Plant Cell Reports

, Volume 9, Issue 6, pp 335–339 | Cite as

Factors affecting PEG-mediated stable transformation of maize protoplasts

  • Charles L. Armstrong
  • William L. Petersen
  • Wallace G. Buchholz
  • Benjamin A. Bowen
  • Sally L. Sulc
Article
Received:
Revised:

Abstract

Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 1∶1 ratio to 65% at a 100∶1 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.

Keywords

Marker Gene Hygromycin Polyethylene Glycol Stable Transformation Plating Condition 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

PEG

polyethylene glycol

BMS

Black Mexican Sweetcorn

MS

salts of Murashige and Skoog (1962) culture medium

MaCa

0.2M mannitol, 80mM calcium chloride

MaMg

0.4M mannitol, 15mM magnesium chloride, 0.1% MES, pH 5.6

CM

conditioned medium

SE

standard error of the mean

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Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Charles L. Armstrong
    • 1
    • 4
  • William L. Petersen
    • 1
    • 4
  • Wallace G. Buchholz
    • 2
    • 4
  • Benjamin A. Bowen
    • 3
    • 4
  • Sally L. Sulc
    • 4
  1. 1.Monsanto Agricultural CompanySt. LouisUSA
  2. 2.Biology DepartmentTexas A & M UniversityCollege StationUSA
  3. 3.Pioneer Hi-Bred InternationalJohnstonUSA
  4. 4.Agrigenetics Advanced Science CompanyMadisonUSA

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