Colchicine induced embryogenesis and doubled haploid production in maize (Zea mays L.) anther culture

Document Type : Research Paper


Department of Plant Breeding, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, I.R. Iran


This study involves in vitro androgenesis of Zea mays L. via anther culture. Combination of two embryo induction media (IMSS & YPm) in presence of different colchicine concentrations (0, 100, 200, 250, 300 and 400 mg/l ) in the pretreatment medium (IML) and pretreatment duration (0, 3, 6 and 9 days) in two genotypes (DH5×DH7 and ETH-M82) were tested. After colchicine pretreatment, anthers were transferred to the induction media without colchicine to induce of embryo like structures (ELSs). The ELSs were then transferred to plant regeneration medium (YPNAS). It was found that in the genotype DH5×DH7, colchicine at a concentration of 100 mg/l significantly induced the number of ELSs (19.6). The control (without colchicine) and 400 mg/l of colchicine resulted in lower levels of ELSs (5.8, 5.7, respectively). In this genotype, colchicine pretreatment for 3 days produced highest number of ELSs (16.83) and a large increase in the ELS yield was observed in the YPm medium (14.4). In ETH-M82 genotype, 6 days of pretreatment with 300 mgl-1 with colchicine, produced highest frequency of ELSs (25). Also, in this genotype, a large increase in ELS yield was observed in the YPm induction medium (22.3). The frequency of spontaneous chromosome doubling in control group was very low for both genotypes (7%), but these genotypes were able to produce doubled haploid plantlets from the ELSs (63% doubled haploid) using a low concentration of colchicine in the pretreatment medium (250 mgl-1 for 6 days). At high concentrations of colchicine (300 and 400 mg/l), more morphological and chromosomal aberrations were observed.


Development of inbred lines by selfing is a time consuming process in maize breeding. Therefore, many
investigations on the use of doubled haploids produced
by androgenesis, have been carried out (Dieu and
Beckert, 1985). Anther culture capacity is characterized by embryo production and plant regeneration.
Embryogenesis can be influenced by genetic and environmental factors such as anther pretreatment and
embryo induction medium.
In cereal anther culture, chromosome doubling of
haploid cells or tissues at certain stages is a critical
step in producing doubled haploid plants (Obert and
Barnabás, 2004). The frequency of spontaneous chromosome doubling occurring during androgenesis of
maize is relatively low, varying between 4.5 to 22%
with an average of 10% (Buter, 1997). The frequency
of spontaneous chromosome doubling exceeding 50%
has been reported in barley (Lyne et al., 1986) and rice
(Rania, 1989). Several investigations investigated the
effect of incorporating colchicine on anther culture
derived plants in several cereals: wheat (Redha et al.,
1998; Zamani et al., 2000), maize (Saisingtong et al.,
1996; Barnabás et al., 1999), rice (Chen et al., 2001)
and triticale (Arzani and Darvey, 2001). Colchicine is
frequently used for chromosome doubling under both
in situ and in vitro conditions, since it disrupts mitotic
cell division by inhibiting the formation of spindle
fibers and polar migration of chromosomes.
Colchicine treatment of maize seedlings or plantlets
may double the chromosome number in the tassel or
the ear, but often not in both, which will make self-pollination impossible (Wan et al., 1989). High mortality
and abnormal plant development can also be observed
in colchicine-treated plant populations. This could
explain the low efficiency of doubled haploid produc

