_??_ 1994 The Japan Mendel Society Cytologia 59: 115-120, 1994

Somatic Chromosome Differentiation in Cypripedium

segawai Masamune and C. japonicum Thunberg

Katsuhiko Kondo, Yoshikazu Hoshi and Ryuso Tanaka

Laboratory of Plant Chromosome and Gene Stock , Faculty of Science, Hiroshima University, Higashi-Hiroshima 724 , Japan

Accepted December 2, 1993

Among the members of the Orchidaceae, Cypripedium of the subfamily Cypripedioideae together with its closely related Paphiopedilum have the largest chromosome (Maekawa 1971, A

twood 1984). Thus, they have been more attractive by chromosome workers. Up to the present 19 species of Cypripedium showed two different chromosome numbers of 2n=20 and 2n=22 (e. g., Karasawa and Atwood 1988), with the exceptions of 2n=30 in C. guttatum (Balaeva and Siplivinsky 1976) and C. formosanum (Karasawa and Aoyama 1986). These references suggested that 2n=20 might be the prototype of the chromosome numbers of the

genus which showed less variability than those of the other genera of the subfamily Cypripedioideae (Karasawa and Aoyama 1986).

Since the karyotypes of the Cypripedium species studied are similar to each other, they cannot lead any important role for taxonomic treatment (Karasawa and Aoyama 1986). Thus, chromosome banding methods should be applied to justify taxonomic treatment of the genus.

Materials and methods

The plants of Cypripedium segawai Masamune used in this study (Fig. 1) were originally

collected in Taiwan, and those of C. japonicum Thunberg (Fig. 2) were collected in Japan .

They were all purchased from a plant nursery and were cultivated in the Laboratory of Plant

Chromosome and Gene Stock, Faculty of Science, Hiroshima University. Each plant produced

only 2-3 roots every year containing a few dividing cells. Those root-tips were collected and

were pretreated in 0.002M 8-hydroxyquinoline for four hours at 18•Ž after cutting long

itudinally and equally into two pieces, before they were fixed in a mixture of ethanol and glacial

acetic acid (3•F1). They were hydrolized in 1 N hydrochloric acid at 60•Ž for five minutes.

Then, they were stained in Feulgen for more than 30 minutes. Since total chromosome length

of single mitotic metaphase cell of respective Cypripedium was 200-300ƒÊm and their cell

diameter was 40-80ƒÊm, those chromosomes were very difficult to be separated from each other

by the mass squash method. Thus, cells of the meristematic tissue were transferred on to hole

glass-slide under dissecting microscope, and only visible, single metaphase cell was isolated,

picked up and dropped on glass slide by pippette 100ƒÊm diameter. A cover slip was placed on

it under dissecting microscope, and the preparation was tapped and squashed.

Karyotype formulas at mitotic metaphase were based on the data of measurements of the

chromosomes taken from the photographs. Position of the primary constriction was estimated

by arm ratio calculated by long arm/short arm. Classification of mitotic metaphase chromo

somes in somatic cells followed Levan et al. (1964): m=median-centromeric chromosome with

arm ratio of 1.0-1.7; sm=submedian-centromeric chromosome with arm ratio of 1.8-3.0; st=

subterminal-centromeric chromosome with arm ratio of 3.1-7.0; and t=terminal-centromeric

chromosome with arm ratio of 7.1 or more.

Chromomycin A3 (CMA) guanine-cytosine-specific (GC-specific) fluorescent-staining

116 K. Kondo, Y. Hoshi and R. Tanaka Cytologia 59

Figs. I and 2. Plants of two species of Cypripedium studied . 1. C. segawai Masamune. 2. C.

japonicum Thunberg. Bar=2cm.

technique followed and modified Hizume et al. (1989): The slides were preincubated for 30

minutes in McIlvaine's buffer [C3H4(OH)(OOOH)3•EH2O+Na2HPO4• 12H2O, pH 7.0] and

treated with 0.1mg/ml distamycin A in the buffer for ten minutes before rinsed with the buffer

containing 5mM MgSO4 for ten minutes. Then, they were stained for ten minutes with 0 .1

mg/ml CMA in the buffer containing 5mM MgSO4 and were mounted with glycerol . They were

stored in a refrigerator until observation was performed. CMA bands were observed under an

epi-fluorescent microscope with B filter cassette.

