Meiotic behaviour and pollen fertility in an open-pollinated population of `Lee' mandarin [Citrus clementina(c. paradisic.

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1 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. Cavalcante a, M.T. Schi no-wittmann b,*, A.L.C. Dornelles a a Departamento de Horticultura e Silvicultura, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Caixa Postal 776, , Porto Alegre, RS, Brazil b Departamento de Plantas Forrageiras e Agrometeorologia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Caixa Postal 776, , 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.: ; fax: address: mtschif@vortex.ufrgs.br (M.T. Schi no-wittmann) /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (00)

2 104 H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103± 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 south latitude and 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

3 H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103± 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 ± 1% Agar ± 15% Sucrose 15% Sucrose 15% Sucrose 15% Sucrose 100 ppm H 3 BO ppm H 3 BO ppm H 3 BO ppm H 3 BO ppm Ca(NO 3 )4H 2 O 1000 ppm Ca(NO 3 )4H 2 O 1000 ppm Ca(NO 3 )4H 2 O 300 ppm Ca(NO 3 )4H 2 O 200 ppm MgSO 4 7H 2 O 300 ppm MgSO 4 7H 2 O 300 ppm MgSO 4 7H 2 O 200 ppm MgSO 4 7H 2 O 100 ppm KNO ppm KNO ppm KNO ppm KNO 3

4 106 H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103± 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 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

5 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 Anaphase telophase Metaphase Anaphase telophase Number of cells analysed Associations a Number of cells analysed b Number of cells analysed Number of cells analysed b II (6), 5II 8I (1) II (13), 7II 4I (1) 32 (3) II (11), 8II 2I (1), 7II 4I (2) II (5), 8II 2I 0 (1) II (11), (41) c 15 (9) 05 (3) II (20), 3II 1IV 8I (1) 16 (5) II (13), 7II 4I (1) 08 (2) II (8), (34) d 05 (3) II (7), 7II 4I(1) 06 (4) (1) II (8), (26) e 04 (2) II (76), 18I (3) 12 (2) All 59 others II 413 (34) (2) 96.0±99.8 Total a No. of cells with each con guration shown in brackets. b 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. H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±

6 108 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

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

8 110 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. < ; ± ; 112; 122; ± ; 076; 077; 080; 081; 120; ± Lee; 023; 025; 028; 030; 031; 034; 038; 043; 044; 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; ± ; 021; 022; 026; 027; 032; 039; 048; 050; 051; 052; 058; 061; 064; 067; 092; 130; 152; 170; 188; 200; 223; 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 hybrid Citrus taxa 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 between Citrus and Poncirus, suggesting a lack of homology of different chromosomes (Iwamasa, 1966), though Raghuvanshi (1962) said this could be due to an early separation of Table 4 Percentage of pollen fertility in 53 plants of a `Lee' mandarin open-pollination population estimated by in vitro germination Pollen fertility (%) No. of plants Percentage of all plants Plant No. < ; 112; ± ; 050; 081; 087; ± ; 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; ± Lee; 022; 025; 026; 038; 043; 049; 069; 078; 094; 099; 110; 130; 152; 170; 202

9 H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103± bivalents. This explanation is also given for the presence of univalents in other genera, for examples, in wild Saccharum species (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 monoembryonic 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 in Actinidia (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% in Leucaena (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 with nˆ9 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

10 112 H.C. Cavalcante et al. / Scientia Horticulturae 86 (2000) 103±114 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 openpollinated 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. References Agarwal, P.K., Cytogenetical investigations in Rutaceae II. Meiotic studies in three intergeneric hybrids of Citrus. Cytologia 52, 757±760. Agarwal, P.K., Cytogenetical investigations in Rutaceae V. Cytomorphology of the three intergeneric hybrids of Citrus and Poncirus. Cytologia 54, 705±708. Agusti, M.F., Almela, V.O., Aznar, M.A., Desarrollo y tamanoä nal del fruto en los agrios. Valencia, pp. 1±71. Burner, D.M., Cytogenetic analyses of sugarcane relatives (Andropogoneae: Saccharinae). Euphytica 54, 125±133. Cameron, J.W., Burnett, R.H., Use of a sexual tetraploid seed parent for production of triploid Citrus hybrids. HortScience 13, 167±169. Cameron, J.W., Soost, R.K., Citrus. In: Moore, J.N., Janick, J. (Eds.), Advances in Fruit Breeding. Purdue University Press, West Lafayette, pp. 507±540. Coelho, Y.S., Tangerina para exportacëaäo: aspectos teâcnicos da producëaäo. EMBRAPA-SPI, 42 pp. (SeÂrie PublicacËoÄes TeÂcnicas FRUPEX, 24).

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