Skip to main content

Production of three new grapefruit cybrids with potential for improved citrus canker resistance

Abstract

Cybrid production, combining the nucleus of one species with alien cytoplasmic organelles of another, is a potentially valuable method used for improvement of various crops including Citrus species. Furthermore, this technology is considered a non-GMO biotechnology strategy. In citrus, cybrid plants can be produced as a by-product of somatic fusion. Host resistance is the most desirable strategy for control of citrus canker. By using a cybridization approach, several putative cybrids were created by protoplast fusion of embryogenic suspension culture-derived protoplasts of canker resistant ‘Meiwa’ kumquat ( Citrus japonica Thunb), with mesophyll-derived protoplasts of three grapefruit (Citrus paradisi Macfad.) cultivars ‘Marsh,’ ‘Flame,’ and ‘N11-11’ somaclone of ‘Ruby Red.’ In an effort to generate new grapefruit cultivars with enhanced canker resistance, putative cybrid grapefruit plants morphologically equivalent to standard grapefruit from all three combinations were produced. Four mitochondrial (mt) introns, a mt ribosomal RNA spacer region, and four chloroplast (cp) DNA regions previously shown to have polymorphism among different Citrus species were tested. Four molecular markers, two mt DNA regions (intron nad7i2 and a rRNA spacer), and two cp DNA regions (NADH dehydrogenase subunit K (ndhk) gene and a trnG-trnR intergenic spacer) revealed polymorphism between kumquat and grapefruit and were used to validate the cybrids. All the cybrids had the mt genome of kumquat, and most had the cp genome of kumquat with a few exceptions. EST-SSR marker analysis confirmed that the nuclear genome in all the generated cybrids came from the grapefruit parent. All the cybrid clones have been propagated and are undergoing extensive canker assays to identify any clones that have improved canker tolerance/resistance. These cybrid populations provide a valuable tool for investigating the contribution of cytoplasmic organelles to plant disease resistance.

This is a preview of subscription content, access via your institution.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

References

  1. Aleza P, Garcia-Lor A, Juárez J, Navarro L (2016) Recovery of citrus cybrid plants with diverse mitochondrial and chloroplastic genome combinations by protoplast fusion followed by in vitro shoot, root, or embryo micrografting. Plant Cell Tissue Organ Cult 126:205–217

    CAS  Article  Google Scholar 

  2. Bayer RJ, Mabberley DJ, Morton C, Miller CH, Sharma IK, Pfeil BE, Rich S, Hitchcock R, Sykes S (2009) A molecular phylogeny of the orange subfamily (Rutaceae: Aurantioideae) using nine cpDNA sequences. Am J Bot 96:668–685

    CAS  Article  PubMed  Google Scholar 

  3. Boscariol RL, Monteiro M, Takahashi EK, Chabregas SM, Vieira MLC, Vieira LGE, Pereira LFP, Mourao Filho FAA, Cardoso SC, Christiano RSC (2006) Attacin a gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis cv. Hamlin. J Am Soc Hortic Sci 131:530–536

    CAS  Google Scholar 

  4. Cabasson CM, Luro F, Ollitrault P, Grosser JW (2001) Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion. Plant Cell Rep 20:604–609

    CAS  Google Scholar 

  5. Chen C, Zhou P, Choi YA, Huang S, Gmitter FG Jr (2006) Mining and characterizing microsatellites from citrus ESTs. Theor Appl Genet 112:1248–1257

    CAS  Article  PubMed  Google Scholar 

  6. Chen C, Grosser JW, Calovic M, Serrano P, Pasquali G, Gmitter J, Gmitter FG Jr (2008a) Verification of Mandarin and Pummelo somatic hybrids by expressed sequence tag–simple sequence repeat marker analysis. J Am Soc Hortic Sci 133:794–800

    Google Scholar 

  7. Chen C, Bowman KD, Choi YA, Dang PM, Rao MN, Huang S, Soneji JR, McCollum TG, Gmitter FG Jr (2008b) EST-SSR genetic maps for Citrus sinensis and Poncirus trifoliata. Tree Genet Genom 4:1–10

    Article  Google Scholar 

  8. Chen CX, Lyon MT, O'Malley D, Federici CT, Gmitter J, Grosser JW, Chaparro JX, Roose ML, Gmitter FG Jr (2008c) Origin and frequency of 2n gametes in Citrus sinensis x Poncirus trifoliata and their reciprocal crosses. Plant Sci 174:1–8

