J. Japan. Soc. Hort. Sci. 45(3) : 225-230. 1976.

Production of Seedless Fruits in Hyuganatsu, Citrus tamurana Hort. ex TANAKA, and Hassaku, Citrus hassaku HAYATA through Pollination with Pollen Grains from the 4x Natsudaidai, Citrus natsudaidai HAYATA

Kensuke YAMASHITA Collegeof Agriculture, Miyazaki University,Miyazaki

Summary 1. The present paper deals with the preliminary efforts to produce seedless fruits in Hyuganatsu and Hassaku using crosses of 2 X X 4 X, a procedure which results in the formation of abortive seeds in many cases. 2. Two hundred flowers of Hyuganatsu (Citrus tamurana Hort. ex TANAKA) were emasculated and pollinated with pollen grains of the 4 X Natsudaidai (Citrus natsudaidai HAYATA). The fruits thus obtained were normal in size, weight and skin color, but almost all the seeds degenerated into rudiments and traces at an early stage of development. They were virtually seedless. 3. The soluble solid and titrable acid contents in the juice of the ripe seedless fruits showed no difference from the control. The edible portion of these fruits increased by 15% as compared with the control. 4. The seedless fruits did not differ from the controls by date of dropping early or late. Also they displayed similar pattern of development of dry juice sacs after freezing in the winter. The fruits after harvest were stored in a carton box under room condition for about two months. They did not display any particular change in the contents of the soluble solids and titrable acids in the juice during storage. The seedless fruits seemed to soften a little faster than that of the control fruits. 5. The flowers of the Hassaku (Citrus hassaku HAYATA) were pollinated with pollen grains of the 4 X Natsudaidai. The fruits thus obtained were virtually seedless and did not differ from the control in fruit growth and size or in other qualities. The results coincided with those for the Hyuganatsu X 4 X Natsudaidai cross. 6. Further investigations are under way for the purpose of finding the proper proportion of the 4 X Natsudaidai tree to be planted as a pollinizer for Hyuganatsu.

average fruit of Hyuganatsu weighs 200 g at Introduction the time of harvest and contains as many as Hyuganatsu (Citrus tamurana Hort. ex TA- 20 seeds in the center of the fruit, and there- NAKA) is one of the late citrus varieties which fore Lie edible portion is displaced considerably. has very good flavor. This is the local variety The auther is looking for any means possible originated in Miyazaki and is now cultivated to produce a seedless fruit. in limiting southern areas of Japan on a In 1971, Naka jima dipped some flowers into small scale, because many fruits drop in a 100 ppm GA3 solution at flowering time and winter and dry juice sacs develop to a consider- succeeded in inducing parthenocarpic fruits (7) . able extent after freezing. Since this variety In 1972, Yamamoto and Yamashita sprayed a is self-incompatible and non-parthenocarpic, 500 ppm water solution of GA3 onto the Natsudaidai (Citrus natsudaidai HAYATA)trees flowers of Hyuganatsu at full bloom and are usually mix-planted as a pollinizer (6) . An succeeded in obtaining seedless fruits, but the Received for publication October 2, 1975. fruits were too small for commercial use(10).

