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 Rough , Volkamer lemon, and 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 is tolerant of citrus blight, but is not Hubei 430070, People’s Republic of China used as a rootstock due to sus- ceptibility. Somatic hybridization of Cleo- Nicasio Tusa4 patra mandarin with , 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 was attempted to gen- Additional index words. citrus blight, citrus breeding, , 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 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 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 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; 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) ‘Hamlin’ sweet orange + Rangpur somatic for the Cleopatra mandarin + Rangpur and hybrid; (1) ‘Hamlin’ sweet orange; (m) ‘Valencia’ sweet orange; (n) ‘Valencia’ + Carrizo citrange sour orange + Volkamer lemon combina- somatic hybrid; (o) Carrizo citrange; (q) Cleopatra mandarin + Volkamer lemon somatic hybrid; (r) tions appeared to be most efficient on DBA3 Volkamer lemon; (s) tetraploid Volkamer lemon zygotic; (t) sour orange + Volkamer lemon zygotic medium, and shoots also developed via ad- somatic hybrid (leaf type I); (u) sour orange + Volkamer lemon zygotic somatic hybrid (leaf type ventitious budding on the cut surfaces of dis- II). sected embryos of these hybrid genotypes on this medium. Embryo germination for the remaining combinations was adequate on B + embryo germination medium. The Cleopatra mandarin + Rangpur and ‘Hamlin’ sweet orange + Rangpur combinations showed vigor throughout the protoplast to plant cycle, as demonstrated by the large populations of hybrid plants recovered (Table 1). The Cleo- patra mandarin and sour orange callus lines apparently lost their regeneration capability, because no plants were regenerated from nonfused protoplasts of these lines. Plant re- generation of ‘Hamlin’ sweet orange from nonfused callus-derived protoplasts was in- hibited by the high-sucrose content of EMEP protoplast culture medium, as previously re- ported (Grosser et al., 1988a, 1988b; Ohga- wara et al., 1985). Numerous diploid plants of sour orange, the zygotic Volkamer lemon, z and a few plants of Rangpur were regener- Indicates the number of plants regenerated but not necessarily the number of plants recovered from ated from nonfused leaf protoplasts. A tetra- individual fusion events. yPhosphoglucose isomerase used to confirm somatic hybridity. ploid plant of the zygotic Volkamer lemon xPeroxidase used to confirm somatic hybridity. was also recovered, apparently resulting from wPhosphoglucomutase used to confirm somatic hybridity. a homokaryotic fusion. These results prob- vIncludes one autotetraploid plant. ably depended on co-culture with embry-

1034 HORTSCIENCE,VOL. 27(9), SEPTEMBER 1992 Table 2. Designated leaf isozyme genotypes of the donor Citrus parents for peroxidase (PER), phos- toplast fusion and Citrus improvement. Plant phoglucose isomerase (PGI), and phosphoglucose mutase (PGM). Breeding Rev. 8:339-374. Grosser, J.W. and F.G. Gmitter, Jr. 1990b. Wide Donor PER PGI PGM hybridization of Citrus via protoplast fusion: Cleopatra mandarin FF MM FF Progress, strategies and limitations, p. 31-41. Sour orange WS FS In: A.B. Bennett and S.D. O’Neill (eds.). Hor- Rough lemon MS FI ticultural biotechnology, Plant biology. vol. 25. Volkamer lemon MS FI Wiley-Liss, New York, Volkamer lemon zygotic SS MS IS Grosser, J.W. and F.G. Gmitter, Jr. 1990c. So- Rangpur SS MS FI matic hybridization of Citrus with wild relatives Hamlin/ FF MS FS for germplasm enhancement and de- Smooth Flat Seville FS MS FS velopment. HortScience 25:147-151. Carrizo citrange FP SS PS Grosser, J.W., F.G. Gmitter, Jr., and J.L. Chan- dler. 1988a. Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus tri- foliata cv. Flying Dragon. Plant Cell Rpt. 7:5- ogenic culture-derived protoplasts and add to and did not show the S allele that we ex- 8. the growing number of genotypes amenable pected to be contributed by both parents. The Grosser, J.W., F.G. Gmitter, Jr., and J.L. Chan- to plant regeneration from leaf protoplasts S allele was either lost or its expression dler. 1988b. Intergeneric somatic hybrid plants (Grosser et al., 1991; Ohgawara et al., 1989; blocked in these plants for reasons we cannot from sexually incompatible woody : Cit- Tusa et al., 1990). explain. All sour orange + Volkamer lemon rus sinensis and Severinia disticha. Theor. Ap- The leaf morphology of all recovered so- zygotic somatic hybrid plants were clearly plied Genet. 75:397-401. matic hybrid plants was intermediate to that hybrid at the PGI locus, showing the ex- Grosser, J.W., F.G. Gmitter, Jr., F. Sesto, X.X. of the parents (Fig. 1). Two distinct mor- pected WMSS banding pattern. We have Deng, and J.L. Chandler. 1991. Six new so- matic citrus hybrids and their potential for cul- phological types were unexpectedly ob- previously observed and explained missing tivar improvement. J. Amer. Soc. Hort. Sci. served in the population of sour orange + isozyme bands in somatic hybrid plants on 117:169-173. Volkamer lemon zygotic somatic hybrid plants two occasions. An expected M band at the Grosser, J,W., F.G. Gmitter, Jr., N. Tusa, and (designated types I and II) (Fig. l), with about PGI locus was not present in Milam lemon J.L. Chandler. 1990. Somatic hybrid plants from three times as many type I as type II plants. (Citrus jambhiri Lush purported hybrid) + sexually incompatible woody species: Citrus Both types exhibited the same isozyme ‘Femminello’ lemon ( C. limon L. Burm. F.) reticulata and Citropsis gilletiana. Plant Cell banding patterns for PER, PGI, and PGM. somatic hybrid plants, but the M band was Rpt. 8:656-659. This is the first time we have observed mor- also missing in the parental Milam lemon Grosser, J.W., G.A. Moore, and F.G. Gmitter, phological variation in somatic hybrid plants callus line and plants regenerated from this Jr. 1989. Interspecific somatic hybrid plants from the fusion of ‘Key’ (Citrus aurantifolia) from a single parental combination. This line (Tusa et al., 1992). An expected S band with ‘Valencia’ sweet orange (Citrus sinensis) variation may be explained by differences in at the PGI locus was not present in ‘Hamlin’ protoplasts. Scientia Hort. 39:23-29. the inheritance of chloroplast and/or chon- sweet orange ( C. sinensis) + Severinia bux- Kobayashi, S., K. Fujiwara, I. Oiyama, T. Ohga- driome genomes in the two types. Kobayashi ifolia Poir. Tenore, but these plants were also wara, and S. Ishii. 1988a. Somatic hybridiza- et al. (1991) showed differential inheritance missing chromosomes (2n = 27, whereas tion between navel orange and ‘’ . of chloroplast genomes in somatic hybrid the expected chromosome number was 2n = 1988 Proc. Intl. Soc. Citriculture 1:135-140. plants of nave1 orange ( C. sinensis) and 4x = 36). Kobayashi, S., T. Ohgawara, K. Fujiwara, and I. ‘Murcott’ tangor, but no morphological var- Plants of the somatic hybrid rootstocks re- Oiyama. 1991. Analysis of cytoplasmic gen- iation was observed. ported herein have been propagated and en- omes in somatic hybrids between navel orange (Citrus sinensis Osb.) and ‘Murcott’ tangor. Chromosome number determinations con- tered into field trials. Determining their Theor. Applied Genet. 82:6-10. firmed tetraploidy (2n = 4x = 36) for all characteristics and the nature of the genetic Kobayashi, S., T. Ohgawara, I. Oiyama, and S. eight somatic hybrid combinations, Leaf iso- control of important traits should provide a Ishii. 1988b. A somatic hybrid plant obtained zyme genotypes (PER, PGI, PGM) for all valuable basis for selecting parents for future by protoplast fusion between navel orange (Cit- donors are listed in Table 2. Expression of somatic hybridizations. rus sinensis) and satsuma mandarin (Citrus un- all parental alleles (complementation) was shiu). Plant Cell, Tissue & Organ Cult. 14:63- 69. observed in the putative somatic hybrids (with Literature Cited one exception described below) and con- Murashige, T. and D.P.H. Tucker. 