Annals of Botany 87: 729±735, 2001

doi:10.1006/anbo.2001.1402, available online at http://www.idealibrary.com on

Molecular Cytogenetic Analysis of Polyploidization in the Anther Tapetum of Diploid and

Autotetraploid Arabidopsis thaliana Plants
H A N N A W EIS S * and JO L ANTA M AL US Z YN S KA
Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland

Received: 31 August 2000 Returned for revision: 22 November 2000 Accepted: 5 February 2001 Published electronically: 5 April 2001

Like those of most angiosperms, vegetative tissues of Arabidopsis thaliana undergo high levels of endopolyploidiza-
tion. One such tissue is the anther tapetum which plays a role in male sporo- and gametogenesis. The degree of
polyploidization of the tapetum varies from species to species. Although the role of this process is not yet fully
understood, it may be linked to functioning of the tapetum, increasing the copy number of genes needed for the
synthesis of speci®c factors required by developing pollen mother cells (PMCs) and pollen grains. The present study
focused on polyploidization during the development of the tapetum of Arabidopsis thaliana. The aim was to outline
the mode of tapetum polyploidization in this model plant species and to establish an ecient method for analysing
ploidy levels in di€erentiated cells. The course and degree of tapetum polyploidization in Arabidopsis was analysed in
interphase nuclei using ¯uorescence in situ hybridization (FISH) with repetitive DNA (45S rDNA). The stages of
development of the tapetum were analysed alongside meiosis in PMCs. The majority of tapetal cells undergo two,
maximally three, rounds of divisions. Tapetal nuclei have usually divided by metaphase I of meiosis of PMCs. The
pattern of tapetum polyploidization was similar in diploid and autotetraploid plants and is thus not a€ected by
increasing amounts of maternal plant DNA. The tapetum of autotetraploid plants exhibits a higher frequency of
additional division than seen in diploid plants. # 2001 Annals of Botany Company

Key words: Arabidopsis thaliana, autotetraploid, FISH, rDNA polyploidization, tapetum.

