Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol86.Issue2.Oct2000:

Scientia Horticulturae 86 (2000) 103±114

Meiotic behaviour and pollen fertility in an
open-pollinated population of `Lee' mandarin
[Citrus clementina(C. paradisiC. tangerina)]
H.C. Cavalcantea, M.T. Schi®no-Wittmannb,*, A.L.C. Dornellesa
a

Departamento de Horticultura e Silvicultura, Faculdade de Agronomia, Universidade Federal do
Rio Grande do Sul, Caixa Postal 776, 91501-970, Porto Alegre, RS, Brazil
b
Departamento de Plantas Forrageiras e Agrometeorologia, Faculdade de Agronomia,
Universidade Federal do Rio Grande do Sul, Caixa Postal 776, 91501-970,
Porto Alegre, RS, Brazil
Accepted 8 February 2000

Abstract
As part of a mandarin genetic breeding programme in Southern Brazil cytogenetic studies of
meiotic behaviour and estimations of pollen fertility were performed in an open-pollinated
population of `Lee' mandarin [Citrus clementina(C. paradisiC. tangerina)]. For meiotic and
pollen staining analysis ¯ower buds and ¯owers in anthesis were, respectively, examined. In most

plants microsporogenesis was regular, with meiotic indexes over 90.0% and pollen viability over
83.0%. We observed meiotic abnormalities including univalents and stickiness in metaphase I,
laggards and bridges in anaphase and telophase I and II and microcytes at the tetrad stage. Dyads
and triads were found in two plants (112 and 122) at the end of microsporogenesis, suggesting
production of unreduced gametes. These plants had a low pollen viability (22.9 and 24.4%,
respectively) probably due to observed meiotic irregularities. One plant (223) presented pollen
grains with a different shape and bigger size, possibly unreduced. Plants 112 and 122 were
monoembryonic and plant 223 polyembryonic with 3.5 embryos per seed. The potential fertilization
ability of the pollen grains was estimated by in vitro germination tests. Pollen germination was
generally high, up to 91.5%, but eight plants (039, 050, 072, 081, 087, 112, 122 and 126) had pollen
germination of less than 59%. We conclude that most plants have the ability to produce viable
gametes and could be used as pollen donors in programmed crosses. The monoembryonic
unreduced-gamete producer plants 112 and 122 are potential progenitors in crosses aimed at
producing triploid seedless fruits. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Mandarins; Meiotic behaviour; Pollen fertility; Unreduced gametes
*
Corresponding author. Tel.: ‡55-51-316-6037; fax: ‡55-51-316-6045.
E-mail address: mtschif@vortex.ufrgs.br (M.T. Schi®no-Wittmann).

0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 3 9 - 4

104

H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114

1. Introduction
Oranges, limes, lemons, grapefruits and pummelos are classi®ed in three
genera, Citrus, Poncirus and Fortunella (Rutaceae, Aurantoidea) (Agusti et al.,
1995).
Citrus is one of the most important tree crops in Brazil, which is the main
world producer of oranges and ranks four in the production of mandarins, after
China, Spain and Japan (Coelho, 1996). Mandarins are cultivated mainly in the
southern region of Brazil.
The ®rst cytogenetic studies in Citrus indicated a basic chromosome number of
xˆ8. Improved techniques allowed a more precise visualization of the chromosomes and it became clear that most citrus species are diploid (2nˆ18) and
xˆ9 (Frost, 1925, 1938; Longley, 1925; Krug and Bacchi, 1943; Raghuvanshi,
1962; Guerra et al., 1997). Meiotic behaviour in Citrus and associated
interspeci®c and intergeneric hybrids is mainly regular; meiotic irregularities
are infrequent (Raghuvanshi, 1962; Iwamasa, 1966; Agarwal, 1989).

Triploidy is considered to be the simplest way to generate Citrus cultivars with
seedless fruits (Iwamasa and Nito, 1988). Triploids and tetraploids are known in
Citrus and the related genera Poncirus and Fortunella (Krug, 1943), and triploids
formed via unreduced female gametes have been described (Lapin, 1937; Frost,
1938; Esen and Soost, 1971). Triploids have also been identi®ed in zygotic
progenies from diploid crosses (Lapin, 1937; Krug and Bacchi, 1943; Quinn et al.,
1974; Cameron and Soost, 1975). Triploids are readily produced in crosses
between diploids, if a monoembryonic unreduced-gamete producing plant is used
as the female progenitor (Esen et al., 1978).
The aim of this work was to study the meiotic behaviour and pollen fertility of
a `Lee' mandarin open-pollinated population in order to characterize and identify
potential male-fertile progenitors, as well as unreduced-gamete producer plants
which could be employed in the mandarin breeding programme that is being
developed at our institution.

2. Materials and methods
Studies were carried out on 72 plants of an open-pollinated population of 316
plants of `Lee' mandarin cultivar grown at the Agronomy Experimental Station of
Universidade Federal do Rio Grande do Sul, Eldorado do Sul township, Rio
Grande do Sul, Brazil, at 308050 2200 south latitude and 518390 0800 west longitude.

