Application of Flow Cytometry for Rapid Determination of Ploidy

.1. Fac . Agr. , Kyushu Univ., 43 (l·2) , 8:3-.. .-88
( 1 99~ )
Application of Flow Cytometry for Rapid Determination of Ploidy
Levels in Asparagus (Asparagus o.fj'icinalis L.)
Yukio Ozaki, Kumiko Narikiyo, Michikazu Hiramatsu*,
Kenji Vreshino and Hiroshi Okubo
[",'1.tJoratOI)' of Hortic ultural Sei eIl c~, Faculty of Agriculi.ure,
Ky\lshu University, FuklJOka 812-8581, J<lpan
(Rrlcei·t1ed July 28, 1.9.98 and accepted Augu st 7, 1998)
Flow cy tomctry of PJ-stainpd n uc:lc i was su bjected t.o f'st imatc the plo id y level:; ill
asparag us. No s ign ificant diffe rence W(lS observed in PI fl uorescent intensity at t he prominent
p eak in diploid cull..ivars/strajn, and thc rc W;lS a ve ry strong positive lin ear c orre lat ion
(r=0.9977) between PI fluorescenl. ill Lt:!llsi rjes and ploidy levels in haploid , diploid, triploid a nd
Lelrilp)oid f·u.ltivarslstrains. F'low cytometry has been proven to he a rapid and cmcient. ploidy
analysis duriug large scale experimt:!nlS,
regenerated plant s in anther culture
~lit": h
as selection of haploid
p laJlt~
from numbers of
INTROD UC TION
Asparagus (A<;1H1Tagus officinalis L.) is a dioccious perennial species, and originated
in Europe and easte rn As ia. The dioecious nature of a.<.;paragus provides many ge netic
and physiological studies on sex determination and differentiation. Sex express ion has
heen reported to be governed by only a single locus (Hick and Hanna, 194:3), which is
located on the L5 chromosome (Lbptien , 1979).
Male (XY) plants give higher yields and more vigorous growth than females (XX) , and
males do not create the 'asparagus weed problem' by producing seeds which germinate
a.nd grow in the production fields (Yeager and Scott, 1938). Thus, an a ll-male population
obtainable h.Y crossing \\<ith sllpermaies (YY) is one of the major objectives in asparagus
breeding. Haploid production by anthe r culture has bee n performed to prepare
sllpermale plants (Fal.vigna et al., 1983; Feng and Wolyn, 1991, 1993). It is, however,
difficult. t.o produce haploid plants by a nth er c ulture b ecause most of regenerat.erl p!ants
are diploids from somatic cells (e. g., anther wall)) not from microspores. The selection of
haploid plants is done by counting th e s omatic chromosomes and by m easuring the length
of stomata. The procedures are still difficult and/or time eonsuming to do in asparagus, so
that it is necessary to establish the efficient analytical methods for rap id and suitable
determination of ploidy levels.
Recently, now cytometry has become a useful tool for rapid and efficient est.imatiun
of genome size and ploidy levels in some crops (Baird et af ., 1994; Martinez et «I., H194;
O'Brien et «I., 1996; Ollitrault-Sanunarcelli et al., 1994; OZlas- Akins and Jarret, 1994).
The objective of this study is to est.ablish the rapid and effi cient method of plOidy
determination with flow cytometer in as paragu s.
*Ilniversity Farrn , F'aeulty of Agricu lture, Kyushu University, fi'lIkllo ka 8 11- 2307, Japan
83
84
Y Ozaki et al
MATERIALS AND METHODS
Plant materials
Seven diploid cultivars, 'eito', 'Franklim', 'Geynlim', 'Hokkai 100', 'Larae', 'f\.lary
Washington 500V/', and 'UC157F I ', one triploid cultivar, 'Hiroshima Green', and one
tetraploid cultivar, 'Seto Green', were used in this investigation. One gynogenetic haploid
strain, 97SA-003, obtained from the cross between diploid and tetraploid plants
(Nakashima et aI., HJ92), and one supermale diploid strain MM2 obtained from the
crosses \vith hermaphrodite plant, from Hokkaido University were also llsed in this study.
