64 1. Introduction Seedlessness is a desirable characteristic for both fresh and processed citrus markets (Vardi et al., 2008). In fact, major citrus cultivars on a global level such as ‘Valencia,’ navel orange [Citrus sinensis (L.) Osbeck] and ‘Marsh’ grapefruit (C. paradisi Macfad.) are seedless (Reuther, 1988). Although a major early-maturing citrus, satsuma mandarin (C. unshiu Marcow.), is seedless, almost all mid- and late-maturing citruses, such as natsudaidai (C. natsu- daidai Hayata), iyo (C. iyo hort. ex Tanaka), and hassaku (C. hassaku hort. ex Tanaka), are seedy in Japan (Iwamasa, 1988). Thus, breeding new seedless cultivars is an urgent issue to develop the Japanese citrus industry. To develop seedless citrus at the diploid (2x=18) level, utilization of sterility is essential (Iwamasa, 1966). Steril- ity can be divided into three types: male sterility, female sterility, and self-incompatibility. In addition, selection of triploid (3x=27) individuals is useful for breeding seed- less cultivars (Ollitrault et al., 2007). Therefore, various kinds of investigations on the mechanism and genetic fac- tors underlying seedlessness in citrus have been conducted and many breeding works have been carried out to develop new seedless cultivars, many of them carried out in Japan over the last few decades. In this review, the progress on studies for seedless breeding of citrus in Japan, with regard to 1) seedlessness at the diploid level and 2) ploidy ma- nipulation for seedless breeding, is discussed. 2. Seedlessness at the diploid level Male sterility The degree of male (pollen) sterility is variable in citrus, and usually pollen sterile accessions produce seedless or low-seedy fruits when cultivated in solid blocks. Male ste- rility couples with parthenocarpy to produce seedless fruits when cross-pollination is prevented. Even in mixed planting with pollen fertile accessions, male sterility reduces seed production and increases the percentage of seedless fruits because those accessions have a smaller chance of fertiliza- tion than male fertile ones (Yamamoto et al., 1993, 1995). Iwamasa (1966) and Ollitrault et al. (2007) summarized the various levels of male sterility at the diploid level in cit- rus (Table 1). Chromosome aberration was one of the most important phenomena causing pollen sterility. Asynapsis in ‘Mukaku Yuzu’ (C. junos Sirbold ex Tanaka) is geneti- cally controlled, while that in ‘Eureka’ lemon (C. limon (L.) Burm. f.) and ‘Mexican’ lime (C. aurantifolia (Cristm.) Swingle) is induced by low temperature (Nakamura, 1943; Iwamasa and Iwasaki, 1962; Iwamasa, 1966). Reciprocal translocation is found to cause pollen sterility of ‘Valencia’ orange (C. sinensis) (Iwamasa, 1966). Inversion is the cause of partial pollen sterility of ‘Mexican’ lime (C. aurantifo- lia) (Iwamasa, 1966). Male sterility that is not caused by chromosome aberration is also well known. Anther abor- Progress on studies for seedless breeding of citrus in Japan M. Yamamoto(*) Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan. Key words: female sterility, male sterility, protoplast fusion, self-incompatibility, triploid. Abstract: Seedlessness is a desirable characteristic for both fresh and processed citrus markets and one of the most im- portant breeding objectives. In this paper, progress on studies for seedless breeding of citrus in Japan is reviewed. Among the several types of male sterility, anther abortion is the strictest male sterility in citrus and was shown to be controlled by both nuclear and cytoplasmic genes. Several seedless cultivars with male sterility have been developed. The mechanism and inheritance of the strictest female sterility derived from ‘Mukaku Kishu’ (Citrus kinokuni hort. ex Tanaka) were clarified and seedless cultivar and parental lines with this female sterility have been released. Some self-incompatible cultivars show seedlessness when coupled with parthenocarpy. S (self-incompatibility) genotypes of several cultivars have been estimated. Tetraploid plants, as parents of triploid offspring, were obtained from nucellar seedlings, and by ploidy mutation, colchicine treatment, and protoplast fusion. Triploid plants were produced from the combination of not only tetraploid and diploid crosses but also diploid and diploid crosses. New triploid