tion in maize after colchicine treatment of regenerated
haploid plantlets. Genome doubling in culture media
before the plantlet regeneration stage might help to circumvent the above-mentioned problems. Wan et al.
(1989) and Wan and Widholm (1995) reported the
recovery of genetically stable doubled-haploid maize
plants at high frequency through the colchicine treatment of embryogenic, microspore-derived haploid
calli. In other experiments, the application of
colchicine treatment together with a 7-days cold shock
to cultured maize anthers resulted in a considerable
increase in chromosome doubling in microsporederived plants (Saisingtong et al., 1996; AntoineMichard and Beckert, 1997).
The present paper reports the effects of colchicine
pretreatment of maize anthers on embryogenesis and
chromosome doubling.
Plant material: Two maize genotypes DH5×DH7,
and ETH-M82 (provided kindly by Dr. M. Beckert,
INRH, Clermont-Ferrand, France and Dr. IE. Aulinger,
Swiss Federal Institute of Technology, Zurich,
Switzerland, respectively) were used as anther donor
plants. They were grown in a growth chamber at 25°C
(day) and 15°C (night), with a photoperiod of 16 h and
a light intensity of 500 µmolm-2s-1.
Anther culture and colchicine pretreatment: Tassels
were collected prior to the emergence of the main leaf
blade and tested for microspore development. As a
cold pretreatment, the tassels were covered with an
aluminum foil and then kept at 7°C for 10 days
(Jumpatong et al., 1996). Tassel fragments containing
anthers in the late-uninuclear microspore developmental stage (determined by acetocarmine squash) were
surface sterilized with 2% w/v sodium hypochlorite
for 10 min and then washed three times with sterile
distilled water. The anthers were then dissected under
sterile conditions and were placed in 55×15 mm plastic petri-dishes containing 8 ml of filter-sterilized liquid pretreatment medium (IML); Saisingtong et al.,
1996) and different concentrations of colchicine, in
order to double the chromosome number of the
embryogenic microspores. Colchicine was dissolved
in distilled water and then added to medium. The cultures were then incubated for 3, 6 and 9 days at 7°C in
the dark. After colchicine pretreatments, the anthers
were transferred to colchicine-free semi-solid induction media (IMSS); (Saisingtong et al., 1996) and
YPm) medium (Genovesi and Collins, 1981) and then
incubated at 28°C in the dark for one month. The number of responding anthers and the frequency of
microspore-derived structures (embryo-like structures;
ELSs) were then calculated as a percentage of the cultured anthers.
The experiment was carried out in a 4-factorial
experiment (based on a completely randomized
design) with 5 replications. An external analysis of
sum of squares (SS) was employed, because of the significance of most of the interactions. Therefore, in
each genotype an independent analysis was carried out
based on a three-factorial experiment. Each replication
consisted of one petri-dish containing 25 anthers.
Three studied factors consisted of colchicine concentration in the pretreatment medium (0, 100, 200, 250,
300 and 400 mg/l), incubation duration in the
colchicine pretreatment medium (3, 6 and 9 days) and
embryo induction medium (IMSS and YPm media).
Analysis of variance (ANOVA) was carried out using
the SPSS statistical software (version 10.0).
Plant regeneration: The produced ELSs were
removed from the one-month cultured anthers and
transferred directly to the 10 cm plastic petri-dishes
containing 15 ml of plant regeneration medium
(YPNAS; Genovesi and Collins, 1981). They were
incubated under a 16 h illumination period (50 µmol
m–2s–1 light intensity) at 25°C. The regenerated
plantlets were then transferred into the 250 ml glass
containers containing 30 ml of basal MS medium for
further growth. Healthy green plantlets were then
transplanted into 10-cm pots containing vermiculite
and peat (1:1). Finally, the plantlets were transferred to
soil and grown to maturity in a growth chamber. Plants
which produced normal male and female inflorescences were self-pollinated and the seed set was
Cytological observations: The ploidy analyzer I
(Partec GmbH, Germany) was used to determine the
ploidy level of regenerated plants. Using a sharp razor
blade, 1 cm2 leaf segment was cut into small pieces in
2 ml of an 8°C, DAPI (4,6-diamidino-2-phenylindole)
staining solution (5 µg/ml, Partec GmbH) and passed
through a 50 µm sized nylon mesh (Aulinger, 2002).
The filtrate was then used for flow cytometric analysis;
at a per gain FL1 of 412 to 420 (relative fluorescence),
a peak set at 100 and 200 FL (corresponding to the G1,
G2 or M-phases, respectively) was interpreted as cor

responding to diploid or doubled haploid plantlets
(Figure 1A). A peak set at 50 and 100 FL was interpreted as corresponding to haploid material (Figure 1B).
Moreover, the chromosome numbers were checked
using the root tips squash method (Figure 2).
The results of ANOVA (Table 1) for ELS production in
the genotype DH5×DH7 showed significant differences among colchicine concentrations in pretreatment
medium (A), the durations of colchicine pretreatment
(B) and embryo induction media (C). In the genotype
ETH-M82; the main effect of the embryo induction
medium (C) and the interaction between colchicine
concentration and its duration in the pretreatment
medium (A×B) were significant. In the genotype
DH5×DH7 (Figure 3), colchicine at a concentration of
100 mg/l produced significantly, the highest number of
ELSs (19.6), while the control (colchicine-free pretreatment medium) and 400 mg/l of colchicine produced the least ELSs (5.8 and 5.7, respectively). Three
days of colchicine pretreatment produced the highest
frequency of ELSs (16.83; Figure 4). Also, a signifcant increase in ELS yield was observed in the YPm
induction medium (14.4; Figure 5) in comparison with
the IMSS medium.
In the ETH-M82 genotype, the interaction between
colchicine concentration in the pretreatment medium
and the incubation duration of anthers in this medium
was highly significant (Figure 6). Anthers pretreated
with 300 mgl-1 of colchicine for 6 days showed the
highest frequency of ELSs (25). Moreover, in this
genotype, a large increase in ELS yield was observed
in the YPm medium (18; Figure 7).
The frequency of spontaneous chromosome doubling in the colchicine-free pretreatment medium was
very low in both genotypes (7%), and according to
the phonological observations, most of the plants

