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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