J. Japan. Soc. Hort. Sci. 64 (1) : 23-29. 1995.

Relationship between Sterility and Seedlessness in Citrus

Masashi Yamamoto, Ryoji Matsumoto and Yoshio Yamada* Kuchinotsu Branch, Fruit Tree Research Station, Kuchinotsu, Nagasaki 859 -25

Summary The relationship of female and male sterility, and self-incompatibility to seed content in citrus were investigated using 22 male sterile and fertile cultivars. The male fertile culti- vars were grouped into self-compatible and self-incompatible. Positive correlation (r = 0.927**, r2=0.859) existed between the average number of seeds per fruit obtained by hand pollination, which indicated the degree of female fertility, and that yielded by open pollination. This result indicates that the degree of female fertility and sterility can be estimated from seeds derived by open pollination. The difference between the average number of seeds per fruit by hand pollination and that by open pollination was greater in male sterile cultivars and in self-incompatible cultivars than it was in self-compatible cul- tivars. This indicates that self-incompatibility as well as male sterility is effective on the reduction of seediness. In open pollination, self-compatible cultivars produced very few seedless fruits, whereas male sterile and self-incompatible cultivars produced many seed- less fruits.

Hanaori (1990) who investigated male sterility Introduction and fertility and seediness of progenies using Seedlessness is a desirable characteristic for satsuma mandarins, 'Kiyomi' and 'Sweet Spring' as both fresh and processed citrus market, thus, it is parents. These results suggest that not only male a major breeding objective. Among the breeding sterility but also female sterility and self-incom- methods for developing seedless cultivars (Spiegel- patibility play important roles in developing seed- Roy, 1988) are the production of triploid hybrid less cultivars. 'Mukaku Kishu' is completely seedless under seedlings (Soost and Cameron, 1969) and creating mutants by irradiation (Hearn, 1986). any conditon. Navel orange and satsuma mandarin While cross breeding to develop diploid seedless are considered to be seedless, but occasionally cutlivars, we found a close relationship in seediness they produce a few seeds when pollinated. On the between hybrid progenies and parents; seedless or other hand, with cross pollination a fruit of some few seeded parents frequently produced seedless pummelos produces more than one hundred seeds. offsprings, whereas seedy or very seedy parents Female fertility and sterility in Citrus vary with produced very few seedless offsprings (Yamamoto the cultivars (Frost, 1948). The close relationship et al., 1992b). Furthermore, the percentage of between female fertility and seediness has long seedless progenies was higher in male sterile prog- been recognized, but there are a few reports which enies than in male fertile progenies in the same attribute the degree of female fertility to seediness. 'Kiyomi' and 'Egami Buntan' (C cross combination (Yamamoto et al., 1993). . grandis) are However, not all male sterile progenies were seed- normally seedless, as a result of male sterility and less and some male fertile progenies were seedless self-incompatibility, respectively (Iwamasa and (Yamamoto et al., 1993). Similar results were Ohba, 1980; Nishiura et al., 1983). Furthermore, reported by Yoshida (1982) and Yoshida and seediness was less in male sterile progenies than in male fertile progenies in the same cross com- Received for publication 24 March 1994. bination (Yamamoto et al., 1993). However, the Contribution No. D-123 of the Fruit Tree Research Station. *Present address: Okitsu Branch effect of male sterility and self-incompatibility on , Fruit Tree Research the reduction of seediness under open field condi- Station, Okitsu, Shimizu, Shizuoka 424-04.

23 24 M. Yamamoto, R. Matsumoto and Y. Yamada

tions where they are surrounded with much pollen 1956; Takahara et al., 1982; Yamada, 1988). from fertile cultivars has never been clarified. On May 9, 1990, 50 to 100 flowers on one tree In the present study, relationship between of each cultivar were hand-pollinated with 'Hyuga- female sterility and seedlessness, and effects of natsu', which is self-incompatible (Miwa, 1951); it male sterility and self-incompatibility on the was pollinated with 'Kawachi Bankan'. The flow- reduction of seediness under open pollination were ers were not emasculated or enclosed in bags to investigated using many cultivars. prevent open pollination. Fruits from hand and 30 to 100 open pollinated fruits were harvested from Materials and Methods each cultivar on November 16 and their seed con- Bearing trees of 22 citrus cultivars grafted on tents counted. trifoliate orange growing in the Kuchinotsu The degree of female fertility was rated on the Branch, Fruit Tree Research Station were used. basis of the average number of seeds per fruit According to pollen yield and viability obtained obtained through hand pollination. under field conditions, all cultivars were divided Results into two groups; male fertile and male sterile. The 'Hayasi Unshiu' male fertile cultivars were divided into two , 'Okitsu Wase', 'Kiyomi' and 'Shirayanagi Navel' belonged to the male sterile groups; self-compatible and self-incompatible according to previous works (Hearn et al., 1969; group because these cultivars produced very few Ito and Ueno, 1986; Mishima and Inaba, 1971; or no pollen. 'T132 Tankan' produced pollen with Miwa, 1951; Mustard et al., 1956; Nishiura and low viability (26.8%). Among 18 male fertile culti- Iwasaki, 1963; Reece and Register, 1961; Soost, vars, 10 were self-incompatible (see Materials and

