Wide Somatic Hybrids of Citrus with Its Related Genera and Their Potential in Genetic Improvement

Home , Aurantioideae, Citropsis, Severinia (plant), Severinia buxifolia

Wide somatic hybrids of Citrus with its related genera and their potential in genetic improvement

W.W. Guo & X.X. Deng∗ National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China; (∗author for correspondence; e-mail: [email protected])

Received 11 November 1999; accepted 11 September 2000

Key words: Aurantioideae, Citrus, genetic improvement, protoplast fusion, sexually compatible / incompatible intergeneric somatic hybrids

Summary Citrus wild relatives are an untapped germplasm reservoir, which possesses many elite resistance traits. Genetic introgression into Citrus by conventional methods has been greatly hindered by polyembryony, pollen / ovule sterility, sexual / graft incompatibility, long juvenility etc. Somatic hybridization via protoplast fusion may make it possible to transfer genes from wild relatives to Citrus. To date, more than sixty sexually compatible or incompatible intergeneric somatic hybrids between Citrus and its various related wild genera have been produced by PEG – or electrically – induced fusion. These wide somatic hybrids were identiﬁed by morphology, cytology, isozyme, RAPD and RFLP analyses. Genetic variation or recombination was also revealed in some of them. Several sexually compatible combinations have ﬂowered and set fruits. The agronomic performance of these wide somatic hybrids is diverse, and the current results are not conclusive. Somatic hybrids are being tested as rootstocks. The prospects of wide somatic hybridization of Citrus with its wild relatives are discussed.

Introduction resistance, and exhibits good cold hardiness (Bitters, 1969). Citropsis offers strong resistance to Phytoph- Citrus wild relatives are an untapped germplasm reser- thora induced diseases (Swingle & Reece, 1967) and voir, which possesses many elite resistance traits. Pon- burrowing nematode (Ford & Feder, 1960). Feronia cirus is highly resistant to citrus tristeza virus (CTV), is known to be drought tolerant, and due to its de- Phytophthora-induced diseases, as well as the citrus ciduous nature, it may be a source of genes for cold nematode (Castle, 1987), and is extensively used as a hardiness (Swingle & Reece, 1967). Swinglea glu- citrus rootstock in China and Japan. Fortunella is cold tinosa grows well in sandy loam and porous limestone hardy (Swingle & Reece, 1967), F. hindsii var. Chin- soils (Swingle & Reece, 1967), and Feroniella lucida tou was verified to be highly resistant to Phytophthora is dwarfing (Swingle & Reece, 1967), resistant to CTV parasitica (Zhu et al., 1991), and F. crassifolia cv. (Yoshida, 1996). Murraya paniculata is immune to Meiwa has been identified to be CTV resistant (Mestre citrus huanglongbing (Chen & Liao, 1982; Guo & et al., 1997). Microcitrus offers resistance to drought, Deng, 1998a), resistant to citrus nematodes (Sykes, flooding, the burrowing nematode (O’Bannon & Ford, 1987), citrus tristeza virus (Yoshida, 1996), and grows 1977), and Phytophthora induced diseases (Carpenter well in alkaline conditions (Sykes, 1987). Clausena is & Furr, 1962). Severinia offers cold resistance, salt tolerant to alkaline conditions (Sykes, 1987). Further and boron tolerance (Cooper, 1961), resistance to study of citrus wild relatives will undoubtedly reveal Phytophthora (Carpenter & Furr, 1962; Grimm & more elite traits. Hutchison, 1977), and nematodes (Baines et al., 1960; However, these citrus wild relatives have not been Hutchison & O’Bannon, 1972). Atalantia is known to commercially used except Poncirus, Fortunella,and perform well in wet soils, suggesting Phytophthora Clausena lansium, and only Poncirus and Fortun- ______中国科技论文在线176 http://www.paper.edu.cn

