Journal of Heredity Advance Access originally published online on January 4, 2006
Journal of Heredity 2006 97(1):39-44; doi:10.1093/jhered/esj007
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Induction of Tetraploid Derivatives of Maize Inbred Lines by Nitrous Oxide Gas Treatment
From 117 Tucker Hall, University of Missouri, Columbia, MO 65211. Akio Kato is now at the Faculty of Agriculture, Kyoto Prefectural University, Kyoto-shi, Sakyo-ku, Shimogamo Hangi-cho 1-5, Kyoto 606-0823, Japan
Address correspondence to Akio Kato at the address above, or e-mail: katoa{at}kpu.ac.jp.
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Abstract |
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Maize (Zea mays L.) is a model organism for various genetic and physiological studies. Induction of autotetraploid lines from elite inbred lines is valuable for investigating gene dosage effects on the molecular level. We applied nitrous oxide gas at the time of fertilization (30–36 h after pollination) for 20 h on maize inbred line Oh43. The nitrous oxide gas treatment between pressures of 600–1000 kPa proved to be effective in inducing tetraploids. The treatment also significantly increased the rates of germless and shriveled kernels. Twelve inbred lines were treated with nitrous oxide gas for 20 h at either 800 or 900 kPa pressures, 30 or 36 h after pollination. Although tetraploid or tetraploid class aneuploid plants from 9 of 12 inbreds tested were successfully generated, only six genotypes produced progenies. The successful tetraploid inbred lines were from the A188, B73, H99, Oh43, Stock 6, and W22 genetic backgrounds. Aneuploids, plants with broken chromosomes and chimeras, were also found among the treated materials.
Induction of autotetraploids using colchicine or other antimicrotubule chemicals has been used as a plant breeding procedure for decades. Plants with elevated chromosome number are known to show larger organ size and superior cold tolerance. For this reason, the procedure is used for root crops, fruit trees, and ornamental flowers. For seed propagating crops, however, autotetraploids generally show lower fertility and allotetraploids are more common (Poehlman 1986).
In maize there are two previously described methods to induce autotetraploids. Heat treatment just after fertilization (Randolph 1932) is one; the other is use of the elongate mutation (Rhoades and Dempsey 1966), which produces unreduced gametes. These maize tetraploid lines show chromosomal instability and lower fertility compared to the diploid counterparts (Alexander 1957; Sockness and Dudley 1989). For this reason, tetraploid maize is mainly utilized for basic research, such as the study of the behavior of meiotic or somatic chromosomes (Mastenbroek et al. 1982; Punyasingh 1947) and gene dosage effects (reviewed by Birchler 1993). Tetraploid maize can also be used for examining mechanisms of endosperm balance number (Cooper 1951; Lin 1984; Sarkar and Coe 1971). With the recent advancement of technology, molecular level investigation became possible. Use of a ploidy series of the same genetic background is desirable to eliminate the effect of heterozygosity.
We attempted to induce tetraploid maize inbreds by heat treatment of elite diploid maize inbreds, and found it was difficult (J. A. B. unpublished). The use of the elongate gene requires successive backcrossing to inbred lines and elimination of the mutation from the tetraploid. It might also be impossible to eliminate residual heterozygosity in these materials.
Here we report the successful use of nitrous oxide gas treatment on the maize zygote just after fertilization to produce tetraploids. The procedure was originally developed by Östergren (1954), and there are a number of successful reports on induction of plants with doubled chromosome number by this procedure in Russian wildrye, barley, wheat, and red clover (Berdahl and Barker 1991; Dvorak and Harvey 1973; Dvorak et al. 1973; Kihara and Tsunewaki 1960; Subrahmanyam and Kasha 1975; Taylor et al. 1976). In this method, the first zygotic division of a fertilized egg cell is arrested. In maize, generation of haploids and triploids from given inbred lines is already established (Coe 1959; Deimling et al. 1997; Kato 1997, 1999b, 2002; Sarkar and Coe 1966). On tetraploid induction from a given inbred line, sets of 1x–4x ploidy series can be generated from the diploid and can be used to investigate a ploidy series for various characteristics.
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Materials and Methods |
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Experiment 1: Determination of Optimal Treatment for the Induction of Tetraploid Maize Seedlings
From our preliminary experiments, fertilized ovules (30–36 h after pollination) of maize survived nitrous oxide gas treatment for at least 20 h and produced germinable seed. The timing is the first to second mitotic division of the zygote (Mol et al. 1994).
