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Isolation of protoplasts from developing barley endosperm: a tool for transient expression studies

Abstract

We have developed a method for the routine isolation of protoplasts from developing starchy endosperm of barley (Hordeum vulgare L.). Preplasmolysis of the intact endosperms, a low concentration of hydrolytic enzymes and gravity sedimentation before any centrifugation step, were crucial factors for a good preparation. Best yields were obtained early after pollination (8–13 days) or with mutants with low starch content. Transient expression of a reporter gene under the control of the 35S promoter, after polyethyleneglycol transfection of endosperm protoplasts, was of the same order as that found in coleoptile derived protoplasts. No significant difference in expression was found for a given tissue between cv. Bomi and its mutant Risø 1508.

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Abbreviations

2, 4D:

2, 4 dichlorophenoxyacetic acid

dap:

days after pollination

MS:

Murashige and Skoog medium

pp:

protoplasts

PEG:

polyethylenglycol

GUS:

ß-glucuronidase

MUG:

4-methylumbelliferyl-ß-D-glucuronide

X-gluc:

5-bromo-4 chloro-3 indolyl glucuronide

References

  • Denecke J, Gossele V, Botterman J, Cornelissen M (1989) Meth Mol Cell Biol 1:19–27

    Google Scholar 

  • Doll H (1984) Philos Trans Royal Soc London Ser B 304:380

    Google Scholar 

  • Entwistle G, Tyson RH, Rees T (1988) Phytochemistry 27:993–996

    Google Scholar 

  • Gopalakrishnan B, Sonthayanon B, Rahmatullah R, Muthukrishnan S (1991) Plant Mol Biol 16:463–467

    Google Scholar 

  • Hähne B, Fleck J, Hähne G (1991) Physiol Plant 82:A7

    Google Scholar 

  • Jacobsen JV, Zwar JA, Chandler PM (1985) Planta 163:430–438

    Google Scholar 

  • Jähne A, Lazzeri PA, Lörz H (1991) Plant Cell Rep 10:1–6

    Google Scholar 

  • Jefferson RA (1987) Plant Mol Biol Rep 5:387–405

    CAS  Google Scholar 

  • Kaneko T, Kuroda H, Hirota N, Kisninami I, Kimura N, Ito K (1991) Proc 6th Intern Barley Genet Symp, Helsingborg, Sweden. Munksgaard International Publishers, Denmark pp 231–233

    Google Scholar 

  • Keeling PL, Baird S, Tyson RH (1989) Plant Sci 65:55–62

    Google Scholar 

  • Krens FA, Molendijk L, Wullems GJ, Schilperoot RA (1982) Nature 296:72–74

    Google Scholar 

  • Lazzeri PA, Biettschneider R, Lührs R, Lörz H (1991) Theor Appl Genet 81:437–444

    Google Scholar 

  • Lee BT, Murdoch K, Topping J, Kreis M, Jones MGK (1989) Plant Mol Biol 13:21–29

    Google Scholar 

  • Lee BT, Murdoch K, Topping J, Jones MGK, Kreis M (1991) Plant Sci 78:237–246

    Google Scholar 

  • Loveys BR, Robinson SP (1987) Plant Sci 49:23–30

    Google Scholar 

  • Lührs R, Lörz H (1988) Planta 175:71–81

    Google Scholar 

  • Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Plant Mol Biol 8:363–373

    Google Scholar 

  • Olsen FL, Stilling B, Mundy J (1990) Abstract VII Inter Congr Plant Tiss and Cell Cult. IAPTC (ed) Amsterdam, Holland pp 71

  • Owen JH (1987) J Plant Physiol 130:385–393

    Google Scholar 

  • Potter R, Melby T, Klemsdal S, Aalan R, Lønneborg A, Olsen OA (1991) Physiol Plant 82:A32

    Google Scholar 

  • Power JB, Chapman JV (1985) In: Dixon RA (ed) Plant Cell Culture. IRL Press, Oxford, Washington DC pp 37–66

    Google Scholar 

  • Salmenkallio M, Hannus R, Teeri TH, Kauppinen V (1990) Plant Cell Rep 9:352–355

    Google Scholar 

  • Schwall M, Feix G (1988) Plant Sci 56:161–166

    Google Scholar 

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Communicated by I. Potrykus

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Diaz, I., Carbonero, P. Isolation of protoplasts from developing barley endosperm: a tool for transient expression studies. Plant Cell Reports 10, 595–598 (1992). https://doi.org/10.1007/BF00232377

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  • DOI: https://doi.org/10.1007/BF00232377

Keywords

  • Starch
  • Reporter Gene
  • Hydrolytic Enzyme
  • Expression Study
  • Polyethyleneglycol