seedless cultivars were bred by pro- grammed cross-breeding and protoplast fusion. Adv. Hort. Sci., 2014 28(2): 64-72 (*) Corresponding author: yamasa@agri.kagoshima-u.ac.jp Received for publication 31 March 2014 Accepted for publication 17 June 2014 Review paper 65 tion in satsuma mandarin (C. unshiu) hybrids is the strictest male sterility in citrus (Iwamasa, 1966). The sterile stamen appears only as the filament, and no pollen grains are pro- duced (Fig. 1). Male sterility of ‘Washington’ navel (C. si- nensis), ‘Tahiti’ lime (C. latifolia Tanaka), and some other hybrids is due to early degeneration of pollen mother cells (PMCs) (Osawa, 1912; Uphof, 1931; Frost, 1948; Iwamasa, 1966). Pollen sterility of satsuma mandarin (C. unshiu) is caused by plural sterility such as abnormal behavior and degeneration of pollen grains (Nakamura, 1943; Yang and Nakagawa, 1969, 1970). To develop new seedless cultivars efficiently, genetic analysis of male sterility has been conducted. Among these studies, genetic analysis of anther abortion has pro- gressed remarkably. This male sterility is due to gene- cytoplasmic interaction [satsuma mandarin (C. unshiu), ‘Encore’ mandarin (C. nobilis Lour. × C. deliciosa Ten.), yuzu (C. junos), and lemon (C. limon) possess sterile cy- toplasm] and is probably controlled by more than one major gene (Iwamasa, 1966; Yamamoto et al., 1992 a, b, 1997; Nakano et al., 2001; Dewi et al., 2013 a). Dewi et al. (2013 a) postulated that a dominant nuclear fertility- restoring gene system comprising one epistatic gene and two complementary genes controls the restoration of male Table 1 - Diagramatic representation of various kinds of the male-sterility in citrus, according to the sequential order of development (Modified from Iwamasa, 1966) Developmental stage Nature of sterility Cultivar or hybrid Reference Initiation of anther development Anther abortion Satsuma mandarin × Sweet orange, etc. Iwamasa, 1966 Archesporial stage Resting stage Degeneration of PMCs Washington Navel Tahiti lime Lemon × Valencia, etc. Satsuma mandarin × Trifoliate orange Osawa, 1912 Uphof, 1931 Frost, 1948 Iwamasa, 1966 Meiosis I-division Asynapsis (genic) Asynapsis (by low temp.) Translocation Inversion Mukaku Yuzu Eureka lemon Mexican lime C. assamensis Valencia orange C. assamensis, etc Mexican lime Iwamasa, 1966 Nakamura, 1943 Iwamasa and Iwasaki., 1962 Naithani and Raghuvanshi, 1958 Iwamasa, 1966 Raghuvanshi, 1962 a Iwamasa, 1966 II-division Failure of spindle Marsh grapefruit Raghuvanshi, 1962 b Liberation from tetrad Degeneration Jaffa orange Oppenheim and Frankel, 1929 Mitotic division Degeneration Satsuma mandarin Nakamura, 1943 Mature pollen grain Fig. 1 - Flowers of male-sterile (aborted anthers, MS) and male-fertile (normal anthers, MF) citrus. 66 fertility and male-sterile anther size in citrus plants with sterile cytoplasm. Nakano et al. (2000) found DNA mark- ers linked to aborted anther for juvenile screening of male- sterile plants. Male-sterile (aborted anther) progenies were also determined at the early period of seedling growth to exhibit precocious flowering, a phenomenon in which very young seedlings have flowers (Dewi et al., 2013 b). Another type of male sterility, inheritance of pollen fertil- ity/sterility, was also studied (Ueno, 1986). Some pollen- sterile progenies arose from two pollen fertile parents. In citrus, not only somatic hybrids but also cybrids were produced by symmetric protoplast fusion (Saito et al., 1993; Moriguchi et al., 1996; 1997; Tokunaga et al., 1999). Yamamoto and Kobayashi (1995) produced a cy- brid having the sweet orange (C. sinensis) nuclear genome and satsuma mandarin (C. unshiu) cytoplasmic genome by fusion between satsuma mandarin protoplasts isolated from embryogenic callus and sweet orange mesophyll pro- toplasts. The cybrid is useful for seedless breeding because it has sterile cytoplasm derived from satsuma mandarin. Several new seedless cultivars with male sterility were released in Japan (Nishiura et al., 1983; Okudai et al., 1991; Matsumoto et al., 1991, 2003; Yoshida et al., 2005 c) (Fig. 2, Table 2). All cultivars with aborted anther possess cytoplasm derived from satsuma mandarin (C. unshiu). Female sterility Female sterility is a very important trait which is close- ly related to seedlessness. Yamamoto et