Scientia Horticulturae 83 (2000) 301±310 Production of doubled haploid plants of carnation (Dianthus caryophyllus L.) by pseudofertilized ovule culture S. Sato *, N. Katoh, H. Yoshida, S. Iwai 1, M. Hagimori Applied Plant Research Laboratory, Japan Tobacco Inc., 1900 Idei, Oyama, Tochigi, 323-0808 Japan Accepted 22 July 1999 Abstract Doubled haploids of carnation (Dianthus caryophyllus L.) were obtained by pseudofertilized ovule culture. Emasculated ower buds of carnation were pollinated with pollen inactivated by X- ray irradiation. After 2±3 weeks, the ovaries were explanted and were cultured on solid MS medium containing 2 mm NAA, 2 mm BAP and 6% sucrose. Regenerated plants(r0) were morphologically different from the mother plants, indicating that they did not originate from their somatic cells. Root tip cells of the R0 plants were chimeric for the chromosome number; both 2n ˆ 30 cells and 2n ˆ 15 cells were observed in root tips. The R0 plants were fertile and selfed seeds (R1) were obtained. The R1 plants of each R0 plant were morphologically uniform and were identical to their respective R0 plants. From these results we conclude that the R0 plants were doubled haploids. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Doubled haploid; Carnation (Dianthus caryophyllus L.); Pseudofertilized ovule culture; Irradiated pollen 1. Introduction In carnation (Dianthus caryophyllus L., 2n ˆ 30), most commercially important varieties are vegetatively propagated, and are not F1 hybrids. Some * Corresponding author. Fax: 81-285-22-2961. E-mail address: jdj05073@nifty.ne.jp (S. Sato). 1 Present address: Faculty of Agriculture, Kagoshima University, Kohrimoto, Kagoshima 890-0065, Japan. 0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0304-4238(99)00090-4
302 S. Sato et al. / Scientia Horticulturae 83 (2000) 301±310 disadvantages exist with clonal propagation compared with seed propagation from the viewpoint of commercial production of seeds and rooted cuttings. First, the production cost per plantlet is much higher in clonal propagation than in seed propagation. Second, perfect control of virus and other diseases in the nursery is essential in clonal propagation. This is, however, rather dif cult and increases the rooted cutting cost. Third, the shelf life of cuttings is far shorter than that of seeds. A change to propagating the F1 variety from propagating the clonal variety would be greatly advantageous in seed and seedling production. To breed F1 varieties, producing inbred lines as the parental lines is necessary. Conventionally, an inbred line is produced by repeating sel ng for several generations. Also, in carnation, inbreeding depression appears in the S3 progeny in most varieties so that it is almost impossible to produce S4 seeds. Production of doubled haploids is another way to produce pure lines. Anther culture of carnation has been tried (Murty and Kumar, 1976; Villalobos, 1981), but to our knowledge, no reports exist of the successful production of haploids or doubled haploids. Likewise, no reports exist of microspore cultures of carnation. Ovules are a possible alternative source material for haploid (or doubled haploid) production. Successful production of haploids or doubled haploids has been reported for several species, such as Nicotiana (Pandey and Phung, 1982), Petunia (Raquin, 1985), apple (Zhang et al., 1988), Brassica (DoreÂ, 1989) and melon (Sauton and Dumas, 1987; Katoh et al., 1993). In this study, we tried the pseudofertilized ovule culture of carnation and succeeded in the reproducible production of doubled haploids. This is the rst report to succeed in producing doubled haploids of carnation. 2. Materials and methods 2.1. Plant materials Commercial cultivars and breeding lines of carnation (Dianthus caryophyllus L., 2n ˆ 30) were used. All plants were grown in a greenhouse in which the temperature was maintained between 188C and 328C. In experiment 1, breeding lines of the standard type and the spray type were used as the ovule donors. Pollen was collected for pseudofertilization from owers of the spray type lines that produce suf cient pollen grains. In experiment 2, the spray type line ``933145'' with purple petals was used as the pollen donor. In petal color genetics, purple is dominant over white and yellow. The line ``933145'' has been con rmed to be homozygous for this trait from our breeding results. Seven lines or cultivars with white or yellow petals including a cultivar ``Tundra'' were used as the ovule donors.