Table 1. Male fertility, pollen viability and self-compatibility of citrus cultivars. J. Japan. Soc. Hort. Sci. 64 (1) : 23-29. 1995. 25

Methods) and 8 were self-compatible. Consequent- yanagi Navel'; 90 in 'Hayashi Unshiu'; and 44 in ly, of 22 cultivars, 4 were male sterile; 8 self- 'Kiyomi' . Out of 10 open pollinated self-incompati- compatible group; 10 self-incompatible group ble cultivars, 7 produced seedless fruits; among (Table 1). which 'Page' had 67%. On the other hand, among The average number of seeds per fruit by open the 8 open pollinated self-compatible cultivars pollination ranged from 0 to 2.7 in all male sterile only 3 produced seedless fruits, but their range of cultivars. In 'T132 Tankan' and 2 self-incompati- seedless fruits were from 1 to 25% (Table 2). ble cultivars; 'Page' and 'Robinson', the seed count The relationship between the average seed num- ranged from 1.6 to 2.1; whereas in 'Kara', ber of open and hand pollinated revealed a posi- 'Trovita' , 'Fukuhara', Ponkan and Clementine, the tive coefficient of correlation, r = 0.927 ** (Fig. range was from 4.2 to 7.3. In two cultivars, 'Hira- 1). However, this coefficient of correlation was do Buntan' and 'Tosa Buntan', the seed counts strongly affected by two very seedy cultivars, 17: 'Tosa Buntan' and 18: 'Hirado Buntan' were 61.9 and 46.1, respectively (Table 2). . A coef- All open pollinated male sterile cultivars pro- ficeint of correlation derived by excluding these duced very high percentage of seedless fruits, cultivars was r=0.788** ranging from 100 in 'Okitsu Wase'; 91 in 'Shira- The ratio of the average seed number from open

Table 2. Number of seeds per fruit and percentage of seedless fruits of open pollination, and number of seeds per fruit and percentage of seedless fruits of hand pollination. 26 M. Yamamoto, R. Matsumoto and Y. Yamada

Fig. 1. Relationship between seed number of hand pollination and open pollination. Cultivar No. (1-22): same as Table 1. Solid line is obtained including cvs. No. 17 and 18, while broken line is obtained excluding cvs. No. 17 and 18.

pollination to that of hand pollination (OP/HP), ex- group; it was lower in the self-incompatible group pressed as a percentage in the self-compatible than in the self-compatible group. group, 1, 4 and 3 cultivars revealed ranges from In male sterile and self-incompatible groups, a 40.0~59.9%, 60.0~79.9% and 80.0~99.9%, re- negative correlation existed between the percent- spectively (Fig. 2). In the self-incompatible group, age of seedless fruits derived from open pollina- 2, 4, 3 and 1 cultivars, the ratio ranges from 0.0 tion and the OP/HP percentage (Fig. 3). — ~19.9%, 20.0~39.9%, 40.0~59.9% to 60.0~ 79.9%, respectively. In the 4 male sterile culti- vars, the ratio ranged from 0.0 to 19.9%. The OP/HP ratio was the lowest in the male sterile

