吉田｜Ushiyama et al. - Effects of Various Phytohormones on Haploid Wheat Production

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本頁は PPS (2006) に掲載したものです．図表一部省略

Effects of various phytohormones on haploid wheat production in wheat x maize crosses

Tomohiko Ushiyama1, Tatsuo Kuwabara and Tomohiko Yoshida
Abbreviations :2,4-D, 2,4-Dichlorophenoxyacetic acid; IAA, indole-acetic acid, NAA, naphthalene acetic acid; BA, 6-benzylaminopurine

Abstract: The effects of phytohormones on the production of haploid wheat were examined in the intergeneric crosses between Japanese wheat cultivar (Triticum aestivum cv. Zenkojikomugi) and maize (Zea mays cv. Pioneer P80 Lisa). The detached wheat spikes pollinated with maize were cultured in a solution containing sucrose and sulfurous acid supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), indole-acetic acid, naphthalene acetic acid, kinetin or 6-benzylaminopurine at 0, 0.1, 1, 10, 100 or 1000 mg L-1. Haploid embryos obtained were cultured on agarose-solidified B5 medium. The frequency of plant regeneration was significantly affected only by the treatment with 100 mg L-1 2,4-D. Thus, the detached spikes were cultured in the media containing 2,4-D at 0, 25, 50, 75, 100, 125, 150, 175, 200 or 400 mg L-1. The treatments with 50 mg L-1 2,4-D increased the embryo size, but the treatments with above 75 mg L-1 2,4-D inhibited the development of the embryo. The percentage of florets developing into haploid plants was increased by the treatment with 100 mg L-1 2,4-D. Therefore, the concentration of 2,4-D in the spikes treated with 2,4-D at 50, 100 and 150 mg L-1 were measured by gas chromatography. The concentration of 2,4-D in the seed was increased to 9.24 ppm by the treatment with 100 mg L-1 2,4-D, a further increase of 2,4-D concentration in the medium having no effect. On the other hand, the concentration of 2,4-D in the glumes and rachis increased up to 12.72 and 41.55 ppm by the treatment with 100 and 150 mg L-1 2,4-D, respectively. The treatments with 2,4-D at a concentration higher than 100 mg L-1 inhibited embryo development. The present results suggested that 2,4-D at the concentrations from 50 to 100 mg L-1 would be optimum for haploid wheat production using maize.
Key words : 2,4-Dichlorophenoxyacetic acid, Haploid, Intergeneric cross, Phytohormone, Plant regeneration, Triticum aestivum, Zea mays,

Intergeneric crossing, wheat (Triticum aestivum L.) x Hordeum bulbosum L. (Barclay, 1975) and wheat x maize(Zea mays L.) crosses (Laurie and Bennett, 1988) have been used to produce haploid wheat. In the crosses of wheat x H. bulbosum, treatment with 2,4-dichlorophenoxyacetic acid (2,4-D) at a concentration lower than 100 mg L-1 was effective for haploid wheat production although 2,4-D treatment did not overcome the cross-incompatibility gene(s), Kr alleles of wheat (Inagaki, 1986; Ushiyama and Kuwabara, 1994). However, the action of Kr alleles cannot prevent fertilization when maize is used as the male plant (Laurie and Bennett, 1987, 1988; Suenaga, 1990). In addition, the variations of agronomic characters of doubled haploids obtained from the crosses of wheat x maize were similar to those found in the crosses of wheat x H. bulbosum. Therefore, we used wheat x maize crosses in this study.

　In the crosses of wheat x maize, 2,4-D treatment promoted seed setting and embryo formation (Kisana et al., 1993; Laurie and Bennett, 1988; Riera-Lizarazu and Mujeeb-Kazi, 1990; Suenaga and Nakajima 1989), but the optimum concentration of 2,4-D for embryo differentiation and the production of haploid wheat have not been determined. In this study, we determined the optimum concentration of 2,4-D and other phytohormones on the embryo formation and haploid plant regeneration in the wheat x maize crosses.

Materials and Methods
　An F1 hybrid of maize, Pioneer P80 Lisa, which heads early time in a greenhouse in winter and has a high capacity of haploid production in the crosses with wheat (Ushiyama et al., 1991), was used in this study as a pollen source. The Japanese wheat cultivar, Triticum aestivum cv. Zenkojikomugi, which showed a medium frequency of haploid production in the crosses of wheat x maize and wheat x Hordeum bulbosum (Ushiyama et al., 1990), was used as a female parent. Wheat and maize were seeded every other day from September to February. The maize seedlings were grown in a greenhouse kept at the minimum temperature of 20℃. For haploid wheat cultivation, we used intergeneric crossing and embryo culture followed by the bulbosum system of Ushiyama et al. (in press). The experiment to determine useful phytohormones was carried out in November and December 1990. After pollination, detached spikes were cultured in the solution, containing 10 mg L-1 ethanol, 0.05% sulfurous acid , 40 g L-1 sucrose and various phytohormones, i.e., 2,4-D, naphthalene acetic acid (NAA), indole acetic acid (IAA), kinetin and 6-benzylaminopline(BA)at various concentrations. Unpollinated spikes were also cultured in the above solution excluding the solutions containing IAA, kinetin and BA. The numbers of seeded florets, florets with embryos formed and florets with regenerated plantlets were counted for every spike. These numbers were compared between treated and untreated plots by the t-test.

