Plant Cell Reports (1998) 18: 297–300 © Springer-Verlag 1998 W. W. Guo · 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 jes- samine [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 – 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 · RAPD · Since the first intergeneric somatic hybrid in Citrus was Citrus huanglongbin · Sexual and graft incompatibility · regenerated (Ohgawara et al. 1985), more than 150 inter- Aurantioideae specific and intergeneric somatic hybrids have been ob- tained by polyethylene glycol or electrically induced fu- Abbreviations GOT Glutamateoxaloacetate transami- sion (Deng et al. 1992; Grosser et al. 1996). Those somatic nase · MT medium Murashige and Tucker medium · 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 auran- tifolia (Christ.) Swing.+Feroniella lucida (Scheff) Swing. Communicated by G. Phillips or Swinglea glutinosa (Blanco) Merr. (Takayanagi et al. W. W. Guo · 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 pro- toplast 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 l–1 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 shak- er (110 rpm). The calli were subcultured every 12 – 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 l–1 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 asep- tically 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 jes- samine grafted onto trifoliate orange Protoplasts from suspension cultures and leaves were isolated ac- cording to the method of Grosser and Gmitter (1990 a). Approxi- mately 1 g of suspension culture after 7 – 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 – 20 h on a rotatory shaker (30 rpm). Two or three aseptically (RAPD) analysis fully expanded leaves of orange jessamine were feathered by a ster- ilized 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 – 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- –1 –1 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 – 5×105 callus proto- × 5 plasts/ml and 10 – 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 be- fore 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 – 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 – 40 days of cul- ture, white globular calli were seen in the petri dishes that Protoplast culture, shoot regeneration and micrografting later developed into green globular embryoids (5 – 10 em- bryoids per dish). These embryoids were transferred to so- The cultures were incubated at 25 – 27 °C in the dark for 4 – 5 weeks –1 before being transferred to solidified MT basal medium containing lidified MT basal medium containing 500 mg l malt ex- 50 g l–1 sucrose and 500 mg l–1 malt extract, in the light (1500 – 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 l–1) kinetin (0.5 mg l–1), and ten turned brown and then died, which could be due to so- 1-naphthylacetic acid (0.1 mg l–1) 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.