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Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film

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Summary.

Somatic hybridization has been identified as one method for the genetic improvement of roses. The success of somatic hybridization programmes relies to a great extent upon efficient protoplast isolation and culture and selection of heterokaryons. This paper reports the isolation of rose cell suspension protoplasts by direct sucrose flotation and demonstrates their culture using extra thin alginate film. A comparative assessment of the efficiency of conventional culture techniques versus those with extra thin alginate film or thin alginate layer is also presented. A very high plating efficiency (80%) was obtained using thin alginate layer or extra thin alginate film techniques with improved media formulations. Protoplasts of Rosa damascena and R. bourboniana were fused by using polyethylene glycol as fusogen and later immobilized in the thin layer of alginate. The fused protoplasts were tracked on the basis of differential fluorescent staining, and the hybridity of heterokaryons following their development to callus was confirmed by molecular characterization. This novel selection strategy has general applicability and is faster and simpler to perform during somatic hybridization experiments.

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Abbreviations

ETAF:

extra thin alginate film

FITC:

fluorescein isothiocyanate

PEG:

polyethylene glycol

RAPD:

random amplified polymorphic DNA

RITC:

rhodamine isothiocyanate

TAL:

thin alginate layer

References

  • S Austin MK Ehlenfeldt MA Baer JP Helgeson (1986) ArticleTitleSomatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions Theor Appl Genet 71 682–690 Occurrence Handle10.1007/BF00263264

    Article  Google Scholar 

  • YPS Bajaj (Eds) (1994) Somatic hybridization in crop improvement SeriesTitleBiotechnology in agriculture and forestry NumberInSeries27 Springer Berlin

    Google Scholar 

  • F Crea O Calderini E Nenz PD Cluster F Damiani S Arcioni (1997) ArticleTitleChromosomal and molecular rearrangements in somatic hybrids between tetraploid Medicago sativa and diploid Medicago falcata Theor Appl Genet 95 1112–1118 Occurrence Handle10.1007/s001220050670 Occurrence Handle1:CAS:528:DyaK1cXhsFWguw%3D%3D

    Article  CAS  Google Scholar 

  • MR Davey P Anthony JB Power KC Lowe (2005) ArticleTitlePlant protoplasts: status and biotechnological perspectives Biotechnol Adv 23 131–171 Occurrence Handle15694124 Occurrence Handle10.1016/j.biotechadv.2004.09.008 Occurrence Handle1:CAS:528:DC%2BD2MXptlCjsw%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • TR Eriksson (1985) Protoplast isolation and culture LC Flowke F Constabel (Eds) Plant protoplasts CRC Press Boca Raton, Fla 1–20

    Google Scholar 

  • N Fish A Karp MGK Jones (1988) ArticleTitleProduction of somatic hybrids by electrofusion in Solanum Theor Appl Genet 76 260–266

    Google Scholar 

  • YY Gleba KM Sytnik (1984) Protoplast fusion R Shoeman (Eds) Genetic engineering in higher plants Springer Berlin 115–162

    Google Scholar 

  • TJ Golds J Babczinsky G Rauscher HU Koop (1992) ArticleTitleComputer controlled tracking of single cell development in Nicotiana tabacum L. and Hordeum vulgare L. protoplasts embedded in agarose/alginate films J Plant Physiol 140 582–587

    Google Scholar 

  • SW Kim SC Oh DS In JR Liu (2003) ArticleTitlePlant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts Plant Cell Tissue Organ Cult 73 15–19 Occurrence Handle10.1023/A:1022693605436 Occurrence Handle1:CAS:528:DC%2BD3sXhslOkt7Y%3D

    Article  CAS  Google Scholar 

  • PJ Larkin (1976) ArticleTitlePurification and viability determinations of plant protoplasts Planta 128 213–216 Occurrence Handle10.1007/BF00393231

    Article  Google Scholar 

  • R Marchant MR Davey JB Power (1997) ArticleTitleIsolation and culture of mesophyll protoplasts from Rosa hybrida Plant Cell Tissue Organ Cult 50 131–134 Occurrence Handle10.1023/A:1005910002625

    Article  Google Scholar 

  • D Matthews J Mottley I Horan AV Roberts (1991) ArticleTitleA protoplast to plant system in roses Plant Cell Tissue Organ Cult 24 173–180 Occurrence Handle10.1007/BF00033473

    Article  Google Scholar 

  • J Mottley K Yokoya D Mathews J Squirrell JE Wentworth (1996) ArticleTitleProtoplast fusion and its potential role in the genetic improvement of roses Acta Hort 424 393–397