2.2. Pseudofertilization Immature ower buds of the ovule donors were emasculated at least 1 week before anthesis and were bagged to prevent outcrossing. After about 2 weeks, the ower buds matured to fertility is indicated by the stigma of the buds; they had curled to the outside by this stage. Anthers from the pollen donor were collected in a petri dish and irradiated with 100 kr (experiment 1) or 200 kr (experiment 2) at 1252 R/min using an X-ray unit (OM-100R, Ohmic, Ltd., Tokyo). The ower buds of the ovule donor were pollinated with the irradiated pollen and bagged again. 2.3. Ovule culture Ovaries were collected 1±4 weeks after pseudofertilization. Their surfaces were sterilized using a spray of 70% ethanol. The ovaries were then submerged in sodium hypochlorite solution (1% available chlorine) for 15 min and they were rinsed with sterilized water three times. Ovules with placenta were isolated from the ovaries and were placed on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with NAA 2 mm, BAP 2 mm, 0.8% agar and 6%, 9% or 12% sucrose. These media were based on Demmink et al. (1987). The ph of the medium was adjusted to 5.8 with 0.1N KOH before autoclaving. The ovules were cultured at 248C under a 12 h light (white uorescent lamp, light intensity of 20 mmol m 2 S 1 ) ±12 h dark cycle for 4 weeks. The shoots regenerated from the ovules were transferred to MS medium without plant growth regulators containing 3% sucrose and 0.8% agar. The plantlets were potted using perlite as the substrate, acclimatized in a mist room (relative humidity 100%) set in the greenhouse at 258C for 2 weeks, and then transferred to soil in pots and grown in a greenhouse. 2.4. Cytological studies S. Sato et al. / Scientia Horticulturae 83 (2000) 301±310 303 The germination of irradiated pollen on stigmas was observed by the aniline blue staining methods of Shivanna and Rangaswamy (1992). One day after pollination the ovaries were collected and were stained by soaking them in a solution containing 1% aniline blue and 0.1N Na 3 PO 4 for 1 h. They were then placed on a glass slide, cut into two pieces and observed using a uorescent microscope. The number of chromosomes in the root tip cells of the plantlets were counted by the method of Nishibayashi and Kaeriyama (1986). 3. Results The irradiated pollen, which had pollinated the stigma, germinated and the pollen tube elongated into the style as normal pollen (Fig. 1). Although the
304 S. Sato et al. / Scientia Horticulturae 83 (2000) 301±310 Fig. 1. X-ray irradiated pollen of carnation germinating on a stigma and their pollen tubes elongating into the style. Scale bar, 100 mm. ovaries pollinated with irradiated pollen, began to swell 1 week after pollination, they eventually aborted after 4 weeks unless they were cultured. Thus we collected ovaries 2±3 weeks after pollination. In experiment 1, about 300 ovaries of various lines were cultured. More than a hundred shoots regenerated from ovules only cultured on a medium containing 6% sucrose (Fig. 2). On the other hand, in the case cultured on media containing 9% or 12% sucrose, shoots were not regenerated. These shoots directly emerged from the ovules without forming a callus. These elongated shoots were obtained at the rate of 1 shoot per 1 ovule. After rooting they were acclimatized and grown in a greenhouse. Flowers of most of the regenerated plants (R0) were found to have normal pollen, so they were self-crossed. Seeds were obtained from 55 regenerated plants. Each S1 progeny of those 55 plants were cultivated and observed for petal color and petal number, both traits segregated in 54 of the S1 progeny, but all individuals of the S1 progeny of one regenerated plant (97KC- R0-1) showed uniform petal color and uniform petal number per ower, indicating that 97KC-R0-1 was genetically xed. The mother plant (ovule donor) of the xed plant was 97KC, which is a line of the double- owered type with lilac petals. In the self-cross progeny of 97KC, the trait of petal number per ower segregated among 5±25 and the petal color changed from light pink to deep purple (Fig. 3). The regenerated plant (97KC-R0-1) was the single- owered type with purple petals. All individuals of the S1 progeny of 97KC-R0-1 were similar in petal color and petal number per ower to those of 97KC-R0-1 (Fig. 4). The chromosome number in the root tip cells of 97KC-R0-1 was counted. Cells with 15 chromosomes and those with 30 chromosomes were found to coexist in a
Fig. 2. Shoot emerged from a cultured ovule. Scale bar, 1 cm. Fig. 3. A ower of the line ``97KC'', center, and those of its S1 progeny around it. Scale bar, 3 cm.