Fig. 3. Relationship between percentage of seed- less fruits of open pollination and percentage of seed number of open pollination to that of hand pollinations in self-incompatible and Fig. 2. Distribution of percentage of seed number of open male sterile cultivars. Cultivar No. (9-22): pollination to that of hand pollination. same as Table 1. J. Japan. Soc. Hort. Sci. 64(1) : 23-29. 1995. 27

smaller chance of fertilization than have the self- Discussion compatible cultivars because the former needs The role of male sterility which is very actively compatible pollen to produce seeds. Thus, a great- investigated in citrus, has resulted in seedless er difference was observed between seed number Japanese cultivars (Matsumoto et al., 1991; of hand pollination and open pollination in male Nishiura et al., 1983). Results of cross pollination sterile and self-incompatible cultivars. Consequent- studies revealed the degree of female fertility and ly, male sterility and self-incompatibility reduce sterility vary greatly among cultivars (Nagai and seediness and increase the percentage of seedless Tanikawa, 1926; Nishiura and Iwasaki, 1963). fruits. However, in some self-incompatible culti- The mechanism of self-incompatibility is also vars, the percentage of OP/HP is as high as that investigated by. Yamashita and Iwanaga (1984). of self-compatible cultivars. It is supposed that in The close relationship between these sterility and those self-incompatible cultivars, the number of seedlessness has been recognized, but there are seedless fruits probably decreased because they few studies which deal with the relationship using do not set parthenocarpically and set only seedy many cultivars. By using 22 cultivars in this fruits. In most cases, the difference 'between seed study, it becomes apparent that female sterility, count from hand pollination and open pollination male sterility and self-incompatibility influence was generally greater as the percentage of seed- seedlessness in fruits derived from open pollinated less fruits by the open pollination was higher in flowers. male sterile and self-incompatible cultivars. Cer- Since seed formation in citrus fruits is affected tainly, parthenocarpy is indispensable because any by both genetic and environmental factors, seedi- cultivar which is both male and female sterile ness varies even in the same cultivar depending on would be incapable of producing seedless fruit. time and planting area. The environmental effects Seedless cultivars such as 'Hayashi Unshiu', 'Okitsu Wase' and 'Shirayanagi Navel' are both on seediness was not clarified in this study be- cause our data were obatined only in one year and female and male sterile. 'T132 Tankan' is female at one place. It is clear, however, that among genetic sterile to a similar degree as the above cultivars, factors influencing seediness, female fertility is but it is self-compatible in spite of low pollen directly related to seediness. Positive correlation viability. Thus, open pollinated fruits of 'T132 (r = 0.927**, r = 0.788**) was found between Tankan' is seedier than those of 'Hayashi Unshiu', 'Okitsu Wase' and 'Shirayanagi Na seed number of hand pollination and that of open vel'. 'Kiyomi', pollination. This indicates that the degree of which is considered to be a seedless cultivar, is female fertility can be estimated from the seed completely male sterile (Nishiura et al., 1983). number of open pollination. Previously (Yamamoto However, seediness of 'Kiyomi' often increases et al., 1993), positive correlation (r = 0.722**) when cross pollinated by male fertile cultivars be- was found between the average number of seeds cause its degree of female fertility is high. Conse- per fruit in hybrids and their parents. Results quently, female sterility coupled with male steril- from this study and previous reports show that ity or self-incompatibility is essential to the female fertility and sterility are heritable charac- development of seedless cultivars. teristics. Thus, we must pay attention to female The results obtained in this study indicate a sterility in the breeding of seedless cultivars. close relationship between sterility and seedless- In all cultivars, average number of seeds per ness. Further investigations to elucidate inherit- fruit was greater by hand pollination than by open ance patterns of sterility are needed to develop pollination. In addition, the percentage of OP/HP seedless cultivars efficiently. Several studies on was smaller in self-incompatible cultivars and inheritance of male sterility have been made (Iwa- male sterile cultivars than it was in self-compati- masa, 1966; Ueno, 1986; Vardi and Spiegel-Roy, ble cultivars. The lower seed number in open pol- 1981; Yamamoto et al., 1992a), and the results of lination than in hand pollination indicates that a which have been applied in selection of parents. low order of pollination occurs under open field Although