　In order to determine the optimum concentration of phytohormones for the haploid production, the following experiment was conducted twice in January 1991. The numbers of seeded florets, florets with embryos and plantlets regenerated per embryo and per floret were counted. These data were transformed by arcsin square root transformation and subjected to analysis of variance. The mean length of embryos was measured two weeks after pollination and subjected to analysis of variance. The concentration of 2,4-D in the spikes was measured (Goto and Kato 1980) by gas chromatography (Shimadzu GC-8A) in March 1991. Five spikes were collected after each treatment with 2,4-D and divided into two parts, i.e., seeds and the other parts(glumes and rachis). The data were transformed and subjected to analysis of variance.

Results
1. The effect of phytohormone on haploid wheat production
Table 1 shows the percentages of seeded florets, florets with embryos formed and florets from which haploid plants developed in the pollinated spikes of the crosses of wheat x maize treated with 2,4-D, NAA, IAA, kinetin and BA at various concentrations 2,4-D at a concentration of 10 and 100mg L-1 significantly increased the percentage of seeded florets to 15.4% and 73.4%, respectively, from 3.8% in the control. The percentage of seeded florets after treatment with other phytohormones were not higher than that in the untreated control (3.8%). The percentages of florets with embryos formed and that with haploid plants developed, significantly increased by the treatment with 2,4-D at the concentration of 100 mg L-1 (21.4% and 8.5% respectively) in comparison with the control (0.4% and 0.4% respectively). In the spikes without pollination, 2,4-D treatments at 10 and 100 mg L-1 significantly increased the percentage of seeded florets (23.5% and 55.2%), compared with untreated control (0.0%). Spikes treated with 1000 mg L-1 NAA set some seeds but the difference of the percentage of seeded florets was not significant. However, seeds formed in the unpollinated florets had no embryo. This indicates that the treatment with various phytohormones induces parthenogenesis.

2. The optimum concentration of 2,4-D for haploid production
　Table 2 shows the frequency of haploid production in the crosses Zenkojikomugi x Pioneer P80 Lisa treated with 2,4-D at various concentrations. Table 3 shows the results of the analyses of variance of these data. Differences among 2,4-D concentrations in the percentages of seeded florets, florets with embryos formed, and florets with haploid plants developed, were significant at the 1 or 0.1% level (Table 3). The percentages of seeded florets in the spikes treated with 2,4,D at the concentrations of 125 and 150 mg L-1, 87.4% and 83.5%, respectively, were significantly higher (P<0.05) than those at the other concentrations (0.0% to 63.9%) by Duncan's multiple range test. The percentage of seeded florets increased with increasing concentration of 2,4,D up to 125 mg L-1 2,4-D, but decreased at higher concentrations.tendency more than 150 mg L-1 2,4-D. The percentage of florets with embryos formed was significantly increased by 2,4-D at the concentration of 25 to 200 mg L-1 2,4-D. Analysis of variance for embryo size showed that there was a significant difference in the embryo size with the concentration of 2,4-D at the 0.1% level. The mean length of the embryo in the spikes with 2,4D at 50 mg L-1 was longer than that at the other concentrations. The size of embryo was largest at 50 mg L-1 2,4-D and reduced at higher concentrations. No significant difference was observed with the concentration of 2,4-D in the percentage of embryos developed into haploid plants (Table 3). The percentages of florets with haploid plants developed was significantly influenced by the concentration of 2,4-D (Table 3), and was the highest (10.0%) at 100 mg L-1. The treatments with 2,4-D at the concentrations from 25 to 150 mg L-1 (6.0% to 10.0%) significantly increased haploid production compared with the untreated control and the treatments at other concentrations of 2,4-D (0% to 1.0%).

3. Concentration of d 2,4-D in the spikes
　Table 4 shows the concentrations of 2,4-D in the seed, and glumes + rachis on the spikes treated with 50, 100 and 150 mgL-1. Table 5 shows the results of the analysis of variance of the data. There were significant differences both with the 2,4-D concentration and among the parts of the spikes. The concentration of 2,4-D in the seed on the spikes treated with 2,4-D at the concentration of 50 mg L-1 (1.15 ppm) was significantly lower than that treated with 100 and 150 mg L-1 2,4-D (9.24 and 6.55 ppm, respectively). The concentration of 2,4-D in the glumes and rachides on the spikes treated with 2,4-D at the concentration of 150 mg L-1 (41.55 ppm), but, was significantly higher than that treated with 2,4-D at 50 mg L-1 2,4-D (5.53ppm) and 100 mg L-1 2,4-D (12.72ppm).

