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Journal of Heredity 2006:97(1):3944 The American Genetic Association. 2006. All rights reserved.

doi:10.1093/jhered/esj007 For permissions, please email: journals.permissions@oxfordjournals.org.


Advance Access publication January 4, 2006

Induction of Tetraploid Derivatives of


Maize Inbred Lines by Nitrous Oxide
Gas Treatment
AKIO KATO AND JAMES A. BIRCHLER
From 117 Tucker Hall, University of Missouri, Columbia, MO 65211. Akio Kato is now at the Faculty of Agriculture, Kyoto

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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@kpu.ac.jp.

Abstract
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 (3036 h after pollination) for 20 h on maize inbred line Oh43. The nitrous oxide gas treatment between
pressures of 6001000 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 anti- genetic background is desirable to eliminate the effect of
microtubule chemicals has been used as a plant breeding heterozygosity.
procedure for decades. Plants with elevated chromosome We attempted to induce tetraploid maize inbreds by heat
number are known to show larger organ size and superior treatment of elite diploid maize inbreds, and found it was
cold tolerance. For this reason, the procedure is used for root difficult ( J. A. B. unpublished). The use of the elongate gene
crops, fruit trees, and ornamental flowers. For seed propa- requires successive backcrossing to inbred lines and elimina-
gating crops, however, autotetraploids generally show lower tion of the mutation from the tetraploid. It might also be
fertility and allotetraploids are more common (Poehlman impossible to eliminate residual heterozygosity in these
1986). materials.
In maize there are two previously described methods to Here we report the successful use of nitrous oxide gas
induce autotetraploids. Heat treatment just after fertilization treatment on the maize zygote just after fertilization to
(Randolph 1932) is one; the other is use of the elongate produce tetraploids. The procedure was originally developed
mutation (Rhoades and Dempsey 1966), which produces by O stergren (1954), and there are a number of successful
unreduced gametes. These maize tetraploid lines show chro- reports on induction of plants with doubled chromosome
mosomal instability and lower fertility compared to the dip- number by this procedure in Russian wildrye, barley, wheat,
loid counterparts (Alexander 1957; Sockness and Dudley and red clover (Berdahl and Barker 1991; Dvorak and Harvey
1989). For this reason, tetraploid maize is mainly utilized 1973; Dvorak et al. 1973; Kihara and Tsunewaki 1960;
for basic research, such as the study of the behavior of mei- Subrahmanyam and Kasha 1975; Taylor et al. 1976). In this
otic or somatic chromosomes (Mastenbroek et al. 1982; method, the first zygotic division of a fertilized egg cell is
Punyasingh 1947) and gene dosage effects (reviewed by arrested. In maize, generation of haploids and triploids from
Birchler 1993). Tetraploid maize can also be used for exam- given inbred lines is already established (Coe 1959; Deimling
ining mechanisms of endosperm balance number (Cooper et al. 1997; Kato 1997, 1999b, 2002; Sarkar and Coe 1966).
1951; Lin 1984; Sarkar and Coe 1971). With the recent On tetraploid induction from a given inbred line, sets of 1
advancement of technology, molecular level investiga- 4 ploidy series can be generated from the diploid and can be
tion became possible. Use of a ploidy series of the same used to investigate a ploidy series for various characteristics.

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Journal of Heredity 2006:97(1)

Materials and Methods


Experiment 1: Determination of Optimal Treatment for
the Induction of Tetraploid Maize Seedlings
From our preliminary experiments, fertilized ovules (3036 h
after pollination) of maize survived nitrous oxide gas treat-
ment 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

