Somatic Hybrid Plantlets Regeneration Between Citrus and Its Wild Relative, Murraya Paniculata Via Protoplast Electrofusion

Plant Cell Reports (1998) 18: 297Ð300 © Springer-Verlag 1998

W. W. Guo á X. X. Deng Somatic hybrid plantlets regeneration between Citrus and its wild relative, Murraya paniculata via protoplast electrofusion

Received: 17 January 1998 / Revision received: 12 June 1998 / Accepted: 14 July 1998

Abstract Protoplasts isolated from ‘Page’ tangelo (Min- identified as a Liberobacter (Jagoueix et al. 1994), and al- neola tangelo × clementine) cell suspension cultures were most all citrus cultivars are susceptible to it. Orange jes- electrically fused with mesophyll protoplasts of orange jessamine [Murraya paniculata (L.) Jack], a remote relative samine [Murraya paniculata (L.) Jack]. Shoots were re- of citrus belonging to another tribe, Clauseneae, is an at- generated after 6 Ð 10 months of culture, but they were ex- tractive ornamental plant because of its fragrant white tremely recalcitrant to producing roots in root-induction flowers and small red fruits (Swingle and Reece 1967). medium. Complete plantlets were formed via micrograft- Orange jessamine is immune to citrus huanglongbin (Chen ing. Chromosome counting of shoot tips revealed they were and Liao 1982), and is resistant to citrus nematode (Sykes tetraploids (2n = 4x = 36). Glutamateoxaloacetate transam- 1987) and citrus tristeza virus (Yoshida 1996). It grows inase isozyme and randomly amplified polymorphic DNA well in alkaline conditions (Sykes 1987). Efforts to create analysis confirmed their hybridity. Orange jessamine is im- sexual progeny and grafted plants between orange jessa- mune to citrus huanglongbin, a severe disease of citrus, but mine and Citrus have failed due to sexual incompatibility sexual incompatibility and limited graft compatibility ex- and limited graft compatibility (Swingle and Reece 1967). ist between Citrus and orange jessamine. The cell fusion However, the established protoplast manipulation tech- technique may make it possible to transfer the huanglong- nique in Citrus may make it possible to create somatic hy- bin resistance trait from orange jessamine to Citrus. brids between orange jessamine and Citrus as a way to combine complementary traits from both genera. Key words Somatic hybridization á RAPD á Since the first intergeneric somatic hybrid in Citrus was Citrus huanglongbin á Sexual and graft incompatibility á regenerated (Ohgawara et al. 1985), more than 150 inter- Aurantioideae specific and intergeneric somatic hybrids have been obtained by polyethylene glycol or electrically induced fu- Abbreviations GOT Glutamateoxaloacetate transami- sion (Deng et al. 1992; Grosser et al. 1996). Those somatic nase á MT medium Murashige and Tucker medium á RAPD hybrids include many sexually and/or graft-incompatible Randomly amplified polymorphic DNA combinations such as Citrus sinensis (L.) Osbeck+Severi- nia disticha (Blanco) Swing. (Grosser et al. 1988), Citrus reticulata Blanco+Citropsis gilletiana Swing. & M. Kell (Grosser et al. 1990), C. sinensis+Severinia buxifolia Introduction (Poir.) Oliv (Grosser et al. 1992), C. sinensis+Feronia li- monia (L.) Swing. (Grosser et al. 1996), C. reticulata+Ci- Citrus huanglongbin is a severe and widespread disease of tropsis gabunensis (Engl.) Swing. & M. Kell (Ling and citrus in the southern part of China. It also exists in some Iwamasa 1994), C. sinensis+Atalantia ceylanica (Arn.) other parts of the world such as South Africa and South- Oliv. (Louzada et al. 1993), ‘Seminole’ tangelo (C. reticu- east Asia (Zhao 1981). The pathogen has recently been lata Blanco×Citrus paradisi Macf)+S. buxifolia or Atlan- tia monophylla DC. (Motomura et al. 1995), Citrus aurantifolia (Christ.) Swing.+Feroniella lucida (Scheff) Swing. Communicated by G. Phillips or Swinglea glutinosa (Blanco) Merr. (Takayanagi et al. W. W. Guo á X. X. Deng (½) 1992). Somatic hybrid plantlets between ‘Trovita’ sweet National Key Laboratory of Crop Genetic Improvement, orange (C. sinensis) and orange jessamine were obtained Huazhong Agricultural University, Wuhan 430070, China (Shinozaki et al. 1992), but unfortunately, the plantlets Fax: +86-27-87396057 were only preserved in test tubes (T. Hidaka, personal com- e-mail: [email protected] munication). Here we report another somatic cell fusion 298 combination between Citrus and orange jessamine via protoplast electrofusion.