Discussion
　The results showed that 2,4-D was more useful for haploid wheat production than either NAA, IAA, kinetin or BA. In the crosses of wheat x maize, the high embryo recovery by 2,4-D treatment was already reported (Kisana et al., 1993; Laurie and Bennett 1988, Riera-Lizarazu and Mujeeb-Kazi 1990, Suenaga and Nakajima 1989). In addition, the present results indicated that 2,4-D and NAA, namely synthetic auxin, might be more effective for haploid wheat production than cytokinin. However, in the previous experiments using wheat x maize crosses, the promotive effect of 2,4-D treatment on the plant regeneration from haploid wheat embryo was not clear. In this study, the frequency of plant regeneration was significantly increased by the treatment with 100 mg L-1 2,4-D. The embryo development was inhibited at the treatment with over 50 mg L-1 2,4-D. In the crosses of wheat x H. bulbosum, similar results was observed by Ushiyama et al. (in preparation), who pointed out that the treatment with 2,4-D at the concentration of 100 mg L-1 or less was effective for haploid wheat production while 2,4-D treatment reduced embryos sizes and arrested embryo development into plant. However, the mechanism of the effect of 2,4-D was not elucidated . The treatment with 2,4-D at 50 mg L-1 was most effective for the development of the embryo into the plant and the increase of embryo size, and that at 100 mg L-1 for haploid production. The optimum concentration of 2,4-D for seed setting was 125 ~ 150 mg L-1. We measured the concentration of 2,4-D in the spike treated with 2,4-D at different concentrations. The increase of the concentration of 2,4-D beyond 100 mg L-1 up to 150 mg L-1, did not increase the concentration of 2,4-D in the seed, but increased the concentration of 2,4-D in the glumes and rachis. Maddock et al. (1983) reported that 1.0 mg L-1 2,4-D was optimum for induction of shoot development from cultured immature embryos, and 5 mg L-1 2,4-D decreased the percentage of shoot-forming cultures, though it increased callus growth. In this study, the concentration of 2,4-D in the seed on the spikes treated with 50 mg L-1 2,4-D was 1.15 ppm, and embryo development was increased under this condition. 2,4-D in the seed at a concentration higher than 5 ppm may inhibit the normal activity of the embryo though it increased seed setting. Riera-Lizarazu and Mujeeb-Kazi (1990) pointed out that the rate of embryo recovery was higher in the detached spikes cultured in the medium containing 2,4-D than in on the tiller injected with 2,4-D solution and in the floret sprayed with 2,4-D. 2,4-D presumably stimulated the development in immature florets development a few days after pollination. The concentrations of 2,4-D in the seeds reached 5ppm earlier on the spikes treated with 150 mg L-1 2,4-D than with 100 mg L-1 2,4-D. Some seeds without embryo were also set by the treatments with 2,4-D (10 and 100 mg L-1) and NAA (1000 mg L-1) in emasculated florets. The frequencies of seed setting in the unpollinated florets treated with these phytohormones paralleled those in the pollinated spikes. Marshall et al. (1983) reported that the application of a single drop of 80 mg L-1 or 160 mg L-1 2,4-D solution to them increased the size of seeds, and Inagaki (1986) reported that 70.2% of the unpollinated florets set seeds without embryos when the culms were injected with 1000 ppm 2,4-D. Falk and Kasha (1982) reported that the unpollinated spikes sprayed with 0.1 and 1.0 mg L-1 IAA did not set seeds. Ushiyama et al. (in press) investigated the influence of various concentrations of 2,4-D on the production with wheat haploid H. bulbosum and pointed out that the treatment with 2,4-D at 125 to 175 mg L-1 increased the percentage of the seed setting, but reduced the formation and development of the embryo. Therefore, it appears that the promoting effect of 2,4-D and NAA on seed setting is not reflected in fertilization and embryo development. The percentage of seeded florets without embryos formed should be low, but that of florets with embryos formed and developed was high in the spikes treated with 2,4-D at the optimum concentration. It is, therefore, concluded that the treatment with 2,4-D at 50 to 100 mg L-1 was useful to produce haploid wheat plant in the crosses of Zenkojikomugi x maize. The embryo development may depend on the concentration of 2,4-D in the seeds.

Acknowledgements
　Thanks are due to Ms. Yuko Tobita (Nagano Agriculture Research Center) for measuring the concentrations of 2,4-D.

Literature Cited
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Table 1. Frequencies of haploid production in the crosses of wheat cultivar Zenkojikomugi x maize cultivar Pioneer P80 Lisa treated with phytohormones at various concentrations.
Table 2. Percentages of florets that formed seeds, embryos and haploid plantlets in the crosses of wheat cultivar Zenkojikomugi x maize cultivar Pioneer P80 Lisa treated with 2,4-D at various concentrations.
Table 3. Analysis of variance of the haploid production.
Table 4. Concentration of absorbed 2,4-D in the parts of wheat spikes in the cross, wheat cultivar Zenkojikomugi x maize cultivar Pioneer P80 Lisa.
Table 5. Analysis of variance of the concentration of 2,4-D in the spikes of wheat.