Jpn. J. Trop. Agr. 48(3):194-197, 2004
Short Report
Improved Method of Chromosome Preparation for Sugarcane (Saccharum spp.)
Seiji FUKUHARA, Yoshifumi TERAJIMA, Akira SUGIMOTO and Kunihiro UJIHARA
Sugarcane Breeding Laboratory, National Agricultural Research Center for Kyushu Okinawa Region, Nishino-omote, Kagoshima, 891-3102, Japan
Key words: Glass slide preparation, High ploidy, Interspecific hybrid
サ トウキビの染色体観察 法 福原誠司 ・寺 島義文 ・杉本 明 ・氏原邦博 九州沖縄農業研究 センター さと うきび育種
研究室 〒891-3102鹿 児島県西之表市安納
キーワー ド ス ライ ドガ ラス処理,高 次倍数性,種間 雑種
Introduction
Sugarcane (Saccharum spp.) consists of complex interspecific and intergeneric hybrids. In spite of the large number of studies (SREENNASAN et al. 1987; D'HONT et al. 1996; PIPERIDIS et al. 2000), the mode of inheritance has not been elucidated due to the small size of the chromo-somes, high ploidy level, and variation in the chromosome numbers within an individual plant
(T ASKAL, et al. 1970), referred to as aneusomaty. Aneusomaty results from mitotic aberration during embryonic development or plant growth
(D'AMATO, 1995). At the meiosis, an uneven chromosome distribution occurs (BURNER and LEGENDRE, 1993), and the chromosome num-bers vary in the pollen. Improved knowledge of the chromosome behavior may enable to promote sugarcane breeding.
Nuclear plates of high quality are essential for chromosome research. Environmental condi-tions, such as humidity and temperature, affect the
quality of the nuclear plates. Recently, HENEGARIU et al. (2001) have reported a detailed, controlled method of preparation for spreading human chromosomes, which basically involves the use of steam-metal plate treatment and chemical aging. For further convenience, the authors tried to simplify and improve the chromosome
preparation method for a high ploidy plant, sugarcane.
Materials and methods
Commercial sugarcane varieties, NiF8, F177
and NCo310 were used as plant materials.
Chromosomes were prepared as described
by FUKUI (1996a) and HENEGAMU et al. (2001),
with slight modifications. Fresh root tips were
collected, pretreated in 4•Ž distilled water for 24
hr, fixed in 3:1 v/v ethanol: acetic acid for 2-3
days, and stored in 70% ethanol at 4•Ž until use.
Stored root tips were washed in distilled water
for 20 min, and transferred to an enzyme
solution (2% w/v Cellulase Onozuka RS; 1% w/v
Macerozyme R200; 0.3% w/v Pectolyase Y-23;
10mM citrate buffer, pH 4.8). Root tips were
kept under a decompressed environment for 5-
10min, followed by maceration at 37•Ž for 50-70
min. Macerated root tips were washed in 10mM
citrate buffer for 20 min, transferred to pre-
cleaned glass slides, treated with drops of a cold
fixative (2:1 v/v ethanol: acetic acid, instead of
3:1 v/v) and tapped with fine forceps. When the
fixative started to dry, the slides were quickly
dried on a hot plate at 45 •Ž for a few seconds.
For comparison, slides were air-dried overnight
instead of being subjected to the hot plate
treatment. Dried slides were dipped into 96%
ethanol, and a 3:1 v/v ethanol-acetic acid fixative
for 10 min, respectively and then air-dried. Slides
were stained with a Giemsa or Orcein solution.
Slides were examined with a Nikon Eclipse
E600 microscope. Images were captured by a
CCD camera (DXM-1200, Nikon, Japan) and
processed with Adobe Photoshop(R) and NIH
Received Feb. 18, 2004
Accepted Jun. 19, 2004
Fukuhara et al.: Improved chromosome preparation for sugarcane 195
Image) software.
At least 30 nuclear plate (chromosome
spreading) diameters were measured for the long
and short axes in every treatment. Statistical
analysis was performed by one-way ANOVA
(analysis of variance).
Results and Discussion
Uneven chromosome spreading predomi-
nated in the air-drying treatment, as shown in
Fig. la and 1b. In contrast, well-spread metaphases
and prometaphases were obtained in the hot
plate treatment, as shown in Fig. lc. Most of the
chromosomes were round- or oval-shaped. Table 1
shows the average values (Av) of the long and
short axes of the nuclear plate diameters, standard
deviations (SD) and coefficients of variance (CV).
The average nuclear plate diameters for the long
and short axes were 34-37 ƒÊm and 29-30 ƒÊm
respectively in the hot plate treatment, while 32-
33ƒÊm and 24-28ƒÊm in the air-drying treatment.