I N T RO D U C T I O N processes involved in polyploidization have been proposed

The anther tapetum is involved in the process of male sporo-
and gametogenesis, playing a key role during microspore
and pollen grain development. Its most signi®cant functions
are nutrition (Maheshwari, 1950; Esau, 1961) and secretion
of enzymes and various substances needed for pollen wall
formation (Bhandari, 1984; D'Amato, 1984; Pacini, 1997).
Two types of tapetum are distinguished in plants: secretory
and ameboidal. The tapetum of Arabidopsis thaliana, typical
of that of all Brassicaceae, is of the secretory type and was
described as a single layer of binucleate cells in a mature
state ( for references see Pacini, 1997). Analyses of anther
development both in wild type A. thaliana plants and in
male-sterile mutants have usually involved ultrastructural,
genetic, molecular and biochemical studies of tapetal cells
(e.g. Dawson et al., 1993; Chaudhury et al., 1994; Owen and
Makaro€, 1995). However, there is still very little infor-
mation available on the cytogenetics of tapetum develop-
ment in A. thaliana.
Cells of the secretory tapetum are usually polyploid and
(or) multinuclear (D'Amato, 1984). The number of nuclei is
species-speci®c and varies depending on the stage of
development of the tapetum and processes involved in its
polyploidization (Pacini, 1997). Numerous cytological
studies have described changes in ploidy level in tapetal
cells in several plant species, and some hypotheses on the
* For correspondence at: Department of Higher Plant Systematics
and Evolution, Institute of Botany, University of Vienna, Rennweg 14,
A-1030, Vienna, Austria. Fax ‡43 1 4277 9541, e-mail hweiss@ s1.
botanik.univie.ac.at
0305-7364/01/060729+07 $35.00/00
(Avanzi, 1950; Berger et al., 1951; Mechelke, 1952; Davis,
1961; Bhandari et al., 1976; Oksala and Therman, 1977;
Bhandari, 1984; D'Amato, 1984).
The present study focuses on polyploidization during the
development of the tapetum of Arabidopsis thaliana plants
with varying ploidy levels (diploid and autotetraploid). This
approach has several aims: (1) to analyse the degree and
mode of polyploidization of the tapetum of Arabidopsis
thaliana; (2) to elaborate the outline of tapetum poly-
ploidization that could be used in the analysis of tapetum
development in mutants, especially those that show
disturbances in male meiosis; (3) to determine whether the
ploidy level of the maternal plant has an e€ect on the
pattern of tapetum polyploidization; and (4) to establish an
ecient method for the determination of ploidy level in
specialized, di€erentiated cell types.
rRNA genes are highly repetitive sequences present in
every plant cell, they are easy to map by in situ
hybridization and are routinely used in plant laboratories
(Schmidt and Heslop-Harrison, 1998). The number of 45S
rDNA sites in the Arabidopsis thaliana genome is constant
in all ecotypes examined to date, in contrast to 5S rDNA
loci which exhibit ecotype-speci®c polymorphism in num-
ber and localization (Fransz et al., 1998). For these reasons,
45S rDNA was chosen in the present study as a marker for
the determination of ploidy level in interphase nuclei of the
tapetum. The application of FISH ( ¯uorescence in situ
hybridization) and analysis of the number and size of in situ
hybridization signals made it possible to outline the details
# 2001 Annals of Botany Company
730 Weiss and MaluszynskaÐCytogenetic Analysis of the Arabidopsis Tapetum
of tapetal nuclei polyploidization in Arabidopsis thaliana.
The preferential fusion of 45S rDNA loci of chromosome 4,
observed in diploid somatic nuclei of Arabidopsis (Weiss
and Maluszynska, 1998; Weiss, 1999), did not a€ect the
precision of the distinction between certain ploidy levels
(2n, 4n, and higher ploidy). The proposed scheme of
tapetum polyploidization may be useful in analysing the
tapetum in developmental mutants of Arabidopsis. Analyses
indicate that the pattern of polyploidization is not
in¯uenced by the ploidy level (2n and 4n) of the maternal
plant.
M AT E R I A L S A N D M E T H O D S
Plants of diploid Arabidopsis thaliana (L.) Heynh. (2n ˆ 10)
and of its autotetraploid line (2n ˆ 4x ˆ 20), ecotype
Wilna, were grown in test tubes on MS medium (Murashige
and Skoog, 1962) at 21 8C, 16/8 h photoperiod. Tetraploid
plants were originally obtained from diploid seedlings,
Wilna ecotype, after colchicine treatment (Maluszynska
et al., 1990). Polyploid plants analysed in the present work
were of a stable tetraploid line obtained from a single C1
plant and cultivated for more than 20 generations. Flower
buds in appropriate stages of development (Ross et al.,
1996; Table 1) were collected from the ®rst in¯orescence
that appeared before stem bolting (Vieira et al., 1990). The
buds were ®xed in ethanol : acetic acid (3 : 1) for 12 h and
stored at ÿ20 8C until use.
Chromosomes and nuclei from ¯ower buds (Table 1) were
prepared according to the method described by Ross et al.