ella are being successfully utilized through sexual ance of plants regenerated from most fusions between hybridization in breeding programs. Other citrus wild sexually incompatible parents was acceptable. Only a relatives performed poor as citrus rootstocks, or are few sexually incompatible fusions regenerated weak sexually / graft incompatible with citrus (Iwamasa et shoots (Motomura et al., 1996a; Shinozaki et al., 1992; al., 1988). Even if they are sexually compatible with Takayanagi et al., 1992). The success in regenera- citrus, the following problems or difficulties still exist tion of these somatic hybrids proved that protoplast (Barrett, 1985): 1) sexual crosses are usually much fusion was effective to bypass the difficulties such more difficult to make, and if seed is obtained at all, as polyembryony, pollen/ovule sterility, or sexual in- the seed yield is often low and it may be nonviable; compatibility encountered in sexual crossing, and it 2) if hybrids are obtained, the progenies ultimately is possible to realize genetic recombination of Citrus available for selection are often few in number due with its wild relatives. For the detailed advantages of to lethals, abnormal recombinants, or poor field sur- somatic hybridization versus sexual hybridization to vival; 3) the surviving hybrids may not flower, those recover hybrids among compatible and incompatible that do may have very long juvenile periods, or may parents, see Grosser & Gmitter, 1990a. be completely to partially ovule or pollen sterile; 4) undesirable traits may be integrated into Citrus.In comparison, somatic hybridization via protoplast fu- Genetic identification of these wide somatic sion is an alternative to circumvent some of these hybrids difficulties, and may pave the way for transferring their resistance genes to Citrus. These somatic hybrids were routinely identified by morphology, cytology, isozyme, RAPD (randomly amplified polymorphic DNA) (Grosser et al., 1996c; Somatic hybrids of Citrus with its related genera Shi et al., 1998b; Xiao et al., 1995) and RFLP (re- produced to date striction fragment length polymorphism) (Ohgawara et al., 1994; Motomura et al., 1996b), and genetic Mesophyll protoplasts isolated from citrus leaves can differences were revealed in some of them. not divide and regenerate into plants under the cur- rently used culture schemes (Tusa et al., 1992), but Leaf morphology protoplasts isolated from Citrus embryogenic callus can do so. Embryogenic calluses have been induced, The leaf of these wide somatic hybrids was generally and their protoplast-derived plants have been regener- wider and thicker than either fusion parent (Grosser ated from many species of Citrus genus and its related et al., 1996c) probably due to ploidy effect. Citrus genera (Huo et al., 1999; Jumin & Nito, 1996; Vardi & species are unifoliate. When citrus wild relatives with Spiegel-Roy, 1982). For protoplast fusion, the model compound leaves were used as the other fusion par- of ‘embryogenic callus + mesophyll protoplasts’ is ex- ent, their corresponding somatic hybrids were also of tensively used in Citrus, and allows the regeneration of compound leaves with bifoliate or trifoliate character, hybrid plants. For methods and procedures of Citrus as can be seen from the somatic hybrids of Citrus + protoplast isolation, protoplast fusion (PEG and elec- Poncirus (Ohgawara et al., 1985), Citrus + Citrop- trofusion) and culture, see Grosser & Gmitter, 1990a; sis (Grosser et al., 1996c), Citrus + Murraya (Guo Guo et al. 1998; Hidaka & Omura, 1992. & Deng, 1998b), Citrus + Clausena (Guo & Deng, Since the first intergeneric somatic hybrid in Citrus 1999b), and Citrus + Feronia (Grosser et al., 1996c). was regenerated (Ohgawara et al., 1985), more than Additionally, plants with unexpected abnormal leaf 60 intergeneric somatic hybrids have been obtained morphology were also found in some fusion combin- by PEG or electrically induced fusion (Table 1). Two ations (Louzada et al., 1993; Motomura et al., 1995, thirds of the existing wide somatic hybrids were cre- 1996b). ated by the research group led by Prof. J.W. Grosser in University of Florida, U.S.A., the rest produced in Ploidy variation France, Israel, Japan, and in our program led by Prof. X.X. Deng in China. From the published papers, it is Most wide somatic hybrids were tetraploids (2n = 4x = noteworthy that fusions between sexually compatible 36) following fusion of diploid + diploid, but un- parents regenerated healthy plants, and the perform- expected ploidy variation also exists in some fusion ______中国科技论文在线 http://www.paper.edu.cn177

Table 1. Somatic hybrids between Citrus and its related genera

Embryogenic parent Mesophyll parent Number of fusion References combinations

Sexually compatible Citrus aurantium Citrangea 2 Grosser et al., 1998, 2000 Poncirus 2 Grosser et al., 1994, 2000 Citrus deliciosa Fortunella japonica 1 Ollitrault et al., 1996 Poncirus 1 Ollitrault et al., 1996 C. jambhiri (hybrid Milam) Citrange 1 Grosser et al., 1998 Citrumelob 1 Grosser et al., 1998 Citrus paradisi Poncirus 3 Grosser et al., 1996c, 2000 Citrus reticulata Citrange 3 Grosser et al., 1996b, 1998; Guo, 1998 Citrumelo 1 Grosser et al., 1992 Fortunella obovata 1 Liu, 1999; Microcitrus papuana 1 Motomura et al., 1996b Poncirus 3 Grosser et al., 1992, 1994; Guo, 1998; Citrus sinensis Citrange 2 Louzada et al., 1992; Orgawara et al., 1991 Fortunella crassifolia 2 Deng et al., 1992; Grosser et al., 1996c Microcitrus papuana 2 Grosser et al., 1996c Poncirus 4 Grosser et al., 1988b, 1994, 2000; Orgawara et al., 1985 F. crassiforlia C. reticulata 2 Grosser et al., 1996c F. hindsii Poncirus 1 Miranda et al., 1997 M. papuana C. jambhiri 1 Liu et al., 1999 Tangeloc & Tangord Citrange 2 Grosser et al., 1996b M. papuana 1 Grosser et al., 1996c; Poncirus 1 Guo et al., 2000b Sexually incompatible C. aurantifolia Feroniella lucida 1 Takayanagi et al., 1992 Swinglea glutinosa 1 Takayanagi et al., 1992 ∗ C. reticulta Citropsis gabunensis 1 Ling & Iwamasa, 1994 Citropsis gilletiana 1 Grosser et al., 1990 Severinia disticha 1 Grosser et al., 1996c C. sinensis Atlantia ceylanica 2 Louzada et al., 1993; Grosser et al., 1996c C. gilletiana 2 Grosser & Gmitter, 1990b; Grosser et al., 1996c Clausena lansium 2 Guo & Deng, 1999b; Louzada & Grosser, 1994 Feronia limonia 1 Grosser et al., 1996c Severinia buxifolia 3 Grosser et al., 1992, 1996c S. disticha# 3 Grosser et al., 1988a, 1996c Murraya paniculata 1 Shinozaki et al., 1992 Tangelo Atalantia monophylla 1 Motomura et al., 1995 C. gilletiana 1 Grosser et al., 1996c M. paniculata 1 Guo & Deng, 1998b S. buxifolia 1 Motomura et al., 1995 S. disticha 1 Grosser et al., 1996c ______中国科技论文在线178 http://www.paper.edu.cn

