J. Japan. Soc. Hort. Sci. 65(4) : 685-692. 1997.

Protoplast Fusion for Production of Hybrid between and Its Related Genera

Toshiaki Motomura1*, Tetsushi Hidaka2* *, Tomoya Akihama1, Shinsaku Katagi3, Mark A. Berhow2* * *, Takaya Moriguchi2 and Mitsuo Omura2* * * * 'Faculty of Agriculture , Meiji University, Tama, Kawasaki, Kanagawa 214 2Depertment of Citviculture, National Institute of Fruit Science, Okitsu, Shimizu, Shizuoka 424-02 3Kanagawa Horticultural Experiment Station , Nebukawa Branch, Nebukawa, Odawara, Kanagawa 250

Summary Electrofusions between embryogenic callus protoplasts from Citrus and mesophyII protoplasts from other representatives, including , Triphasiinae, Balsamocitrinae, and Citrinae, were performed to estimate the range within which fusion could produce embryos. Production and regeneration of hybrids between Citrus and allied species depended on the taxonomical distance of the parents; the closer the mesophyII pa- rent were taxonomically to Citrus, the greater the potential of forming viable hybrid plants. The most successful fusions were obtained between Citrus and other Citrinae spe- cies. Most of these combinations produced embryos that generated normal plantlets with good rooting ability. Embryoids from fusions with Balasamocitrinae species initiated numerous adventitious buds, but they were incapable of rooting. When these shoots were grafted onto existing stocks, some produced deformed leaves. In fusions between Citrus and Clausenae species, most of the regenerated shoots were weak and abnormal, but a few normal plantlets were rescued. In fusions with Triphasiinae species, the embryos which survived failed to develop shoots. Thus, from fusions involving Triphasiinae spe- cies, which considered closer to Citrus than are Clausenae species, we were unable to re- generate any somatic hybrids.

Raf. through protoplast fusion. Protoplast fusion Introduction between Citrus and allied species has since been Since the first successful regeneration of soma- applied to obtain useful plants for breeding prog- tic hybrids within the Nicotiana species (Carlson et rams with emphasis on the improvement of disease al., 1972), many protoplast fusion studies have resistance, rootstocks or chemical constituents been carried out to overcome sexual incompatibil- (Vardi et al., 1987, 1989; Grosser et al., 1988, ity among species. Ohgawara et al. (1985) 1990, 1992; Grosser and Gmitter, 1990; Deng et obtained 'Oretachi', a somatic hybrid between Cit- al., 1992; Shinozaki et al., 1992; Takayanagi et rus sinensis (L.) Osbeck and Poncirus trifoliata (L.) al., 1992; Louzada et al., 1993; Saito et al., 1993, 1994; Ling and Iwamasa, 1994; Motomura et al., Received; January 18, 1996. Accepted; June 28, 1996. 1995, 1996; Moriguchi et al., 1996). Many desir- Contribution No. 1050 of the NIFTS. able characteristics are present in wild relatives *Present address: Shoda Shoyu Co . Ltd. Tate- of Citrus. Consequently, establishing the range of bayashi, Gunma 374. * * P species within which somatic hybrids and cybrids resent address: Jpn. Int. Res. Center for Agr. Sci., can be produced may yield some valuable informa- Okinawa Subtropical Station, Ishigaki, Okinawa tion not only for the production of germplasm with 907. * * * Present address: Natl . Center for Agr. Utilization potential for breeding programs, but also for a bet- Res. ARS, USDA, Illinois 61604, U. S. A.. ter understanding of their taxonomic relationships. ** * *A uthor to whom correspondence should be addres- Based on embryogenesis, shoot formation, and sed. rooting ability of the fusion products between Cit-

685 686 T. Motomura, T. Hidaka, T. Akihama, S. Katagi, M. A. Berhow, T. Moriguchi and M. Omura rus and other Auratioideae representatives, an ap- Protoplast isolation, fusion and culture plicable range of combinations was evaluated and some taxonomic aspects are discussed. Approximately 6 x 105 callus protoplasts and

106 mesophyll protoplasts prepared from suspen- Materials and Methods sion cultures and leaves, respectively, according to

Hidaka and Omura (1992) were mixed in 0.35 M Plant materials mannitol, 0.35 M sorbitol and 0.25 mM calcium

Embryogenic callus protoplasts from Citrus and chloride; 8 ml aliquots were then transferred to leaf protoplasts from Citrus or its relatives were individual plastic Petri dishes. Electrical fields fused. Mexican lime (Citrus aurantifolia [Christm.] were generated by a RSG-1000 (Hoei Sci. Co. Ltd.