The inbred line Oh43 was used to determine the optimal treatment timing and pressure. Each plant was grown in a pot (18 cm diameter 25 cm height) that fit the airtight nitrous oxide gas chamber (20 cm diameter and 2 m long, made of iron flanges and pipes, bolts and nuts, sealed with silicone rubber gasket, gas pressure meter, and valve) (Kato 2002) and treated with nitrous oxide gas for 20 h at two different timings (30 or 36 h after pollination) with one of four different pressures (600, 800, 900, 1000 kPa, + 1 atm air, room temperature). The chamber accommodates two mature maize plants at once, and, in total, two to six plants in each batch were treated using the chamber. The kernels from one or two representative ears in each batch were dissected and germinated in moist vermiculite (30°C). Root tip chromosome counts were performed according to the enzymatic maceration–air-drying procedure (Kato 1999a).
Experiment 2: Induction of Tetraploids From Maize Elite Inbred Lines
Twelve diploid inbred lines (A188, A632, B37, B55, B73, H99, Mo17, Oh43, Pa91, Stock 6, W22, and W23) were treated for 20 h with nitrous oxide gas (900 kPa), with treatment starting 36 h after pollination. Some lines (B73, H99, Oh43) were also treated with 800 kPa, 30 h after pollination, for 20 h to compare the possible difference between the 900-kPa, 36- to 56-h, and 800-kPa, 30- to 50-h, gas treatments. These inbreds were selected based on their use in various biological experiments, such as tissue culture, genome analysis, dosage gene expression analysis, and investigation of fertilization processes. Two to 15 plants were treated in each genotype, and the germinating kernels from one representative ear in each genotype were analyzed by the same chromosome counting method used in Experiment 1. The identified tetraploids were transplanted to pots and grown in a winter greenhouse to maturity. The temperature during growth was maintained between 20°C–25°C, and the day length had been adjusted to 16 h by timer-controlled sodium lamps. Plants were self-pollinated using standard procedures.
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Results |
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Experiment 1
The kernels produced on the nitrous oxide–treated plants exhibited a high occurrence of shriveled or germless kernels (Figure 1 and Table 1). The rate of shriveled kernel was highest when the plants were treated with nitrous oxide at 600 kPa. In these treatments, 65%–70% of the kernels became shriveled. Germless kernels with apparently normal endosperm development occurred at the rates of 7%–44%, and the 900- to 1000-kPa, 30- to 50-h treatments showed significantly (chi-square test, P < .01) lower germless kernel rates than others. Chromosome counting of 757 Oh43 germinated kernels revealed that most (94%) were diploid; however, a total of 9 tetraploids (4x = 40) and 10 tetraploid class aneuploids (chromosome numbers = 36–39) were recovered. Other classes include one haploid, one monosomic, one triploid, and 21 aneuploids with chromosome numbers ranging between 21 and 28. Two plants were diploid tetraploid chimeras, and one was a diploid-trisomic chimera (Table 2). The self-pollinated ears of Oh43 tetraploids showed irregular kernel rows with larger kernel size (Figure 2). Among the treatments, the 800 kPa (treated between 30–50 h after pollination) treatment and the 900 kPa (treated between 36–56 h after pollination) treatment produced four tetraploid class plants each; however, the difference of tetraploid plus tetraploid class aneuploid induction rates was not statistically significant among treatments.
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Experiment 2
The 12 inbred lines were treated with nitrous oxide gas with the 900-kPa, 36- to 56-h condition or 800-kPa, 30- to 50-h condition. The tetraploid induction rates were between 0%–20% (average 4.7 %) (Table 3). We obtained fertile tetraploid progenies from six inbred lines: A188, B73, H99, Oh43, Stock 6, and W22 (Figure 3). The tetraploid plants induced from B37, B55, and Mo17 were completely sterile. Plants of Pa91 appeared to be killed by the treatment, based on multiple trials. All the ears produced on the 15 treated W23 plants set no kernels. The inbred line A632 did not produce any tetraploid progeny. We further treated 10 A632 plants with 800 or 900 kPa nitrous oxide and obtained about 2000 kernels. We germinated them and selected 100 seeds with apparently thicker root tips; chromosome counts revealed that none of them was tetraploid (data not shown).