were chimeric, having sectorial fertility in the tassels.
On the other hand, both the genotypes were able to
produce doubled haploid plantlets from ELSs (63%
doubled haploids) using a low concentration of
colchicine (250 mg/l for 6 days). By increasing the
concentration of colchicine (300 and 400 mg/l), more
morphological and chromosomal aberrations were
observed. Both haploids and doubled haploids
showed variation in agronomic characters including
plant height, ear height, days to anthesis and days to
silking (data not presented). Abnormal morphological characters were also observed in both haploids
and doubled haploids. Generally, they were short in
plant stature with some narrow leaves and had abnormal reproductive organs such as tassel seeds with
varying degree of pistillate and staminate flowers,
very small ears and tassels, prolific ears, terminal
ears, no tassel and/or ear formation, non-shedding
tassels and non-silking ears. However, the degree of
abnormality was higher in haploids than doubled
Self-pollination of each doubled haploid plant was
attempted in order to produce selfed seeds for inbred
line formation. Only a few doubled haploids (13 plants
in ETH-M82 and 19 plants in DH5×DH7) could produce selfed seeds due to the abnormality of reproductive organs and the non-synchronization of pollen
shedding and ear silking.DISCUSSION
In this study, the pretreatment medium containing
colchicine at a concentration of 100 mg/l increased
significantly the number of ELSs in the genotype
DH5×DH7 (Figure 8), while in genotype ETH-M82
the concentration of 300 mg/l produced the highest frequency of ELS. The negative effect of 400 mg/l of
colchicine on ELSs formation in genotype DH5×DH7
might be due to the toxic effect of colchicine on
microspores. In fact, two main physiological factors
are important for the achievement of a successful
androgenic response in maize: the developmental
stage of the microspores and the exogenous stimulus
inducing an androgenic response (Reynolds, 1997). In
maize anther culture, the first asymmetric division of
the androgenic microspore increases embryogenesis
(Szakács and Barnabás, 1995). The signal responsible
for the switch in the genetic programme of the
microspores from gametophytic to the sporophytic
developmental pathways is stress. Stress acts as a
launching mechanism for redirection to embryogenesis and stops the development of the fertile pollen
grain (Touraev et al., 1997). Anther and microspore
culture could be affected by colchicine treatment.
Increase in the embryo frequency due to colchicine
treatment has already been reported in maize genotypes (Barnabás et al., 1999).
One factor, which is limiting in the application of
doubled haploid plants to maize breeding, is low
induction of spontaneous chromosome doubling. A
low rate of spontaneous chromosome doubling yields
only a small number of doubled haploid plants useful
for breeding purposes. In the present study, the frequency of spontaneous chromosome doubling in the
control treatment was very low for the two studied
genotypes (7%). In a review by Buter (1997) the frequency of spontaneous chromosome doubling in
maize was reported to range from 4.5 to 22%, with an
average frequency of approximately10%. While,
Antoine-Michard and Beckert (1997) reported that
spontaneous chromosome doubling occurred at the
rate of 26.92% during direct embryogenesis of maize
genotypes F1937× DH229 and DH147× L1. Both
endoreduplication and nuclear fusion of the
microspores occurring during anther culture are presumed to be the cause of spontaneous chromosome
doubling (Chen and Wu, 1983).
Use of antimitotic drugs at the beginning of cultivation of the anthers or microspores, for the direct
doubling of chromosomes in the genome of haploid
microspores, seems to be very effective for stable
dihaploid offspring production in cereals (Barnabás et
al., 1991 and 1999; Saisingtong et al., 1996; AntoineMichard and Beckert, 1997). In the present research,
the studied genotypeds produced doubled haploid
plantlets when their anthers were pretreated with 250
mgl-1 of colchicine for 6 days, at 7°C. Saisingtong et
al., (1996) observed that the maximum frequency of
chromosome doubling (49%) was accomplished when
anthers of the maize genotype ETH-M36 were treated
with 250 mg/l of colchicine for 7 days at 14°C. In the
present research, many of the produced plants from
pretreatment media containing high concentrations of
colchicine (300 and 400 mg/l) showed morphological
abnormality and chromosomal aberrations. Wan et al.
(1989) faced the problem that some doubled haploid
plants derived from colchicine-treated anthers could
not set seed after self-pollination which was mainly
caused by delayed silk emergence or the lack of ear
formation. The abnormality of reproductive organ and
the non-synchronization of pollen shedding and ear
silking are common phenomena among tissue culturederived maize plants (Miao et al., 1978; Petolino and
Jones, 1986; Wan et al., 1989). Under the experimental conditions used in the present study, some seeds
were set on the plants showing normal morphological
feathers to allow the further cultivation of doubled
haploid plants in the field.
The results presented here indicate that the best
embryo induction can be achieved by combination of
all four factors (genotype, colchicine concentration in
the pretreatment medium, duration of colchicine pretreatment and the embryo induction medium) and that
the colchicine pretreatment of maize anthers can be
used to induce in vitro chromosome doubling.

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