Swing.), 'Hazzara (Abohar)' (C. reticulata Blanco), Japan) power supply and the fusion was carried 'Ohta' ponkan (C. reticulata Blanco), 'Saruwatari' out using an electrode chamber with concentric unshu (C. unshu Marc.), 'Kara' mandarin (C. unshu circles (Hidaka et al., 1995). The electrical para-

x C. nobilis Lour.), 'Valencia' orange (C. sinensis meters were as follows: AC fieds, 1 MHz, [L.] Osbeck), and 'Seminole' tangelo (C. reticulatax 125-150 V/cm, 50-75 sec; DC field, 1000-1150 C. paradisi Macf.) were the source of embryogenic V/cm square-wave 50 ,sec in duration, 4-5 times calli, whereas leaf protoplasts were derived from at 1 sec intervals. After fusion, the mixtures were the following species: i) Tribe Clauseneae ( centrifuged at 100 x g for 7 min, the supernatant paniculata [L.] Jack, Murraya koenigii [L.] discarded, and the fusion products resuspended at Spreng., pentaphylla [Retz.] Correa and a density of 2 x 105 cells in MS medium containg

Merrillia caloxylon [Ridl.] Swing.); ii) Subtribe 0.15 M sucrose and 0.45 M glucose (Motomura et Triphasiinae (Triphasia trifolia [Burm. f.] P. al., 1995), for selection of heterokaryons. De-

Wils.); iii) Subtribe Balsamocitrinae (Feroniella veloped embryoids were transferred onto a 0.2% lucida [Scheff.] Swing., Aegle marmelos [L.] and Gelrite (Kelco, Merck & Co. Inc. USA) -solidified Swinglea glutinosa [Blanco] Merr.); and iv) Sub- MS medium containing 0.1 M galactose, 0.1 M sor- tribe Citrinae (rough lemon [Citrus jambhiri bitol, and 1 ƒÊM gibberellic acid (GA3). Regener-

Lush.], yuzu [Citrus junos Sieb. ex Tan.] and ated shoots incapable of rooting were grafted on 'Miho Wase' [Citrus unshu Marc .] bux- rootstocks of 'Troyer' citrange (Citrus sinensis x ifolia [Poir.] Tenore, Atalantia monophylla DC., Poncirus trifoliata). Acclimatization was carried and Microcitrus australis [Planch.] Swing.). out as described by Hidaka and Kajiura (1989). Embryogenic calli were maintained on solidified Results Murashige and Skoog's (1962) medium (MS) con- taining 0.2 M sucrose and 50 AiMkinetin. Prior to The number of embrvoids that developed after isolation of protoplasts, callus cultures were trans- electrofusion in various combinations is shown in ferred to a liquid MS medium containing the same Table 1. In all combinations, the first cell division components and subcultured, for at least 3 times of the fused protoplasts occured within 2-3 weeks at 2-week intervals. Suspension cultures were then after plating. Micro-colonies and green globular kept on a hormone-free liquid MS medium contain- embryoids formed in about 6 and 8 to 12 weeks ing 0.1 M galactose and 0.1 M sorbitol for 1-2 after plating, respectively. When 'Hazzara (Abo- weeks. They were subcultured for up to 3 times in har)' protoplasts as the embryogenic callus parent the same medium at intervals of 2 weeks. These were fused to leaf protoplasts from sexually-com- calli were used for preparing callus protoplasts as patible species, such as rough lemon, yuzu, and previously described (Hidaka and Omura, 1992; Microcitrus australis, more than 150 embryoids Motomura et al., 1995). were generated out of approximately 2x105 proto- Plants for producing leaf protoplasts were plasts plated per Petri dish. However, in combina- grown in a greenhouse at 25-35 °C without sup- tions with sexually-incompatible species, the re- plemental lighting. Leaf protoplasts were prepared sulting number of embryoids was considerably from fully expanded upper leaves. lower, i. e. as low as one-fourth of those from the sexually-compatible combinations. The same tendency was observed when Mexican lime, 'Semi- J. Japan. Soc. Hort. Sci. 65(4) : 685-692. 1997. 687

Table 1. Number of surviving green embryoids in each fusion combination2.

