Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Abstract

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

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

Fig. 1
Fig. 2

References

  1. Alan AR, Mutschler MA, Brants A, Cobb ED, Earle ED (2003) Production of gynogenic plants from hybrids of Allium cepa L. and A. roylei Stearn. Plant Sci 165:1201–1211. doi:10.1016/S0168-9452(03)00327-3

    Article  CAS  Google Scholar 

  2. Almeida WAB, Mourão Filho FAA, Mendes BMJ, Pavan A, Rodriguez APM (2003) Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments. Scientia Agricola 60:23–29. doi:10.1590/S0103-90162003000100005

    Article  Google Scholar 

  3. Benelli C, Germanà MA, Ganino T, Beghe D, Fabbri A (2010) Morphological and anatomical observations of abnormal somatic embryos from anther cultures of Citrus reticulate. Biol Plant 54:224–230. doi:10.1007/s10535-010-0040-0

    Article  Google Scholar 

  4. Cabral GB, Carneiro VTC, Lacerda AL, do Valle CB, Martinelli AP, Dusi DMA (2011) Somatic embryogenesis and organogenesis in apomictic and sexual Brachiaria brizantha. Plant Cell Tiss Organ Cult. doi:10.1007/s11240-011-9978-7

  5. Cai XD, Liu X, Guo WW (2006) GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage. Plant Cell Tiss Organ Cult 87:245–253. doi:10.1007/s11240-006-9158-3

    Article  CAS  Google Scholar 

  6. Cao H, Biswas MK, Lu Y, Amar MH, Tong Z, Xu Q, Xu J, Guo W, Deng X (2011) Doubled haploid callus lines of Valencia sweet orange recovered from anther culture. Plant Cell Tiss Organ Cult 104:415–423. doi:10.1007/s11240-010-9860-z

    Article  Google Scholar 

  7. Carimi F (2005) Somatic embryogenesis protocol: Citrus. In: Jain SM, Gupta PK (eds) Protocol for somatic embryogenesis in woody plants. Springer, The Netherlands, pp 321–343

    Chapter  Google Scholar 

  8. Carimi F, Tortorici MC, Pasquale FD, Crescimanno FG (1998) Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group (Citrus sinensis (L.) Osbeck.). Plant Cell Tiss Org Cult 54:183–189. doi:10.1023/A:1006113731428

    Article  Google Scholar 

  9. Carimi F, De Pasquale L, Crescimanno FG (1999) Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus. Plant Cell Rep 18:935–940. doi:10.1007/s002990050687

    Article  CAS  Google Scholar 

  10. Cerezo S, Mercado JA, Pliego-Alfaro F (2011) An efficient regeneration system via somatic embryogenesis in olive. Plant Cell Tiss Organ Cult 106:337–344. doi:10.1007/s11240-011-9926-6

    Article  CAS  Google Scholar 

  11. Chen J, Cui L, Malik AA, Mbira KG (2011) In vitro haploid and dihaploid production via unfertilized ovule culture. Plant Cell Tiss Organ Cult 104:311–319. doi:10.1007/s11240-010-9874-6

    Article  Google Scholar 

  12. Chu C (1978) The N6 medium and its applications to anther culture of cereal crops. In: Proceedings of symposium on plant tissue culture. Science Press, Peking, pp 43–50

    Google Scholar 

  13. Creste S, Tulmann Neto A, Figueira A (2001) Detection of simple sequence repeats polymorphisms in denaturating polyacrylamide sequencing gels by silver staining. Plant Mol Biol Rep 19:299–306. doi:10.1007/BF02772828

    Article  CAS  Google Scholar 

  14. Cunha A, Fernandes-Ferreira M (1999) Influence of medium parameters on somatic embryogenesis from hypocotyl explants of flax (Linum usitatissimum L.): effect of carbon source, total inorganic nitrogen and balance between ionic forms and interaction between calcium and zeatin. J Plant Physiol 155:591–597

    Article  CAS  Google Scholar 

  15. D’Onghia AM, Carimi F, De Pasquale F, Djelouah K, Martelli GP (2001) Elimination of citrus psorosis virus by somatic embryogenesis from stigma and style cultures. Plant Pathol 50:266–269. doi:10.1046/j.1365-3059.2001.00550.x

