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Eight New Somatic Hybrid Citrus Rootstocks with Potential For

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Eight New Somatic Hybrid Citrus Rootstocks with Potential For HORTSCIENCE 27(9):1033-1036. 1992. pest resistance into horticulturally desirable rootstocks adapted to the various environ- mental conditions that exist in Florida. Eight New Somatic Hybrid Citrus Rough lemon, Volkamer lemon, and Rangpur have been used as rootstocks, be- Rootstocks with Potential for cause trees on these stocks are vigorous, high- yielding, and tristeza- and drought-tolerant Improved Disease Resistance (Castle, 1987; Castle et al., 1989). How- ever, their use has declined because they are Eliezer S. Louzada1, Jude W. Grosseti, Frederick G. Gmitter, Jr.2, highly susceptible to citrus blight. Cleopatra Beatriz Nielsen3, and J.L. Chandler3 mandarin has become an increasingly im- portant rootstock that is less susceptible to Department of Horticultural Sciences, Citrus Research and Education citrus blight than the rootstocks named above, Center, Institute of Food and Agricultural Sciences, University of tolerant to tristeza, and cold-hardy. Trees Florida, 700 Experiment Station Road, Lake Alfred, FL 33850 grafted on Cleopatra mandarin are moder- ately vigorous but are slow to reach full 2 Xiu Xin Deng bearing potential (Castle et al., 1989). Sweet Department of Horticulture, Huazhong Agricultural University, Wuhan orange is tolerant of citrus blight, but is not Hubei 430070, People’s Republic of China used as a rootstock due to Phytophthora sus- ceptibility. Somatic hybridization of Cleo- Nicasio Tusa4 patra mandarin with rough lemon, Volkamer lemon, and Rangpur; ‘Hamlin’ sweet orange Centro di Studio per il Miglioramento Genetico degli Agrumi, Consiglio with Rangpur; and ‘Valencia’ sweet orange Nazionale delle Ricerche, Male delle Scienze, Palenno 90128, Sicily with Carrizo citrange was attempted to gen- Additional index words. citrus blight, citrus breeding, citrus tristeza virus, protoplast erate vigorous productive rootstocks with ac- fusion, tetraploid, tissue culture, Citrus ceptable horticultural characteristics but improved blight tolerance. Abstract. Protoplast culture following polyethylene glycol-induced fusion resulted in Sour orange, formerly the most important the regeneration of vigorous tetraploid somatic hybrid plants from eight complemen- rootstock in almost every citrus-growing re- tary parental rootstock combinations: Citrus reticulata Blanco (Cleopatra mandarin) gion of the world, is still important in some + C. aurantium L. (sour orange), C. reticulata (Cleopatra mandarin) + C. jambhiri areas because of its yield potential, favorable Lush (rough lemon), C. reticulata (Cleopatra mandarin) + C. volkameriana Ten. & influence on fruit quality, wide soil adapta- Pasq. (Volkamer lemon), C. reticulata (Cleopatra mandarin) + C. limonia Osb. (Rang- bility, and tolerance to cold, Phytophthora- pur), C. sinensis (L.) Osb. (Hamlin sweet orange) + C. limonia (Rangpur), C. auran- induced diseases, and citrus blight (Castle, tium (sour orange) + C. volkameriana (Volkamer lemon) zygotic seedling, C. auruntium 1987; Castle et al., 1989). However, its ex- hybrid (Smooth Flat Seville) + C. jambhiri (rough lemon), and C. sinensis (Valencia treme susceptibility to tristeza-induced de- sweet orange) + Carrizo citrange [C. paradisi Macf. × Poncirus trifoliata (L.) Raf.]. cline with sweet orange or grapefruit scions Diploid plants were regenerated from nonfused callus-derived protoplasts of Valencia has drastically reduced or eliminated its use sweet orange and Smooth Flat Seville and from nonfused leaf protoplasts of sour in most citrus-growing regions, including orange, Rangpur, rough lemon, and Volkamer lemon. Regenerated plants were class- Florida and Brazil. Somatic hybridization of ified according to leaf morphology, chromosome number, and leaf isozyme profiles. sour orange with tristeza-tolerant Cleopatra All somatic hybrid plants were tetraploid (2n = 4x = 36). One autotetraploid plant mandarin and Volkamer lemon, and Smooth of the Volkamer lemon zygotic was recovered, apparently resulting from a homokar- Flat Seville with tristeza-tolerant rough lemon yotic fusion. These eight new citrus somatic hybrids have been propagated and entered was attempted to generate sour orange-type into field trials. rootstocks that are tolerant to tristeza virus and citrus blight. Somatic hybridization via protoplast fu- Grosser et al., 1988a, 1991; Ohgawara et Leaf protoplasts of sour orange, rough sion has become a powerful tool in Citrus al., 1985) and sexually incompatible parents lemon, Volkamer lemon, and Carrizo ci- for combining superior genomes to produce (Grosser and Gmitter, 1990c; Grosser et al., trange were isolated from young nucellar viable allotetraploid plants. New genetic 1988b, 1990, 1991), and cybrids (Vardi et seedlings maintained in a growth chamber combinations produced by protoplast fusion al., 1987, 1989). Recent reviews of proto- (16-h photoperiod, 300 µmol·m-2·s-1 light include interspecific somatic hybrids (Gros- plast culture (Vardi and Galun, 1988) and intensity, 26 to 30C; Grosser and Chandler, ser et al.; 1989, 1991; Kobayashi et al., somatic hybridization in Citrus (Grosser, 1987). Protoplasts of Cleopatra mandarin, 1988a, 1988b; Ohgawara et al., 1989; Tusa 1992; Grosser and Gmitter, 1990a, 1990b) ‘Hamlin’ and ‘Valencia’ sweet oranges, sour et al., 1990), intergeneric somatic hybrids are available. orange, and Smooth Flat Seville were iso- from sexually compatible parents (Deng et Primary pathological factors that dictate lated from friable, nucellus-derived, embry- al., 1991; Grosser and Gmitter, 1990a; citrus rootstock choice in Florida are citrus ogenic callus cultures maintained on either blight, citrus tristeza virus, Phytophthora- in- EME or H + H solid medium (Grosser and Received for publication 27 Jan. 1992. Accepted duced disease, and nematodes (Castle, 1987; Gmitter, 1990a). All protoplasts were puri- for publication 13 Apr. 1992. Research conducted Castle et al., 1989; Whiteside et al., 1988). fied by passage through a 4.5pm stainless at the Citrus Research and Education Center, Lake Horticultural performance and resistance to steel filter followed by centrifugation on a Alfred, Fla. Florida Agricultural Experiment Sta- abiotic stresses must also be adequate. No 25% sucrose-13% mannitol gradient before tion Journal Series no. R-02133. We thank S. commercial rootstock is completely satisfac- mixing (Grosser and Gmitter, 1990a; Tusa Huang and X.L. Hu for technical assistance. The tory for all selection criteria. One strategy et al., 1990). cost of publishing this paper was defrayed in part that applies somatic hybridization techniques Approximately equal volumes of embry- by the payment of page charges. Under postal reg- to rootstock improvement is the production ogenic callus-derived protoplasts of one par- ulations, this paper therefore must be hereby marked of tetraploid somatic hybrid rootstocks that ent and leaf protoplasts of the second parent advertisement solely to indicate this fact: 1Visiting scientist from the Univ. of Rio de Ja- combine the intact genomes of complemen- were mixed and fused for each parental com- neiro, Brazil. tary rootstock parents (Grosser and Gmitter, bination (Table 1) by use of the polyethylene 2Associate Professor. 1990b). The objective of our research was glycol (PEG) method of Grosser and Gmitter 3Biological Scientist. to produce a population of somatic hybrids (1990a). Following fusion, protoplasts were 4Researcher. that combine all of the necessary disease and cultured directly in 60 × 15mm fusion petri HORTSCIENCE, VOL. 27(9), SEPTEMBER 1992 1033 dishes (Falcon, Lincoln Park, N.J.) in either EMEP protoplast culture medium (for ‘Ham- lin’ sweet orange + Rangpur) or a 1 EMEP : 1 BH3 (v/v) protoplast culture media (for all remaining combinations), according to Grosser and Gmitter (1990a). Recovered so- matic embryos were germinated on either B + embryo germination medium, DBA3 me- dium (Grosser and Gmitter, 1990a), or MT basal medium (Murashige and Tucker, 1969) containing (mg·liter-1): 0.5 6-benzyladenine (BA), 1.0 gibberellic acid (GA3), and 0.01 1-napthaleneacetic acid (NAA) (designated BGN). Chromosome numbers in root-tip cells from regenerated plants were determined with the modified hematoxylin staining method of Grosser and Gmitter (1990a). Isozyme band- ing patterns were determined and compared with crude leaf tissue extracts of Cleopatra mandarin, ‘Hamlin’ and ‘Valencia’ sweet oranges, sour orange, Smooth Flat Seville, Carrizo citrange, rough lemon, Volkamer lemon, Rangpur, and regenerated plants. Peroxidase (PER), phosphoglucomutase (PGM), and .phosphoglucose isomerase (PGI) isozymes were separated by horizontal starch gel electrophoresis on 10% Connaught starch gels with the pH 5.7 histidine citrate buffer of Cardy et al. (1981). Electrophoresis was carried out for 3 h at 4C and constant 60 mA current. Gels were stained as described by Vallejos (1983). Plants regenerated following protoplast fusion are summarized in Table 1. The shapes of recovered somatic embryos were often ab- normal, but this did not interfere with ger- mination and plant recovery. For the ‘Hamlin’ Fig. 1. Leaf morphology of: (a) Smooth Flat Seville; (b) Smooth Flat Seville + rough lemon somatic sweet orange + Rangpur combination, em- hybrid; (c) rough lemon; (d) Cleopatra mandarin + rough lemon somatic hybrid; (e, h, p) Cleopatra bryo germination appeared to be most effi- mandarin; (f) Cleopatra mandarin + sour orange somatic hybrid; (g, v) sour orange; (i) Cleopatra cient on BGN medium. Embryo germination mandarin + Rangpur somatic hybrid; (j) Rangpur; (k)
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