Table 2. Perfomance of the hybrids resulting from protoplast fusion between Citrus and related spe-

cies. 688 T. Motomura, T. Hidaka, T. Akihama, S. Katagi, M. A. Berhow, T. Moriguchi and M. Omura

nole' tangelo or 'Saruwatari' unshu was used as the not survive. The other two combinations diffe- embryogenic callus parent. rentiated shoots but not roots. Hence, plantlets Shoot formation, rooting, and graft compatibility were obtained by grafting. Shoots from the com- were analyzed taxonomically (Table 2). When spe- bination between 'Saruwatari' unshu and Aegle cies of the tribe Clauseneae (, marmelos developed abnormal, small contorted caloxylon, Murraya paniculata), which are leaves (Fig. 1 F), but those from 'Hazzara (Abo- the remotest Citrus relatives among the Auran- har)' and Swinglea glutinosa underwent normal tioideae Subfamily, were hybridized with Citrus morphogenesis (Fig. 1 G). callus protoplasts, green embryoids were obtained When species from the Subtribe Citrinae (Sever- in all 8 combinations, but none of the embryoids inia buxifolia, Atalantia monophylla and Microcitrus from the combination between 'Saruwatari' unshu australis) were used as mesophyll parents, normal and Glycosmis pentaphylla survived a year after shoots formed in all combinations. Root formation plating. Moreover, in the remaining combinations, and vigorous growth, either with or without graft- embryoids developed into a mass of small vitrified ing procedures, were commonly observed (Fig. 1 contorted leaves. Plantlets, resulting from com- H). The combinations between 'Hazzara (Abohar)' binations beween Mexican lime and Murraya pani- and Severinia buxifolia, and 'Seminole' tangelo and culata (Fig. 1 A) or Murraya kioenigii, and between Atalantia monophylla, developed numerous shoots 'Hazzara (Abohar)' and M urraya koenigii (Fig. 1 B) but no roots. Grafting was successfully employed or Glycosmis pentaphylla, did not develop beyond for rescuing shoots from the latter combination this abnormal stage. Occasionally, shoot elongation (Fig. 1 I), but not from the former. occured in fused hybrids of 'Kara' mandarin and Discussion Merrillia caloxylon, 'Hazzara (Abohar)' and Merrillia caloxylon, and 'Ohta' ponkan and Glycosmis pen- These experiments demonstrate the difficulties taphylla, however, only the shoots from the last in inducing plantlet formation following protoplast combination could be rescued by grafting (Fig. 1 fusion between Citrus and more distant relatives C); others were too weak to undergo this proce- in the Aurantioideae Subfamily. In the combina- dure (Fig. 1 D,E). tions with Citrus and Clauseneae species, only the 'Ohta' ponkan and Gl Although fusions of Triphasia trifolia, with ycosmis pentaphylla combina- either 'Seminole' tangelo or 'Hazzara (Abohar)' as tion formed normal shoots, whereas all the others callus parents, developed some embryoids, no showed abnormal organogenesis. Considering the shoot differentiation occurred, so that no somatic use of a selecting medium with high sugar concen- hybrid was generated. tration (Ohgawara et al., 1985; Motomura et al., Following fusions with Balsamocitrinae species 1995) and also morphological features of the re- (Feroniella lucida, Aegle marmelos and Swinglea glu- generated shoots, we may assume that the regener- tinosa), green embryoids were obtained in all 3 ated plants between `Ohta' ponkan and Glycosmis combinations, but those from the combination be- pentaphylla are indeed somatic hybrids. Shinozaki tween 'Valencia' orange and Feroniella lucida did et al. (1992) reported shoot regeneration after pro-

