CHANGES IN CHRQMQSQME NUMBER IN MICRQSgQRE

CALLUS OF RICE DURING SUCCESSIVE SLTBCULTURES

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CHI-CWANG
CHEN AND CHUNG-MQNG
CHEN
Deparfmenr of Botany, National Taiwan University, Taipei, Taiwan, Republic of China

Forty-six callus cultures of rice (Oryza sativa L., 2n - 241, each presumably origi-
nating from a single microspore, were established and maintained on a medium containing
2,4-B. At the end sf the first transfer 24% of the cultures were nonhaploid consisting of
only diploid or poIypIoiJ cells, or of cells of two ploidy levels. Nuclear fusion and
endomitosis occurring during the initial stages of in vitro microspore development were
postulated to account for the fomation of nonhaploid callus. Seventeen cultures were
studied cytologically through 19 transfers. Only in one tetraploid and one hexaploid callus
did the ploidy levels of cells remain unchanged during culture. Chromosome numbers in
13 cultures fell into a geometric series. Since no diplo- and quadruplochromosomes were
observed, it was inferred that endomitosis rather than endoteduplication was responsible
for the changes. In spite of the tendency for chromosome doubling, the proportions of
cells of different ploidy levels were fixed in the 13 cultures at later transfers. Haploid
cells were eliminated from all cultures. Diploid cells became predominant in eight cultures
and tetraploid cells in five, suggesting a selection for either cell type. Triploids appeared
in two cultures which initially did not contain this type of cell. Limited cytological
information indicated that triploid cells might have originated from tetraploid cells through
reductional grouping of chromosomes accompanied by multipolar formation.
For personal use only.

Quarante-six cultures de riz en forme de calus (Oryza safiva L., 2n = 241, chacune
originant probablement d'un seul microspore, fumnt 6tablies et maintenues dans un milieu
contenant du 2,4-B. A la fin du premier transfat, 24% des cultures itaient nonkaplo'ides
consistant seulement de cellules diploides ou polyploides, ou de cellules a deux niveaux
ploidaux. Qn peut supposer que la fusion nucliaire et l'endomitose survenant pendant les
stages initiaux du dtveloppement des microspores in vitrs sont responsables de la formation
du calus nonhaploi'de. Durent dix-neuf transferts on a ttudii dix-sept C U ~ N ~ cytologi-
~ S
quement. Sauf dans un caius tttraploTde et un calus hexaploYde, il ne s'est produit aucun
changement dans les niveaux ploidaux des cellulea durant la culture. Le nombre chro-
mosomique chez 13 cultures se conformait B une d r i e giumiuique. Cornme on n'a observt
aucun diplo- et quadmplo- chromosome, on en a dtduit que l'endomitose pluti3t que l'endo-
reduplication etait responsable &s changements. Malgre la tendance aru d6doublement des
chromosomes, les proportions des cellules des diffirents niveaux ploidaux furent f i d e s
dms Ies 13 cultures au cours des derniers transferts. Les cellules haploides furent bliminies
de toutes les cultures. Les cellules diploides devinrent predominantes dam huit cultures
et les cellules t6traploides dans cinq cultures, ce qui suggitre une stlection an faveur de
l'on ou de l'autre genre de cellule. Les uiploides sont apparus clans deux cultures qui ne
contenaient pas initialement ce genre de cellule. L9information cytologique restreinte a
montrk la possibiliti que les cellules triploides tirent leur origine de cellules dtraploides
par la fomation des groupes rt5ductionnels de chmmosomes accompagnis par formation
multipolaire. [Traduit par le journal]

Introduction
Anther cultures of rice (Oryza sativa L., 2n = 24) have given rise to plants
of various ploidy levels ranging from haploid to pentaploid (Nishi and Mitsuoka,
1969; Niizeki and Oono, 1971; Chen and Lin, 1976; Chen, 1977). Histological and
genetic evidence nevertheless indicated that under certain culture conditions the
plants derived from rice anthers were of microsgore origin (Harn, 1969; Wsu and
Chen, 1977). In a previous paper (Chen, 1977) we reported the occurrence of
abnormal nuclear events in the developing microspores in cultured rice anthers and
postulated that this would lead to the formation of callus consisting of nonhaploid
Manuscript received January 14, 1980; accepted April 8, 1988.
Can. J. Genet. Cytol. 22: 607-614, 1980.
 608 c.-c.CHEN AND c.-M. CHEN

cells. Nuclear behavior in the microspore callus of rice during subculture has not
been investigated. However, it has been shown in other plant species that cultured
cells and tissues, especially those initiated from microspores and somatic tissues of
haploid plants, are generally characterized by instability of chromosome number and
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structure (Bennici et a l . , 1968; SacristBn, 1971; Guo, 1972; LRvenko et a l . , 1977).