Materials and methods

Plant materials

Embryogenic callus of ‘Page’ tangelo (Minneola tangelo × Clemen- tine) was kindly provided by Prof. J. W. Grosser (University of Flor- ida) and preserved on solidified Murashige and Tucker (MT) basal medium (Murashige and Tucker 1969) containing 500 mg lÐ1 malt extract. The callus was subcultured on the same medium at 1- to 2- month intervals. For protoplast isolation, the callus was transferred to liquid medium containing the same components on a rotatory shaker (110 rpm). The calli were subcultured every 12 Ð 14 days at least three times before being used for protoplast isolation. Seeds of mature orange jessamine fruits were collected and sur- face sterilized with 1 mol lÐ1 NaOH for 2 min, then immersed in a 0.5% sodium hypochlorite solution for 10 min, and washed at least three times with sterilized distilled water. The seeds were then aseptically germinated in vitro in test tubes on MT basal medium, and fully expanded leaves were used for mesophyll protoplast isolation.

Protoplast isolation and electrofusion Fig. 1 A somatic hybrid plant between ‘Page’ tangelo+orange jessamine grafted onto trifoliate orange Protoplasts from suspension cultures and leaves were isolated according to the method of Grosser and Gmitter (1990 a). Approxi- mately 1 g of suspension culture after 7 Ð 11 days into a 14-days subculture cycle was macerated in the enzyme solution (Grosser Chromosome counting, glutamateoxaloacetate transaminase and Gmitter 1990 a). The mixed culture was kept at 28 ¡C for (GOT) isozyme and randomly amplified polymorphic DNA 16 Ð 20 h on a rotatory shaker (30 rpm). Two or three aseptically (RAPD) analysis fully expanded leaves of orange jessamine were feathered by a sterilized razor blade and then transferred into a 60×15 mm petri dish Chromosome counting of regenerated embryoids and shoot tips was in the same enzyme solution as that of suspension cultures. The conducted according to the hematoxylin staining technique (Sass cultures were maintained at 28 ¡C for 20 Ð 24 h on a rotatory shak- 1958) with slight modification (Grosser and Gmitter 1990 a). For iso- er (30 rpm). zyme analysis, crude leaf tissue extracts from somatic hybrids and Following filtration through a 45-µm stainless steel mesh, both both parental genotypes were electrophoresed by PAGE, and gels suspension- and mesophyll-derived protoplasts were purified by 25% were stained for GOT (Xiao et al. 1989). Total DNA was extracted sucrose-13% mannitol gradient centrifugation. They were then from leaves according to the SDS method (Xiao et al. 1995). The washed twice by centrifugation at 100 g for 10 min in electrofusion DNA amplification apparatus was a DNA Thermal Cycler 480 (Per- Ð1 Ð1 kin Elmer, USA). The following random primers were used: AN-07, solution containing 0.6 mol l mannitol, and 0.25 mmol l CaCl2. The fusion was conducted using an SSH-2 instrument (Shimad- W-02, W-03, V-06, A-04, A-05, A-07, A-08, A-10, A-19, and A-20 zu Somatic Hybridizer-2, Japan). The electrofusion chamber was (Operon Technologies, Alameda, Calif.). Reaction products were FTC-03 with 0.8 ml volume. The electrical parameters were careful- electrophoresed in agarose gels (1.6%) and visualized with UV light ly determined and used here as follows: a.c. field, 1 MHz, 125 V/cm, after staining with ethidium bromide. 60 s; d.c. pulse field, 1250 V/cm, 40 µs in duration, five times at 0.5-s intervals; final time, 5 s. A protoplast mixture (0.8 ml) containing 3 Ð 5×105 callus proto- × 5 plasts/ml and 10 Ð 15 10 mesophyll protoplasts/ml was transferred Results and discussion to chamber FTC-03, incubated for 5 min and then fused. After the fusion treatment, the mixture was incubated for at least 10 min before being transferred to 10-ml centrifuge tubes and then centrifuged The mesophyll protoplasts of orange jessamine were easy at 100 g for 4 min. The supernatant was discarded and the fusion to isolate, and easy to fuse with protoplasts from embryo- × 5 products were resuspended at a density of 1 Ð 2 10 cells/ml in BH3 genic callus of ‘Page’ tangelo. The rate of binuclear het- medium (Grosser and Gmitter 