Number of investigated plants with different genotypes in each cultivarlstrain is
presented ill Table I,
1
Flow cytometric analysis
Young cladphylls were cut into pieces in chopping buffer containing 1.0% Triton
X-IOO, 140 roM 2-mereaptoethanol, GOmM Na$O), GOmM Tris-Hel (PH 7.5) and 25 .ug/ml
propidium iodide (PI), ",ith a razor for releasing nuclei from cells. The suspension
solution was filtered through a 25,urn nylon mesh to remove debris, and the filtrate was
centrifuged at 12,000rpm for one minute. Nuclei in the residue \vere re-suspended in the
chopping buffer, and the suspension solution was subjected to flmv cytometric analysis
\-vith EPICS XL (Coulter, Tokyo, Japan). Helative nuclear DNA content ,vas estimated by
measuring fluorescent intensity of 5,000 nuclei in each sample. The data obtained were
analyzed by the XL SYSTEM II (Coulter, Tokyo, ,Japan).
RESULTS AND DISCUSSIONS
Figure 1 5ho\-vs a typical result of flow cytornetric analysis in diploid asparagus
cultivar '1lary Washington 500W'. This histogram shO\ved a prominent peak of nuclei at
about 300 in PI fluorescent intensit.y in Gil and G1 stages of interphase during the cell
cycle. These interphase nuclei in a diploid plant are at the 2C (a double genome) level of
DNA content. A minor peak at. about t,vice the intensity value of the prominent peak
(about 600) \vas also distinguished. The minor peak is interpreted as being composed of
nuclei primarily in the G~ stage of interphase and in the early and mid stages of mitosis
during the cell cycle. Nuclei in this small peak have an average DNA content. of 4C (a
quadruple genome) in a diploid. Extensive fluorescent emissions at higher intensities,
indicative of populations of nuclei at increased ploidy levels (e. g., 8n, 16n) or nuclear
adhesion (e. g., artificial aggregat.ion of individual nuclei forming triplets ancl quadnlplets),
,vere not observed.
Average and range of PI fluorescent intensities at prominent peaks in diploid
asparagus cultivars and strain are described in Table 1. The values of individual
measurements in diploid plants ranged from 2GG.O to :346.0. The variation of average PI
fluorescent intensities in each diploid cultivarlstrain including the supermale 1Uvl2 was
not significantly different statistically (P<0.05). Average value of all diploid plants was
283.7. Intraspecific variation in DNA content has been reported in Zea mays, and a
positive correlation between genome size and altitude of the habitat places of the plant
was recognized (Rayburn el al., 1989). It was also descr:ibed that the correlation might be
Flow Cytometry inAsparagus
85
Table 1. PI t1uorescent intensity of the prominent peak ill haploid. diploid, t.riploid and tetraploid
clllt.ivars/sLrains in asparagus,
Ploidy
Cultivar or strain
No. of plants
x
97S/\-003
Cito
FrankUm'"
Geynli.m'"
Hokkai 100
2x
(Range)
I
185.0 a
3
3
284,7 b
(180.0-190.0)
(278.0-2930)
(2760-3460)
(286.0-316.0)
(296.0--.303.0)
(266.0-319.0)
(:1ll2.0-314.0)
(277.0-295.0)
3
3
;3
3
3
Lara(
Mary Washington 500 \1./
UClmF
MM2"'*
(Mean in diploids)
Hiroshima Green
Seto Green
3x
4x
PI nuorescen\. intensity
Average
investigated
level
10
8
a01.2 b
301.0 b
30(U b
287.3 b
:30G.0 b
288.7 b
280.011
293.7
(2RO'(l)
(;)80.0-428.0)
(502.0-562.0)
4lHL3c
,528.1 d
Mean separation \.,'.ithin a column by DUIlcan's lIlultiple range lest. (5% level),
>I< All-male cultivars., ** Supcnnalc strain.
150
"Qi
U
::J
C
....