    Article  Google Scholar 

  16. Dhekney SA, Li ZT, Gray DJ (2011) Factors influencing induction and maintenance of Vitis rotundifolia Michx embryogenic cultures. Plant Cell Tiss Organ Cult 105:175–180. doi:10.1007/s11240-010-9849-7

    Article  Google Scholar 

  17. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  18. Fiore S, De Pasquale F, Carimi F, Sajeva M (2002) Effect of 2, 4-D and 4-CPPU on somatic embryogenesis from stigma and style transverse thin cell layers of Citrus. Plant Cell Tiss Organ Cult 68:57–63. doi:10.1023/A:1012944100210

    Article  CAS  Google Scholar 

  19. Froelicher Y, Bassene JB, Jedidi-Neji E, Dambier D, Morillon R, Bernardini G, Constantino G, Ollitraut P (2007) Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analyses of plantlets. Plant Cell Rep 26:937–944. doi:10.1007/s00299-007-0314-y

    PubMed  Article  CAS  Google Scholar 

  20. Germanà MA, Chiancone B (2001) Gynogenetic haploids of Citrus after in vitro pollination with triploid pollen grains. Plant Cell Tiss Organ Cult 66:59–66. doi:10.1023/A:1010627310808

    Article  Google Scholar 

  21. Germanà MA, Chiancone B (2003) Improvement of Citrus Clementine Hort. ex Tan. microspore-derived embryoid production and regeneration. Plant Cell Rep 22:181–187. doi:10.1007/s00299-003-0669-7

    PubMed  Article  Google Scholar 

  22. Germanà MA, Micheli M, Chiancone B, Macaluso L, Standardi A (2011) Organogenesis and encapsulation of in vitro-derived propagules of Carrizo citrange [Citrus sinensis (L.) Osb. x Poncyrus trifoliata (L.) Raf]. Plant Cell Tiss Organ Cult 106:299–307. doi:10.1007/s11240-011-9921-y

    Article  Google Scholar 

  23. Gill MIS, Singh Z, Dhillon BS, Gosal SS (1995) Somatic embryogenesis and plantlet regeneration in mandarin (Citrus reticulata Blanco). Scientia Horticult 63:167–174. doi:10.1016/0304-4238(95)00812-8

    Article  Google Scholar 

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

    Google Scholar 

  25. Grosser JW, Gmitter FG Jr (2011) Protoplast fusion for production of tetraploids and triploids: application for scions and rootstock breeding in citrus. Plant Cell Tiss Organ Cult 104:343–357. doi:10.1007/s11240-010-9823-4

    Article  CAS  Google Scholar 

  26. Hu J, Ochoa OE, Truco MJ, Vick BA (2005) Application of the TRAP technique to lettuce (Lactuca sativa L.) genotyping. Euphytica 144:225–235. doi:10.1007/s10681-005-6431-1

    Article  CAS  Google Scholar 

  27. Hu J, Mou B, Vick BA (2007) Genetic diversity of 38 spinach (Spinacia oleracea L.) germplasm accessions and 10 commercial hybrids assessed by TRAP markers. Genet Resour Crop Evol 54:1667–1674. doi:10.1007/s10722-006-9175-4

    Article  CAS  Google Scholar 

  28. Huang P, Liao L, Tsai C, Liu Z (2011) Micropropagation of bromeliad Aechmea fasciata via floral organ segments and effects of acclimatization on plantlet growth. Plant Cell Tiss Organ Cult 105:73–78. doi:10.1007/s11240-010-9843-0

    Article  CAS  Google Scholar 

  29. Kayim M, Koc NK (2006) The effects of some carbohydrates on growth and somatic embryogenesis in citrus callus culture. Scientia Horticult 109:29–34. doi:10.1016/j.scienta.2006.01.040

    Article  CAS  Google Scholar 

  30. Kobayashi S, Ikeda I, Nakatani M (1981) Role of the primordium cell in nucellar embryogenesis in Citrus. Proc Int Soc Citricult 1:44–48

    Google Scholar 

  31. Kobayashi AK, Bespalhok JC, Pereira LFP, Vieira LGE (2003) Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments. Plant Cell Tiss Organ Cult 74:99–102. doi:10.1023/A:1023375526781