The present study was undertaken to determine the chromosome numbers of cells
in newly established rice microspore callus and the subsequent changes occurring
in the callus cultures during subculturing.
Material and Methods
The plant material used to establish microspore callus was a pure line of Orjza sativa
L. cv. Tainan 5 (2rz = 24) which was obtained through self-fertilization of a doubled haploid
plant (Lin, 1979). Anthers at the mid-uninucleate microspore stage were excised and cultured
according to the method described previously (Chen, 1977, 1978). After 20 to 25 days of
culture, anthers were removed from culture tubes and opened with two fine-tip hooked needles
under a dissecting microscope. Calli of about 0.3 mm in diameter were dissected and
transferred individually to single culture tubes containing Murashige and Skoog9s (1962) basal
agar medium supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Cultures of
the microspore callus were maintained in an incubator at 25&1°C and under 16 h daily
illumination with cool-white fluorescent light at 25 p Einsteins m-2sec-1. They were sub-
cultured every 28 days through 19 transfers.
For cytological observations, samples of the callus cultures were collected at regular
intervals during subculturing. Because the callus was very small when it was initially dissected,
For personal use only.

sufficient material could be obtained only at the end of the first transfer. For the subsequent
subcultures, samples were collected ten days after each transfer. Samples of the callus cultures
were pretreated in 0.002 M 8-hydroxyquinoline at 18 to 20°C for 3 h, fixed in ethanol-glacial
acetic acid ( 3 ~ 1 )overnight, stained in Feulgen reagent for 1 to 2 h, and then treated with
5% gectinase (ICN Pharmaceuticals, Inc.) for 2 h. In the preparation of slides, samples were
taken at random from all parts of the callus and squashed in diluted propiono-carmine. Four
to six slides per culture were prepared and at least 180 metaphases were examined.
Results
After 20 to 25 days of cultivation about 25% of the anthers contained calli of
various sizes, ranging from that barely recognizable under 3 0 ~ magnification to
approximately 0 . 3 rnm in #diameter. The calli were initially round, but they became
somewhat irregular due to differential growth of the tissue during later development.
Although the number of calli per responding anther varied from one to nine, most
anthers contained two or three calli. The calli appeared to maintain individuality in
the anthers; there was no indication of fusion up to this stage of development. Forty-
six callus cultures were established by dissecting the individual callus from the
anthers and transferx-ing to the medium containing 2,4-B.
At the end of the first transfer only four of the 46 callus cultures were found
to consist exclusively of haploid cells (Fig. 1). The majority of the cultures (67.4%)
contained, in addition to the predominant haploid cells, small fractions of cells with
2n (Fig. 2), 4n (Fig. 3 ) , and 8n chromosomes (Table I). Some cultures (24%) had
no haploid cells; they were composed of wholly diploid or polyploid cells, or of
cells of two ploidy levels. No aneuploid cells were observed in any of these cultures.
Seventeen callus cultures were studied cytologically through 19 transfers. During
this period of subculturing, changes in chromosome number were detected in 15
cultures; only one tetraplsid and one hexaploid (Fig. 4) callus retained their respective
initial chromosome number during the culture history. The chromosome numbers in
13 cultures were present in a geometric series ( n , 2n, 491, and 8n); cells with
numbers intermediary in the series or with aneuploid numbers were not observed
(Figs. 7A-F). The proportions of cells of different ploidy levels in each of these
 CHROMOSOMES IN MICROSPORE CALLUS 689

Chumussme numbers in 46 microspore callus cultures of rice determined 20 days after the first transfer