Fig. 1. A; Abnormal organogenesis in embryoids from Mexican lime and Murraya paniculata protoplast fusion. B; Abnormal organogenesis in embryoids from 'Hazzara (Abohar)' and Murraya koenigii protoplast fusion. C; Regenerated shoot of somatic hybrid of 'Ohta' ponkan and Glycosmis pentaphylla grafted on a rootstock. D, E; Abnormal shoot formation after protoplast fusion between 'Hazzara (Abohar)' and Merrillia caloxylon. F; Deformed leaves in a growing shoot from 'Saruwatari' unshu and Aegle marmelos protoplast fusion, after grafting on a rootstock. G; Normal leaf morphology in a growing shoot from 'Hazzara (Abohar)' and Swinglea glutinosa protoplast fu- sion, after grafting on a rootstock. H; Growing shoot from 'Ohta' ponkan and Severinia buxijolia protoplast fusion, after grafting on a rootstock. I; Growing shoot from 'Seminole' tangelo and Severinia buxifolia protoplast fusion, after grafting on a root- stock. J. Japan. Soc. Hort. Sci. 65(4) : 685-692. 1997. 689 690 T. Motomura, T. Hidaka, T. Akihama, S. Katagi, M. A. Berhow, T. Moriguchi and M. Omura toplast fusion between sweet orange (Citrus sinen- volve other Balsamocitrinae species, even though sis) and Orange jessamine (Murraya paniculata), the prospect of improving the yield of viable hyb- but the weak plantlets did not survive in the rids is very low with the current methodology. De- greenhouse. riving somatic hybrids Citrus and other remote Shoots in hybrids between Citrus and Balsamo- species with present fusion techniques seems to be citrinae formed more readily than did in hybrids very difficult. between Citrus and Clauseneae. Shoots from both Acknowledgement combinations could be rescued by grafting, but had deformed leaves. Embryoids derived from The authors are grateful to Prof. N. Nito of the 'Valencia' orange and Feroniella lucida developed Faculty of Agriculture, Saga University for pro- deformed leaves and did not survive. In our pre- viding Murraya koenigii and Glycosmis pentaphylla vious experiments, shoots were regenerated from samples. embryoids between Mexican lime (Citrus aurantifo- Literature Cited lia) and Java feroniella (Feroniella lucida) (Takayanagi et al., 1992). Moreover, hybrid plants Carlson, P. S., H. H. Smith and R. D. Dearing. 1972. between Citrus sinensis and Feronia limonia (L.) Parasexual interspecific plant hybridization. Proc. Nat. Acad. Sci. (U. S. A.) 69 : 2292-2294. Swing. were recently obtained (Grosser et al., Da Silva, M. F. G. F., 0. R. Gottlieb and F. Ehrendor- 1996), indicating that somatic hybrids can be suc- fer. 1988. Chemosystematics of the : sug- cessfully produced between Citrus and Balsamocit- gestions for a more natural and evolu- rinae. tionary interpretation of the family. Plant Syst. Although some embryoids survived, no normal Evol. 161 : 97-134. somatic hybrids between Citrus and Triphasiinae Deng, X. X., J. W. Grosser and F. G. Gmitter Jr. 1992. were produced, despite the fact that the latter is Intergeneric somatic hybrid plants from protoplast more closely related to Citrus than to Clauseneaeis. fusion of Fortunella crassifolia cultivar 'Meiwa' It is difficult to draw any conclusion concerning with Citrus sinensis cultivar 'Valencia' . Sci. Horti- culturae 49 : 55-62. the genetic distance between Citrus and Tripha- Grosser, J. W., F. G. Gmitter Jr. and J. L. Chandler. siinae species because only one accession was 1988. Intergeneric somatic hybrid plants from used as the leaf parent. Based on chemotaxonomic- sexually incompatible woody species: Citrus sinen- al data, Da Silva et al. (1988) placed Triphasiinae sis and Severinia disticha. Theor. Appl. Genet. 75 : into the Subtribe Citrinae, the 'True Citrus Fruit 397-401. ', which includes Citrus, Severinia, A talantia, Grosser, J. W. and F. G. Gmitter Jr. 1990. Somatic and Microcitrus among others. However, our failure hybridization of Citrus with wild relatives for to produce these hybrids evokes questions con- germplasm enhancement and cultivar development. cerning this new taxonomic proposal. Further hyb- HortScience 25 : 147-151. Grosser, J. W., F. G. Gmitter Jr., N. Tusa and J. L. ridization experiments with Triphasiinae species Chandler. 1990. Somatic hybrid plants from sex- from other genera, such as Pamburus and Luvunga, ually incompatible woody species: Citrus reticulata using improved fusion methods, are needed to and Citropsis gilletiana. Plant Cell Rept. 8 : obtain more of these hybrids. 656-659. Our results agree with the taxonomical inter- Grosser. I. W.. F. G. Gmitter Jr.. F. Sesto. X. X. Deng pretation of the Aurantioideae Subfamily by and J. L. Chandler. 1992. Six new somatic citrus Swingle and Reece (1967). The Citrinae Subtribe hybrids and their potential for cultivar improve- species might be a genetic donor for Citrus im- ment. J. Amer. Soc. Hort. Sci. 117 : 169-173. Grosser, J. W., F. A. A. Mourao-Fo, F. G. Gmitter Jr., provement through somatic hybridization and, in- E. S. Louzada, J. Jingrui, K. Baergen, A. Quiros, C. deed, several somatic hybrids have been obtained Cabasson, J. Schell and J. L. Chandler. 1996. within this subtribe (Grosser et al., 1988, 1990; Allotetraploid hybrids between Citrus and seven Grosser and Gmitter, 1990; Deng et al., 1992; Shi- related genera produced by somatic hybridization. nozaki et al., 1992; Takayanagi et al., 1992; Theor. Appl. Genet 92 : 577-582. Louzada et al., 1993; Ling and Iwamasa, 1994; Hidaka, T and I. Kajiura. 1989. A simple method for Motomura et al., 1995, 1996). Other possible acclimatization of in vitro plantlets of citrus. Bull. means of gaining genetic diversity may be to in- Fruit Tree Res. Sta. B16 : 19-28 (In Japanese J. Japan. Soc. Hort. Sci. 65(4) : 685-692. 1997. 691