    Article  CAS  Google Scholar 

  32. Li GY, Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor Appl Genet 103:455–461. doi:10.1007/s001220100570

    Article  CAS  Google Scholar 

  33. Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Int Plant Propagation Soc Proc 30:421–427

    Google Scholar 

  34. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  35. Niedz RP, Evens TJ (2008) The effects of nitrogen and potassium nutrition on the growth of nonembryogenic and embryogenic tissue of sweet orange [Citrus sinensis (L.) Osbeck]. BMC Plant Biology 8:126. doi:10.1186/1471-2229-8-126

    PubMed  Article  Google Scholar 

  36. Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87

    PubMed  Article  CAS  Google Scholar 

  37. Pan Z, Zhu S, Guan R, Deng X (2010) Identification of 2, 4-D responsive proteins in embryogenic callus of Valencia sweet orange (Citrus sinensis Osbeck) following osmotic stress. Plant Cell Tiss Organ Cult 103:145–153. doi:10.1007/s11240-010-9762-0

    Article  CAS  Google Scholar 

  38. Peña L, Cervera M, Juarez OrtegaC, Pina JA, Durán-Villa N, Navarro L (1995) High efficiency Agrobacterium-mediated transformation and regeneration of Citrus. Plant Sci 104:183–191. doi:10.1016/0168-9452(94)04021-8

    Article  Google Scholar 

  39. Pinto DLP, Barros BA, Viccini LF, Salabert de Campos JM, Lemes da Silva M, Ottoni W (2010) Ploidy stability of somatic embryogenesis-derived Passiflora cinccinata Mast. Plants as assessed by flow citometry. Plant Cell Tiss Organ Cult 103:71–79. doi:10.1007/s11240-010-9756-y

    Article  Google Scholar 

  40. Prakash MG, Gurumurthi K (2010) Effects of type of explants and age, plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in Eucalyptus camaldulensis. Plant Cell Tiss Organ Cult 100:13–20. doi:10.1007/s11240-009-9611-1

    Article  CAS  Google Scholar 

  41. Slama-Ayed O, Slim-Amara H (2007) Production of doubled-haploids in durum wheat (Triticum durum Desf.) through culture of unpolinnated ovaries. Plant Cell Tiss Organ Cult 91:125–133. doi:10.1007/s11240-007-9281-9

    Article  Google Scholar 

  42. Srivastava P, Singh M, Mathur P, Chaturvedi R (2009) In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica. Biol Plant 53:360–364. doi:10.1007/s10535-009-0067-2

    Article  CAS  Google Scholar 

  43. Tomaz ML, Mendes BMJ, Mourão Filho MAA, Demetrio CGB, Jansakul N, Rodriguez APM (2001) Somatic embryogenesis in Citrus spp.: carbohydrate stimulation and histodifferentiation. In Vitro Cell Dev Biol Plant 37:446–452. doi:10.1007/s11627-001-0078-y

    Article  CAS  Google Scholar 

  44. Wu XB, Wang J, Liu JH, Deng XX (2009) Involvement of polyamine biosynthesis in somatic embryogenesis of Valencia sweet orange (Citrus sinensis) induced by glycerol. J Plant Physiol 166:52–62. doi:10.1016/j.jplph.2008.02.005

    PubMed  Article  CAS  Google Scholar 

  45. Yue B, Vick BA, Cai X, Hu J (2010) Genetic mapping for the Rf1 (fertility restoration) gene in sunflower (Helianthus annuus L.) by SSR and TRAP markers. Plant Breed 129:24–28. doi:10.1111/j.1439-0523.2009.01661.x

    Article  CAS  Google Scholar 

Download references

Acknowledgments

JCC and RRL acknowledges Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for research funds and a doctorate scholarship for JCC. APM acknowledges Conselho Nacional de Desenvolvimento Científico, CNPq, for a research fellowship.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Jean C. Cardoso.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cardoso, J.C., Martinelli, A.P. & Latado, R.R. Somatic embryogenesis from ovaries of sweet orange cv. Tobias. Plant Cell Tiss Organ Cult 109, 171–177 (2012). https://doi.org/10.1007/s11240-011-0073-x

Download citation

Keywords

  • Citrus sinensis
  • Pistil culture
  • Callus
  • Culture medium
  • Plantlet regeneration