Table 1. Continued

Embryogenic parent Mesophyll parent Number of fusion References combinations

Cybrids produced by donor-recipient fusion ∗ M. papuana C. aurantium 1 Vardi et al., 1989 ∗ C. jambhiri 1 Vardi et al., 1989

a Citrange: sexual hybrid of Citrus sinensis and Poncirus. b Citrumelo: sexual hybrid of Citrus grandis and Poncirus. c Tangelo: sexual hybrid of Citrus paradisi and Citrus reticulata. d Tangor: sexual hybrid of Citrus sinensis and Citrus reticulata. ∗ Embryogenic callus, instead of mesophyll tissue. # Non-embryogenic callus, instead of mesophyll tissue was used in one fusion (Grosser et al., 1988a).

combinations. All the plants from fusion of Micro- plast isolation, was conducted. The results confirmed citrus papuana with rough lemon (Liu et al., 1999), that Clausena lansium was actually diploid (2n = 2x = and some plants from Bahia navel + Troyer citrange 18) as expected (Guo et al., 2000a). (Ohgawara et al., 1991) were diploid mesophyll par- From the above results, the possible reasons for ent type regenerants. The somatic hybrids between hexaploid regeneration from this fusion combination Fortunella crassifolia cv. Meiwa and Citrus sinensis could be hypothesized as (Guo & Deng, 1999b): 1) cv. Valencia grew less vigorously with uneven can- From the fusion of three protoplasts, and trinuclear opy size and some new flushes frequently died back. heterokaryons were probably more competitive than Chromosome counting showed that the plants were binuclear ones following fusion and during subsequent chimeras containing non-tetraploid cells besides am- culture; 2) Even though the embryogenic parent (i.e. phidiploid (Shi et al., 1998a). The somatic hybrids of Bonanza navel orange) was verified to be true dip- Hamlin sweet orange with Severenia buxifolia were loid, the possibility that tetraploid cells could exist triploids (Grosser et al., 1992). Besides hexaploid in it can not be excluded; 3) Chromosome doub- plants, pentaploid plants were regenerated from fusion ling of either parent may have occurred rather than of tetraploid Fortunella hindissi cv. Mame kumquat chromosome elimination following fusion and dur- with diploid Poncirus trifoliata as revealed by flow ing the subsequent culture, which could have resulted cytometry (Miranda et al., 1997). in genetic and physiological harmony of the somatic All plants regenerated from the fusion between hybrids, providing another possible reason for the diploid Citrus sinensis cv. Bonnaza navel orange and hexaploid plants since the regenerants are morpholo- diploid Clausena lansium cv. chicken heart sweet gically normal and growing. Further efforts to fuse wampee were not tetraploids (2n = 4x = 36), but diploid Chinese wampee mesophyll protoplasts with hexaploids (2n = 2x = 56), which was the first re- embryogenic protoplasts of other diploid citrus spe- port so far on the regeneration of hexaploid hybrid cies, and to check the chromosome number of the plants from fusion between diploids in Aurantioideae regenerants to verify hexaploidy, will be conducted to (Guo & Deng, 1999a, 1999b). We verified that ‘Bon- clarify the reason for hexaploid regeneration. anza’ navel orange was truly diploid (2n = 2x = 18), therefore it was highly unlikely for ‘Bonanza’ navel Genetic differences revealed by Isozyme, RAPD and orange to be tetraploidized at the time when fusion was RFLP analysis conducted. In various isozyme systems, most wide somatic hy- For many years, Citrus wild relatives including the brids of different fusions combined the bands of genus Clausena were considered to be diploids (2n = both parents, and the bands were identical among 2x = 18). However, Clausena excavata was recently plants from the same combination (Deng et al., 1992; identified to be tetraploid by flow cytometry (2n = Grosser et al., 1992). However, the EST isozyme pat- 4x = 36) (Froelicher & Ollitrault, 1998). To confirm tern between the somatic hybrids of Valencia sweet whether Clausena lansium was diploid, chromosome orange with Meiwa kumquat was different and most observation of Clausena lansium cv. chicken heart plants have a new band that was absent in both parents. sweet wampee, the cultivar used for mesophyll proto- RAPD analysis further revealed that some plants lose ______中国科技论文在线 http://www.paper.edu.cn179