No. of callus 5% s f callus

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Chrumosome number cultures cultures

cultures varied early during culture. The general trend of the variations was toward
a decrease in the frequencies of haploid cells and a corresponding increase in those
of diploid and tetraploid cells. The rate of the decrease varied from one culture to
another, but eventually haploid cells were eliminated from all cultures. In eight
cultures diploid cells became the predominant type, in which, with one exception
(Fig. 7A), a few tetraploid cells were also present (Pigs. 7B-D). Tetraploid cells
completely replaced other types of cells in four cultures (Fig. 7E). Octoploid cells
occurred in low frequencies in one culture predominated by tetraploid cells (Fig. 7F).
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The changes that occurred in two other cultures did not fall into the pattern
described above. Culture 39-1 initially contained 98% haploid and 2% diploid cells
(Fig. 7G). At the fourth transfer, the frequency of haploid cells decreased to 69%
and that of diploid cells increased to 24%. In addition to these two cell types, a
smdl fraction (7%) of tetraploid cells also appeared. However, triploid cells (Fig, 5)
became predominant at the 7th and 10th transfers, and from the 16th transfer on,
triploid cells were completely replaced by 26-chromosome aneuploid line (Fig. 6).
The changes that occurred in culture 64-2 were even more complicated. The fre-
quencies of n, 2~2, 32,and 4n cells fluctuated from one transfer to another with
no definite pattern sf change being evident (Fig. 7H).
All the metaphase chromosomes examined in this study were normal, with each
consisting of two chromatids; diplo- and quizdruplochromosornes were not observed.
All the prophase cells observed contained only one nucleus. In one tetraploid cell
of culture 64-2, in which pretreatment did not appear to be quite effective, the 48
metaphase chromosomes were found to be arranged into three groups whose chro-
mosome numbers were 25, H 2, and 11, respectively.
Discussion
Judging from the size and shape of the callus at the time of dissection and
from the number and individuality of the calli in the anthers, it seems reasonable
to assume that each of the 46 cultures established in this study represents a callus
line of single microspore origin. The lines which co,ntained only diploid and polyploid
cells at the end of the first transfer must have arisen through nuclear changes that
occurred during the very early stage of in vifrs microspore development. In a
previous report (Chen, 197'7) we proposed two events involving fusion as the
mechanism for formation of diploid and polyploid microspore-derived rice callus:
one resulting from failure of wall formation in early microspore divisions and the
other from fusion of a vegetative and a generative nucleus. Convincing cytological
evidence for fusion of the vegetative and generative nucleus has been obtained by
Sunderland ef a / . (1974) in microspores of Datuaa innoxia. Recently, Wilson et al.
 618 C.-C. CMEN A N D C . - M . CHEN
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Figs. 1-6. Mitotic metaphses in the microspore callpas cultures of rice. Fig. 1 . Haplploid with 12
chromosomes. Fig. 2. Dipbid with 24 chromosomes. Fig. 3. Tetraploid with 48 chromossmes. Fig. 4 .
Hexaploid with 72 chromosomes. Fig. 5 . Triploid with 36 chromosomes. Fig. 6 . Aneuploid with 26
c h o m s o m e s . AII photomicmgraphs at X 2500.

(1978) reported that wall formation was not visible in barley microspores until the
tri- to tetranucleate stage. Some of the calli they obtained contained only diploid
or polyploid cells. The occurrence of a hexaploid callus in this study indicated that
nuclear fusion had indeed taken place in the developing microspores of rice. However,
this by no means implies that other mechanisms such as endomitosis and endore-
duplication are excluded. In fact, as discussed in the following section, endomitosis
appeared to be widespread in the established microspore callus cultures. Therefore,
it is probable that this process may also occur early in microspore development,
thus resulting in the formation of callus consisting of nonhapfoid cells.
The occurrence of a geometric series of ploidies in 13 callus lines indicates
that the changes induced by in 17ifr-Q culture are mainly a repeated doubling of the
chromosome numbers of cells initially present in the populations. This type of
change may arise either through endomitosis or endoreduplication (Sunderland, 1973).
Direct evidence for endomitosis has not been obtained in this study because the
material used for cytological examination was treated with 8-hydroxyquinoline to
 CHROMOSOMES IN MICROSPORE CALLUS 61 1
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TRANSFERS
Fig. 7 . Chmrnosome number changes in some representative microspore callus cultures of rice
during successive subcultures. A. Culture 21-1. B . Culture 55-1. 6 . Culture 62-1. D. Culture 5-1. E.
Culture 1 1 - 1 . P. Culture 89-1. G . Culture 39-1. H . Culture 64-2.

