Meiotic behaviour and pollen fertility in an
open-pollinated population of `Lee' mandarin
[
Citrus clementina
(
C. paradisi
C. tangerina
)]
H.C. Cavalcante
a, M.T. Schi®no-Wittmann
b,*, A.L.C. Dornelles
aa
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 Scientia Horticulturae 86 (2000) 103±114
*
Corresponding author. Tel.:55-51-316-6037; fax:55-51-316-6045.
E-mail address: [email protected] (M.T. Schi®no-Wittmann).
0304-4238/00/$ ± see front matter#2000 Elsevier Science B.V. All rights reserved.
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 inCitrusindicated a basic chromosome number of
x8. Improved techniques allowed a more precise visualization of the chro-mosomes and it became clear that most citrus species are diploid (2n18) and
x9 (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 generateCitruscultivars with seedless fruits (Iwamasa and Nito, 1988). Triploids and tetraploids are known in
Citrusand the related generaPoncirusandFortunella(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 3080502200 south latitude and 5183900800west longitude.
This is a region with a humid subtropical climate. Seventy two plants that ¯owered during 1997 and 1998 were included in this study.
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: mi(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): % URG(2DTr)/(2D3Tr4T), whereDis the number of dyads, Tr the number of triads andT 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 forPersea. 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 ± 1% Agar ±
15% Sucrose 15% Sucrose 15% Sucrose 15% Sucrose
100 ppm H3BO3 100 ppm H3BO3 100 ppm H3BO3 100 ppm H3BO3
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 (2n18). 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
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 Meiosis II Meiotic index (%)
Metaphase Anaphasetelophase Metaphase Anaphasetelophase
Number of cells
All 59 others 1229 9II 413 (34) 68 146 (2) 96.0±99.8
Total 1520 526 71 226
a
No. of cells with each con®guration shown in brackets.
b
No. of cells with bridges and laggards shown in brackets.
c11 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.
dEight 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
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
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;
(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.
Fig. 1. (Continued).
4. Discussion
The meiotic abnormalities we found in the `Lee' mandarin population have been previously reported in Citrus and other genera. Agarwal (1987) suggested that the presence of tetravalents in four hybridCitrustaxa indicated homology (or homoeology) among different genomes as well as the absence of large chromosomal differences. Univalents are common meiotic irregularities in
Citrus: Raghuvanshi (1962) found univalents in 17 out of 25 taxa analysed. Up to 18 univalents were detected in intergeneric hybrids betweenCitrusandPoncirus, suggesting a lack of homology of different chromosomes (Iwamasa, 1966), though Raghuvanshi (1962) said this could be due to an early separation of
Table 3
Percentage pollen fertility in 72 plants of a `Lee' mandarin open-pollinated population estimated by acetic carmine staining
049; 057; 065; 069; 074; 078; 087; 094; 099; 100; 108; 109; 110; 113; 115; 121; 126; 129; 131; 143; 150; 153; 154; 201; 202; 204
90±100 23 31.9 019; 021; 022; 026; 027; 032; 039; 048; 050; 051;
052; 058; 061; 064; 067; 092; 130; 152; 170; 188; 200; 223; 251
Table 4
Percentage of pollen fertility in 53 plants of a `Lee' mandarin open-pollination population estimated by in vitro germination
60±79 29 54.7 019; 021; 023; 028; 031; 034; 048; 051; 057; 061;
062; 064; 065; 067; 074; 076; 077; 080; 100; 108; 109; 113;129; 131; 140; 143; 153; 154; 223
80±100 16 30.2 Lee; 022; 025; 026; 038; 043; 049; 069; 078; 094;
bivalents. This explanation is also given for the presence of univalents in other genera, for examples, in wildSaccharumspecies (Burner, 1991). Univalents may lead to unequal segregation at anaphase and consequently a decrease in fertility (Khazanehdari and Jones, 1997). Our studies of meiotic behaviour showed that three plants (072, 112 and 122) had a high frequency of cells with univalents, low meiotic indexes and low pollen fertility.
Our values for meiotic indexes (over 90.0% in 94.1% of the plants) are much higher than those reported by Sellito-Boaventura and Pio (1989) in three cultivars of sweet oranges (75.2 and 56.4%). Pollen fertility was more than 80.0% in most of our plants. Similar values have been reported in Citrus deliciosa cultivars (Donadio et al., 1995).
According to Esen and Soost (1971), most of the spontaneous triploids found in
Citrus are due to unreduced gametes. They pollinated three diploid monoem-bryonic Citrus varieties, known to produce unreduced gametes, with a diploid hybrid. The resulting embryos were triploid with a pentaploid endosperm. Similar data have also been reported by Cameron and Burnett (1978) and Esen et al. (1978).
Unreduced gametes occur in almost all plants at a low rate, normally less than 1.0%. However percentages of up to 10.3% have been reported inActinidia(Yan et al., 1997), up to 28.0% in Lotus tenuis (Negri, 1992), 20.0% in Trifolium pratense(Mousset-DeÂclas et al., 1992) and 12% inLeucaena(Schi®no-Wittmann and Simioni, 1999).
The percentage of unreduced gametes in plants 112 and 122 was 11.3 and 8.1%, respectively. The presence of dyads and triads at the end of telophase II and of cells withn9 at anaphase II suggests that the unreduced grains were probably formed by a second division restitution. The low in vitro germination of pollen grains in these two plants may be explained by the presence of the unreduced grains. According to SusõÂn and AÂ lvarez (1997) unreduced grains normally have low in vitro germination and hypertrophy of the pollen tube.
The formation of unreduced gametes is widely known to be under genetic control and the manipulation of this characteristic may be a useful tool in many breeding projects (Mariani et al., 1992; Ortiz, 1997; Sorensson, 1997).
Considering that: the formation of unreduced gametes is also most probably under genetic control in Citrus; that plants 112 and 122 are monoembryonic; if we accept that a similar production of unreduced gametes also occurs in their female side, plants 112 and 122 are excellent potential progenitors in crosses aiming at developing seedless mandarin cultivars and selection in their progenies would lead to individuals with enhanced production of unreduced gametes.
The large pollen grains seen in plant 223 also hint that this plant produces unreduced gametes. However, no dyads or triads were observed, and it is possible that polyploidization of the gametes occurred at the ®rst pollen grain mitosis. No
abnormalities during the meiotic process were found and meiotic index and pollen fertility were high. As this plant is polyembryonic it would be inef®cient as a parent in crosses to obtain triploids.
5. Conclusions
The population of `Lee' mandarin analysed has a general high male fertility and almost all plants could be used as pollen donors in programmed crosses aiming at combining desirable characteristics.
The two monoembryonic and unreduced-gamete producing plants identi®ed, 112 and 122, are the best potential female progenitors in crosses to produce triploid seedless plants.
Future work should examine the other individuals of this `Lee' mandarin open-pollinated population to identify other plants that could be used as progenitors to generate triploids.
Acknowledgements
We acknowledge Dr. Otto Carlos KoÈller and Dr. SeÂrgio Francisco Schwarz, Departamento de Horticultura e Silvicultura, Universidade Federal do Rio Grande do Sul, Brazil, who established the studied population. We also thank ComissaÄo de AperfeicËoamento de Pessoal de Ensino Superior (CAPES), Brazil, for the ®rst author's studentship during her M.Sc. research and Dr. Eric Boa, CABI Bioscience, UK, for the revision of the English version.
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