Sequential staining with Hoechst 33258 and quinacrine mustard (QM) was performed

essentially according to Carlin and Rao (1982). The slides were preincubated for 30 minutes

in McIlvaine buffer (pH 4.5) and were stained with 0.5g/ml Hoechst 33258 in the buffer for 15

minutes at 37•Ž before rinsed with the buffer for ten minutes. Then , they were restained with

0.5g/ml QM in the buffer and were mounted with glycerol. They were immediately observed

under the epi-fluorescent microscope with U filter cassette.

Figs. 3 and 4. Karyotypes of two species of Cypripedium. 3. C. segawai . 4. C. japonicum.

Bar=10ƒÊm.

1994 Somatic Chromosome Differentiation in Cypripedium 117

Results and discussion

Cypripedium segawai showed the chromosome number of 2n=20 and the chromosome

complement at mitotic metaphase consisted of 16 median-centromeric chromosomes and four

terminal-centromeric chromosomes (the 11th, 12th, 17th and 18th chromosomes) (Fig. 3).

Size of the largest chromosome was 18.2ƒÊm, while that of the smallest chromosome was 9.4

ƒÊ m. This karyotype result was a little different from that of Karasawa and Aoyama (1986);

that karyotype consisted of 17 median-centromeric chromosomes and three terminal

centromeric chromosomes (the 14th, 17th and 18th chromosomes).

Cypripedium japonicum had the chromosome number of 2n=20 and the chromosome

complement at mitotic metaphase consisted of 14 median-centromeric chromosomes and six

submedian-centromeric chromosomes (the 13th, 14th and 17th-20th chromosomes) (Fig. 4).

Size of the largest chromosome was 12.9ƒÊm, while that of the smallest chromosome was 5.3

ƒÊ m. This chromosome count confirmed that reported by Mutsuura and Nakahira (1958),

Tanaka (1965, 1971) and Karasawa and Aoyama (1986). Karasawa and Aoyama (1986) also

reported the karyotype of 12 median-centromeric chromosomes and eight subterminal

centromeric chromosomes (the 11th, 12th and 15th-20th chromosomes) in this species, which

was a little different from the present result. Thus, certain chromosomes of these two species

of Cypripedium, especially C. japonicum seemed to perform different condensation of chroma

tins during some period of the metaphase or different base composition of DNA. The

chromosome sets of the two species showed a significant difference in C-band: C. segawai had

61 C-bands (68.5%) at the interstitial region, 24 C-bands (27.0%) at the distal region, four

C-bands (4.5%) at the proximal region, and two heterozygous C-bands (2.2%), while C.

japonicum had no C-band (Hoshi et al. 1994).

The chromosomes of Cypripedium segawai exhibited CMA negative bands at the intersti

tial region (Fig. 5) and the double-staining positive bands at the positions of CMA negative

band (Fig. 6), which were also recognized with the C-bands. In contrast, the chromosomes of

C. japonicum displayed two bright CMA positive dots at the interstitial region of the short arm

of two submedian-centromeric chromosomes and two bright CMA positive bands at the

secondary constriction (Fig. 7), and thin positive bands of Hoechst 33258-QM-double staining

at the distal region of all of the chromosomes (Fig. 8). Hoechst 33258-QM-double staining

made bands deeper contrast and longer duration of brighting than the single staining did.

Qinacrine and Hoechst 33258 were considered specific for adenine-thymine-rich (AT-rich)

region in the chromosomal DNA (Weisblum and Haseth 1972, Weisblum and Haenssler 1974).