1990 a) by liquid thin layer culture. erokaryons was as high as 15%. After 30 Ð 40 days of culture, white globular calli were seen in the petri dishes that Protoplast culture, shoot regeneration and micrografting later developed into green globular embryoids (5 Ð 10 embryoids per dish). These embryoids were transferred to so- The cultures were incubated at 25 Ð 27 ¡C in the dark for 4 Ð 5 weeks Ð1 before being transferred to solidified MT basal medium containing lidified MT basal medium containing 500 mg l malt ex- 50 g lÐ1 sucrose and 500 mg lÐ1 malt extract, in the light (1500 Ð 2000 tract, where they enlarged and became malformed. During lux). The developed embryoids were transferred to MT basal medi- subculture on shoot induction medium, the embryoids of- um containing 6-benzyladenine (0.5 mg lÐ1) kinetin (0.5 mg lÐ1), and ten turned brown and then died, which could be due to so- 1-naphthylacetic acid (0.1 mg lÐ1) for shoot induction. Multiple shoots were aseptically micrografted onto trifoliate orange [Ponci- matic incompatibility. Shoots regenerated from these mal- rus trifoliata (L.) Raf.], Swingle citrumelo, and citrange (a local formed embryoids after several subcultures on shoot in- strain, C. sinensis × P. trifoliata). duction medium. Some shoots were malformed, but the 299 and Swingle citrumelo. One plantlet on trifoliate orange was transplanted to the greenhouse with no growth in the first 2 months after transplantation. However, it then grew out a new leaf within 4 days, but later stopped growing and finally died. To date, we have only four plantlets grafted on trifoliate orange and two plantlets on citrange alive in test tubes in vitro, with extremely weak growth. More than ten newly germinated shoots from embryoids are growing vigorously in 100-ml flasks in vitro, and efforts will be made to micrograft them on rough lemon, Page tangelo, or orange jessamine, if seeds of those species are available. Chromosome counting of several randomly selected embryoids and eight shoot tips revealed that they have 36 chromosomes (2n = 4x = 36) (Fig. 2), suggesting that chromosome elimination did not occur in this wide combination. Leaf GOT isozyme analysis revealed that the putative somatic hybrids had the specific bands of both parents (data not shown). RAPD analysis showed that AN-07, Fig. 2 Shoot tip chromosome number of the regenerants between W-02, W-03, A-04, A-05, A-08, A-10, and A-20 can iden- ‘Page’ tangelo+orange jessamine (2n = 4x = 36; ×1000) tify somatic hybrids with specific bands from both parental genotypes (A-20 and A-04 shown in Fig. 3). The above results revealed that allotetraploid somatic hybrids majority were morphologically normal with thick wide between Citrus and orange jessamine had been obtained. leaves, suggesting they were putative somatic hybrids. Citrus wild relatives are an untapped germplasm bank The effort to induce roots from the regenerated shoots which possesses many elite resistance traits. Among them, failed. One month after being transferred to root induction orange jessamine is unique, with high resistance to Citrus medium, the unrooted shoots gradually turned yellow from huanglongbin. Here we obtained intertribal somatic hybrid the bottom up and then died. More than 40 shoots were plantlets between orange jessamine and Citrus; this is the grafted onto trifoliate orange, Swingle citrumelo, and ci- second report of such hybrids. From the results, we can in- trange. Two months after grafting, the survival rate was fer that cell fusion, to some extent, is an alternative to cir- 100% (Fig. 1). Unfortunately, their growth was extremely cumvent sexual and/or graft incompatibility, but the in- slow and 2 Ð 3 months later, some shoots gradually turned compatibility phenomenon still exists, as can also be seen yellow from the graft union up and then died, which could from other wide combinations (Grossen and Gmitter be due to graft incompatibility. The growth of plantlets on 1990 b). Genotype interaction which may be predicted by citrange was generally better than that on trifoliate orange graft compatibility could be one reason for the poor per-