bands of their parents (Shi et al., 1998a). PGI isozyme sequently, an alternative propagating method by cut- analysis of the triploid plants between Hamlin sweet ting in vivo or root induction in vitro is necessary. orange and Severinia buxifolia revealed that S allele The updated evidence shows that the rooting ability of PGI specific to Hamlin sweet orange was lost in the of somatic hybrids between Citrus and its sexually somatic hybrids (Grosser et al., 1992). compatible genera (i.e. Poncirus, citrange, citrumelo, Cytoplasmic genome analysis of wide somatic hy- Microcitrus) was acceptable while that of the somatic brids by RFLP technique showed that, for most com- hybrids between Citrus and its sexually incompat- binations, their mitochondrial DNA was identical to ible genera was generally unsatisfactory (Deng et al., their corresponding embryogenic parent while their 1997; Guo, 1998; Motomura et al., 1996a; Sabbah chloroplast DNA was randomly inherited (Ohgawara et al., 1991). There are also several exceptions, for et al., 1994), which was the same for most citrus instance, both somatic hybrids of Citrus sinensis cv. interspecific cybrids (Grosser et al., 1996d; Guo & Bonnaza navel + Clausena lansium cv. chicken heart Deng, 2000). The mtDNA of the tested 10 plants from sweet wampee (Guo & Deng, 1999b), and Citrus Citrus reticulata cv. Hazzara + Microcitrus australis sinensis + Severinia disticha (Grosser et al., 1988a) was identical to Hazzara, the embryogenic parent; but rooted well. the cpDNA of six plants was from Hazzara, and four The effort to induce rooting from the regenerated plants from both parents. Diversity was also revealed shoots of Citrus + Murraya paniculata failed. One in the remaining four plants (Motomura et al., 1996b). month after being transferred to root-induction me- The cpDNA of the somatic hybrids between Semin- dium, the unrooted shoots gradually turned yellow ole tangelo + Severinia buxifolia, Seminole tangelo from the bottom up and then died (Guo & Deng, + Atalantia monophylla respectively was identical to 1998b). Even grafted, some somatic hybrids still their corresponding mesophyll parents, and recombin- did not survive probably due to graft incompatibility ation of mtDNA was found in both combinations (Mo- (Louzada & Grosser, 1994; Guo & Deng, 1998b). tomura et al., 1995). The cytoplasmic analysis of one pentaploid and two hexaploid plants from tetraploid Reproductive ability Mame kumquat + diploid Poncirus trifoliata revealed Generally, the fertility of citrus autotetraploids is that their cpDNA and mtDNA were all identical to the lower than that of its corresponding diploid species, embryogenic parent (i.e. Mame kumquat) (Miranda et al., 1997). while that of citrus allotetraploid somatic hybrids was higher than the mean value of both parents (Deng et The possible explanations for genetic variation of these wide somatic hybrids include: somaclonal vari- al., 1995a). To date, only several (not exceeding ten) ation (possibly induced by plant growth regulators somatic hybrids of Citrus + Poncirus, Citrus + citrange, Citrus + citrumelo, Citrus + Microcitrus have during culture); genetic variability in the embryogenic callus parent; chromosome elimination or polyploidiz- flowered and set fruits (Grosser et al., 2000; Ohgawara & Ishii, 1991; Ohgawara et al., 1991). Their pollen ation following wide protoplast fusion; or preferential viability was acceptable, and the fruits had abundant organelle inheritance (Grosser et al., 1992; Guo & polyembryonic seeds, which would be conducive to Deng, 1999b; Louzada et al., 1993). producing uniform rootstock seedlings. Most somatic hybrids between citrus and its related genera have not yet flowered though they have been created for many The agronomic performance of wide somatic years. This is probably due to somatic incompatibility hybrids between both fusion parents.

Rooting ability Performance as rootstocks

Rooting ability of wide somatic hybrids in vitro and Dwarfing rootstocks are the objective of many im- in vivo is possibly an index for whether or not they provement programs. Due to a ploidy effect, tetraploid could be utilized as rootstocks. As a rootstock, its citrus rootstocks can have a dwarfing effect on scion root system should be strong. Most citrus wide so- cultivars (Lee et al., 1990). Citrus wide somatic hy- matic hybrids have not flowered yet, and even if brid rootstocks generally produced trees smaller than flowered, whether they would have seeds and whether presently used commercial rootstocks (Grosser et al., seeds would be polyembryonic is still unknown. Con- 2000), and could be tested for dwarfing rootstock ______中国科技论文在线180 http://www.paper.edu.cn