with English summary). and S. Ishii. 1985. Somatic hybrid plants obtained Hidaka, T and M. Omura. 1992. Regeneration of soma- by protoplast fusion between Citrus sinensis and tic hybrid plants obtained by electrical fusion be- Poncirus trifoliata. Theor. Appl. Genet. 71 : 1-4. tween satsuma mandarin (Citrus unshiu) and Saito, W., T. Ohgawara, J. Shimizu, S. Ishii and S. rough lemon (C. jambhiri) or yuzu (C. junos). Kobayashi. 1993. Citrus cybrid regeneration fol- Japan. J. Breed. 42 : 79-89. lowing cell fusion between nucellar cells and Hidaka, T., T. Moriguchi, T. Motomura, S. Katagi and mesophyll cells. Plant Sci. 88 : 195-201. M. Omura. 1995. Developement of a new electrode Saito, W., T. Ohgawara, J. Shimizu and S. Kobayashi. chamber and its efficiency in protoplast fusion in 1994. Somatic hybridization in Citrus using Citrus. Breed. Sci. 45 : 237-239. embryogenic cybrid callus. Plant Sci. 99 : 89-95. Ling, J-T. and M. Iwamasa. 1994. Somatic hybridiza- Shinozaki, S., K. Fujita, T. Hidaka and M. Omura. tion between Citrus reticulata and Citropsis 1992. Plantlet formation of somatic hybrids of gabunensis through electrofusion. Plant Cell Rept. sweet orange (Citrus sinensis) and its wild rela- 13 : 493-497. tive, orange jessamine (Murraya paniculata), by Louzada, E. S., J. W. Grosser and F. G. Gmitter Jr. electrically-induced protoplast fusion. Japan. J. 1993. Intergeneric somatic hybridization of sex- Breed. 42 : 287-295. ually incompatible parents: Citrus sinensis and Swingle, W. T. and P. C. Reece. 1967. The botany of Atalantia ceylanica. Plant Cell Rept. 12 : 687-690. Citrus and its wild relatives. p. 190-430. In: W. Moriguchi, T., T. Hidaka, M. Omura, T. Motomura and Reuther, H. J. Webber and L. D. Batchelor (eds.). T. Akihama. 1996. Genotype and parental com- The citrus industry Vol. I. Univ. Calif. Press, Ber- bination influence efficiency of cybrid induction in keley, Calif., U. S. A. Citrus by electrofusion. HortScience. 31 : 275-278. Takayanagi, R., T. Hidaka and M. Omura. 1992. Re- Motomura, T., T. Hidaka, T. Moriguchi, T. Akihama generation of intergeneric somatic hybrids by elec- and M. Omura. 1995. Intergeneric somatic hybri- trical fusion between Citrus and its wild relatives: dization between Citrus and Atalantia or Severinia Mexican lime (Citrus aurantifolia) and Java fero- by electrofusion, and recombination of mitochond- niella (Feroniella lucida) or tabog (Swinglea gluti- rial genomes. Breed. Sci. 45 : 309-314. nosa). J. Japan. Soc. Hort. Sci. 60 : 799-804. Motomura, T., T. Moriguchi, T. Akihama, T. Hidaka Vardi, A., A. Breiman and E. Galun. 1987. Citrus cyb- and M. Omura. 1996. Analysis of cytoplasmic rids: production by donor-recipient protoplast-fu- genomes in somatic hybrids between 'Hazzara sion and verification by mitochondrial-DNA res- (Abohar)' (Citrus reticulata Blanco) and Microcitrus triction profiles. Theor. Appl. Genet. 75 : 51-58. australis (Planch.) Swingle. J. Japan. Soc. Hort. Sci. Vardi, A., P. Arzee-Gonen, A. Frydman-Shani, S. 65 : 497-503. Bleichman and E. Galun. 1989. Protoplast-fusion- Murashige, T. and F. Skoog. 1962. A revised medium mediated transfer of organelles from Microcitrus for rapid growth and bioassays with tobacco tis- into Citrus and regeneration of novel alloplasmic sue culture. Physiol. Plant. 15 : 473-497. trees. Theor. Appl. Genet. 78 : 741-747. Ohgawara, T., S. Kobayashi, E. Ohgawara, H. Uchimiya 692 T. Motomura, T. Hidaka, T. Akihama, S. Katagi, M. A. Berhow, T. Moriguchi and M. Omura