al. (1995) reported that the degree of female fertility/sterility is rated on the basis of the average number of seeds per fruit obtained through hand pollination. A high positive correlation (r = 0.93**) was found between the number of seeds of hand- pollinated fruits and that of open-pollinated fruit. This result indicated that female sterility is directly related to seediness. Female sterility estimated by the above-men- tioned method was revealed to be a heritable characteristic (Yamamoto et al., 2001). Table 2 - Seedless cultivars and parental lines of citrus released in Japan Cultivar or Parental line Cross combination Note Male sterility Kiyomi Miyagawa wase (Citrus unshiu) × Trovita (C. sinensis) Seiho Kiyomi × Minneola (C. paradisi × C. tangerina) Tsunokaori Kiyomi × Okitsu wase (C. unshiu) Amaka Kiyomi × Encore (C. nobilis × C. deliciosa) Setoka (Kiyomi × Encore) No. 2 × Murcott (probably tangor) Harehime E647 (Kiyomi × Osceola) × Miyagawa wase (C. unshiu) Tsunokagayaki (Kiyomi × Okitsu wase) No. 14 × Encore (C. nobilis × C. deliciosa) Female sterility Southern Yellow Tanikawa Buntan (C. maxima) × Mukaku Kishu (C. kinokuni) Citrus parental line Norin No. 5 Lee (Clementine × Orlando) × Mukaku Kishu (C. kinokuni) Citrus parental line Norin No. 6 King mandarin (C. nobilis) × Mukaku Kishu (C. kinokuni) Self-incompatibility Ariake Seike navel (C. sinensis) × Clementine (C. clementina) Triploid Puchimaru Oval kumquat (Fortunella margarita) × tetraploid Meiwa kumquat (C. crassifolia) White Love Cybrid having lemon (C. limon) Somatic hybrid nuclear and sudachi (C. sudachi) cytoplasmic genome + Haploid clementine (C. clementina) Tokushima 3X No. 1 Tetraploid sudachi HS4 (C. sudachi) × sudachi Ryokuko-kei (C. sudachi) Yellow Bell Open-pollinated seedling of diploid Michitani-line Villafranca (C. limon) Fig. 2 - Male-sterile seedless citrus ‘Setoka’. 67 ‘Mukaku Kishu’, a bud variant of the seedy kinokuni mandarin (Citrus kinokuni hort. ex Tanaka), is completely seedless and considered to have the strictest female sterility in citrus. Yamasaki et al. (2007, 2009) studied the mecha- nism of expression of seedlessness derived from ‘Mukaku Kishu’. In fruits of ‘Mukaku Kishu’, specific very small and swollen seeds called “type A seeds” were observed. The ex- pression of ‘Mukaku Kishu’-type seedlessness is character- ized by formation of “type A seed” with an immature seed coat and an embryo arrested at an early stage. However, arrested embryo development in the “ type A seed” is not caused by endosperm abortion. This female sterility is con- trolled by two major genes: sterility and fertility are domi- nant and recessive, respectively (Nesumi et al., 2001). New seedless cultivar and parental lines with this sterility were bred in Japan (Yoshida et al., 2005 a, b) (Fig. 3, Table 2). Navel orange (C. sinensis) and satsuma mandarin (C. unshiu) have strong female sterility; only a few seeds were developed when they were hand-pollinated (Miki, 1921; Nagai and Tanikawa, 1926; Nishiura and Iwasaki, 1963; Ya- mamoto et al., 1995). Osawa (1912) observed degeneration of the embryo sac in both navel orange and satsuma manda- rin. Nesumi et al. (2000) assumed that the female sterility of satsuma mandarin is controlled by two major genes: sterility and fertility are recessive and dominant, respectively, and they were mapped on a linkage map (Omura et al., 2000). Self-incompatibility Self-incompatibility is a genetically controlled phenom- enon preventing seed set in self-pollinated plants producing functional gametes. Self-incompatibility in citrus is a very important trait for fruit production. Without parthenocarpy, it requires cross pollination to achieve stable fruit produc- tion (Nagai and Tanikawa, 1926; Miwa, 1951). However, its coupling with parthenocarpy could produce seedless fruit (Iwamasa and Oba, 1980; Yamamoto et al., 1995; Yama- moto and Tominaga, 2002). Thus, much research has been conducted to determine self-incompatibility of many acces- sions (Nagai and Tanikawa, 1926; Miwa, 1951; Nishiura and Iwasaki, 1963; Iwamasa and Oba, 1980; Yamamoto and Tominaga, 2002; Yamamoto et al., 2006, 2012). The incompatibility system of citrus is of the gameto- phytic type and Soost (1965, 1969) proposed S (self-in- compatibility) genotypes of some accessions. Since then, the source