Fig. 3 RAPD pattern of somatic hybrids and their parental genotypes (A primer OPA-20; B primer OPA-04; lanes 1 Ð 4 somatic hybrids, lane 5 ‘Page’ tangelo, lane 6 orange jessamine, lane 7 1-kb ladder) 300 formance of the somatic hybrids, since only three kinds of Motomura T, Hidaka T, Moriguchi T, Akihama T, Omura M (1995) rootstocks were tried. Grafting the somatic hybrids on ei- Intergeneric somatic hybrids between Citrus and Atalantia or Severinia by electrofusion, and recombination of mitochondrial ther parental genotype would be helpful to elucidate this. genomes. Breed Sci 45: 309 Ð 314 The incompatibility or low vigor may also be solved by the Murashige T, Tucker DPH (1969) Growth factor requirements of cit- selection of the embryogenic callus parent. A case in point rus tissue culture. Proc 1st Int Citrus Symp 3: 1155 Ð 1161 was the contrasting vigor of the somatic hybrids between Ohgawara T, Kobayashi S, Uchimiya H, Ishii S (1985) Somatic hybrid plants obtained by protoplast fusion between Citrus sinen- ‘Hamlin’ sweet orange (C. sinensis)+S. buxifolia, and sis and Poncirus trifoliata. Theor Appl Genet 7: 1 Ð 4 ‘Succari’ sweet orange (C. sinensis)+S. buxifolia, with the Sass JE (1958) Botanical microtechnique. Iowa State University former hybrid showing lower vigor (Grosser et al. 1996). Press, Ames, pp 73 Ð 75 The combination reported here was better than that previ- Shinozaki S, Fujira K, Hidaka T, Omura M (1992) Plantlet forma- ously reported (Shinozaki et al. 1992). This suggests that tion of somatic hybrids of sweet orange (Citrus sinensis) and its wild relative, orange jessamine (Murraya paniculata), by electri- the selection of fusion parents may play a role, especially cally-induced protoplast fusion. Jpn J Breed 42: 287 Ð 295 the embryogenic callus parents. More vigorous callus par- Swingle WT, Reece PC (1967) The botany of citrus and its wild rel- ents will be used to fuse with mesophyll protoplasts of atives. In: Reuther W, Webber HJ, Batchelor LD (eds) The citrus orange jessamine in order to regenerate vigorous and hor- industry, vol 1. University of California Press, Berkeley, pp 190 Ð 430 ticulturally acceptable somatic hybrids. Sykes SR (1987) An overview of the family Rutaceae. In: Walker RR (ed) Citrus breeding work shop. CSIRO, Melbourne, Acknowledgements This project was partially funded by the Na- pp 93 Ð 100 tional Commission of Science and Technology and the National Nat- Takayanagi R, Hidaka T, Omura M (1992) Regeneration of interge- ural Science Fundation of China. Great appreciation is given to Prof. neric somatic hybrids by electrical fusion between Citrus and its J. W. Grosser, University of Florida, for his valuable advice and crit- wild relatives: Mexican lime (Citrus aurantifolia) and Java fer- ical reading of the manuscript. oniella (Feroniella lucida) or tabog (Swinglea glutinosa). J Jpn Soc Hort Sci 60: 799 Ð 804 Xiao SY, Zhang WC, Chen