(1996) with minor modi®cations. For each preparation one
¯ower bud was used. Brie¯y, after washing o€ the ®xative,
the material was digested with 0.4 % cytohelicase (w/v;
Sigma, Taufkirchen, Germany), 0.4 % cellulase Onozuka
(w/v; Serva, Heidelberg, Germany) and 0.4 % pectolyase
(w/v; Sigma, Germany) for 5 h at 37 8C. Squash prep-
arations were made in a drop of 60 % acetic acid. After
removing coverslips, slides were air-dried and checked for
quality of spreading and developmental stages after staining
with DAPI (40 ,6-diamidino-2-phenylindole; 2 mg ml ÿ1). The
slides chosen were destained, re®xed with ethanol : acetic
acid (3 : 1) to maintain chromosome structure and stored at
4 8C until use.
Fluorescence in situ hybridization (FISH) followed the
method developed for Arabidopsis by Maluszynska and
Heslop-Harrison (1991) with minor modi®cations. The
probe used for FISH was 45S (18S±25S) rDNA from
A. thaliana, directly labelled with Cy3 by nick translation
(Amersham, Oxford, UK) according to the manufacturer's
instructions. The denaturation of slides and the hybridiza-
tion mixture was carried out separately. Chromosome
preparations were denatured in 70 % formamide in 2 
SSC (standard saline citrate) on a hot plate (Hybaid
Thermal Cycler PCR-in situ, Hybaid Ltd, Ashford, UK)
at 86 8C for 5 min. The hybridization mixture (60 %
formamide, 10 % dextran sulfate, salmon sperm blocking
DNA in 100  the excess of the labelled probe, 2  SSC
and 2 ng ml ÿ1 of labelled probe) was denatured for 10 min
at 95 8C in a waterbath and kept on ice for at least 5 min.
Hybridization was carried out overnight (18±20 h) at 37 8C.
After stringent washes (2  SSC, 0.1  SSC, 2  SSC at
40 8C, 5 min each) the preparations were counterstained
with DAPI (2 mg ml ÿ1) and mounted in antifade bu€er
CITIFLUOR (AF1, Pelco Int. Redding, USA). Analyses
were made with an epi¯uorescent microscope (OLYMPUS)
using appropriate ®lters. Pictures were taken with CCD
camera and processed with image analysis software
(AnalySIS; OLYMPUS) using only functions that are
applied equally to all pixels in the image.
Stages of tapetum development were determined and
related to development of pollen mother cells (PMCs) and
pollen (Ross et al., 1996). Of the six stamens present in
Arabidopsis ¯owers, meiosis occurs earlier in the two outer
(shorter) ones and later in four inner (longer) ones, but
within an individual stamen, development of tapetal cells
and PMCs is highly synchronized. Therefore, each prep-
aration consisted of one ¯ower bud with additional
separation of individual anthers from the given ¯ower
bud over the slide. This allowed us to analyse separately the
tapetum of shorter and longer stamens, which appeared to
di€er in developmental stage. Anthers from ten ¯ower buds
from ®ve randomly selected plants and 200 tapetal cells
were analysed for each stage of tapetum development both
for diploids and tetraploids. The size of ¯ower buds in
diploid and tetraploid plants with corresponding stages of
male meiosis in anthers is given in Table 1. These
parameters were taken into account during the ®rst
selection of buds for tapetum analysis. To mark more
precisely the developmental stages of the tapetum, the stage
of PMC development was determined in stamens in which
the tapetum was analysed.
R E S U LT S
Tapetal cell development was related to the developmental
stages of meiocytes (PMCs). The tapetal cells of A. thaliana
were distinguishable from other somatic cells in ¯ower buds
already in the premeiotic stage. Their nuclei were round and
surrounded by a layer of cytoplasm, and their chromatin
was denser and more intensely stained with DAPI than that
in other cells. The tapetum began to divide before meiosis
started in PMCs, when ¯ower buds were smaller than 0.04
and 0.09 mm in diploid and tetraploid plants respectively
(Table 1).
Chromosomes of Arabidopsis are small and dicult to
separate out from tapetal cells, especially in material that is
not pre-treated with 8-hydroxyquinoline so as not to
disturb meiotic division in PMCs. Mitotic chromosomes
of the tapetum are not very informative with regard to
ploidy level determination since they are sticky and clumped
together (Fig. 1C, D); neither do interphase nuclei stained
with DAPI provide enough information about ploidy level.
However, when combined with FISH using repetitive 45S
rDNA, tapetal interphase nuclei appeared to be convenient
and informative for the determination of tapetum poly-
ploidization.
Two pairs of NOR chromosomes are present in somatic
cells of ¯ower buds in diploid Arabidopsis (Wilna ecotype)
(Fig. 1A) and four pairs in the tetraploid genome (Fig. 1D).
The number of FISH signals during interphase in
 Weiss and MaluszynskaÐCytogenetic Analysis of the Arabidopsis Tapetum 731
T A B L E 1. The size (mm) of Arabidopsis thaliana ¯ower buds, Wilna ecotype, in subsequent developmental stages correlated
with the stage of male meiosis in pollen mother cells