of S genotypes has been less well elucidated. Recently, however, research has progressed remarkably via certain methods. Pollen tube growth was strongly in- hibited in incompatible pollination; those pollen tubes ex- hibited abnormal behaviors, namely twisted and heavy and irregular callose deposition. On the other hand, in com- patible pollination, many normal pollen tubes penetrated into the style (Ngo et al., 2001). Self-incompatibility S genotypes of several citrus cultivars were estimated by the observation of pollen tube behavior in the styles after controlled pollination with a restricted number of pollen grains on their stigmas (Ngo et al., 2010). Cross-incom- patible, cross-semi-compatible, and cross-full-compatible relationships were clarified based on the results of the number of pollen tubes that reached the base of the style. From these results, S genotypes of several cultivars were estimated. The S genotype could be estimated with the aid of allozymes produced by the glutamate oxaloacetate transaminase isozyme gene (Got-3), which appeared to be linked to the S gene (Ngo et al., 2011). The most efficient way to determine the S genotype is considered to be pol- lination with pollen homozygous for the S genotype. Kim et al. (2010, 2011) revealed the S genotype of some culti- vars by pollination of homozygous S 1 seedlings of ‘Hirado buntan’ [C. maxima (burm.) Merr.] and ‘Banpeiyu’ (C. maxima). There are no differences in estimated S geno- types among the three above-mentioned methods. Table 3 shows the estimated S genotypes of several accessions. Incompatibility S alleles are distributed widely, not only in self-incompatible accessions but also self-compatible ones such as satsuma mandarin (C. unshiu), grapefruit (C. paradi- si), and ‘Dancy’ (C. tangerina hort. ex Tanaka) (Soost, 1965, 1969; Vardi et al., 2000). Thus, self-incompatible individuals can be produced from cross combinations between two self- Fig. 3 - Female-sterile seedless citrus ‘Southern Yellow’. Table 3 - Estimated S genotype of citrus accessions (Kim et al., 2011; Ngo et al., 2010, 2011) Accession Latin name Estimated S genotype (z) Banpeiyu Citrus maxima (Burm.) Merr. S 1 S 2 Tosa Buntan C. maxima (Burm.) Merr. S 1 S 3 Iriki Buntan C. maxima (Burm.) Merr. S 1 S 2 Kaopang C. maxima (Burm.) Merr. S 1 S 2 Soyu C. maxima (Burm.) Merr. S 1 S 2 Hassaku C. hassaku hort. ex Tanaka S 4 S 5 Yuge-hyokan C. yuge-hyohan hort. ex Yu. Tanaka S 6 S 7 Shishiyuzu C. pseudogulgul hort. ex Shirai S 1 S 6 Hyuganatsu C. tamurana hort. ex Tanaka S 1 S 8 Tachibana No. 1 C. tachibana (Makino) Tanaka SfS 8 Rough lemon C. jambhiri Lush. SfS 1 Zadaida C. aurantium L. SfS 1 Kinukawa C. glaberima hort. ex Tanaka SfS 2 Kawano Natsudaidai C. natsudaidai hort. ex Tanaka SfS 2 (z) Sf: self-compatible. 68 compatible parents, for example, ‘Orlando’ and ‘Minneola’ arose from ‘Duncan’ grapefruit and ‘Dancy’ combination (Swingle et al., 1931). In Japan as well, self-incompatible seedless ‘Ariake’ was bred by crossing self-compatible ‘Seike’ navel orange (C. sinensis (L.) Osbeck) and self-in- compatible clementine (C. clementina hort. Tanaka) (Yama- da et al., 1995; Yamamoto et al., 2006) (Fig. 4, Table 2). 3. Ploidy manipulation for seedless cultivar breeding Although spontaneous triploid (3x=27) accessions were very rare in citrus (Krug, 1943; Krug and Bacchi, 1943; Noro and Kajimoto, 1955), many triploid hybrids have been produced by artificial hybridization. Since these triploids are seedless, it could be considered that producing triploids is a useful way to promote seedless breeding in citrus efficiently. Tachikawa et al. (1961) conducted one of the earliest pro- grammed triploid breeding projects. First, they produced tet- raploid materials (4x=36) by colchicine treatment and then triploid (2x=18) hybrids were obtained from these tetraploid and diploid cross combinations. As they showed, since trip- loids arise from tetraploid and diploid crossing, tetraploid plants are important for triploid breeding. Therefore, various tetraploid accessions were obtained by certain methods. Oiyama et al. (1980) selected spontaneous autotetra- ploids from nucellar seedlings of polyembryonic cultivars. They revealed the leaf morphological characteristics of tet- raploids: thick and broad leaves and reduced number of sto- mata per