there is a close relationship between conditions compared with hand pollination. The female sterility and seedlessness, comparatively male sterile and self-incompatible cultivars have a little is known about the inheritance of female 28 M. Yamamoto, R. Matsumoto and Y. Yamada sterility (Nesumi et al., 1992). The inheritance (In Japanese). pattern of self-incompatibility has already been re- Miwa, T. 1951. Pollination, fertilization, and fruit drop in Citrus tamurana Hort. Bull. Miyazaki Univ. ported, e.g., some self-compatible cultivars such as (Natural Science) 2 : 1-67. (In Japanese with grapefruit and 'Dancy' tangerine carry a self- English summary). incompatible gene (Soost, 1965). However, the dis- Mustard, M. J., S. Y. Lynch and 0. Nelson. 1956. Pol- tribution of the self-incompatible gene is not well lination and floral studies of the Minneola tangelo. known. Parthenocarpy is necessary for developing Proc. Fla. State Hort. Soc. 69 : 277-281. seedless cultivars; very little information regard- Nagai, K. and T. Tanikawa. 1926. On citrus pollina- ing its inheritance pattern is available (Ueno, tion. Proc. Third Pan-Pacific Soc. Congress 1976). Hence, further investigation must be con- 2023-2029. ducted to elucidate the inheritance of the sterility Nesumi, H., Y. Ito, T. Yoshioka and T. Yoshida. 1992. system and the parthenocarpy. Female sterility of 'Mukakukishu' and its inherit- ance. J. Japan. Soc. Hort. Sci. 61 (Suppl. 1): 36-37. Acknowledgments (In Japanese). Nishiura, M. and T. Iwasaki. 1963. Studies on the cit- We thank trainees of Kuchinotsu branch, Fruit rus breeding. I. Variation of seed formation in cit- Tree Research Station for their assistance and also rus crossing. Bull. Hort. Res. Sta. B2 : 1-13. (In thank to Dr. S. Iwahori, Professor of the Institute Japanese with English summary). of Agriculture and Forestry, University of Tsuku- Nishiura, M., T. Shichijo, I. Ueno, M. Iwamasa, T. ba, for reading the manuscript. Kihara, Y. Yamada, T. Yoshida and T. Iwasaki. 1983. New citrus cultivar 'Kiyomi' tangor. Bull. Literature Cited Fruit Tree Res. Stn. B10 : 1-9. (In Japanese with Frost, H. B. 1948. Seed production: development of ga- English summary). metes and embryos. In: H. J. Webber and L. D. Reece, P. C. and R. 0. Register. 1961. Influence of pol- Batchelor (eds.). The Citrus industry. Vol. 1. linators on fruit set on Robinson and Osceola 767-815. Univ. Calif. Press, Berkeley and Los tangerine hybrids. Proc. Fla. State Hort. Soc. 74 : Angeles. 104-106. Hearn, C. J. 1986. Development of seedless grapefruit Soost, R. K. 1956. Unfruitfulness in the Clementine cultivars through budwood irradiation. J. Amer. mandarin. Proc. Amer. Soc. Hort. Sci. 67 : Soc. Hort. Sci. 111 : 304-306. 171-175. Hearn, C. J., P. C. Reece and R. Fenton. 1969. Self- Soost, R. K. 1965. Incompatibility alleles in genus incompatibility and the effect of different pollen Citrus . Proc. Amer. Soc. Hort. Sci. 87 : 176-180. sources upon fruit characteristics of four Citrus Soost, R. K. and J. W. Cameron. 1969. Tree and fruit hybrids. Proc. First Int. Citrus Symp. 1 : characters of Citrus triploid from tetraploid by 183-187. diploid cross. Hilgardia 39 : 569-579. Ito, Y. and I. Ueno. 1986. Studies on the incompatibil- Spiegel-Roy, P. 1988. Citrus breeding past, present ity in citrus. Annual Rep. Okitsu Branch, Fruit and future. Proc. Sixth Int. Citrus Congress 1 : Tree Res. Stn. 1986. 12. (In Japanese). 9-17. Takahara, T., I. Oiyama and N. Okudai. 1982. Effect of Iwamasa, M. 1966. Studies on the sterility in genus Citrus with special reference to the seedlessness. tetraploid as the production of seedless fruits in Bull. Hort. Res. Sta. B6 : 1-82. self-incompatible cultivars in citrus. Bull. Fruit Iwamasa, M. and Y. Ohba. 1980. Seedlessness due to Tree Res. Stn. D4 : 11-24. (In Japanese with En- self-incompatibility in Egami-Buntan, a Japanese glish summary). Ueno, I. 1976. Parthenocarpy of tangor and tangelo pummelo cultivar. Bull. Fac. Agr., Saga Univ. 49 : 39-45. (In Japanese with English summary). using satsuma mandarin as seed parent. Abstr. Matsumoto, R., N. Okudai, I. Oiyama, T. Takahara, M. Japan. Soc. Hort. Sci. Spring Meet. 1976. 94-95. Yamamoto, K. Asada, D. Ishiuchi and H. Murata. (In Japanese). 1991. New citrus cultivar 'Tsunokaori'. Bull. Ueno, I. 1986. Studies on the inheritance of citrus Fruit Tree Res. Stn. 21 : 59-65. (In Japanese with flower characteristics. I. Segregation of viable pol- English summary). len production in hybrid seedlings. Bull. Fruit Mishima, K. and I. Inaba. 1971. Studies on the ecolo- Tree Res. Stn. B13 : 1-9. (In Japanese with English summary). gical adaptation of Kawachi-Bankan. [1] Influence of the cross pollination on the form, seeded and Vardi, A. and P. Spiegel-Roy. 1981. Gene controlled dropping. Kyushu Agr. Research 33 : 273-274. meiosis in Citrus reticulata . Proc. Int. Soc. Citri- culture 26-27. J. Japan. Soc. Hort. Sci. 64(1) : 23-29. 1995. 29