Plant Premeiosis Prophase I Metaphase IÐdiads Diads-tetrads

Diploid 50.04 + 0.01 0.04±0.1 + 0.02 0.11±0.16 + 0.03 0.16±0.25 + 0.02

Tetraploid 50.09 + 0.01 0.09±0.20 + 0.03 0.20±0.30 + 0.05 0.30±0.50 + 0.1

For each type of plant, 100 ¯ower buds were measured for each class (marked by meiotic stages).

of the cells were binucleate. Both nuclei were diploid with

F I G . 1. Fluorescence in situ hybridization (FISH) with 45S rDNA in
chromosomes and interphase nuclei of Arabidopsis thaliana plants. A,
Mitotic chromosomes in somatic tissues of diploid ¯ower buds; B,
interphase nucleus in a somatic cell of a diploid plant; C and D, sticky
chromosomes in a tapetal cell of a diploid (C) and tetraploid (D) plant.
Bar ˆ 5 mm.
three to four signals of 45S rDNA-FISH (Fig. 2B). In about
3 % of cells, a single tetraploid nucleus with ®ve to eight 45S
rDNA signals was observed. This ®rst, very synchronized
division seemed to occur in all tapetal cells.
Most of the binucleate tapetal cells underwent a second,
also synchronous cell cycle, during or shortly after
pachytene. As a result, two tetraploid nuclei were observed
in each cell which indicates that this cycle was endomitosis
or restitution mitosis (Fig. 2C). rDNA-FISH revealed the
presence of ®ve to eight signals in each of the nuclei. These
signals were similar in size to those in unicellular tapetal
cells in the premeiotic stage. Six and a half percent of
tapetal cells had three nuclei, two of which were the same
size and diploid, and one which was bigger and tetraploid.
Cells with one nucleus with a ploidy level reaching 16n were
observed rarely (2.5 %). About 30 % of tapetal cells
analysed remained binucleate with two diploid nuclei.
After this division the majority of cells remained at a stable
ploidy level until disintegration, which took place during
pollen grain maturation. An additional third cycle occurred
in about 0.05 % of cells. This third division resulted in cells
with two octaploid nuclei with ten to 16 rDNA-FISH
signals each. Fragments and micronuclei were not observed
after any of the divisions in the tapetum of Arabidopsis. The
results are summarized in Fig. 3A.

Arabidopsis nuclei can be lower than the total number of
rDNA loci because of 45S rDNA loci association (see also
Weiss and Maluszynska, 1998; Bauwens et al., 1991). Due
to this association, diploid Arabidopsis tapetum nuclei have
two to four signals (with three signals being observed most
often; Fig. 1B) and tetraploid nuclei have ®ve to eight
signals. In the octaploid nuclei, ten to 16 signals of FISH
rDNA are present. Therefore, despite the tendency for
association, the range of rDNA signals distinguishes the
di€erent ploidy levels of tapetal nuclei of Arabidopsis.
Diploid plants
During the premeiotic stage when PMCs started to
enlarge and di€ered distinctly from other ¯ower bud cells,
the cells bordering the PMCs were uninucleate. At this stage
tapetal cell nuclei were round and showed strong, uniform
DAPI ¯uorescence indicating a higher degree of chromatin
condensation than found in nuclei of other vegetative cells.
There were usually three 45S rDNA signals after FISH
(Fig. 2A). Beginning at the leptotene, tapetal cells under-
went mitosis without cytokinesis. By pachytene, almost all
Tetraploid plants
The stages of tapetum development described above for
diploid Wilna genotypes were also observed in tetraploid
plants. During the premeiotic stage, uninucleate tapetal
cells were larger than both other somatic cells and PMCs.
This di€erence in size was more distinct than in diploid
plants. At this stage, tapetal nuclei were tetraploid with six
to eight signals of 45S rDNA-FISH (Fig. 2D). About 95 %
of tapetal cells entered mitosis before or during pachytene
and this division led to the formation of binucleate cells.
Each of the two nuclei was tetraploid with six, seven or
eight rDNA hybridization signals (Fig. 2E). In 5 % of the
cells, one octaploid nucleus was present. In pachytene or
early diplotene, 83 % of tapetal cells synchronously entered
the second cycle and became binucleate with two octaploid
nuclei (Fig. 2F). Eleven percent of the tapetal cells became
trinucleate (4n ‡ 4n ‡ 8n), 2.5 % of cells had one nucleus
with a ploidy level reaching 16n and 3 % of cells remained
with two tetraploid nuclei. Nuclei of 0.5 % of the tapetal
cells underwent additional division after which two 32n
nuclei were observed. The timing and mode of tapetal cell
disintegration seemed to be similar to that in diploid plants.
732 Weiss and MaluszynskaÐCytogenetic Analysis of the Arabidopsis Tapetum

F I G . 2. The most frequently observed course of tapetal cell development in Arabidopsis thaliana plants analysed after FISH with 45S rDNA. A±C,
Diploid plants; D±F, tetraploid plants. Bar ˆ 10 mm.