306 S. Sato et al. / Scientia Horticulturae 83 (2000) 301±310 Fig. 4. The owers of S1 progeny plants of 97KC-R0-1 that regenerated from a cultured pseudofertilized ovule of line ``97KC''.Scale bar, 1 cm. root tip indicating the root tips were chimeric consisting of both haploid and doubled haploid cells (Fig. 5). The chromosome number of each ploidy levels was counted over 10 cells. In experiment 2, pollen was irradiated with 200 kr X-ray to inactivate the pollen completely. Almost a hundred ovaries were cultured. One plantlet was regenerated from an ovary of ``Tundra'' cultured on a medium containing 6% sucrose. The plantlet was acclimatized and grown in a greenhouse. In the measurement of nuclear DNA content of leaf samples by using ow cytometry, the ploidy level of the regenerated plant (Tundra-R0-1) was the same as the Tundra (data not shown). The ower of the regenerated plant (Tundra-R0-1) was the single- ower type with ve white petals per ower (Fig. 6), whereas Tundra, the mother plant, had 50±70 yellow petals with a pink edge (Fig. 7). Tundra-R0-1 yielded abundant normal pollen grains. They were self-crossed and S1 seeds were obtained. The S1 progeny were grown and their morphology observed. All the observed individuals of the S1 progeny of Tundra-R0-1 were similar to each other not only in ower color and petal number, but also in other traits, such as formation of the plant to Tundra-R0-1. From these results we conclude that Tundra-R0-1 and 97KC-R0-1 were doubled haploids. 4. Discussion In this study, we obtained two regenerated plants, 97KC-R0-1 and Tundra-R0-1, which we conclude to be doubled haploids. They were fertile without any chromosome doubling treatment. In studies of pseudofertilized ovule culture, as well as of anther culture, the origin of the regenerated plant should be demonstrated early. The regenerated plants have three potential origins in addition to real fertilized ovules.
S. Sato et al. / Scientia Horticulturae 83 (2000) 301±310 307 Fig. 5. Chromosomes in root apex cells of a plantlet regenerated from the cultured pseudofertilized ovule of line ``97KC'': (a) haploid cell (2n ˆ 15); (b) diploid cell (2n ˆ 30). Scale bar, 10 mm. The rst potential origin is from the somatic cells of the mother plant, which, if true, means that the regenerated plants would be identical to the mother plant. Both 97KC-R0-1 and Tundra-R0-1 were morphologically different from their respective mother plants. Also, when the spontaneous somaclonal variation was induced from regeneration process, the S1 progeny would segregate by many phenotypes. However, The S1 progeny of both 97KC-R0-1 and Tundra-R0-1 were uniform. So, the rst potential origin is rejected. The second potential origin is an ovule fertilized with active pollen escaped from X-ray irradiation. If true, dominant traits of the pollen donor must be expressed in the regenerated plants. In experiment 2, the ower color of the
308 S. Sato et al. / Scientia Horticulturae 83 (2000) 301±310 Fig. 6. The owers of S1 progeny plants of Tundra-R0-1 that regenerated from a cultured pseudofertilized ovule of the cultivar ``Tundra''. Scale bar, 1 cm. pollen donor ``933145'' was purple and that of the mother plant ``Tundra'' was yellow. Purple is dominant over yellow in carnation. If the origin of the regenerated plant ``Tundra-R0-1'' had been an ovule fertilized with active ``933145'' pollen, the ower color should have been purple. Because the ower color of Tundra-R0-1 was white, the second potential origin is rejected. The third potential origin is an ovule self-fertilized with the pollen of the mother plant that had not been emasculated. If true, the regenerated plant would not be xed because the material plants are highly heterozygous and the S1 Fig. 7. The ower of cultivar ``Tundra''. Scale bar, 1 cm.