225 226 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE

The author's intention to use the 4 X flowers were pollinated with pollen grains of Natsudaidai as a pollinizer was based on the the 4 X Natsudaidai and bagged for the follow- observation that crosses of 2 X><4 X result in ing 7 days. Thirty flowers left for open the formation of abortive seeds in many pollination were used for the control. The cases (1, 5, 9). In the present experiment, the transverse diameter of the fruits was measured emasculated flowers of Hyuganatsu were every two weeks until harvest. Several fruit pollinated with pollen grains of the 4 X characteristics as given in Figs. 8 and 9, and Natsudaidai, and this procedure succeeded in Table 2 were investigated for the harvested yielding seedless fruits of normal size. The fruits. same experiment was also carried out with Results and Discussion Hassaku (Citrus hassaku HAYATA), which, like Hyuganatsu, is a self-incompatible variety. 1. Fruit growth and development Figs. 1 and 2 show pictures of transverse Materials and Methods sections of the seeded and seedless fruits of The 4 X tree of Natsudaidai used in the the Hyuganatsu and Hassaku harvested in present experiment was found as a chance September; in each figure, the left is the seedling by Dr. Furusato some decades ago(5), control and contains many seeds, while the and has been grown in the orchard of Mr. right, being vertually seedless, is the fruit S. Mizuta in Manazuru, Kanagawa Prefecture. obtained through pollination with pollen grains In April, a branch of the 4 X tree was of the 4 X Natsudaidai. There was almost no covered with a vinyl sheet for the purpose difference in fruit size between them. Figs. 3 of speeding-up the flowering time, and pollen and 4 show the growth curves showed the grains were collected a few days before pollination and brought to Miyazaki in a small bottle in which a small volume of silica gel was contained as a desicant. The germination percentage of these pollen grains was 5% on a 1/ agar medium with 10% sucrose. The characteristics of floral organs as well as leaves of the 4 X Natsudaidai are shown in Figs. 11 and 12, and Table 4.

At flowering time, 200 emasculated flowers Fig. 1. Transverse sections of seeded and seedless of Hyuganatsu were pollinated with pollen fruits in Hyuganatsu (Photo. Sept. 23, '74) grains of the 4 X Natsudaidai in the experi- left : Seeded fruit bred by pollination with mental orchard of Miyazaki University. As a pollen grains of the 2 X Natsudaidai (control) cantrol, the same number of emasculated right : Seedless fruit bred by pollination with pollen grains of the 4 X Natsudaidai flowers were pollinated with pollen grains of the 2 X Natsudaidai. All the flowers were covered after pollination with paper bags for about one week to prevent contamination by foreign pollen grains. The transverse diameter of the fruits was measured every two weeks until harvest. At harvest, several fruit charac- teristics as given in Figs. 5, 6 and 7, and Table 1 were observed for these fruits. Frequencies of fruit drop early in the season as well as during winter were also investigated. Of these harvested fruits, the proprotion of Fig. 2. Transverse sections of seeded and seedless dry juice sac was graded into 5 categories fruits in Hassaku (Photo. Sept. 23. '74) left : Seeded fruit bred by open pollination from 0 to 4 (Fig. 10). right : Seedless fruit bred by pollination with In the case of the Hassaku, 30 emasculated pollen grains of the 4 X Natsudaidai YAMASHITA : PRODUTION OF SEEDLESS FRUITS IN HYUGANATSU 227

Fig. 3. Growth curves of seeded and seedless fruits in Hyuganatsu bred by pollination with pollen grains Fig. 4. Growth curves of seeded and seedless fruits in of the 2 X and 4 X Natsudaidai (Each value is the average of 30 fruits) Hassaku bred by pollination in the open and with pollen grains of the 4 X Natsudaidai (Each value is the average of 30 fruits) tendency that the seedless fruits were a little larger than the seeded ones from May to July, but the reverse became true after August. In Hassaku, the growth curves show- ed the tendency that the seedless fruits were observed to be larger than the seeded ones during the whole period of development. It is said that the growth and development of a fruit is influenced by the function of plant hormones produced in the seeds. In fact, in Hyuganatsu, a high correlation of 0.82 was observed between fruit weight and seed number (10) . But in fruits obtained through pollination with pollen grains of the 4 X Natsudaidai, only a few seeds were found Fig. 7. Seed clusters of seeded and seedless fruits in together with a large number of undeveloped '75) A Hyuganatsu at harvest (Photo. Jan. 13, seeds. The reason why these seedless fruits Seeded fruit (control) B : Seedless fruit grew to normal size may be partly due to the hormone stimulus given by pollen grains tion of the embryo due to the disproportion of the 4 X Natsudaidai, but mostly due to the in the number of chromosomes between function of plant hormones which young ovules embryo and endosperm in their early develop- may have produced until they became abortive. ment (2, 3, 4, 5) . In fact, the author observed that some of 2. Fruit characteristics at harvast the ovules developed into young seeds until The characteristics of the fruits of the August and then degenerated into abortive Hyuganatsu at harvest are given in Figs. 5, 6 seeds. It has been clarified, furthermore, that and 7, and Table 1. Average weight of the such abortive seeds are formed by the degenera- seedless and seeded fruit was 189.5 g and 228 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE

Table 1, Characteristics of seeded and seedless fruits in Hyuganatsu bred by pollination with pollen grains of the 2 X and 4X Natsudaidai (Investigated on Jan. 13, 1975*)

Table 2. Characteristics of seeded and seedless fruits in Hassaku bred by pollination in the open and with pollen grains of the 4 X Natsudaidai (Investigated on Jan. 13, 1975*)

195.4g, respectively. Concerning pericarp Table 3. Change of fruit quality during storage color, there was no difference between the in Hyuganatsu seedless fruits and the seeded ones, but the flesh color of the former was citron yellow while the latter was dirty yellow. The content of soluble solids and titrable acids in the juice were nearly the same. In the Hyuganatsu, the edible portion was considerably reduced because of the large cluster of seeds ; as many as 30 seeds per fruit were formed in the control in the present experiment. Subsequently, the edible portion of the seedless fruit was increased by 15% as compared with that of the control. As to development of dry juice sacs after The results obtained in the Hassaku are freezing in winter, there seemed to be no given in Figs. 8 and 9, and Table 2. The difference between the seeded fruits and seedless fruits contained only a few perfect seedless ones. According to Nakajima, this seeds and the edible portion increased as defect occurred more frequently in the smaller compared with the control. fruits outside the canopy than in the larger 3. Fruit drop, development of dry juice fruits inside the canopy; the symptoms of the sac and change of fruit quality disorder were much more severe in the seedy during storage segments on the sunscald side than in those The number of fruit dropped early was on the healthy side of the fruits (8). counted on September 10 th. It was 48 for the About the change of the soluble solid and control, while it was 58 for the case of titrable acid contents in the juice during pollination with pollen grains of the 4 X storage, no peculiarity was found in the Natsudaidai. seedless fruits. On March 12 th, the number of remaining 4. Further considerations for the prac- fruits was counted. It was 121 in the former, tical application of the 4 X Natsu- and 111 for the latter. The result corresponds daidai as a pollinizer to that of Miwa in that there was no difference Concerning mix-planting of the 4 X Natsu- between the late drop and seed number in the daidai tree as a pollinizer for Hyuganatsu, Hyuganatsu (5). the following points are left for further YAMASHITA : PRODUTION OF SEEDLESS FRUITS IN HYUGANATSU 229