In relationship between GC-specific CMA and AT-specific 4•Œ,6-diamidino-2-phenylindole

(DAPI), the large band stained with one fluorochrome was negatively stained with the other

fluorochrome (Hizume et al. 1983). In contrast, in a case of the C-banding pattern of Pinus

densiflora Sieb. et Zucc. it seemed to coincide generally in location and number with thick

CMA and DAPI-bands (Hizume et al. 1989). Thus, the present result of fluorescent banding

in C. segawai followed Hizume et al. (1983, 1989). However, the positions of thin CMA and

DAPI-bands might not be detected by the C-banding technique (Hizume et al. 1989). The

present result shown at the distal region of the chromosomes in C. japonicum (Fig. 8) followed

the phenomenon observed by Hizume et al. (1989).

On the other hand, although the nucleolar organizer regions (NORs) are considered to

appear at the secondary constriction of mitotic metaphase chromosomes (Kusanagi 1966,

Tanaka 1980), all of the NORs might not be detected at the secondary constrictions by the

conventional karyotype analysis (Hizume et al. 1989). NORs in higher plants can be generally

stained with CMA (Schweizer 1976). Thus, the present result on NOR detection followed

Schweizer (1976) and Hizume et al. (1989): Two secondary constrictions in C. japonicum were

118 K. Kondo, Y. Hoshi and R. Tanaka Cytologia 59

Figs. 5-8. Fluorescent-banded chromosomes in two species of Cypripedium. 5 and 6. C.

segawai. 7 and 8. C. japonicum. 5 and 7. Chromosomes stained with CMA. Bright, constantly

stable bands and dots (arrows) and faint, unstable bands (arrowheads) are present. 6 and 8.

Chromosomes double-stained with Hoechst 33258 and quinacrine mustard. Bar=10ƒÊm.

stained positively and deeply, while those in C. segawai were not observed. Cypripedium

japonicum could have different base composition of mostly DNA and in its chromosomes from that of C. segawai.

Cypripedium segawai and C. japonicum are morphologically quite different from each other: C. segawai sets one or sometimes two yellow-colored flowers and three to five alternate leaves (Fig. 1), while C. japonicum sets a flower with yellowish green-colored sepals and petals and crimson-pinkish white-colored, brownish pink-veined lip and 3-4 small leaves at the lower

portion of the stem and two large, sessil, crescent, alternate leaves at the upper portion of the stem (Fig. 2). Cypripedium segawai was sometime treated taxonomically as conspecific to C. macranthum Swartz (Su 1986). Although the two species are taxonomically placed in Section

Cypripedium (Chen and Xi 1987), C. segawai is considered to be placed in Subsection Cypripedium and C. japonicum is placed in Subsection Flabellinervia. Among the species of

Cypripedium studied here and elsewhere (Yamasaki 1956, 1959, 1961, 1965, 1971, 1973, Karasawa and Aoyama 1986, Hoshi et al. 1994), C. japonicum showed the highest ratio of the largest chromosome to the smallest chromosome, 2.4, variable karyotype, no C-band and less fluorescent banding. Imai (1991) stated that elimination of constitutive heterochromatin might

be evolutionary advantage. These phenomena suggest that C. japonicum might be the most advanced species among the species of the genus studied here and elsewhere. Cypripedium

japonicum may not be placed in section Cypripedium.

Summary

The chromosome complement of Cypripedium segawai at somatic metaphase carried

numerous C-bands and various CMA-negative bands at the interstitial region and positive and

1994 Somatic Chromosome Differentiation in Cypripedium 119

deep bands of the Hoechst 33258 and quinacrine mustard (QM) double-staining at the positions where were of the C-bands and the CMA-negative bands . The chromosome complement of Cypripedium japonicum at somatic metaphase exhibited no C-band and a few CMA-positive , bright bands and dots in certain chromosomes and positive but faint bands of the Hoechst 33258 and QM double-staining at the distal region . Chromosome differentiation of C. segawai and C. japonicum could be different from each other .

This paper is constributed from Laboratory of Plant Chromosome and Gene Stock , Faculty of Science, Hiroshima University (Contribution Number 27) .

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