    Google Scholar 

  • T Murashige F Skoog (1962) ArticleTitleA revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol Plant 15 473–479 Occurrence Handle10.1111/j.1399-3054.1962.tb08052.x Occurrence Handle1:CAS:528:DyaF3sXksFKm

    Article  CAS  Google Scholar 

  • T Nagata I Takebe (1970) ArticleTitleCell wall regeneration and cell division in isolated tobacco mesophyll protoplasts Planta 92 301–308 Occurrence Handle10.1007/BF00385097

    Article  Google Scholar 

  • SJ Ochatt JB Power (1988a) ArticleTitleAn alternative approach to plant regeneration from protoplasts of sour cherry (Prunus cerasus L.) Plant Sci 56 75–79 Occurrence Handle10.1016/0168-9452(88)90188-4

    Article  Google Scholar 

  • SJ Ochatt JB Power (1988b) ArticleTitlePlant regeneration from mesophyll protoplasts of Williams’ Bon Chretien (syn. Bartlett) pear (Pyrus communis L.) Plant Cell Rep 7 587–589 Occurrence Handle10.1007/BF00272764

    Article  Google Scholar 

  • PK Pati M Sharma PS Ahuja (2001) ArticleTitleMicropropagation, protoplast culture and its implications in the improvement of scented rose Acta Hortic 547 147–158 Occurrence Handle1:CAS:528:DC%2BD3MXltFShs74%3D

    CAS  Google Scholar 

  • PK Pati M Sharma PS Ahuja (2005) ArticleTitleExtra thin alginate film: an efficient technique for protoplast culture Protoplasma 226 217–221 Occurrence Handle16244810 Occurrence Handle10.1007/s00709-005-0096-4 Occurrence Handle1:STN:280:DC%2BD2Mnlt1altw%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • E Pehu M Thomas T Poutala A Karp MGK Jones (1990) ArticleTitleSpecies-specific sequences in the genus Solanum: identification, characterization, and application to study somatic hybrids of S. brevidens and S. tuberosum Theor Appl Genet 80 693–699 Occurrence Handle10.1007/BF00224231 Occurrence Handle1:CAS:528:DyaK3MXisVWqtLs%3D

    Article  CAS  Google Scholar 

  • D Pental A Mukhopadhyay A Grover AK Pradhan (1988) ArticleTitleA selection method for the synthesis of triploid hybrids by fusion of microspore protoplasts (n) with somatic cell protoplasts (2n) Theor Appl Genet 76 237–243 Occurrence Handle10.1007/BF00257851

    Article  Google Scholar 

  • M Saghai Maroof KM Solaman RA Jorgenson RW Allard (1984) ArticleTitleRibosomal DNA spacer length polymorphism in barley: Mendelian inheritance, chromosomal location and population dynamics Proc Natl Acad Sci USA 81 8014–8018 Occurrence Handle6096873 Occurrence Handle10.1073/pnas.81.24.8014 Occurrence Handle1:CAS:528:DyaL2MXptlWitg%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • MW Saul I Potrykus (1984) ArticleTitleSpecies-specific repetitive DNA used to identify interspecific somatic hybrids Plant Cell Rep 3 65–67 Occurrence Handle10.1007/BF00270973 Occurrence Handle1:CAS:528:DyaL2cXksV2nt7o%3D

    Article  CAS  Google Scholar 

  • A Schum K Hofman N Ghalib A Tawfik (2001) ArticleTitleFactors affecting protoplast isolation and plant regeneration in Rosa spp Gartenbauwissenschaft 66 115–122 Occurrence Handle1:CAS:528:DC%2BD3MXovVGisb8%3D

    CAS  Google Scholar 

  • A Strauss I Potrykus (1980) ArticleTitleCallus formation from protoplasts of cell suspension cultures of Rosa ‘Paul’s Scarlet’ Physiol Plant 48 15–20 Occurrence Handle10.1111/j.1399-3054.1980.tb03212.x

    Article  Google Scholar 

  • S Waara K Glimelius (1995) ArticleTitleThe potential of somatic hybridization in crop breeding Euphytica 85 217–233 Occurrence Handle10.1007/BF00023951

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Biotechnology, Guru Nanak Dev University, Amritsar

    P. K. Pati

  2. Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur

    Madhu Sharma & P. S. Ahuja

Authors
  1. P. K. Pati
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  2. Madhu Sharma
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  3. P. S. Ahuja
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Correspondence to P. K. Pati.

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Correspondence: P. K. Pati, Department of Biotechnology, Guru Nanak Dev University, Amritsar 143 005, India.

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Pati, P., Sharma, M. & Ahuja, P. Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film. Protoplasma 233, 165–171 (2008). https://doi.org/10.1007/s00709-008-0297-8

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