of the 4 X Natsudaidai will be lower than that of the 2 X Natsudaidai. But, if the sedless Hyuganatsu compensates the loss, it could be sufficiently profitable on a commercial scale. There are some problems to be solved before practical application, but the results Fig. 10. Grade of dry juice sac after freezing in of the present experiment indicates the pos- Hyuganatsu fruit (Photo. April 14, '75) sibility of using the 4 X Natsudaidai as a Table 4. Characteristics of floral organs and leaves pollinizer in the practical production of seedless of the 4 X Natsudaidai in comparison with fruits in Hyuganatsu and Hassaku. those of the 2 X Natsudaidai Acknowledgement The author wishes to express his deep gratitude to Dr. Kazuo Furusato, Director of Hamamatsu Flower Park, for his sugges- tions throughout the course of this study. The author is greatly indebted to Mr. Susumu Mizuta for his kindness in giving me access to his invaluable 4 X Natsudaidai. The autho wishes to express whole-hearted thanks to Dr. Akira Kobayashi, President of Agricul- tural Junior College in Ishikawa Prefecture, for his kind advice and constant encourage- consideration. ment. The author is very grateful to Dr. Presently, Natsudaidai or Hassaku is mix- Takashi Tomana, Professor of Kyoto Univer- planted as a pollinizer in a proportion from sity, for his invaluable guidance and sugges- 10% to 15%. There is some question whether tions throughout the course of this study. this proportion is proper for the 4 X Natsu- The author wishes to thank to Dr. Kiyoshi daidai as a pollinizer since the quantity of Gomi, Professor of Miyazaki University, for pollen grains of a tetraploid is generally less his constant encouragement. than that for a diploid. But, the 4 X Natsu- daidai used in the present experiment did not Literature Cited differ from the 2 X Natsudaidai in both the 1. CAMERON, J. W. and R. K. SOOST.1969. Charac- ters of new populations of Citrus polyploids, quantity of pollen grains per flower and the numder of flowers per unit shoot length. and the relation between tetraploidy in the Furthermore, the pollen fertility of the former pollen parent and hybrid tetraploid progeny. Proc. 1st Int. Citrus Symp. 1 : 199-205. did not differ from the latter so far as could 2. ESEN, A. and R. K. SOOST. 1971. Unexpected be observed by the acetocarmine and cotton triploid in Citrus : their origin, identification, blue staining methods. The proper proportion and possible use. J. Hered. 62 : 329-333. of the 4 X Natsudaidai to be planted as a 3. and . 1972. Tetraploid proge- pollinizer for Hyuganatsu will be found nies from 2 X X 4 X crosses of Citrus and their through further in vestigations. origin. J. Amer. Soc. Hort. Sci. 97 : 410-414. According to Dr. Furusato, the fruit of the 4. and . Relation of unexpected 4 X Natsudaidai was a little smaller than polyploids to diploid megagametophytes and that of the 2 X Natsudaidai ; its flesh was embryo : endosperm ploidy ratios in Citrus. Abstract. of Proc. 1973. Int. Citrus Cong. slightly less voluminous than that of the 2 X 5. FURUSATO, K. 1961. Studies on cytoplasmic Natsudaidai because of its thick pericarp ; inheritance and polyembryony in Citrus. its flesh was less juicy and a little harder Doctral Thesis at Kyoto Univ. than that of the 2 X Natsudaidai. Since the 6. MIWA, T. 1951. Pollination, fertilization, fruit quality of the 4 X Natsudaidai seems to and fruit drop in Citrus tamurana Hort. Bull. be a little inferior to that of the 2 X Natsu- Miyazaki Univ. (Natural Science) 2 : 1-67. daidai, it is assumed that the retail value 7. NAKAJIMA, Y. 1973. Studies on the fruit 230 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE

development and physical disorders in the 9. TACHIKAWA, T., Y. TANAKA, and S. HARA. late sta ges of development of Hyuganatsu 1961. Investigations on the breeding of Citrus (Citrus tamurana Hort.) in Kochi. Mem. Fac. trees. 1. Study on the breeding of triploid Cit- Agr., Kochi Univ. 22 : 1-53. rus varieties. Shizuoka Pref. Citrus Exp. Sta. 8 T. MIYAMOTO, Y. ANDO, and K. 4 : 33-44. SHINKAI. 1974. Studies on dry juice sac of 10. YAMAMOTO, S. and K. YAMASHITA, Jr. 1972. Hyuganatsu (Citrus tamurana Hort.) occurred Parthenocarpy of Hyuganatsu (preliminary by freezing. Res. Rep. Kochi Univ. 23 : 147- Report) Induction of parthenocarpy by GA3. 153. Agr. and Hort. 47 : 635-636.