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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 tim-
ings (30 or 36 h after pollination) with one of four different
pressures (600, 800, 900, 1000 kPa, 1 atm air, room tem-
perature). 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 (30C). Root tip chromo-
some counts were performed according to the enzymatic Figure 1. Mature ears from plants treated with nitrous oxide
macerationair-drying procedure (Kato 1999a). gas between 3050 h after pollination. The ear produced by the
plant treated at the pressure of 600 kPa (left), 800 kPa (middle),
Experiment 2: Induction of Tetraploids From Maize Elite and 1000 kPa (right) nitrous oxide gas. The sterility level was
Inbred Lines highest in the 600-kPa treatment.
Twelve diploid inbred lines (A188, A632, B37, B55, B73,
H99, Mo17, Oh43, Pa91, Stock 6, W22, and W23) were came shriveled. Germless kernels with apparently normal en-
treated for 20 h with nitrous oxide gas (900 kPa), with treat- dosperm development occurred at the rates of 7%44%, and
ment starting 36 h after pollination. Some lines (B73, H99, the 900- to 1000-kPa, 30- to 50-h treatments showed signif-
Oh43) were also treated with 800 kPa, 30 h after pollination, icantly (chi-square test, P , .01) lower germless kernel rates
for 20 h to compare the possible difference between the than others. Chromosome counting of 757 Oh43 germinated
900-kPa, 36- to 56-h, and 800-kPa, 30- to 50-h, gas treat- kernels revealed that most (94%) were diploid; however, a
ments. These inbreds were selected based on their use in var- total of 9 tetraploids (4 40) and 10 tetraploid class an-
ious biological experiments, such as tissue culture, genome euploids (chromosome numbers 3639) were recovered.
analysis, dosage gene expression analysis, and investigation Other classes include one haploid, one monosomic, one trip-
of fertilization processes. Two to 15 plants were treated in loid, and 21 aneuploids with chromosome numbers ranging
each genotype, and the germinating kernels from one repre- between 21 and 28. Two plants were diploid tetraploid
sentative 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 Table 1. Kernel development after nitrous oxide gas treatment
(20 h) at various pressures just after fertilization on maize inbred
in a winter greenhouse to maturity. The temperature during line Oh43
growth was maintained between 20C25C, and the day
length had been adjusted to 16 h by timer-controlled sodium Presumably
lamps. Plants were self-pollinated using standard procedures. normal Germless Shriveled
Treatment No. (%) No. (%) No. (%)
600 kPa 3050 h 47 (17.7) 31 (11.7) 188 (70.7)
Results 3656 h 57 (8.5) 171 (25.6) 439 (65.8)
Experiment 1 800 kPa 3050 h 325 (69.1) 144 (30.6) 1 (0.2)
3656 h 214 (52.5) 178 (43.6) 16 (3.9)
The kernels produced on the nitrous oxidetreated plants 900 kPa 3050 h 312 (91.2) 24 (7.0) 6 (1.8)
exhibited a high occurrence of shriveled or germless kernels 3656 h 275 (64.9) 136 (32.1) 13 (3.1)
(Figure 1 and Table 1). The rate of shriveled kernel was 1000 kPa 3050 h 318 (92.4) 25 (7.3) 1 (0.3)
3656 h 299 (74.6) 100 (24.9) 2 (0.5)
highest when the plants were treated with nitrous oxide at
Control 356 (99.2) 0 (0.0) 3 (0.8)
600 kPa. In these treatments, 65%70% of the kernels be-

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Kato and Birchler  Tetraploid Induction in Maize

Table 2. Chromosome number of seedlings obtained from ears treated with nitrous oxide gas (20 h) at various pressures and timing
after fertilization in maize inbred line Oh43

Chromosome number
Chimeras
Treatment 10 19 20 21 22 24 25 28 30 36 37 39 40 20 40 20 21
600 kPa 3050 h 38 2
3656 h 37 8 2 1 2
800 kPa 3050 h 115 4
3656 h 95 2 3 1 1 1 1
900 kPa 3050 h 110 1 1 1
3656 h 100 1 1 2 1
1000 kPa 3050 h 1 116 2
3656 h 1 100 1 1 2 2

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Control 100

chimeras, and one was a diploid-trisomic chimera (Table 2). condition. The tetraploid induction rates were between 0%
The self-pollinated ears of Oh43 tetraploids showed irregular 20% (average 4.7 %) (Table 3). We obtained fertile tetraploid
kernel rows with larger kernel size (Figure 2). Among the progenies from six inbred lines: A188, B73, H99, Oh43,
treatments, the 800 kPa (treated between 3050 h after pol- Stock 6, and W22 (Figure 3). The tetraploid plants induced
lination) treatment and the 900 kPa (treated between 3656 h from B37, B55, and Mo17 were completely sterile. Plants of
after pollination) treatment produced four tetraploid class Pa91 appeared to be killed by the treatment, based on mul-
plants each; however, the difference of tetraploid plus tetra- tiple trials. All the ears produced on the 15 treated W23 plants
ploid class aneuploid induction rates was not statistically sig- set no kernels. The inbred line A632 did not produce any
nificant among treatments. tetraploid progeny. We further treated 10 A632 plants with
800 or 900 kPa nitrous oxide and obtained about 2000 ker-
nels. We germinated them and selected 100 seeds with appar-
Experiment 2 ently thicker root tips; chromosome counts revealed that
The 12 inbred lines were treated with nitrous oxide gas with none of them was tetraploid (data not shown).
the 900-kPa, 36- to 56-h condition or 800-kPa, 30- to 50-h 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
(3656 h, 900 kPa) showed the chimeric structure of tetra-
ploid and octoploid cells (Figure 5).