カンキツ とその類縁種 問の細胞融合 による雑種作 出

本 村 敏 明1*・ 日 高 哲 志2**・ 秋 濱 友 也1・ 片 木 新 作3・Mark A, Berhow2***・ 森 口 卓 哉2・ 大 村 三 男2 1明治大学農学部 214 神奈川県川崎市

2果 樹 試 験 場 カ ンキツ 部 興 津 424 -02 静 岡 県 清 水 市 3神奈川県園芸試験場根府川分場250神 奈川県小田原市

摘 要

カンキツ類縁種 には病害虫抵抗性等の有用遺伝子 を ツ亜 連 内 の カ ン キツ と他 の 種 と の 電 気 融 合 で は,多 く 有 す る もの が 多 く,育 種 素 材 と して の 活 用 が 期 待 され の 組 合 わ せ で 融 合 後 の胚 様 体 形 成,シ ュ ー トの 再 分 化, て い る.し か し,カ ンキ ツ と の 類 縁 関 係 が 遠 く な る と 発 根 が 容 易 で あ っ た.カ ン キ ツ と カ ン キ ツ連 バ ル サ モ 交 雑 は 困 難 と な る.こ れ を 克 服 す る手 段 と して 細 胞 融 シ トラ ス亜 連 の 電 気 融 合 で は,比 較 的 シ ュ ー トの 形 成 合 が 考 え られ る.こ こ で は 細 胞 融 合 法 の 適 用 限 界 を 知 は 容 易 で あ っ た.し か しな が ら,そ の 発 根 は 困 難 な た る た め に,カ ンキツ と カ ンキツ 類 縁 種 の 電 気 融 合 を行 め に,接 ぎ木 した と こ ろ,一 部 は 植 物 体 に ま で 生 長 し い,融 合 後 の 胚 様 体 の 発 育 に つ い て 調 査 を行 っ た. た が,奇 形 葉 を 呈 す る もの も多 か っ た.ワ ン ピ 連 との 材 料 に は,ミ カ ン亜 科 カ ンキツ 連 の ト リフ ァ シ ァ亜 組 合 わ せ の 電 気 融 合 で は,個 体 再 生 は 極 め て 困 難 で, 連,カ ン キ ツ亜 連(カ ン キ ツ を 含 む),バ ル サ モ シ ト 分 化 して も異 常 な 個 体 しか 得 ら れ な か っ た.カ ン キツ ラ ス亜 連 お よ び ワ ン ピ連 の ワ ン ピ亜 連 と メ リ リ ア 亜 連 と カ ンキツ 連 ト リ フ ァシ ア 亜 連 と の 電 気 融 合 で は,カ 内 の 種 を用 い た. ン キ ツ と ワ ン ピ連 よ り も類 縁 関 係 が 近 い に もか か わ ら 一般 的 に カ ンキツ と分 類 的 に 近 縁 な組 合 わ せ に お い ず 個 体 再 生 は 困 難 で あ り,体 細 胞 雑 種 作 出 の 可 能 性 は

て 雑 種 個 体 の 作 出 が 容 易 で あ っ た.カ ンキツ 連 カ ンキ 低 か っ た. *現 在 :正 田醤 油 ㈱ **現 在 :国 際 農林 水 産 業研 究 セ ン ター 沖縄 支 所 ***現 在;Natl . Center for Agr.Utilization Res.ARS, USDA, Illinois, 61604, U. S. A.