4倍 体 ナ ツダイ ダ イ花 粉 の受 粉 に よ る無種 子 の ヒ ュ ーガ ナ ツ お よび ハ ッサ ク果 実 の 生 産

山 下 研 介 (宮崎大学農学部)

摘 要

4倍 体 ナ ツダ イ ダ イを 授 粉 樹 と し て ヒ ュー ガ ナ ジな ら られ ず,可 食 部 が15%も 増 加 した.つ ぎに 収 穫 果 を ダ び に ハ ッサ ク の無 種 子 果 を生 産 す る方 法 につ い て,実 用 ンボ ール 箱 に い れ て室 温 下 に お き,貯 蔵 中 の果 実 の形 質 化 の 可 能 性 を探 るた め に 本 実 験 を行 な っ た. の 変 化 を2ケ 月 間 に わ た って 調 査 した.そ の結 果,果 汁 1・ ヒ ュー ガ ナ ッ の成 木 を材 料 と して,開 花 時 に 除 雄 ・ 中 の 可 溶 性 固 形 物 含 量 な らび に 滴 定 酸 含:量の変 化,ス ア 袋 か け を行 っ て4倍 体 ナ ッ ダ イダ イの 花 粉 を 人 工 授 粉 し ガ リの 進 行 に つ い ては,対 照 区 と の間 に差 は み られ なか た.こ の よ うに して得 られ た果 実 は,果 実 発 育 の 初 期 に った が,果 皮 の軟 化 は や や 早 い よ うに観 察 され た. 種 子 が完 全 に退 化 して 無種 子果 とな った が,早 期 落 果 ・ 2.ハ ヅサ ク若 木 を材 料 とし て,開 花 時 に除 雄 ・袋 か 後 期 落 果 の程 度 は 対 照 区(2倍 体 ナ ツ ダ イ ダ イ 花 粉 授 け を 行 っ て倍 体 ナ ッ ダ イ ダ イ の花 粉 を人 工 授 粉 した.得 粉)と ほ とん ど変 わ らな か っ た.収 穫 時 に この 無 種 子 果 られ た 無 種 子 果 の肥 大 発 育 な らび に収 穫 時 の形 質 は 対 照 の形 質 を調 査 した と ころ,果 径,果 重,着 色 度,果 汁 中 区(放 任 受 粉)と 比 べ て ほ とん ど差 が なか った. の可 溶 性 固形 物 含 量 な らび に 滴 定 酸 含 量,凍 害 ス アガ リ な お授 粉 樹 とし て の4倍 体 ナ ッ ダ イダ イの 適 正 混 植 率 の発 生 程 度 に つ い て は,対 照 区 との 間 に ほ とん ど差 がみ につ い て検 討 を加 え る た め実 験 を継 続 中 で あ る. Fig. 5. Appearance of seeded and seedless fruits in Fig. 6. Transverse sections of seeded and seedless Hyuganatsu (Photo. Jan. 13, '75) fruits in Hyuganatsu at harvest (Photo. Jan. A : Seeded fruit (control) B : Seedless fruit 13, '75) A : Seeded fruit (control) B : Seedless fruit

Fig. 8. Appearance of seeded and seedless fruits in Fig. 9. Transverse sections of seeded and seedless Hassaku at harvest (Photo. Jan. 13, '76) fruits in Hassaku at harvest (Photo. Jan. 13, A : Seeded fruit (control) B : Seedless fruit '75) A : Seeded fruit (control) B : Seedless fruit Fig. 11. Floral organs and leaves of the 4X Natsudaidai as compared with those of the 2X Natsudaidai I: Petal 2 : Stamen 3 Pistil 4 : Leaf

Fig. 12. Pollen grains of the 4X Natsudaidai observed by a scanning electron microscope 1 : Pollen grains of the 2X Natsudaidai (x 1000) 2 : ditto. (x 3000) 3 : Pollen grains of the 4X Natsudaidai (x 1000) 4 : ditto. (x 3000)