breeding. For most wide somatic hybrids, their resist- sexual and/or graft incompatibility, but the incompat- ance to various biotic or abiotic factors were generally ibility phenomenon still exists, as can be seen from intermediate between both parents (Deng et al., 1995b; many wide fusion combinations. Graft compatibility Deng & Zhang, 1995; Grosser et al., 1996a; Zhou et between two fusion parents seems to be a good in- al., 1997). Trees budded to Citrus + Severinia, Citrus dicator as to whether or not a somatic hybrid can be + Citropsis, Citrus + Fortunella crassifolia somatic produced (Louzada & Grosser, 1994). hybrids performed poorly, while the field perform- Asymmetric fusion or donor-recipient fusion, in ance of trees budded to the somatic hybrids between which highly irradiated donor protoplasts are fused Succari sweet orange + Atalantia ceylanica and Nova with nonirradiated cells, can reduce or in some tangelo + Citropsis gilletiana is encouraging (Grosser cases circumvent the somatic incompatibility barriers et al., 2000). Wide somatic hybrids are also being between two species (Hinnisdaels et al., 1994). How- tested as interstocks to improve tree size and possibly ever, our understanding of the genetic background of disease resistance. Other promising somatic hybrid Citrus and its related genera is presently too limited. rootstocks include sour orange + Carrizo citrange, and Furthermore, natural aneuploids of citrus are very few Cleopatra mandarin + Argentina trifoliate orange for and could not survive, and citrus asymmetric somatic commercial plantings, and sour orange + flying dragon hybrids (excluding cybrids) are actually aneuploids, trifoliate orange, and Cleopatra mandarin + flying which will be recalcitrant to regenerate and survive dragon trifoliate orange which are dwarfing for door- transplantation making citrus asymmetric fusion even yard use. All these four somatic hybrids are producing more difficult. nucellar seeds abundantly (Grosser, 2000, personal Wide somatic hybrids between Citrus and its re- commu.). lated genera are being tested directly as rootstocks We have previously created the somatic hybrid or being propagated by rooted cuttings. If they are between Poncirus trifoliata and Citrus reticulata cv. fertile, they may be also useful as bridge plants to Hongju (Guo, 1998). Poncirus trifoliata,whichis move wild relative genes into horticulturally more immune to citrus tristeza virus (CTV), is the predom- acceptable material by breeding at the tetraploid level. inantly used rootstock in China. Its only defect is the GISH (genomic in situ hybridization) technique susceptibility to citrus exocortis virus (CEV), which has been extensively used to detect genetic interac- resulted in great economic losses. In the citrus pro- tion in wide somatic hybrids of other higher plants duction area affected by CEV, Hongju (C. reticulata) (Buiteveld et al., 1998; Escalante et al., 1998; which is tolerant to CEV has been used as rootstock in Parokonny et al., 1994), but has not yet been ap- China; however, the fruit quality on it is not as good plied to study citrus wide somatic hybrids. Compared as that on Poncirus trifoliata. We hope the somatic with somatic hybrids of other higher plants, citrus hybrid of Hongju with trifoliate orange could be tol- is a perennial woody plant, and can be vegetatively erant to both CTV and CEV. It is now being tested as propagated. The citrus wide somatic hybrids will be rootstock in the CEV infected citrus production area. long preserved whether they can root, flower, set fruits Actually, many wide somatic hybrids in the world are or not, which is beneficial to conduct basic genetic still under test for their performance as rootstocks, in- research. The GISH analysis of citrus wide somatic cluding behavior against stresses, dwarfing effect, and hybrids will undoubtedly enrich the knowledge of their effect on scion fruit quality. It seems too early plant somatic cell genetics. to conclude whether any somatic hybrids will become successful commercial rootstocks.

Acknowledgements Conclusions and prospects This project was funded by the National Natural Sci- To date, more than sixty wide somatic hybrids between ence Foundation of China (NSFC), the International Citrus and its related genera have been created in Foundation for Science (IFS) (Stockholm, Sweden) the world. The performance of sexually compatible through a grant to Dr W.W. GUO, and the Chenguang somatic hybrids was generally better than that of sexu- Youth Project of Wuhan City in China. Great appreci- ally incompatible ones. It can also be inferred that cell ation is extended to Prof. Jude W. Grosser, University fusion, to some extent, is an alternative to circumvent of Florida, U.S.A. for his providing of new informa- ______中国科技论文在线 http://www.paper.edu.cn181