No fragments or micronuclei were observed. Results are
summarized in Fig. 3B.
DISCUSSION
The pattern of Arabidopsis thaliana tapetum
polyploidization
Previous studies of changes in the nuclear compartment
during tapetum development in A. thaliana have indicated
that the ploidy level and number of nuclei change (Dawson
et al., 1993; Zajac, 1997). However, there has been no report
of ploidy level changes in the tapetum with reference to the
stage of microsporocyte development. By analysing rDNA
signal numbers in interphase nuclei, we hereby con®rm
earlier general indications that tapetal cells of Arabidopsis
are uninucleate during the premeiotic and early meiotic
stages of PMC development (Dawson et al., 1993; Zajac,
1997). Most changes in ploidy level of nuclei of tapetal cells
occurred during prophase I of male meiosis and were
®nished by metaphase I. A similar situation has been
observed in tapetal cells of other plant species (reviewed in
Bhandari, 1984; D'Amato, 1984) e.g. Eremurus (Oksala and
Therman, 1977) and Antirrhinum majus (Berger et al.,
 Weiss and MaluszynskaÐCytogenetic Analysis of the Arabidopsis Tapetum 733
A B

2n 4n
Leptotene
AM 100% RM/EM -pachytene AM 100% EM/RM

2n 4n 4n 4n 8n
2n
95% 5% Pachytene 5%
-metaphase I 95%

AM+SF EM/RM AM EM/RM AM+SF EM/RM AM EM/RM

2n 4n 4n
4n 4n 4n 8n 16n
2n 4n 4n 8n 8n
2n 2n 8n
30% 6.5% 63% 0.5% 3.5% 11% 83% 2.5%
EM/RM EM/RM
8n 16n
8n
0.05% 16n
0.5%
F I G . 3. Schematic representation of polyploidization and development of nuclei of tapetal cells in Arabidopsis thaliana as related to developmental
stages of pollen mother cells. A, Diploid plants; B, tetraploid plants. RM/EM, Restitution mitosis/endomitosis; AM, acytokinetic mitosis; SF,
spindle fusion.