S. Sato et al. / Scientia Horticulturae 83 (2000) 301±310 309 progeny would segregate by many characteristics. The S1 progeny of both 97KC- R0-1 and Tundra-R0-1 were uniform, not only in ower color and petal number per ower, but also in other characteristics, indicating both 97KC-R0-1 and Tundra-R0-1 are xed. So, the third potential origin is also rejected. Thus, we conclude that 97KC-R0-1 and Tundra-R0-1 are doubled haploids. Both 97KC-R0-1 and Tundra-R0-1 were fertile indicating they were diploids. Spontaneous chromosome doubling of haploids is observed in several species (Hamaoka et al., 1991; Miyoshi, 1996). It is considered to occur in the early stages of regeneration of both 97KC-R0-1 and Tundra-R0-1. The fact that haploid cells coexisted with diploid cells in the root tips of 97KC-R0-1 supports the view that 97KC-R0-1 had been originally a haploid. This is the rst report to succeed in producing doubled haploids of carnation. Although our results indicate that the doubled haploid plants were obtained by pseudofertilized ovule culture, the ef ciency of doubled haploid production from ovules was not so high. Therefore, haploid induction may be improved by the manipulation of physical, chemical and physiological conditions especially in the maternal materials. And then, the modi cation of irradiate condition and embryo rescue might be effective for haploid plants production. A greater understanding of factor that are involved in morphogenic competence is still needed to realize the full potential of in vitro parthenogenesis in carnation. References Demmink, J.F., Custer, J.B.M., Bergervoet, J.H.W., 1987. Gynogenesis to bypass crossing barriers between diploid and tetraploid Dianthus species. Acta Hort. 216, 343±344. DoreÂ, C., 1989. Obtention de plantes haploõèdes de chou cabus (Brassica oleracea L. spp. capitata) apreás culture in vitro d'ovules polliniseâs par du pollen irradieâ. C.R. Acad. Sci. Paris, ser. III, pp. 729±734. Hamaoka, Y., Fujita, Y., Iwai, S., 1991. Effect of temperature on the mode of pollen development in anther culture of Brassica campestris. Physiol. Plant. 82, 67±72. Katoh, N., Hagimori, M., Iwai, S., 1993. Production of haploid plants of melon by pseudofertilized ovule culture. Plant Tissue Culture Lett. 10, 60±66. Miyoshi, K., 1996. Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L.). Plant Cell Rep. 15, 391±395. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiol. Plant 15, 473±497. Murty, Y.S., Kumar, V., 1976. In vitro production of plantlets from the anthers of Dianthus caryophyllus L., Acta Bot. Indica 4, 172±173. Nishibayashi, S., Kaeriyama, J., 1986. Structural stability of chromosomes in rice (Oryza sativa L.) plants regenerated from somatic tissue culture. Plant Tissue Culture Lett. 3, 31±34. Pandey, K.K., Phung, M., 1982. ``Hertwig effect'' in plants: induced parthenogenesis through the use of irradiated pollen. Theo. Appl. Genet. 62, 295±300. Raquin, C., 1985. Induction of haploid plants by in vitro culture of petunia ovaries pollinated with irradiated pollen. Z. P anzenzucht 94, 166±169.
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