The hot plate treatment effectively increased
the length of both long and short axes. The SD
and CV values were lower in the hot plate
treatment, and statistically different from those in
the air-drying treatment, indicating that chromo-
some spreading was constant in the hot plate
treatment, and that nuclear plates of high quality
could be obtained, irrespective of environmental
conditions.
Colchicine or 8-hydroxyquinoline, disrupts
spindle fibers, and is frequently used for
accumulating metaphases. Such chemicals are
not suitable for observing small chromosomes
because condensed chromosomes are too small
to be distinguished. Cold water treatment is an
efficient method not only for large chromosomes
such as wheat, barley and rye, but also for small
chromosomes, such as alfalfa (BAUCHAN and
HOSSAIN, 2001) and S. spontaneum (HA et al.
1999), in order to accumulate metaphases and
prometaphases. In plants with small chromosomes,
the prometaphase offers more information than
the metaphase (FuKuI, 1996b), especially for
karyotype analysis.
The air-drying treatment is commonly used
for spreading and aging plant chromosomes
(Fuxui, 1996a). The problem with the air-drying
treatment is that chromosome spreading cannot
be controlled (Table 1 and Fig. la and 1b),
namely nuclear plates vary in shape or tend to
display overspreading. Some environmental factors,
Fig. 1. a, b: Unevenly spread chromosomes were
dominant in the air-drying treatment. c:
Chromosomes were well spread in the hot
plate treatment. Most of the chromosome
spreads were round-or oval-shaped. The
scale bar indicates 10ƒÊm.
196 Jpn. J. Trop. Agr. 48 (3) 2004
Table 1. Nuclear plate diameters (ƒÊm) in airdrying and hot plate treatments.
LA: long axis; SA: short axis; Av: average; SD: standard deviation; CV: coefficient of variance; SS: statistical significance
(p<0.05);•¦: significant difference; ns: no significant difference;-: not examined.
especially temperature and humidity, affect
chromosome spreading (HENEGARIU et al. 2001).
During air-drying, the fixative evaporates too
fast under dry conditions and too slowly under
the wet conditions, resulting in the formation of
poor nuclear plates. HENEGARIU et al. (2001)
employed the steam-hot plate treatment for
human chromosomes. It is considered that can
be applied to plant pachytene chromosomes
(KOUMBARIS and BASS, 2003). CLAUSSEN et al.
(2002) reported the occurrence of water-induced
cell swelling and chromosome stretching. After
preferential evaporation of ethanol, the remaining
acetic acid absorbs water from the atmosphere.
The 2:1 v/v ethanol-acetic acid fixative evaporated
more slowly and absorbed more water than the
conventionally used 3:1 v/v ethanol-acetic acid
fixative. Fixative evaporated constantly in the hot
plate treatment. The nuclear plates were round or
oval, and the chromosomes could be distinguished
based on their condensation pattern (Fig. lc). In
the air-drying treatment, water evaporated
irregularly, resulting in inadequate chromosome
spreading (Fig. la and lb).
After chromosome spreading, slides were
usually aged. Omitting this step resulted in a
loss of chromosomes during further preparation.
The ordinary method for aging plant chromosomes
involves air-drying at room temperature (FUKUI,
1996a). In another aging method, the slides are
kept at 65C overnight, at 94 •Ž for 30 min, or in
2X SSC at 37 •Ž for 30-60 min. These methods
are time-consuming and alter the chromosome
structure. HENEGAMU et al. (2001) developed a `chemical aging' method
. Slides were treated
with ethanol at 94•Ž for 2-20 seconds for use for
the next preparation within 30 minutes. Our
alternative treatment of dipping slides into ethanol
and fixative was simple, did not require any
special equipment within a similar time to that of
chemical aging, and succeeded in shortening the
time of preparation, and in improving chromosome
attachment to the glass slides compared with
the air-drying treatment.
Conclusion
Nuclear plates of high quality are essential
for chromosome research. Some key treatments
include cell cycle synchronization, hypotonic
treatment, use of fixative for spreading chromo-
somes, and flame or air-drying treatment. However,
only in a few reports have these aspects been
considered in detail so far. We succeeded in
developing a simpler and more effective treatment.
Therefore, nuclear plates of high quality were
obtained under desirable environmental condi-
tions combined with cold water treatment, rapid
drying on a hot plate at 45•Ž followed by ethanol-
fixative treatment. This improved method will
be applied in cytogenetic studies to further
promote sugarcane breeding.
Acknowledgement
The authors thank Dr. Shigeki Nakayama
for the valuable suggestions and Dr. Constancio
A. Asis Jr. for the critical reading of the manuscript.
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