Discussion
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, 3656 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
Figure 2. Oh43 mature ears. The diploid ear (left) and the the seven out of eight experimental conditions at similar rates
tetraploid counterpart (right) induced by the nitrous oxide gas in Experiment 1 (Table 2). The trial to reveal the difference
treatment. between the 900-kPa, 36- to 56-h treatment and the 800-kPa,

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Journal of Heredity 2006:97(1)

Table 3. Chromosome number of seedlings obtained from the ears treated with nitrous oxide gas just after fertilization in 12 maize
inbred lines

Chromosome number
Chimeras
Nitrous oxide gas
Inbred line treatment condition 10 18 19 20 21 22 23 24 30 33 34 35 36 37 38 39 40 23 40 40 80
A188 3656 h 900 kPa 1 218 5 1 2 1 2
A632 3656 h 900 kPa 1 113 2
B37 3656 h 900 kPa 1 193 1
B55 3656 h 900 kPa 46 2 1
B73 3050 h 800 kPa 51 1 2 2 1 2
B73 3656 h 900 kPa 1 88 1 5 1 11
H99 3050 h 800 kPa 134 1 4 31
H99 3656 h 900 kPa 1 99

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Mo17 3656 h 900 kPa 2 163 1 1
Oh43 3050 h 800 kPa 1 2 136 15 5 1 1 2 1
Oh43 3656 h 900 kPa 82 1 1 1 2 1 7 3 3 1
Pa91 3656 h 900 kPa
Stock 6 3656 h 900 kPa 1 97 2 1 1
W22 3656 h 900 kPa 108 2 1 1 1 3
W23 3656 h 900 kPa

The Pa91 plants did not survive the nitrous oxide gas treatment, and W23 plants produced no seed set.

30- to 50-h gas treatment on the three genotypes did not give induced by the treatment failed to develop. The development
a consistent tendency (Table 3). It is possible to conclude failure effect of nitrous oxide gas was also observed in the
from the current experiments that the 800- to 900-kPa treat- previous study of chromosome doubling of haploid maize
ments are better than that of 600 kPa because the rates of seedlings (Kato 2002).
germinable seed are higher than the 600-kPa treatments The rates of shriveled and germless kernels are higher
(Table 1). Also a lesser number of plants had to be treated in the 600-kPa treatment than the 1000-kPa treatment. This
to generate the same number of tetraploids compared to the seems counterintuitive. One possible explanation is that the
600-kPa treatment. higher gas pressure may extend the duration of the cell cycle
The tetraploid occurrence is sometimes very low or block the cell cycle at certain stages. If this is the case,
(,0.5%) even on plants that were treated for 20 h. Combined under lower gas pressure, more stages of the cell cycle of
with the observation of the high level of aborted embryos and zygotic cells in the 600-kPa treatment will be affected by ni-
endosperm, it is possible that most of the tetraploid zygotes trous oxide than those treated with higher gas pressures.

Figure 3. Ears of six tetraploid inbred lines established by the nitrous oxide gas treatment. From the left, A188, B73, H99, Oh43,
Stock 6, and W22 tetraploids. The ears of W22 and Stock 6 were highly sterile.

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Kato and Birchler  Tetraploid Induction in Maize

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Figure 4. (a) A chromosome spread of a euploid tetraploid induced by the nitrous oxide gas treatment (4 40, W22).
(b) An individual with chromosome number 20 with a broken telocentric chromosome (arrow, W22). (c) An individual with 22
chromosomes (W22), a broken chromosome with small short arm (arrow) is observed. (d) An individual with 40 chromosomes
(A188), a broken chromosome (arrow) with tiny short arm is observed. Bar 10 lm.

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 aneu-
ploids 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
Figure 5. Chromosome spread of the octoploid cell in the the tetraploid plants induced (Mo17, B37, and B55). In the
tetraploid-octoploid chimera root tip of maize inbred line last two cases, large-scale induction experiments might make
Oh43 obtained from Experiment 2 (36- to 56-h, 900-kPa it possible to obtain fertile tetraploid progenies. Preselecting
nitrous oxide gas treatment). Bar 10 lm. seedlings with larger roots (by 10%20%) will aid in reducing