Yamada, Y. 1988. Characteristics of mid and late Sci. 61 : 757-762. (In Japanese with English sum- maturing citrus and their cultivation. Abstr. mary). Symp. Japan. Soc. Hort. Sci. Autumn Meet. 1988. Yamashita, K. and H. Iwanaga. 1984. Physiological 1-19. (In Japanese). characteristics of immature flower buds as con- Yamamoto, M., N. Okudai and R. Matsumoto. 1992a. cerned with self-incompatibility of Hyuganatsu, Studies on the inheritance of aborted anthers in Citrus tamurana Hort. ex Tanaka. J. Japan. Soc. citrus using seed parents having aborted anthers. Hort. Sci. 53 : 6-12. (In Japanese with English J. Japan. Soc. Hort. Sci. 60 : 791-797. (In summary). Japanese with English summary). Yoshida, T. 1982. Pollen viability and seed content in Yamamoto, M., Y. Yamada, R. Matsumoto, H. Ikemiya hybrid seedlings using 'Sweet Spring' or 'Kiyomi' and N. Okudai. 1992b. Inheritance of seed number as seed parents. Abstr. Japan. Soc. Hort. Sci. in citrus. Bull. Fruit Tree Res. Stn. 23 : 47-56. Spring Meet. 1982. 10-11. (In Japanese). (In Japanese with English summary). Yoshida, T. and H. Hanaori. 1990. Pollen viability and Yamamoto, M., N. Okudai, R. Matsumoto and Y. Yama- seed content in satsuma mandarin hybrids. J. da. 1993. Seed number of hybrid plants in citrus, Japan. Soc. Hort. Sci. 59 (Suppl. 1): 8-9. (In with special reference to seed number of parents Japanese). and pollen yield of individuals. J. Japan. Soc. Hort.

カ ンキツ におけ る不稔性 と含核数 との関係

山本雅 史 ・松 本亮司 ・山田彬 雄

果 樹 試 験 場 口 之 津 支 場 859-25 長 崎 県 南 高 来 郡 口 之 津 町

摘 要

カ ンキ ツ に お け る雌 性 不 稔 性,雄 性 不 稔 性 お よ び 自 び 自家 不 和 合 性 品 種 で 低 く,雄 性 不 稔 性 お よ び 自 家 不 家 不 和 合 性 と 含 核 数 と の 関 係 を カ ン キツ22品 種 を 用 和 合 性 は,含 核 数 を減 少 させ る効 果 が あ る こ とが 確 認 い て 調 査 した. で き た. 1.自 然 受 粉 区 と 人 工 受 粉 区 の 含 核 数 と の 間 に は 正 3.一 部 の 自 家 不 和 合 性 品 種 の 人 工 受 粉 果 に 対 す る の 相 関(r=0.927**,r2=0.859)が 認 め ら れ た.人 自然 受 粉 果 の 含 核 数 の 割 合 は,自 家 和 合 性 品 種 と 同 程 工 受 粉 区 の 含 核 数 は雌 性 不 稔 性 の 程 度 を 表 わ して い る 度 で あ っ た.こ れ らの 品 種 に お い て は無 核 果 が ほ と ん と 考 え ら れ る こ と か ら,雌 性 不 稔 性 は 自 然 受 粉 果 の 含 ど結 実 して い な か った.こ れ は,単 為 結 果 性 の 欠 如 の 核 数 に 強 い 影 響 を 及 ぼ して い る こ と が 推 察 で き た. た め,有 核 果 の み が 結 実 した の で,こ の よ うな 結 果 に 2.人 工 受 粉 果 の 含 核 数 に 対 す る 自 然 受 粉 果 の 含 核 な っ た も の と考 え られ た. 数 の 割 合 は,自 家 和 合 性 品 種 よ り雄 性 不 稔 性 品 種 お よ