    CAS  Article  Google Scholar 

  9. Cheng YJ, Guo WW, Deng XX (2003) Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids. Plant Cell Rep 21:445–451

    CAS  Article  PubMed  Google Scholar 

  10. Cheng YJ, De Vicente MC, Meng H, Guo WW, Tao N, Deng X (2005) A set of primers for analyzing chloroplast DNA diversity in Citrus and related genera. Tree Physiol 25:661–672

    Article  PubMed  Google Scholar 

  11. Deng XX, Grosser JW, Gmitter FG Jr (1992) Intergeneric somatic hybrid plants from protoplast fusion of Fortunella crassifolia cultivar meiwa with Citrus sinensis cultivar Valencia. Sci Hort 49:55–62

    Article  Google Scholar 

  12. Duminil J, Pemonge M-H, Petit RJ (2002) A set of 35 consensus primer pairs amplifying genes and introns of plant mitochondrial DNA. Mol Ecol Notes 2:428–430

    CAS  Article  Google Scholar 

  13. Fu CH, Chen CL, Guo WW, Deng XX (2004) GISH, AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and Poncirus trifoliata. Plant Cell Rep 23:391–396

    CAS  Article  PubMed  Google Scholar 

  14. Fu XZ, Gong XQ, Zhang YX, Wang Y, Liu JH (2012) Different transcriptional response to Xanthomonas citri subsp. citri between kumquat and sweet orange with contrasting canker tolerance. PLoS One 7:e41790

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Ge H, Li Y, Fu H, Long G, Luo L, Li R, Deng Z (2015) Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS. Plant Cell Tissue Organ Cult 123:29–38

    CAS  Article  Google Scholar 

  16. Graham JH, Gottwald TR, Cubero J, Achor DS (2004) Xanthomonas axonopodis pv. citri: factors affecting successful eradication of citrus canker. Mol Plant Pathol 5:1–15

    Article  PubMed  Google Scholar 

  17. Green RM, Vardi A, Galun E (1986) The plastome of Citrus: physical map, variation among Citrus cultivars and species and comparison with related genera. Theor Appl Genet 72:170–177

    CAS  Article  PubMed  Google Scholar 

  18. Grosser JW, Gmitter FG Jr (1990) Protoplast fusion and citrus improvement. Plant Breed Rev 8:339–374

    Google Scholar 

  19. Grosser JW, Gmitter FG Jr (2005) Applications of somatic hybridization and cybridization in crop improvement, with citrus as a model. In Vitro Cell Dev Biol-Plant 41:220–225

    Article  Google Scholar 

  20. Grosser JW, Gmitter FG Jr (2011) Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus. Plant Cell Tissue Organ Cult 104:343–357

    CAS  Article  Google Scholar 

  21. Grosser JW, Ollitrault P, Olivares-Fuster O (2000) Somatic hybridization in citrus: an effective tool to facilitate variety improvement. In Vitro Cell Dev Biol-Plant 36:434–449

    Article  Google Scholar 

  22. Grosser JW, Calovic M, Louzada ES (2010a) Protoplast fusion technology—somatic hybridization and cybridization. In: Davey MR, Anthony P (eds) Plant cell culture: essential methods. Wiley-Blackwell, Oxford, pp 175–198

    Chapter  Google Scholar 

  23. Grosser JW, Gmitter FG Jr, Tusa N, Recupero GR, Cucinotta P (1996) Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion. Plant Cell Rep 15:672–676

    CAS  Article  PubMed  Google Scholar 

  24. Grosser JW, An HJ, Calovic M, Lee DH, Chen CX, Vasconcellos M, Gmitter FG Jr (2010b) Production of new allotetraploid and autotetraploid citrus breeding parents: focus on zipperskin mandarins. Hortscience 45:1160–1163

    Google Scholar 

  25. Grosser M (2011) Plant mitochondrial introns as genetic markers. Horticulture Sciences. University of Florida, Gainesville, Florida (undergraduate thesis). http://ufdc.ufl.edu/ufhonors

  26. Guo W-W, Xiao S-X, Deng X-X (2013) Somatic cybrid production via protoplast fusion for citrus improvement. Sci Hort 163:20–26

    Article  Google Scholar 

  27. Guo WW, Cai XD, Grosser JW (2004a) Somatic cell cybrids and hybrids in plant improvement. In: Daniell H, Chase CD (eds) Molecular biology and biotechnology of plant organelles. Kluwer Academic Publisher, Dordrecht, pp 635–659