1969. Growth firmed their hybridity. For the monomers Cardy, B.J., C.W. Stuber, and M.M. Goodman. factor requirements of Citrus tissue culture. Proc. 1981. Techniques for starch gel electrophoresis First Intl. Citrus Symp. 3:1155-1161. (PER, PGM), complementation was ob- of enzymes for maize ( Zea maize L.). Inst. Sta- Ohgawara, T., S. Kobayashi, S. Ishii, K. Yosh- served simply as additive expression of pa- tistics Mimeogr. Series 1317. North Carolina inoga, and I. Oiyama. 1989. Somatic hybridi- rental bands; for PGI (a dimeric enzyme), State Univ., Raleigh. zation in Citrus: Navel orange ( C. sinensis Osb.) complementation resulted in the appearance Castle, W.S. 1987. Citrus rootstocks, p. 361-399. and grapefruit ( C. paradisi Macf.). Theor. Ap: of all parental bands plus unique heterodi- In: R.C. Rom and R.F. Carlson (eds.). Root- plied Genet. 78:609-612. mers not present in either parent. The en- stocks for fruit crops. Wiley, New York. Ohgawara, T., S. Kobayashi, E. Ohgawara, H. zyme activity staining systems diagnostic of Castle, W.S., D.P.H. Tucker, A.H. Krezdom, and Uchimiya, and S. Ishii. 1985. Somatic hybrid hybridity for each combination are noted in C.O. Youtsey. 1989. Rootstocks for Florida cit- plants obtained by protoplast fusion between Table 2. rus. Univ. of Florida, Coop. Ext. Publ. SP 41. Citrus sinensis and Poncirus trifoliata. Theor. Deng, X.X., J.W. Grosser, and F.G. Gmitter, Jr. Applied Genet. 71:14. Isozyme analysis indicated that the Vol- 1991. Intergeneric somatic hybrid plants from Tusa, N., J.W. Grosser, and F.G. Gmitter, Jr. kamer lemon seedling that provided the leaf protoplast fusion of Fortunella crassifolia 1990. Plant regeneration of ‘Valencia’ sweet or- protoplasts used in the fusions with sour or- ‘Meiwa’ with Citrus sinensis ‘Valencia’. Scien- ange, ‘Femminello’ lemon, and the interspe- ange was of zygotic origin. This original tia Hort. 49:55-62. cific somatic hybrid following protoplast fusion. Volkamer lemon seedling and plants regen- Grosser, J.W. 1991. In vitro methods for tropical J. Amer. Soc. Hort. Sci. 115:1043-1046. erated from nonfused leaf protoplasts of this fruit improvement. In: I.K. Vasil and T.A. Tusa, N., J.W. Grosser, F.G. Gmitter, Jr., and plant displayed an IS isozyme banding pat- Thorpe teds.). Plant tissue culture: Theory and E.S. Louzada. 1992. Production of tetraoloid tern at the PGM locus, whereas true-to-type applications. Kluwer Academic Publishers, somatic hybrid breeding parents for use in lemon Volkamer lemon is FI. The zygotic Volka- Dordrecht, The Netherlands. (In Press.) cultivar improvement. HortScience 27:445-447. mer lemon plants displayed the typical Vol- Grosser, J.W. and J.L. Chandler. 1987. ‘Aseptic Vallejos, C.E. 1983. Enzyme staining activity, p. isolation of leaf protoplasts from Citrus, Pon- 469-516. In: S.D. Tanksley and T.J. Orton kamer lemon patterns for the PER and PGI cirus, Citrus × Poncim hybrids and Severinia (eds.). Isozymes in plant genetic and breeding, loci (Table 2). The isozyme banding pattern for use in somatic hybridization experiments. part A. Elsevier, Amsterdam. of the sour orange + Volkamer lemon zyg- Scientia Hort. 31:253-257. Vardi, A., P. Arzee-Gonen, A. Frydman-Shani, otic somatic hybrid plants for PGM was FI Grosser, J.W. and F.G. Gmitter, Jr. 1990a. Pro- S. Breichman, and E. Galun. 1989. Protoplast-

HORTSCIENCE, VOL. 27(9), SEPTEMBER 1992 1035 fusion-mediated transfer of organelles from Mi- rus cybrids: Production by donor-recipient pro- in protoplast culture of horticultural crops. crocitrus into Citrus and regeneration of novel toplast-fusion and verification by mitochondrial- Scientia Hort. 37:217-230. alloplasmic trees. Theor. Applied Genet. 78:741- DNA restriction profiles. Theor. Applied Ge- Whiteside, J.O., SM. Garnsey, and L.W. Tim- 747. net. 75:.51-58 mer (eds.). 1988. Compendium of citrus dis- Vardi, A., A. Breiman, and E. Galun. 1987. Cit- Vardi, A. and E. Galun. 1988. Recent advances eases. APS Press, St. Paul.

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