area. Kawase et al. (2005) obtained Meiwa kum- quat (Fortunella crassifolia Swingle) autotetraploid from 500 seedlings. An autotetraploid also arose as a bud sport (Yamao et al., 1993). Colchicine treatment is useful for the production of autotetraploids in many higher plants. In cit- rus in particular, various tetraploids were produced by this treatment. Colchicine treatment of seeds was effective in polyembryonic cultivars (Yahata et al., 2004). However, this treatment of monoembryonic seeds is a problem because the tetraploids obtained by this treatment are not true-to-type. Oiyama and Okudai (1986) resolved this problem through a combination of colchicine treatment of isolated small buds and their micrografting. They successfully produced auto- tetraploids from three monoembryonic cultivars. Moreover, in another eight autotetraploids, monoembryonic citrus was produced using the same method (Kaneyoshi et al., 2008). It has been revealed that although tetraploids arise from both diploid × tetraploid and tetraploid × diploid crosses, the latter combination is more effective (Cameron and Burnett, 1978; Kaneyoshi et al., 2008) and an unbalanced ploidy ra- tio between embryo and endosperm is considered to cause this phenomenon (Esen and Soost, 1973). Thus, artificially produced monoembryonic autotetraploids were important as seed parents for triploid breeding. On the other hand, triploid hybrids sometimes appeared from diploid-diploid crosses (Esen and Soost, 1971; Oiyama and Okudai, 1983; Yasuda et al., 2010); the appearance of triploids is due to the unre- duced gametophyte of one parent (Esen and Soost, 1971). Biotechnological methods such as protoplast fusion have contributed to the progress of citrus triploid breeding. Since somatic hybrids are tetraploids in general (Ohgawara et al., 1985), they are important parents for triploid breed- ing. Somatic hybrids derived from crosses between navel orange (C. sinensis) + satsuma mandarin (C. unshiu), grape- fruit (C. paradisi), yuzu (C. junos) and ‘Murcott’ (artificial hybrid) were registered as parental lines (Kobayashi et al., 1995). Triploid hybrids could be produced directly by means of protoplast fusion. Somatic hybrids produced from diploid and haploid fusion became triploids (Kobayashi et al., 1997). The haploid parents were obtained by diploid × triploid cross (Oiyama and Kobayashi, 1993). Table 2 shows the triploid cultivars released in Japan. Among the four cultivars, ‘Puchimaru’ (Yoshida et al., 2003) (Fig. 5), ‘White love’, Tokushima 3X No. 1’ (Toku- Fig. 5 - Triploid seedless kumquat ‘Puchimaru’ and diploid seedy Meiwa kumquat and Oval kumquat. P= Puchimaru, M= Meiwa kumquat, and O= Oval kumquat. Fig. 4 - Self-incompatible seedless citrus ‘Ariake’. 69 naga et al., 2005), and ‘Yellow Bell’ (Kaneyoshi et al., 2014) were derived from diploid × tetraploid cross, pro- toplast fusion between diploid and haploid, tetraploid × diploid cross, and diploid × diploid cross, respectively. 4. Conclusions Seedless breeding of citrus has progressed rapidly in Ja- pan over the last few decades. The production of new seed- less cultivars is increasing in contrast to the decrease in production of seedy conventional cultivars such as ‘Kawa- no Natsudaidai’ (C. natusdaidai) and ‘Miyauchi Iyokan’ (C. iyo). Various kinds of cross combinations using cul- tivars or parental lines with sterility have been conducted actively and further polyploid breeding is being carried out to produce new triploid plants. In addition, methods to shorten the long juvenile period of citrus have developed in Japan (Okudai et al., 1980; Mitani et al., 2008), making it easier to breed, compared to a few decades ago, various types of new seedless cultivars. Understanding the mechanism and hereditary mode of each type of sterility will contribute to produce seed- less individuals efficiently and effectively. Biotechnologi- cal techniques are also very useful for producing seedless materials. The results of genome analysis have provided useful information for breeding new seedless citrus with female sterility (Garcia et al., 2000). Moreover, owing to the progress of recent DNA analysis technologies, the draft whole genome of sweet orange (C. sinensis) has already been reported (Xu et al., 2013). 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