    Chapter  Google Scholar 

  28. Guo WW, Prasad D, Cheng YJ, Serrano P, Deng XX, Grosser JW (2004b) Targeted cybridization in citrus: transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness. Plant Cell Rep 22:752–758

    CAS  Article  PubMed  Google Scholar 

  29. Khalaf A, Moore GA, Jones JB, Gmitter FG Jr (2007) New insights into the resistance of Nagami kumquat to canker disease. Physiol Mol Plant Pathol 71:240–250

    CAS  Article  Google Scholar 

  30. Khalaf AA, Gmitter FG Jr, Conesa A, Dopazo J, Moore GA (2011) Fortunella margarita transcriptional reprogramming triggered by Xanthomonas citri subsp. citri. BMC Plant Biol 11:159

    Article  PubMed  PubMed Central  Google Scholar 

  31. Laroche J, Li P, Maggia L, Bousquet J (1997) Molecular evolution of angiosperm mitochondrial introns and exons. Proc Natl Acad Sci U S A 94:5722–5727

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Li D, Xiao X, Guo WW (2014) Production of transgenic ‘Anliucheng’ sweet orange (Citrus sinensis Osbeck) with Xa21 gene for potential canker resistance. J Integr Agric 13:2370–2377

    CAS  Article  Google Scholar 

  33. Lorz H, Paszkowshi J, Dierks-Ventling C, Potrykus I (1981) Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells. Physiol Plant 53:385–391

    CAS  Article  Google Scholar 

  34. Maliga B, Lorz H, Larzar G, Nagy F (1982) Cytoplast-protoplast fusion for interspecific chloroplast transfer in Nicotiana. Mol Gen Genet 185:211–215

    CAS  Article  Google Scholar 

  35. Mendes BMJ, Cardoso SC, Boscariol-Camargo RL, Cruz RB, Mourao Filho FAA, Bergamin Filho A (2010) Reduction in susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis expressing the rice Xa21 gene. Plant Pathol 59:68–75

    CAS  Article  Google Scholar 

  36. Moreira CD, Chase CD, Gmitter FG Jr, Grosser JW (2000) Inheritance of organelle genomes in citrus somatic cybrids. Mol Breed 6:401–405

    CAS  Article  Google Scholar 

  37. Moreira CD, Gmitter FG Jr, Grosser JW, Huang S, Ortega VM, Chase CD (2002) Inheritance of organelle DNA sequences in a Citrus-Poncirus intergeneric cross. J Hered 93:174–178

    CAS  Article  PubMed  Google Scholar 

  38. Murashige T, Tucker DP (1969) Growth factor requirements of citrus tissue culture. In: Chapman HD (ed) Proc 1st Internat citrus Symp. University of Claifornia, Riverside, pp 1155–1161

    Google Scholar 

  39. Navarro L (1992) Citrus shoot tip grafting in vitro. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, no 18 high-tech and micropropagation II. Springer, Berlin Heidelberg, Berlin, pp 327–338

    Google Scholar 

  40. Omar AA, Song WY, Grosser JW (2007) Introduction of Xa21, a Xanthomonas-resistance gene from rice, into ‘Hamlin’ sweet orange [Citrus sinensis (L.) Osbeck] using protoplast-GFP co-transformation or single plasmid transformation. J Horticult Sci Biotechnol 82:914–923

    CAS  Article  Google Scholar 

  41. Peng A, Xu L, He Y, Lei T, Yao L, Chen S, Zou X (2015) Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system. Plant Cell Tissue Organ Cult 123:1–13

    CAS  Article  Google Scholar 

  42. Penjor T, Yamamoto M, Uehara M, Ide M, Matsumoto N, Matsumoto R, Nagano Y (2013) Phylogenetic relationships of Citrus and its relatives based on matK gene sequences. PLoS One 8:e62574

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Petit RJ, Duminil J, Fineschi S, Hampe A, Salvini D, Vendramin GG (2005) Comparative organization of chloroplast, mitochondrial and nuclear diversity in plant populations. Mol Ecol 14:689–701

    CAS  Article  PubMed  Google Scholar 

  44. Rao MN, Soneji JR, Chen C, Huang S, Gmitter FG Jr (2007) Characterization of zygotic and nucellar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers. Tree Genet Genom 4:113–124

    Article  Google Scholar 

  45. Sakai T, Imamura J (1990) Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (CMS line) and Brassica napus through cytoplast-protoplast fusion. Theor Appl Genet 80:421–427