This is a region with a humid subtropical climate. Seventy two plants that
¯owered during 1997 and 1998 were included in this study.
For meiotic analysis, young ¯ower buds (from 2.5 to 4.5 mm length) were
collected between 9:00 a.m. and 10:30 a.m., ®xed in 3:1 ethanol±acetic acid for

105

H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114

24 h and stored in 70% alcohol at about 48C. Anthers were squashed in acetic
carmine and examined. The number of anthers per plant was recorded. All
dividing cells with good chromosome spreading and visibility were analysed.
Special attention was given to chromosome associations at metaphase I and
chromosome disjunction at anaphase and telophase I and II. Meiotic indexes (mi)
were calculated from 400 tetrads per plant in 68 plants according to Love's
(1949) formula: miˆ(number of normal tetrads/total of tetrads)100. Normal
tetrads were considered as those with four equal-sized cells. Percentage of
unreduced gametes (% URG) was calculated according to Yan et al. (1997): %
URGˆ(2D‡Tr)/(2D‡3Tr‡4T ), where D is the number of dyads, Tr the number
of triads and T the number of tetrads.

Pollen fertility was estimated by two methods: ®rstly, uptake of stain by grains
from ¯owers at anthesis ®xed in 3:1 ethanol±acetic acid for 24 h and stored in
70% alcohol at about 48C, and secondly, by in vitro germination of freshly
collected pollen grains. Fertility estimation by staining was performed in all 72
plants, for 1000 pollen grains per plant. Normal, viable, potentially fertile grains
were normal-sized, or double this size in the case of those plants with unreduced
gametes, and fully stained. Partially stained, empty, shrunken, macro and
micropollens were recorded.
In vitro germination was tested on four culture mediums (Table 1). We included
two solid media: M1, from Sahar and Spiegel-Roy (1980), recommended for
Citrus, and M3, from Sahar and Spiegel-Roy (1984), recommended for Persea.
The two liquid media (M2 and M4) were modi®ed from M1 and M3, respectively.
Germination was examined at a controlled temperature of 268C and at room
temperature (approximately 21±238C) and incubation periods of 4, 6 and 10 h.
Four hundred grains per plant were observed.
In the three plants where the potential production of unreduced gametes was
observed, the number of embryos was determined by dissecting the seeds. Six
fruits produced by plants 122 and 223, and one by plant 112 were analysed.

Table 1

Chemical composition of culture media tested for in vitro germination of pollen
Medium 1

Medium 2

Medium 3

Medium 4

1% Agar
15% Sucrose
100 ppm H3BO3
300 ppm Ca(NO3)
4H2O

±
15% Sucrose
100 ppm H3BO3
1000 ppm
Ca(NO3)
4H2O
300 ppm

MgSO4
7H2O
100 ppm KNO3

1% Agar
15% Sucrose
100 ppm H3BO3
1000 ppm
Ca(NO3)
4H2O
300 ppm
MgSO4
7H2O
100 ppm KNO3

±
15% Sucrose
100 ppm H3BO3
300 ppm
Ca(NO3)
4H2O
200 ppm
MgSO4
7H2O
100 ppm KNO3

200 ppm MgSO4
7H2O
100 ppm KNO3

106

H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114

3. Results
3.1. Meiotic behaviour
Seventy plants were analysed for meiotic behaviour (Table 2). Most phases of
meiosis I were found in buds ranging from 2.6 to 3.7 mm length and phases from
the beginning of meiosis II until the end of microsporogenesis were found in buds
ranging from 3.0 to 4.4 mm length. Anther size and number per plant varied;
some anthers were twice the size of the smallest observed whilst number per plant
ranged from 9 to 23.
All plants were diploid (2nˆ18). Most of the plants showed a regular meiotic
behaviour with 9II at metaphase I (Fig. 1a) and a regular disjunction in anaphase
and telophase I and II. However, we also observed meiotic irregularities including
univalents, quadrivalents, laggards, bridges and chromosome stickiness though at

low incidences (Table 2). In plants 026, 048, 069 and 075, the 9II were often
adhered to each other. Notable meiotic irregularities were seen in plants 072, 122
and 112. In plant 072, 41 out of the 52 pollen mother cells had some kind of
abnormality at metaphase I, seven of which had 18 univalents. In plant 112, 34
out of the 42 cells analysed presented abnormalities at metaphase, 14 with 18I.
Irregular metaphase I behaviour was also observed in 26 out of 34 cells of plant
122. Univalents were the most frequent meiotic abnormality at metaphase I.
Bridges and laggards were observed at anaphase and telophase I at very low
levels except for plants 072 and 112 (60%), 109 and 122 (50%) and 120 (67%). In
anaphase and telophase II, abnormalities were seldom found except for plant 072
(60%). In 47% of the plants examined, diploid and tetraploid tapetum cells were
observed.
Only four plants (072, 112, 122 and 153) had a meiotic index less than 90.0%.
The remaining 94.5% of the population (Table 2) can be considered as
meiotically stable according to Love's (1949) de®nition. The abnormal tetrads
showed a varying number of microcytes, up to six in plant 072. Plants 112 and
122 presented dyads and triads at the end of telophase II (Fig. 1b) and the
percentage of unreduced gametes was 11.3 and 8.1%, respectively.

