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Mechanical release and lectin labeling of maize root protoplasts

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Summary

An improved method for the mechanical release of protoplasts from plant tissues is described. The historically-low yield of mechanically-released protoplasts is greatly increased by use of a simple electrically-driven tissue sheer and by optimization of various other steps in the procedure. As counted by light microscopy of a purified preparation, the number of mechanically-released protoplasts obtained is about 6×104 per gram fresh weight of cortical tissue from the primary root of maize (Zea mays L. WF9×Mo 17) seedlings. Nuclear staining of the preparation, however, shows that about half of these protoplasts lack a nucleus and thus are actually subprotoplasts. Comparison of lectin binding to the plasma membranes of mechanically-and enzymatically-released protoplasts shows that both types contain binding sites forRicinus communis agglutinin. Binding sites for peanut (Arachis hypogaea) agglutinin are not naturally present on mechanically-released protoplasts but are generated by exposure to a mixture of Cellulysin and Pectolyase Y-23, the cell wall-degrading enzymes used to prepare enzymatically-released protoplasts.

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Abbreviations

BSA:

bovine serum albumin

DDT:

dithiothreitol

gfw:

gram fresh weight

Mes:

2-(N-morpholino) ethanesulfonic acid

PNA:

peanut (Arachis hypogaea) agglutinin

RCA:

Ricinus communis agglutinin

Tris:

tris(hydroxymethyl)aminomethane

References

  • Bilkey PC, Cocking EC (1982) A non-enzymatic method for the isolation of protoplasts from callus ofSaintpaulia ionantha (African violet). Z Pflanzenphysiol 105: 285–288

    Google Scholar 

  • Chambers R, Höfler K (1931) Microsurgical studies on the tonoplast ofAllium cepa. Protoplasma 12: 338–355

    Google Scholar 

  • Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187: 962–963

    Google Scholar 

  • Cocking EC (1965) Plant protoplasts. Viewpoints Biol 4: 170–203

    Google Scholar 

  • Dugas CM, Li Q, Khan IA, Nothnagel EA (1989) Lateral diffusion in the plasma membrane of maize protoplasts with implications for cell culture. Planta 179: 387–396

    Google Scholar 

  • Eriksson TR (1985) Protoplast isolation and culture. In: Fowke LC, Constabel F (eds) Plant protoplasts. CRC Press, Boca Raton, pp 1–20

    Google Scholar 

  • Furtula V, Walko RM, Nothnagel EA (1987) Direct covalent linkage of fluorescent probes to the plant protoplast surface. Protoplasma 139: 117–129

    Google Scholar 

  • —, Khan IA, Nothnagel EA (1990) Selective osmotic effect on diffusion of plasma membrane lipids in maize protoplasts. Proc Natl Acad Sci USA 87: 6532–6536

    Google Scholar 

  • Galbraith DW, Mauch TJ, Shields BA (1981) Analysis of the initial stages of plant protoplast development using 33258 Hoechst, reactivation of the cell cycle. Physiol Plant 51: 380–386

    Google Scholar 

  • Gronwald JW, Leonard RT (1982) Isolation and transport properties of protoplasts from cortical cells of corn roots. Plant Physiol 70: 1391–1395

    Google Scholar 

  • Huang CN, Cornejo MJ, Bush DS, Jones RL (1986) Estimating viability of plant protoplasts using double and single staining. Protoplasma 135: 80–87

    Google Scholar 

  • Imbrie-Milligan C, Hodges TK (1986) Microcallus formation from maize protoplasts prepared from embryogenic callus. Planta 168: 395–401

    Google Scholar 

  • Ishii S (1988) Factors influencing protoplast viability of suspension-cultured rice cells during isolation process. Plant Physiol 88: 26–29

    Google Scholar 

  • Klercker J (1892) Eine Methode zur Isolierung lebender Protoplasten. Ofvers Vetensk Akad Forh Stock 49: 463–475

    Google Scholar 

  • Knox JP, Roberts K (1989) Carbohydrate antigens and lectin receptors of the plasma membrane of carrot cells. Protoplasma 152: 123–129