GO/G1
0
0 100
Z
50
o
200
400
600
800
PI fluorescent intensity
Fig. 1. Flmv cytomctric histogram pattern in 'MaJy' Washington GOO W'
1,000
r.
86
Ozaki el a l.
due to in cr easi ng knob (C-b andcd) hete roch romatin 01' additio nal intra- or super1I11II1erary chromosomal DNA sequences in proportion to increasing altitude (Ra,yburn and
Auge r , 1990). In con trast, insignifi can t variation r ecognized in diploid asparagus
cultivarsistrain, proved that there mighl he li ttle in traspecific varia tion of genome size in
cultivated diploid asparagus.
Silene IUl iJolia (=Melandriwn uUnon) is one of th e model plan ts as di ocdoLls
species (Maegher and Costich, 19'J4; Veus kens cf aI., 1992), and sex determinant of the
plant was reported t.o be localized on the strongly heteromorphic ChrOITlOSOme pair '(small
X and large Y). Flo\v eytometry is useful for rapid identification of sexes in 8'ilw'r/,e
latijolia because of the significant difference in nuclear DNA conten t betvveen females
(2n =24, XX) alld males (2n=24, XV) (Dolezel and Gohde , 19'J5). There are some
confused discussions of sex chromosome ana lysis in asparagus. Although Zilm ( 1966)
and L6ptien ( 1979) reported t.hat as paragus is a homomorphic sex chromosome plant, An
et al. (1992) and Kitazawa cf a.1. ( 1991\) report.ed it heteromorphic. In t.IJe presenr, st.udy ,
there \vere no ~ignifica nt differences ill flow cytomctrie data between male (XY) and
supermale (l Y) plants. The present result supports that the sex ehrornosomc is homomorphic, othcnvise the difference ill DNA content between X and Y chromosomes might
be quite a li ttle if t.he sex chromosome js heteromorphic in asparagus.
Average aud range of PI tluoreSC€Ilt intensities in diffe rent plOidy cultivars or strains
a re also presented in Table I . The more the ploidy level 'WllS from haploid to tetraploid ,
1x
2x
3x
4x
1x: 97SA-003
2x: Mary Washington
500W
3x : Hiroshima Green
4x: Sato G raen
o
o
Z
o
200
400
600
800
1,000
PI fluorescent intensity
Fig. 2. Flow CYl .omclric histogram patterns in ha ploid . rl iploid, triplOid and lelmploici plants
o f asparagus.
Flow Ci/[ometry in Asparagus
600
87
r-------------------------
500 I-
.•
y=11S.2x+64.26
.... .
(r=O.9977)
400 I300 I-
'
.~ ..
..
200 tCl..
100 t-
o
,
o
2
,
3
4
Ploidy level
Fig. 3. Relationship between ploidy level and average PI fluorescent
intensity in asparagus cultivars/strains.
the larger was the PI fluorescent intensity. Significant differences (P< 0.05) of PI
fluorescent intensities were recognized between different ploidy cultivars/strains. There
were no overlapping values in individual measurements bet\veen different ploidy plants.
Differences in the values of PI fluorescent intensities between haploid and diploid,
between diploid and triploid, and between triploid and tetraploid, were approximal.ely the
same ranging from llO to 120 (Table 1 and Fig. 2). Thus, a very strong positive linear
correlation (r=0.9977) between PI fluorescent intensities and ploidy levels in asparagus
was recognized (Fig. 3).
Our study demonstrated that flow cytometry makes rapid and efficient determination
of ploidy levels possible without chromosome cOlmting and stomata length measurement
in asparagus.
ACKNOWLEDGMENTS
The authors wish to express their sincere appreciation t.o Ms. Mika In (Cytometry
Division, Fukuoka Branch, Coulter K K.) for her teclulical advice during this experiment.
We are also grateful to My. Toshiki Nakashima (Laboratory of Plant Biotechnology, Saga
Prefeclural Agricultural Research Center) and Professor, Dr. Takashi Harada (Hokkaido
University) for providing us the haploid and supermale plants, respectively.
88
Y Ozaki et at
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