JS, Wan SY (1989) Studies on the iden- tification and genetic analysis of citrus hybrids by isozyme analysis (in Chinese). Acta Horticulturae Sinica 16: 255 Ð 260 References Xiao SY, Gmitter FG Jr, Grosser JW, Huang S (1995) RAPD analysis Ð a rapid method to identify citrus somatic hybrids (in Chi- Chen XY, Liao CQ (1982) Observation of biological characteristics nese). Hereditas (Beijing) 17 (4): 40 Ð 42 of Citrus psyllid, Diaphorina citri and its relationship with cit- Yoshida T (1996) Graft compatibility of citrus with plants in the Au- rus huanglongbin (in Chinese). China Citrus 4: 14 Ð 15 rantioideae and their susceptibility to citrus tristeza virus. Plant Deng XX, Grosser JW, Gmitter FG Jr (1992) Intergeneric somatic Dis 80: 414 Ð 417 hybrid plants from protoplast fusion of Fortunella crassifolia cul- Zhao XY (1981) Citrus yellow shoot disease (huanglongbin) in Chi- tivar ‘Meiwa’ with Citrus sinensis cultivar ‘Valencia’. Sci Hort na Ð a review. Proc Int Soc Citricult 1: 466 Ð 469 49: 55 Ð 62 Grosser JW, Gmitter FG Jr (1990a) Protoplast fusion and citrus improvement. Plant Breed Rev 8: 339 Ð 374 Grosser JW, Gmitter FG Jr (1990b) Wide-hybridization of citrus via Note added in proof: protoplast fusion: progress, strategies, and limitations. In: Ben- nett AB, O’Neil SD (eds) Horticultural biotechnology, plant bi- Some shoots were initially unifoliate, then exhibited bifoliate lea- ology, vol 11. Wiley-Liss, New York, pp 31 Ð 41 ves, which probably inherited from the compound leaf character of Grosser JW, Gmitter FG Jr, Chandler JL (1988) Intergeneric somat- Murraya paniculata. ic hybrid plants from sexually incompatible woody species: Cit- rus sinensis and Severinia disticha. Theor Appl Genet 75: 397 Ð 401 Grosser JW, Gmitter FG Jr, Tusa N, Chandler JL (1990) Somatic hybrid plants from sexually incompatible woody species: Citrus reticulata and Citropsis gilletiana. Plant Cell Rep 8: 656 Ð 659 Grosser JW, Gmitter FG Jr, Sesto F, Deng XX, Chandler JL (1992) Six new somatic citrus hybrids and their potential for cultivar improvement. J Am Soc Hort Sci 117: 169 Ð 173 Grosser JW, Mourao-Fo FAA, Gmitter FG Jr, Louzada ES, Jiang J, Baergen K, Ouiros A, Cabasson C, Schell JL, Chandler JL (1996) Allotetraploid hybrids between Citrus and seven related genera produced by somatic hybridization. Theor Appl Genet 92: 577 Ð 582 Jagoueix S, Bove JM, Carnier M (1994) The phloem-limited bacte- rium of greening disease of citrus is a member of the α subdivi- sion of the Proteobacteria. Natl J System Bacter 44: 379 Ð 386 Ling JT, Iwamasa M (1994) Somatic hybridization between Citrus reticulata and Citropsis gabunensis through electrofusion. Plant Cell Rep 13: 493 Ð 497 Louzada ES, Grosser JW, Gmitter FG Jr (1993) Intergeneric somatic hybridization of sexually incompatible parents: Citrus sinensis and Atalantia ceylanica. Plant Cell Rep 12: 687 Ð 690 Fig. 4 A regenerated shoot exhibited bifoliate leaves