    Google Scholar 

  • Komalavilas P, Zhu JK, Nothnagel EA (1991) Arabinogalactanproteins from the suspension culture medium and plasma membrane of rose cells. J Biol Chem 266: 15956–15965

    Google Scholar 

  • Laloue M, Courtois D, Manigault P (1980) Convenient and rapid fluorescent staining of cell nuclei with 33258 Hoechst. Plant Sci Lett 17: 175–179

    Google Scholar 

  • Laver DR (1991) A surgical method for accessing the plasmamembrane ofChara australis. Protoplasma 161: 79–84

    Google Scholar 

  • Lee-Stadelmann O, Chung I, Stadelmann EJ (1985) Plasmolysis ofGlycine max mesophyll cells, the use of octylguanidine and its implications in protoplasts isolation. Plant Sci 38: 1–7

    Google Scholar 

  • Leigh R, Branton D (1976) Isolation of vacuoles from root storage tissue ofBeta vulgaris L. Plant Physiol 58: 656–662

    Google Scholar 

  • Nagata T, Ishii S (1979) A rapid method for isolation of mesophyll protoplasts. Can J Bot 57: 1820–1823

    Google Scholar 

  • Nolte KD, Nothnagel EA, Coggins CW Jr (1990) Electrolyte leakage and protoplast viability of pummelo mesocarp tissue as influenced by exogenous GA3. J Am Soc Hortic Sci 115: 592–597

    Google Scholar 

  • Patnaik G, Wilson D, Cocking EC (1982) Importance of enzyme purification for increased plating efficiency and plant regeneration from single protoplasts ofPetunia purodii. Z Pflanzenphysiol 102: 199–205

    Google Scholar 

  • Pilet PE (1972) Transaminase activities of root protoplasts. Experientia 28: 638–639

    Google Scholar 

  • —, Prat R, Roland JC (1972) Morphology, RNase and transaminase of root protoplasts. Plant Cell Physiol 13: 297–309

    Google Scholar 

  • Schwenk FW, Pearson CA, Roth MR (1981) Soybean mesophyll protoplasts. Plant Sci Lett 23: 153–155

    Google Scholar 

  • Shea EM, Gibeaut DM, Carpita NC (1989) Structural analysis of the cell walls regenerated by carrot protoplasts. Planta 179: 293–308

    Google Scholar 

  • Tornava SR (1939) Expansion capacity of naked plant protoplasts. Protoplasma 32: 329–341

    Google Scholar 

  • Tribe HT (1955) Studies on the physiology of parasitism. XIX. On the killing of plant cells by enzymes fromBotrytis cinera andBacterium aroideae. Ann Bot NS 19: 351–368

    Google Scholar 

  • Walko RM, Furtula V, Nothnagel EA (1987) Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins. Protoplasma 141: 33–46

    Google Scholar 

  • Weaver ML, Breda V, Gaffield W, Timm H (1990) Nonenzymatic release of intact protoplasts from mature pollen of bean. J Am Soc Hortic Sci 115: 640–643

    Google Scholar 

  • Whatley FR (1956) Cytochemical methods. In: Perch K, Tracey MV (eds) Modern methods of plant analysis, vol I. Springer, Berlin Göttingen Heidelberg, pp 452–467

    Google Scholar 

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Author notes

  1. V. Furtula

    Present address: Cancer Imaging, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada

Authors and Affiliations

  1. Department of Botany and Plant Sciences-072, University of California, 92521-0124, Riverside, CA, USA

    S. Sun, V. Furtula & E. A. Nothnagel

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  1. S. Sun
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  2. V. Furtula
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  3. E. A. Nothnagel
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Sun, S., Furtula, V. & Nothnagel, E.A. Mechanical release and lectin labeling of maize root protoplasts. Protoplasma 169, 49–56 (1992). https://doi.org/10.1007/BF01343369

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  • DOI: https://doi.org/10.1007/BF01343369

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