    CAS  Article  PubMed  Google Scholar 

  46. Satpute AD, Chen C, Gmitter FG Jr, Ling P, Yu Q, Grosser MR, Grosser JW, Chase CD (2015) Cybridization of grapefruit with ‘Dancy’ mandarin leads to improved fruit characteristics. J Am Soc Hortic Sci 140:427–435

    CAS  Google Scholar 

  47. Schubert TS, Rizvi SA, Sun X, Gottwald TR, Graham JH, Dixon WN (2001) Meeting the challenge of eradicating citrus canker in Florida-again. Plant Dis 85:340–356

    Article  Google Scholar 

  48. Spangenberg G, Osusky M, Oliveira MM, Freydl E, Nagel J, Pais MS, Potrykus I (1990) Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: morphological, genetic and molecular characterization. Theor Appl Genet 80:577–587

    CAS  Article  PubMed  Google Scholar 

  49. Tsuruta S, Fumiko H, Otabara T, Hashiguchi M, Yamamoto T, Akashi R (2008) Genetic diversity of chloroplast DNA in Zoysia and other warm-season turfgrasses. Grassl Sci 54:151–159

    CAS  Article  Google Scholar 

  50. Tusa N, Grosser JW, Gmitter FG Jr (1990) Plant regeneration of ‘Valencia’ sweet orange, ‘Femminello’ lemon, and the interspecific somatic hybrid following protoplast fusion. J Am Soc Hortic Sci 115:1043–1046

    Google Scholar 

  51. Vardi A, Breiman A, Galun E (1987) Citrus cybrids: production by donor-recipient protoplast-fusion and verification by mitochondrial-DNA restriction profiles. Theor Appl Genet 75:51–58

    CAS  Article  Google Scholar 

  52. Vardi A, Arzee-Gonen P, Frydman-Shani A, Bleichman S, Galun E (1989) Protoplast-fusion-mediated transfer of organelles from Microcitrus into Citrus and regeneration of novel alloplasmic trees. Theor Appl Genet 78:741–747

    CAS  Article  PubMed  Google Scholar 

  53. Wallin A, Glimelius K, Eriksson T (1978) Enucleation of plant protoplasts by cytochalasin B. Z Pflanzenphsiol 78:333–340

    Article  Google Scholar 

  54. Wang Y, Fu X-Z, Liu J-H, Hong N (2011) Differential structure and physiological response to canker challenge between ‘Meiwa’ kumquat and ‘Newhall’ navel orange with contrasting resistance. Sci Hort 128:115–123

    CAS  Article  Google Scholar 

  55. Xu S-X, Cai D-F, Tan F-Q, Fang Y-N, Xie K-D, Grosser JW, Guo W-W (2014) Citrus somatic hybrid: an alternative system to study rapid structural and epigenetic reorganization in allotetraploid genomes. Plant Cell Tissue Organ Cult 119:511–522

    CAS  Article  Google Scholar 

  56. Xu XY, Liu JH, Deng XX (2005) FCM, SSR, and CAPS analysis of intergeneric somatic hybrid plants between Changshou kumquat and Dancy tangerine. Bot Bull Acad Sin 46:93–98

    CAS  Google Scholar 

  57. Xu XY, Liu JH, Deng XX (2006) Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast-protoplast fusion. Plant Cell Rep 25:533–539

    CAS  Article  PubMed  Google Scholar 

  58. Yamamoto M, Kobayashi S, Nakamura Y, Yamada Y (1993) Phylogenic relationships of citrus revealed by RFLP analysis of mitochondrial and chloroplast DNA. Jpn J Breed 43:355–365

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Authors would like to thank the Krezdorn Memorial Fund at University of Florida, the Citrus Research and Development Foundation (CRDF), New Varieties Development and Management Corporation (NVDMC) for financial support, and CAPES—Science without Borders Program—which provided funding for a 3-yr (2013-2016) Graduate Scholarship for 2nd author at University of Florida.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Jude W. Grosser.

Additional information

Editor: Wenhao Dai

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Omar, A.A., Murata, M., Yu, Q. et al. Production of three new grapefruit cybrids with potential for improved citrus canker resistance. In Vitro Cell.Dev.Biol.-Plant 53, 256–269 (2017). https://doi.org/10.1007/s11627-017-9816-7

Download citation

Keywords

  • Disease resistance
  • Microsatellite marker
  • Organelle inheritance
  • Mitochondrial introns
  • Chloroplast genome