3.2. Pollen fertility

Seventy two plants were analysed for pollen fertility. Plants were divided into
®ve classes (Table 3) according to the percentage of pollen fertility determined by
stainability (Fig. 1c). The range of viability was from 98.0% for plant 058 to
9.9% for plant 072. However, only six plants had a pollen fertility less than 70.0%
and just two under 20.0%. A total of 81.9% of the population had a pollen fertility
greater than 80.0%. Plant 223 had pollen grains with unusual shape and large size

Table 2
Meiotic behaviour and meiotic indexes in 70 plants of a `Lee' mandarin open-pollinated population. (II: bivalents; IV: tetravalents; I: univalents)
Plant No.

Meiosis I

Anaphase‡telophase Metaphase

Anaphase‡telophase

Number of cells Associationsa
analysed

Number of
cells analysedb

Number of
cells analysed

Number of
cells analysedb

11
32 (3)

01

05
03
08

1520

9II
9II
9II
9II
9II
9II
9II
9II
9II
9II
9II
9II

(6), 5II 8I (1)
(13), 7II 4I (1)
(11), 8II 2I (1), 7II 4I (2)
(5), 8II 2I 0 (1)
(11), (41)c
(20), 3II 1IV 8I (1)
(13), 7II 4I (1)
(8), (34)d
(7), 7II 4I(1)
(8), (26)e
(76), 18I (3)

04
15 (9)
16 (5)
08 (2)
05 (3)
06 (4)
04 (2)
12 (2)
413 (34)

68

16
06
09 (1)
05
05
146 (2)

526

71

226

05 (3)

01
01

98.8
98.0
99.2
99.5
38.0
98.5
97.5
59.2
97.8
78.5
71.2
96.0±99.8

No. of cells with each con®guration shown in brackets.
No. of cells with bridges and laggards shown in brackets.
c
11 cells with 9II and 41 cells with abnormal associations: (1II 17I: 1), (7II 4I: 2), (7II 1IV: 3), (2IV 10I: 1), (6II 6I: 1), (8II 2I: 2), (1II 16I: 2), (2II
2IV 6I: 1), (18I: 7), (3II 12I: 4), (4II 2IV 2I: 1), (2II 14I: 10), (1II 16I: 3), (4II 10I: 2), (1IV 1II 12I: 1) shown in brackets.
d
Eight cells with 9II and 34 cells with abnormal associations: (5II 8I: 2), (2II 14I: 5), (18I: 14), (1II 16I: 1), (4II 10I: 5), (3II 12I: 7) shown in
brackets.
e
Eight cells with 9II and 26 cells with abnormal associations: (4II 10I: 2), (5II 8I: 2), (1II 16I: 7), (18I: 3), (2II 14I: 5), (3II 12I: 6), (7II 1IV: 1),
shown in brackets.
b

H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114

a

Meiotic index (%)

Metaphase

034
07
039
14
048
14
062
06
072
52
075
21
078
14
112
42
120
08
122
34
155
79
All 59 others 1229
Total

Meiosis II

107

108

H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114

Fig. 1. Meiotic cells and products in various progeny of `Lee' mandarin: (a) metaphase I with 9II;
(b) dyads at the end of microsporogenesis; (c) fertile (stained) and unfertile (empty) pollen grains;
(d) germinating pollen grains. Scale: 10 mm.

but normal fertility. The size of the pollen grains could indicate unreduced
gametes.
The best in vitro germination of pollen grains was obtained with the M3
medium, with incubation for 6 h at 268C. In vitro germination of pollen grains
was examined in 53 plants, which were grouped into four classes according to the
percentage of germinated grains (Table 4). A total of 30.2% of the plants analysed
had values over 80.0%, and 84.9% over 60.0%. The highest value observed was
91.5% in plants 026 and 151 and the lowest values were found in plants 072

H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114

109

Fig. 1. (Continued ).

(1.5%), 112 (5.05%) and 122 (1.25%). Hypertrophy of the pollen tube was
observed in plants 112 and 122. Plant 075 presented shrunken grains.
There were high positive correlations between the different predictors of male
fertility: 0.86 between meiotic index and pollen stainability, 0.77 between meiotic
index and pollen in vitro germination and 0.80 between pollen stainability and
pollen in vitro germination.
The determination of embryo number per seed showed that plants 112 and 122
are monoembrionyc and plant 223 is polyembryonic with 3.5 embryos per seed.
Therefore, the ®rst two are suitable to be used as female progenitors in
programmed crosses.

110

H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114

Table 3
Percentage pollen fertility in 72 plants of a `Lee' mandarin open-pollinated population estimated by
acetic carmine staining
Pollen
fertility (% )

No. of
plants

Percentage of
all plants

Plant No.