tion related to the performance of citrus wide somatic Grosser, J.W., S.M. Garnsey & C. Halliday, 1996a. Assay of sour or- hybrids. ange somatic hybrid rootstocks for quick decline disease caused by citrus tristeza virus. Proc Int Soc Citriculture: 353–356. Grosser, J.W. & F.G. Gmitter Jr., 1990a. Protoplast fusion and Citrus improvement. Plant Breed Rev 8: 339–374. Grosser, J.W. & F.G. Gmitter Jr., 1990b. Somatic hybridization References of Citrus with wild relatives for germplasm enhancement and cultivar development. HortSci 25(2): 147–151. Grosser, J.W., F.G. Gmitter Jr., W.S. Castle & J.L. Chandler, 1996b. Baines, R.C., W.P. Bitters & O.F. Clark, 1960. Susceptibility of Production and evaluation of citrus somatic hybrid rootstocks: some species and varieties of citrus and some other rutaceous progress report. Proc Int Soc Citriculture: 1246–1250. plants to the citrus nematode. Plant Dis Rep 44: 281–285. Grosser, J.W., F.G. Gmitter Jr. & J.L. Chandler, 1988a. Intergeneric Barrett, H.C. 1985. Hybridization of Citrus and related genera. Fruit somatic hybrid plants from sexually incompatible woody spe- Var J 39(2): 11–16. cies: Citrus sinensis and Severinia disticha. Theor Appl Genet Bitters, W.P., D.A. Cole & J.A. Brusca, 1969. The Citrus Relatives 75: 397–401. as Citrus Rootstocks. Proc 1st Int Citrus Symp 1: 411–415. Grosser, J.W., F.G. Gmitter Jr. & J.L. Chandler, 1988b. Intergeneric Buiteveld, J., Y. Suo, M.M. van Lookeren Campagne & J. Creemers- somatic hybrid plants of Citrus sinensis cv Hamlin and Poncirus Molenaar, 1998. Production and characterization of somatic trifoliata cv Flying Dragon. Plant Cell Rep 7: 5–8. hybrid plants between leek (Allium ampeloprasum L.) and onion Grosser, J.W., F.G. Gmitter Jr., F. Sesto, X.X. Deng & J.L. Chand- (Allium cepa L.). Theor Appl Genet 96: 765–775. ler, 1992. Six new somatic citrus hybrids and their potential for Carpenter, J.B. & J.R. Furr, 1962. Evaluation of tolerance to root cultivar improvement. J Amer Soc Hort Sci 117(1): 169–173. rot caused by Phytophthora parasitica in seedlings of citrus and Grosser, J.W., F.G. Gmitter Jr., N. Tusa & J.L. Chandler, 1990. related genera. Phytopathology 52: 1277–1285. Somatic hybrid plants from sexually incompatible woody spe- Castle, W.S., 1987. Citrus rootstocks. In: R.C. Rom & R.F. Carlson cies: Citrus reticulata and Citropsis gilletiana. Plant Cell Rep 8: (Eds.), Rootstocks for Fruit Crops, pp. 361–399. Wiley, New 656–659. York. Grosser, J.W., J. Jiang, E.S. Louzada, J.L. Chandler & F.G. Gmitter Chen, X.Y. & C.Q. Liao, 1982. Observation of biological character- Jr., 1998. Somatic hybridization, an integral component of citrus istics of Citrus psyllid, Diaphorina citri and its relationship with cultivar improvement: II. Rootstock improvement. HorSci 33(6): citrus huanglongbing (in Chinese). China Citrus 4: 14–15. 1060–1061. Cooper, W.C., 1961. Toxicity and accumulation of salts in citrus Grosser, J.W., E.S. Louzada, F.G. Gmitter Jr. & J.L. Chandler, 1994. trees on various rootstocks in Texas. Proc Fla State Hort Soc 74: Somatic hybridization of complementary citrus rootstocks: ﬁve 95–104. new hybrids. HortSci 29(7): 812–813. Deng, X.X., J.W. Grosser & F.G. Gmitter Jr., 1992. Intergeneric so- Grosser, J.W., F.A.A. Mourao-Fo, F.G. Gmitter Jr., E.S. Louzada, matic hybrid plants from protoplast fusion of Fortunella crassi- J. Jiang, K. Baergen, A. Ouiros, C. Cabasson, J.L. Schell & folia cultivar ‘Meiwa’ with Citrus sinensis cultivar ‘Valencia’. J.L. Chandler, 1996c. Allotetraploid hybrids between citrus and Scientia Hort 49: 55–62. seven related genera produced by somatic hybridization. Theor Deng, X.X., F.G. Gmitter Jr. & J.W. Grosser, 1995a. Pollen fertility Appl Genet 92: 577–582. of auto- and allo-tetraploids of Citrus. Acta Hort Sin 22(1): 16– Grosser, J.W., F.G. Gmitter Jr, N. Tusa, G. Reforgiato Recupero & 20. P. Cucinotta, 1996d. Further evidence of a cybridization require- Deng, X.X., Z.H. Sun, L. Gan & W.C. Zhang, 1995b. The agro- ment for plant regeneration from citrus leaf protoplasts following nomic performance of citrus somatic hybrids and their parents. somatic fusion. Plant Cell Rep 15: 672–676. Acta Hort 392: 69–75. Guo. W.W. & X.X. Deng, 1998a. Citrus huanglongbing and its Deng, X.X. & W.C. Zhang, 1995. Protoplast culture and fusion in resistance breeding (in Chinese). J Agric Biotech 6(1): 37–41. Citrus. Progress in Natural Science 5(3): 307–315. Guo, W.W. & X.X. Deng, 1999a. Unexpected ploidy variation of Deng, X.X., X.T. Ma, Z.H. Sun & S.G. Guan, 1997. Studies on the the somatic hybrid plants between Citrus sinensis and Clausena propagation capacity via cutting and some of the related traits of lansium (in Chinese). Acta Bot Sin 41(4): 439–441. citrus somatic hybrids (in Chinese). J Fruit Science 14(2): 77–81. Guo, W.W. & X.X. Deng, 1998b. Somatic hybrid plantlets regener- Escalante, A., S. Imanishi, M. Hossain, N. Ohmido & K. Fukui, ation between Citrus and its wild relative, Murraya paniculata 1998. RFLP analysis and genome in situ hybridization (GISH) via protoplast electrofusion. Plant Cell Rep 18: 297–300. in somatic hybrids and their progeny between Lycopersicon es- Guo, W.W. & X.X. Deng, 1999b. Intertribal hexaploid hybrid plants culentum and Solanum lycopersicoides. Theor Appl Genet 96: regeneration from electrofusion between diploids of Citrus sin- 719–726. ensis and its sexually incompatible relative, Clausena lansium. Ford, H.W. & W.A. Feder, 1960. Citropsis gilletiana, a citrus relat- Theor Appl Genet 98: 581–585. ive resistant to the burrowing nematode in laboratory tests. Proc Guo, W.W., 1998. Protoplast Electrofusion and Genetic Variation of Fla State Hort Soc 73: 60–64. the Regenerants in Citrus (in Chinese). Ph D thesis, Huazhong Froelicher, Y. & P. Ollitrault, 1998. Androgenesis on Clausena ex- Agricultural University, Wuhan, China. cavata. Program and Abstracts in: 1st Int Citrus Biotech Symp Guo, W.W., X.X. Deng & Y.Z. Shi, 1998. Optimization of proto- (Israel), pp. 32. plast electrofusion parameters and interspeciﬁc somatic hybrid Grimm, G.R. & D.J. Hutchison, 1977. Evaluation of Citrus spp. regeneration in Citrus (in Chinese). Acta Bot Sin 40: 417–424. relatives, and hybrids for resistance to Phytophthora parasitica Guo, W.W., X.M. Pang, H.Q. Huo & X.X. Deng, 2000a. Chro- Dastur. Proc Int Soc Citriculture 3: 863–865. mosome observation of Chinese wampee (Clausena lansium). J Grosser, J.W., P. Ollitrault & O. Olivares-Fuster, 2000. Somatic Huazhong Agric Univ 19(2): 166–167. hybridization in Citrus: An effective tool to facilitate variety improvement. In Vitro Cell Dev Biol, in press. ______中国科技论文在线182 http://www.paper.edu.cn