1951). The role of polyploidization of di€erentiated tissues
is not yet fully understood. It is generally assumed that
polyploidization occurs to amplify desirable genes necess-
ary for the synthesis of products in a given tissue without
cell division, which otherwise requires additional energy
and time (Leitch, 2000). We speculate that in a short-lived
and specialized tissue such as the tapetum, polyploidization
needs to be accomplished quickly, before the tapetum starts
to di€erentiate and secrete substances that support pollen
grain development, as also concluded by Oksala and
Therman (1977) for Eremurus.
Several studies have suggested a relationship between
tapetal cells and PMCs (see Bhandari, 1984; Leitch, 2000).
The association of homologous chromosomes in tapetal
cells has been shown to occur in parallel with pairing of
homologues in PMCs in a wheat cultivar carrying a barley
substitution. Thus, the existence of `di€usable' factors,
originating either from PMCs or from the tapetum, which
a€ect pairing of homologues in both types of cells has been
suggested. These factors seem to be unique since the pairing
of homologous chromosomes has not been observed in
other somatic, dividing tissues, except for the tapetum and
PMCs at the early premeiotic stage and prophase I
(Aragon-Alcaide et al., 1997). Extrapolating these results,
it can be speculated that other factors may also exist that
correlate the pattern of divisions in tapetal cells with PMC
development during prophase I of meiosis. This hypothesis
can be tested by analysing development of the tapeum in
male meiotic mutants arrested in di€erent stages of
prophase I. The presented outline of wild-type Arabidopsis
tapetum polyploidization may be useful for such analyses.
Processes involved in tapetal cell polyploidization
Somatic tissues of Arabidopsis thaliana show high levels
of endopolyploidization (Galbraith et al., 1991). The
number and size of centromeric heterochromatin signals
detected by FISH in interphase nuclei indicated that
endoreduplication is a major process involved in somatic
cell polyploidization in Arabidopsis (Maluszynska and
Heslop-Harrison, 1991). Both endoreduplication and endo-
mitoses were found in callus in vitro culture (Fras and
Maluszynska, 1995). The polyploidization of tapetal nuclei
involves several types of mitotic divisions (reviewed by
Bhandari, 1984; D'Amato, 1984). Analysis of interphase
nuclei does not permit one to draw direct conclusions about
the cell processes involved in polyploidization, however it
does exclude some of them. The increased number but
similar size of FISH-rDNA signals observed in tapetal
nuclei of all ploidy levels suggests that no endoreduplication
takes place during polyploidization. This is in agreement
with analysis of the Eremurus tapetum by Oksala and
Therman (1977).
734 Weiss and MaluszynskaÐCytogenetic Analysis of the Arabidopsis Tapetum
The ®rst division in a secretory tapetum is usually mitosis
not followed by cytokinesis. The doubling of ploidy level
within each of the nuclei in the tapetum after the second cell
division suggests endomitosis or restitution mitosis. This
may also hold true for the third and ®nal cycle which occurs
occasionally in the Arabidopsis tapetum. Endomitosis was
also considered to take place during the second division in
the tapetum of Eremurus (Oksala and Therman, 1977) and
Antirrhinum majus (Berger et al., 1951; Mechelke, 1952).
Thus, development of the tapetum of Arabidopsis follows
the scheme suggested by Oksala and Therman (1977) for
Eremurus, and accepted by many scientists (Bhandari,
1984). The nature of the second and third divisions may be
connected to mitotic spindle disturbance (Oksala and
Therman, 1977; D'Amato, 1984). Additionally, the lack
of chromosome segregation and cytokinesis saves energy
and time. These factors are undoubtedly important for
short-lived and specialized tissues.
The cytogenetic analyses did not enable us to distinguish
between restitution mitosis and endomitosis, since the result
of both is an increase in DNA amount and an increase in
chromosome-speci®c FISH signal number within the
nucleus. Two contradictory hypotheses have been proposed
favouring one or the other process (Oksala and Therman,
1977; Bhandari, 1984; D'Amato, 1984). Stickiness and
clumping of chromosomes in tapetal cells (Berger et al.,
1951) were proposed to favour spindle disturbances
(D'Amato, 1984) and supported the hypothesis that the
last cell cycle is restitution mitosis rather than endomitosis.
Additionally, spindle fusion might be considered as a factor
promoting formation of Arabidopsis tapetal cells with more
than two nuclei having di€erent ploidy levels, as indicated
in other species (D'Amato, 1984).
The mode of tapetum polyploidization does not depend on the
ploidy level of the maternal plant
From the outset, tapetal nuclei of tetraploid plants carry
doubled amounts of DNA and thus copies of genes. During
meiosis in PMCs, the tapetum of both diploid and tetraploid
plants (2n and 4n) undergoes the same number of cell cycles
and does not di€er in the general pattern of polyploidiza-
tion. The ®nal ploidy level of the majority of tapetal cells of
diploid and tetraploid plants is four-times higher than the
plant's ploidy level. This indicates that autopolyploid plants
follow, at least in some aspects, patterns of development
typical of their diploid progenitors. The only detectable
di€erence between tapetal nuclei of diploid and tetraploid
plants concerns the frequency of cells with di€erent patterns
of polyploidization after each division.
An ecient method for the determination of ploidy level in
interphase nuclei of di€erentiated cell types in Arabidopsis
The determination of ploidy level on the basis of number
and size of hybridization signals in interphase nuclei after
FISH with 45S rDNA appeared to be e€ective and
informative, especially for species with small chromosomes
and/or di€erentiated tissues with low mitotic activity.
Despite the tendency for association of 45S rDNA loci in
interphase (Bauwens et al., 1991; Weiss, 1999), the number
of loci was stable in all Arabidopsis accessions analysed
(Fransz et al., 1998), making it possible to distinguish
between di€erent ploidy levels. Therefore, 45S rDNA-FISH
may successfully be used to determine ploidy level in
mixoploid, di€erentiated tissues of Arabidopsis.
AC K N OW L E D G E M E N T S
The study was supported by a grant from the Polish
National Committee of Scienti®c Research, No. 6PO3C
06613. The authors thank Prof. J.S. Heslop-Harrison and
two anonymous referees for valuable comments on a
previous version of the manuscript.
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