C 2 Callus induction was evaluated 2 to 4 months after culturing 3 Regeneration of shoots was evaluated 2 to 5 months after culturing Table 3. Chromosome doubling efficiency by colchicine treatment of callus of Gladiolus tristis. Colchicine concentration (%) treatment (hr) No. of treated Ploidy level (No. of plants (%) ) plants 2x 4x 2x + 4x (100.0) 0 (0.0) 0 (0.0) (100.0) 0 (0.0) 0 (0.0) (100.0) 0 (0.0) 0 (0.0) (100.0) 0 (0.0) 0 (0.0) (62.2) 13 (35.1) 1 (2.7) (100.0) 0 (0.0) 0 (0.0) 179

6 Table 4. Chromosome doubling efficiency by colchicine treatment of callus of Gladiolus virescens. Colchicine concentration (%) treatment (hr) No. of treated Ploidy level (No. of plants (%) ) plants 2x 4x 2x + 4x (100.0) 0 (0.0) 0 (0.0) (72.7) 2 (18.2) 1 (9.1) (100.0) 0 (0.0) 0 (0.0) (77.8) 2 (22.2) 0 (0.0) (100.0) 0 (0.0) 0 (0.0) Table 5. Chromosome doubling efficiency by colchicine treatment of callus of Gladiolus priorii. Colchicine concentration (%) treatment (hr) No. of treated Ploidy level (No. of plants (%) ) plants 2x 4x 2x + 4x (57.1) 9 (42.9) 0 (0.0) (100.0) 0 (0.0) 0 (0.0) Figures A B Fig. 1. Callus induction from corm slices of wild Gladiolus species. A. Friable callus induced in the MS medium supplemented with BA. B. Compact callus induced in the MS medium supplemented with 2,4-D. 180

7 2n = 2x A 2n = 4x B Fig. 2. Flow cytometric analysis of wild Gladiolus species. A. Pattern of diploid (2n = 2x) in original wild species. B. Pattern of tetraploid (2n = 4x) in a regenerant treated with colchicine. Fig. 3. A regenerant of a wild Gladiolus species with doubled chromosome number induced by colchicine treatment. 181