Guo, W.W., C.H. Zhou, H.L. Yi & X.X. Deng, 2000b. Intergeneric Motomura, T., T. Moriguchi, T. Akihama, T. Hidaka & M. Omura, somatic hybrid plants between Citrus and Poncirus trifoliata and 1996b. Analysis of cytoplasmic genomes in somatic hybrids evaluation of their root rot resistance. Acta Bot Sin 42: 668–672. between ‘Hazzara (Abohar)’ (Citrus reticulata Blanco) and Mi- Guo, W.W. & X.X. Deng, 2000. Citrus cybrids and their cytoplas- crocitrus australis (Planch) Swingle. J Japan Soc Hort Sci 65(3): mic genetic recombination. Acta Hort Sin 27 (Suppl.): 487–491. 497–503. Hidaka, T. & M. Omura, 1992. Regeneration of somatic hybrid O’Bannon, J.H. & H.W. Ford, 1977. Resistance in citrus root- plants obtained by electrical fusion between Satsuma mandarin stocks to Rodopholus similis and Tylenchulus semipenetrans (Citrus unshiu) and rough lemon (C. jambhiri)orYuzu(C. (Nematoda). Proc Int Soc Citriculture 2: 544–549. junos). Japan J Breed 42: 79–89. Ohgawara, T. & S. Ishii, 1991. Fertility in an intergeneric somatic Hinnisdaels, S., M. Jacobs & I. Negrutiu, 1994. Asymmetric hybrid plant of Rutaceae. HortSci 26(2): 207. somatic hybrids. In: Y.P.S. Bajaj (Ed.),Biotechnology in Agri- Ohgawara, T., S. Kobayashi, S. Ishii, K. Yoshinaga & I. Oiyama, culture and Forestry, Vol 27, pp. 57–71. Springer-Verlag, Berlin 1991. Fertile fruit trees obtained by somatic hybridization: na- Heidelberg. vel orange (Citrus sinensis) and Troyer citrange (C.sinensis × Huo, H.Q., Y.J. Hao & X.X. Deng, 1999. Induction of embryogenic Poncirus trifoliata). Theor Appl Genet 81: 141–143. callus of loose skin mandarins (in Chinese). Acta Biol Exp Sin Ohgawara, T., S. Kobayashi, E. Ohgawara, H. Uchimiya & S. 32: 289–295. Ishii, 1985. Somatic hybrid plants obtained by protoplast fu- Hutchison, D.J. & J.H. O’Bannon, 1972. Evaluation the reaction of sion between Citrus sinensis and Poncirus trifoliata. Theor Appl citrus selections to Tylenchulus semipenetrans. Plant Dis Rep 56: Genet 71: 1–4. 747–751. Ohgawara, T., H. Uchimiya, S. Ishii & S. Kobayashi, 1994. Somatic Iwamasa, M., N. Nito & J.T. Ling, 1988. Intra- and intergeneric hy- hybridization between Citrus sinensis and Poncirus trifoliata.In: bridization in the orange subfamily, Aurantioideae.In:R.Goren Y.P.S. Bajaj (Ed.), Somatic Hybridization in Crop Improvement, & K. Mendel (Eds.), Proc VI Int Citrus Cong, pp. 123–130. Biotechnology in Agriculture and Forestry, Vol 27, pp. 439–454. Jumin, H.B. & N. Nito, 1996. Plant regeneration via somatic Springer-Verlag Berlin Heidelberg. embryogenesis from protoplasts of six plant species related to Ollitrault, P., D. Dambier, Sudahono & F. Luro, 1996. Somatic hy- Citrus. Plant Cell Rep 15: 332–336. bridisation in citrus: some new hybrid and alloplasmic plants. Lee, L.S., D. Gillepie & R. Shaw, 1990. Prospects for using Citrus Proc Int Soc Citriculture: 907–912. tetraploids as rootstocks, pp. 198–203. Proc 3th World Cong Int Parokonny, A.S., A. Kenton, Y.Y. Gleba & M.D. Bennett, 1994. Soc Citrus Nurserymen, Australia. The fate of recombinant chromosomes and genome interaction in Ling, J.T. & M. Iwamasa, 1994. Somatic hybridization between Nicotiana asymmetric somatic hybrids and their sexual progeny. Citrus reticulata and Citropsis gabunensis through electrofusion. Theor Appl Genet 89: 488–497. Plant Cell Rep 13: 493–497. Sabbah, S.M., J.W. Grosser, J.L. Chandler & E.S. Louzada, 1991. Liu, J.H., 1999. Studies on Protoplast Symmetric and Asym- The effect of growth regulators on the rooting of stem cuttings of metric Fusion in Citrus (in Chinese). Ph D thesis, Huazhong citrus, related genera and intergeneric somatic hybrids. Proc Fla Agricultural University, Wuhan, China. State Hort Soc 104: 188–191. Liu, J.H., C.G. Hu & X.X. Deng, 1999. Regeneration of diploid in- Shi, Y.Z., X.X. Deng & Y.L. Yi, 1998a. Variation of intergeneric tergeneric somatic hybrid plants between Microcitrus and Citrus somatic hybrids between Citrus sinensis cv. Valencia and For- via electrofusion. Acta