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Journal of Heredity 2006:97(1)

the work required to identify the lower number of tetraploids. Kato A, 1999b. Induction of bicellular pollen by trifluralin treatment and
The tetraploid inbred line W22 is highly sterile, and only un- occurrence of triploids and aneuploids after fertilization in maize. Genome
42:154157.
der a very limited environment (winter greenhouse, 20C
25C temperature, additional illumination of sodium lamp, Kato A, 2002. Chromosome doubling of haploid maize seedlings using
nitrous oxide gas at the flower primordial stage. Plant Breed 121:370377.
and use of large pots with highly fertile soil) will the plants
produce kernels. Samples of the tetraploids produced are Kihara H and Tsunewaki K, 1960. Production of polyploid wheat by nitrous
oxide. Proc Jpn Acad 36:658663.
available on request.
Lin B-Y, 1984. Ploidy barrier to endosperm development in maize. Genetics
107:103115.
Mastenbroek I, Dewet JMJ, and Chieh-Yin LU, 1982. Chromosome behavior
Acknowledgments in early and advanced generation of tetraploid maize. Caryologia 35:463470.
The authors thank E. H. Coe and S. Melia-Hancock for providing the maize Mol R, Matthys-Rochon E, and Dumas C, 1994. The kinetics of cytological
inbred lines. This work was performed in the Ernie & Lotti Sears Plant events during double fertilization in Zea mays L. Plant J 5:197206.
Growth Facility of University of Missouri-Columbia. This work was sup-
stergren G, 1954. Polyploids and aneuploids of Crepis capillaris produced by

Downloaded from http://jhered.oxfordjournals.org/ at Universidade Estadual de Maring on August 28, 2016


ported in part by grants from the U.S. Department of Energy and the O
National Science Foundation (DBI 0077774) to J.A.B. treatment with nitrous oxide. Genetica 27:5464.
Poehlman JM, 1986. Variations in chromosome number. In: Breeding field
crops, 3rd ed. Westport, CT: Avi Publishing Company, Inc; 87108.

References Punyasingh K, 1947. Chromosome numbers in crosses of diploid, triploid


and tetraploid maize. Genetics 32:541554.
Alexander DE, 1957. The genetic induction of autotetraploidy: a proposal for Randolph LF, 1932 Some effects of high temperature on polyploidy and
its use in corn breeding. Agron J 49:4043. other variations in maize. Proc Natl Acad Sci USA 18:222229.
Berdahl JD and Barker RE, 1991. Characterization of autotetraploid Russian Rhoades MM and Dempsey E, 1966. Induction of chromosome doubling at
wildrye produced with nitrous oxide. Crop Sci 31:11531155. meiosis by the elongate gene in maize. Genetics 54:505522.
Birchler JA, 1993. Dosage analysis of maize endosperm development. Annu Sarkar KR and Coe EH, 1966. A genetic analysis of the origin of maternal
Rev Genet 27:181204. haploids in maize. Genetics 54:453464.
Coe EH, 1959. A line of maize with high haploid frequency. Am Nat 93: Sarkar KR and Coe EH, 1971. Anomalous fertilization in diploid-tetraploid
381382. crosses in maize. Crop Sci 11:539542.
Cooper DC, 1951. Caryopsis development following matings between dip- Sockness BA and Dudley JW, 1989. Morphology and yield of isogenic diploid
loid and tetraploid strains of Zea mays. Am J Bot 38:702708. and tetraploid maize inbreds and hybrids. Crop Sci 29:10291032.
Deimling S, Rober F, and Geiger HH, 1997. Mehodik und genetik der in vivo Subrahmanyam NC and Kasha KJ, 1975. Chromosome doubling of barley
haploideninduktion bei mais. Vortr Pflanzenzuchtg 38:203224. haploids by nitrous oxide and colchicine treatments. Can J Genet Cytol.
Dvorak J and Harvey BL, 1973. Production of aneuploids in Avena sativa L. 17:573583.
by nitrous oxide. Can J Genet Cytol 15:649651. Taylor NL, Anderson MK, Quesenberry KH, and Watson L, 1976. Doubling
Dvorak J, Harvey BL, and Coulman BE, 1973. The use of nitrous oxide for the chromosome number of Trifolium species using nitrous oxide. Crop Sci
producing eupolyploids and aneuploids in wheat and barley. Can J Genet 16:516518.
Cytol 15:205214.
Kato A, 1997. Induced single fertilization in maize. Sex Plant Reprod 10: Received July 12, 2005
96100. Accepted October 11, 2005
Kato A, 1999a. Air drying method using nitrous oxide for chromosome
counting in maize. Biotech Histochem 74:160166. Corresponding Editor: Susan Gabay-Laughnan

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