facilitate chromosome counting. However, the absence of diplo- and guadruplo-

chromosomes in the many rnetaphases examined suggests that endoreduplication is
not a likely mechanism for the increase of ploidy level observed in this material.
In spite of the tendency for chromosome doubling, the proportions of cells of
different ploidies appeared fixed in all 13 lines at later transfers. Elimination of
 612 e.-c.CHEN AND c.-M. CHEN

haploid cells from these lines suggests that haploid cells are not only unstable during
culture but also unable to compete with cells of other ploidy levels in the mixoploid
populations. The predominance of diploid cells in a majority of these lines (8 out
of 13) indicates a selection for diploid cells. However, since seven of these eight
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lines contained low frequencies of tetraploid cells, the diploid cells must also be
unstable during culture and they continuously give rise to tetraploid cells through
chromosome doubling. The predominance of tetraploid cells in five lines suggests
that diploid cells me not necessarily the most successful competitors. Tetraploid cells
may be less predisposed to chromosome doubling compared with haploid and diploid
cells, as only one of the five lines contained some octoploid cells and the remaining
four consisted of only tetraploid cells.
Competition between diploid and polyploid cells in a mixoploid population has
been observed in tissue and cell cultures of a number of plant species. It has been
demonstrated by Singh and Harvey (197%) and Bayliss (1975; 1977a,b) that the
competitive interactions can be altered by factors such as medium composition and
cdture environment. In Haplopapp~sgracikis, for example, a selection for diploid
cells was observed in liquid suspension cultures, but for tetraploid cells in callus
cultures on agar medium (Singh and Harvey, 197%).In Daucus carota the selective
advantage of a tetraploid line over a diploid line in the mixed suspension cultures
was attributed to its genotypically determined greater ability to use the phosphate
supply in the nutrient medium (Bayliss, 1977b). The underlying causes for com-
petition between cells of different ploidy levels have not been investigated in this
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study. Since all the lines were cultured in the same medium and under identical
conditions, the differences in competitive ability of diploid and polyploid cells in
different callus lines may have a genetic basis. It is possible that genic and undetected
chromosomal changes have occurred in some cells during culture, and as a result
of these changes the callus lines and even cells within the same lines may differ
genetically.
The most significant feature of the changes in cultures 39-1 and 64-2 is the
appearance of triploid cells. Odd-ploid chromosome numbers may result either from
nuclear fusion occurring in lower ploid cells or from reductional grouping of
chromosomes accompanied by multipolar formation in higher ploid cells (Partanen,
1963; B'Amato, 1977). The fusion events proposed by us to account for the formation
of nonhaploid callus are characteristic of the developing microspores (Chen, 1977).
Nuclear fusion probably did not occur in the established callus during subculture
since all the cells examined were uninucleate. The significance of reductional
grouping in the origin of haploid from diploid cells was first recognized by Huskins
and associates while working on mitosis in various plant roots (Huskins, 1948;
Wilson and Cheng, 1949; Huskins and Cheng, 1950). Since then it has been observed
in polyploid tissues of both plants (Huskins and Chouinard, 1950) and animals
(Glass, 1957) and has been considered a probable mechanism for somatic reduction
of a whole set($) of chromosomes in polyploid cells. Reductional grouping of
chromosomes has also been observed in animal cells (Pera and Rainer, 1973) and
plant tissues (Mar'yakhina and Butenko, 1974) cultured Irz t ~ i ~ r Although
o. the squash
method and pretreatment used in this study tend to obscure the real picture of the
arrangement of chromssomes at metaphase, a grouping of 25- 12- 11 was observed
in one tetraploid cell in culture 64-2. Division of this type of cell with a multipolar
spindle would give rise to cells with chromosome numbers varying from haploid to
triploid or with numbers close to these ploidy levels. The aneuploid cells in culture
39-1 probably arose through a similar mechanism. The predominance of aneuploid
cells in this culture at later transfers suggests that they are at a selective advantage
 CHROMOSOMES IN MICROSPORE CALLUS 613
over other types of cells. The changes that occurred in culture 64-2 may be attributed
to the interactions of chromosome doubling, reductional grouping, and selection for
some specific cell types.
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Acknowledgment
This study was supported by the National Science Council, Republic of China,
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