J. Japan. Soc. Hort. Sci. 65(4) : 685-692. 1997. Protoplast Fusion for Production of Hybrid Plants between Citrus 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 Tree Science, Okitsu, Shimizu, Shizuoka 424-02 3Kanagawa Horticultural Experiment Station , Nebukawa Branch, Nebukawa, Odawara, Kanagawa 250 Summary Electrofusions between embryogenic callus protoplasts from Citrus species and mesophyII protoplasts from other Aurantioideae representatives, including Clauseneae, 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 plant 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 (Murraya centrifuged at 100 x g for 7 min, the supernatant paniculata [L.] Jack, Murraya koenigii [L.] discarded, and the fusion products resuspended at Spreng., Glycosmis 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 .] Severinia 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 AiM kinetin. 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 (Glycosmis pentaphylla, marmelos developed abnormal, small contorted Merrillia 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