An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses

Steven C Huber, G. E. EDWARDS

Research output: Contribution to journalArticle

Abstract

Variable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C3 gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C4 grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl2, 1 mM KH2PO4, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO2 fixation by the C4 mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C3 mesophyll protoplasts supported CO2 fixation without added substrates. Rates of CO2 fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C4 mesophyll protoplasts were stable when stored at 25°C.

Original languageEnglish (US)
Pages (from-to)203-209
Number of pages7
JournalPhysiologia Plantarum
Volume35
Issue number3
DOIs
StatePublished - Nov 1975

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Protoplasts
Cell Separation
Poaceae
protoplasts
mesophyll
Digitaria
grasses
Triticum
leaves
cells
wheat
Digitaria sanguinalis
Polygalacturonase
Light
Magnesium Chloride
cutters
Sorbitol
Cellulase
Photosynthesis
Enzymes

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science
  • Cell Biology

Cite this

An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses. / Huber, Steven C; EDWARDS, G. E.

In: Physiologia Plantarum, Vol. 35, No. 3, 11.1975, p. 203-209.

Research output: Contribution to journalArticle

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