Bot Sin 41: 1177–1182. tunella crassifolia cv. Meiwa (in Chinese). Acta Bot Sin 40: Louzada, E.S. & J.W. Grosser, 1994. Somatic hybridization of Cit- 1060–1066. rus with sexually incompatible wild relatives. In: Y.P.S. Bajaj Shi, Y.Z., W.W. Guo & X.X. Deng, 1998b. The establishment of (Ed.), Biotechnology in Agriculture and Forestry, Vol 27, pp. RAPD technique and veriﬁcation of Citrus somatic hybrids (in 427–438. Springer-Verlag, Berlin Heidelberg. Chinese). Acta Hort Sin 25: 105–110. Louzada, E.S., J.W. Grosser & F.G. Gmitter Jr, 1993. Intergeneric Shinozaki, S., K. Fujira, T. Hidaka & M. Omura, 1992. Plantlet somatic hybridization of sexually incompatible Citrus sinensis formation of somatic hybrids of sweet orange (Citrus sinensis) and Atalantia ceylanica. Plant Cell Rep 12: 687–690. and its wild relatives, orange jessamine (Murraya paniculata)by Louzada, E.S., J.W. Grosser, F.G. Gmitter Jr., B. Nielsen, J.L. electrically-induced protoplast fusion. Japan J Breed 42: 287– Chandler, X.X. Deng & N. Tusa, 1992. Eight new somatic hybrid 295. citrus rootstocks with potential for improved disease resistance. Swingle, W.T. & P.C. Reece, 1967. The botany of citrus and its wild HortSci 27(9): 1033–1036. relatives. In: W. Reuther, H.J. Webber & L.D. Batchelor (Eds.) Mestre, P.F., M.J. Asins, J.A. Pina & L. Navarro, 1997. Efﬁcient The Citrus Industry, Vol 1, pp. 190–430. University of California search for new resistant genotypes to the citrus tristeza closter- Press, Berkeley. ovirus in the orange subfamily Aurantioideae. Theor Appl Genet Sykes, S.R., 1987. An overview of the family Rutaceae. In: R.R. 95: 1282–1288. Walker (Ed.), Citrus breeding workshop, pp. 93–100. CSIRO, Miranda, M., T. Motomura, F. Ikeda, T. Ohgawara, W. Saito, T. Melbourne, Australia. Endo, M. Omura & T. Moriguchi, 1997. Somatic hybrids ob- Takayanagi, R., T. Hidaka & M. Omura, 1992. Regeneration of in- tained by fusion between Poncirus trifoliata (2x) and Fortunella tergeneric somatic hybrids by electrical fusion between Citrus hindsii (4x) protoplasts. Plant Cell Rep 16: 401–405. and its wild relatives: Mexican lime (Citrus aurantifolia)and Motomura, T., T. Hidaka, T. Akihama, S. Katagi, M.A. Berhow, T. Java feroniella (Feroniella lucida) or tabog (Swinglea glutinosa). Moriguchi & M. Omura, 1996a. Protoplast fusion for production J Japan Soc Hort Sci 60(4): 799–804. of hybrid plants between Citrus and its related genera. J Japan Tusa, N., G. Ferrauto & E. Calderaro, 1992. Investigation on Soc Hort Sci 65(4): 685–692. protoplast regeneration from leaves of monoembryonic and poly- Motomura, T., T. Hidaka, T. Moriguchi, T. Akihama & M. Omura, embryonic citrus species. Proc Int Soc Citriculture: 180–182. 1995. Intergeneric somatic hybrids between Citrus and Atalantia Vardi, A. & P. Spiegel-Roy, 1982. Plant regeneration from proto- or Serverinia by electrofusion, and recombination of mitochon- plasts: variability in methodological requirements among cul- drial genomes. Breed Sci 45: 309–314. tivars and species. Theor Appl Genet 62: 171–176. ______中国科技论文在线 http://www.paper.edu.cn183

Vardi, A., P. Arzee-Gonen, A. Frydman-Shani, S. Bleichman & Zhou, C.H., X.X. Deng & J.W. Grosser, 1997. The resistance of E. Galun, 1989. Protoplast-fusion-mediated transfer of organ- Citrus somatic hybrids to Citrus foot rot (in Chinese). Acta Phyto elles from Microcitrus into Citrus and regeneration of novel Sin 27(4): 361–364. alloplasmic trees. Theor Appl Genet 78: 741–747. Zhu, W.S., Z.S. Chen, X.Y. Lan, H. Chen & Y. Ji, 1991. Studies on Xiao, S.Y., F.G. Gmitter Jr., J.W. Grosser & S. Huang, 1995. RAPD the resistance of some citrus biotypes to foot rot (Phytophthora analysis: a rapid method to identify citrus somatic hybrids (in parasitica) (in Chinese). China Citrus 20(1): 11–13. Chinese). Hereditas (Beijing) 17 (4): 40–42. Yoshida, T., 1996. Graft compatibility of citrus with plants in the Aurantioideae and their susceptibility to citrus tristeza virus. Plant Disease 80(4): 414–417.