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A total of 14 plants contained a broken chromosome of telocentric morphology or a chromosome with a tiny short arm (five in Oh43 in Experiment 1, one in A188, two in B37, one in Mo17, two in Oh43, and three in W22 in Experiment 2, Figure 4). One Oh43 plant obtained from Experiment 2 (36–56 h, 900 kPa) showed the chimeric structure of tetraploid and octoploid cells (Figure 5).
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Discussion |
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The nitrous oxide treatment was proved to be effective to induce tetraploid or tetraploid class aneuploids from multiple maize inbred lines in this experiment. In Experiment 1, the range of tetraploid induction was 0%–4% (average 2.5%). Those tetraploid aneuploids are useful to generate tetraploid euploids by selfing. In Experiment 2, the induction range was expanded to 0.5%–20% (average 4.7%). The Oh43 plants were treated in both experiments in the same manner (900 kPa, 36–56 h), although the incidence of the tetraploid plus tetraploid class aneuploids is significantly different (2% and 14%, respectively, chi-square test, P < .01). The difference may result from uncontrollable fluctuation of temperature just after pollination, which might affect the number of zygotes at a favorable cell stage for tetraploid induction. The tetraploids or tetraploid class plants were obtained from the seven out of eight experimental conditions at similar rates in Experiment 1 (Table 2). The trial to reveal the difference between the 900-kPa, 36- to 56-h treatment and the 800-kPa, 30- to 50-h gas treatment on the three genotypes did not give a consistent tendency (Table 3). It is possible to conclude from the current experiments that the 800- to 900-kPa treatments are better than that of 600 kPa because the rates of germinable seed are higher than the 600-kPa treatments (Table 1). Also a lesser number of plants had to be treated to generate the same number of tetraploids compared to the 600-kPa treatment.
The tetraploid occurrence is sometimes very low (<0.5%) even on plants that were treated for 20 h. Combined with the observation of the high level of aborted embryos and endosperm, it is possible that most of the tetraploid zygotes induced by the treatment failed to develop. The development failure effect of nitrous oxide gas was also observed in the previous study of chromosome doubling of haploid maize seedlings (Kato 2002).
The rates of shriveled and germless kernels are higher in the 600-kPa treatment than the 1000-kPa treatment. This seems counterintuitive. One possible explanation is that the higher gas pressure may extend the duration of the cell cycle or block the cell cycle at certain stages. If this is the case, under lower gas pressure, more stages of the cell cycle of zygotic cells in the 600-kPa treatment will be affected by nitrous oxide than those treated with higher gas pressures.
Occurrence of aneuploids is a common feature in nitrous oxide gas treatment (Dvorak and Harvey 1973; Dvorak et al. 1973). The presence of lower chromosome number aneuploids indicates that their zygotic division was affected by nitrous oxide gas although tetraploid induction did not occur. A few chimeric plants were recovered. One chimeric plant with tetraploid and octoploid cells was intriguing because the original cell was influenced by nitrous oxide gas twice at mitosis.
Although tetraploid or tetraploid class aneuploid plants were obtained from 9 out of 12 genotypes, only 6 genotypes produced kernels. The six successful tetraploid inbred lines were A188, B73, H99, Oh43, Stock 6, and W22 backgrounds. There were genotypes for which tetraploid induction or propagation is not possible or very difficult. The reasons are various: death of the plants by nitrous oxide gas (Pa91), no seed set on the ear from the treatment (W23), no tetraploid progenies among the treated kernels (A632), and sterility of the tetraploid plants induced (Mo17, B37, and B55). In the last two cases, large-scale induction experiments might make it possible to obtain fertile tetraploid progenies. Preselecting seedlings with larger roots (by 10%–20%) will aid in reducing the work required to identify the lower number of tetraploids. The tetraploid inbred line W22 is highly sterile, and only under a very limited environment (winter greenhouse, 20°C–25°C temperature, additional illumination of sodium lamp, and use of large pots with highly fertile soil) will the plants produce kernels. Samples of the tetraploids produced are available on request.
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Acknowledgments |
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The authors thank E. H. Coe and S. Melia-Hancock for providing the maize inbred lines. This work was performed in the Ernie & Lotti Sears Plant Growth Facility of University of Missouri-Columbia. This work was supported in part by grants from the U.S. Department of Energy and the National Science Foundation (DBI 0077774) to J.A.B.
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Footnotes |
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Corresponding Editor: Susan Gabay-Laughnan
Received July 12, 2005
Accepted October 11, 2005
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References |
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