Plant Molecular Biology Manual A6: 1-10 (1988)

Kluwer Academic Publishers, Dordrecht

Extraction of DNA from plant tissues

SCOTT O. ROGERS 1 & ARNOLD J. BENDICH 2

I Department of Botany, KB-15, University of Washington, Seattle, WA 98195, USA;
2 Departments of Botany and Genetics, University of Washington, Seattle, WA 98195, USA

Introduction

Extraction procedures for plant DNA in general must accomplish the following.
(1) The cell walls must be broken (or digested away) in order to release the
cellular constituents. This is usually done by grinding the tissue in dry ice
or liquid nitrogen with a mortar and pestel or a food grinder.
(2) The cell membranes must be disrupted, so that the DNA is released into the
extraction buffer. This is accomplished by using a detergent, usually SDS
(sodium dodecyl sulfate) or CT AB (cetyltrimethylammonium bromide).
(3) The DNA must be protected from the endogenous nucleases. The detergents
are used for this purpose, as is EDT A (ethylenediaminetetraacetic acid). It
is a chelating agent that binds magnesium ions, generally considered a
necessary cofactor for most nucleases (but see note f, below). In addition,
the buffer/tissue mixture is emulsified with either chloroform or phenol to
denature and separate the proteins from the DNA.
(4) Shearing of the DNA should be minimized. DNA in solution can be broken
by exposure to turbulence (e.g., being quickly drawn through a small orifice).
Typically, DNA 50-100 kb in length can be obtained without great care
being taken.
(5) The time between thawing of the frozen, pulverized tissue and its exposure
to the extraction buffer should be minimized to avoid nucleolytic degradation
of the DNA.
There is one other major consideration associated with the isolation of DNA
from higher plants that is not encountered with most other organisms. Enzyme-
inhibiting polysaccharides are often present in the 'purified' DNA. Most ex-
traction methods have employed the expensive and time-consuming cesium
rhloride density gr:1oient technique to eliminate the polysaccharides (e.g
[1,6,9]). Other methods have been reported that do not utilize density gradients,
but they have been described for only a limited number of species and tissue
types [4, 10]. The method presented here is based on the CT AB nucleic acid
extraction procedures of Murray and Thompson [6] and Taylor and Powell [9]
that make it possible to extract purified high molecular weight (> 50 kb) plant

PMAN-A6/1
DNA without the use of expensive equipment and/or time-consuming procedures.
The basis for the separation of polysaccharides from nucleic acids is their
differential solubilities in the presence of CTAB. In most cases only three
disposable microcentrifuge tubes are required for all operations from tissue
homogenization to DNA of sufficient purity to be digested by most restriction
endonucleases. Tissues as small as individual ovules and embryos, or small pieces
of tissue from various parts of the same plant, can be used. In addition, DNA
can be obtained from milligram amounts of herbarium and mummified tissues.
We have used this procedure on over 60 types of tissues from more than
30 species and have almost always been able to obtain DNA that can be digested
by most restriction endonucleases in a few hours. Approximately 2-3 h are
needed to process 1-12 samples. The original method [7] is presented here with
a few refinements and is described for tissue amounts smaller than about 500 mg.
(Typical yields are shown in Table 1.)

Table 1. DNA yields from fresh plant tissues

Tissue Species* Genome size DNA yield

(pg) (ng/mg)

Leaves Nicotiana tabacum (young) 3.9 38

Petunia hybrida (young) 2.0 14
Triticum aestivum (flag) 15.7 45
Triticum aestivum (mature) 15.7 48
Triticum aestivum (young) 15.7 52
Vicia faba (young) 13.2 40
Vitis vinifera (senescing) 0.4
Zea mays (mature) 4.0 40
Zea mays (seedling) 4.0 49
Whole seedling Arabidopsis thaliana 0.08 8.0

Suspension culture Nicotiana tabacum (NT-I) 3.9 10

Whole seeds/grains Allium cepa 16.5 55

Citrullus vulgariS 1.1 1.0
Glycine max 0.9 5.5
Hordeum vulgare 5.5 9.6
Linum usitatissimum 0.7 28
Phaseolus vulgaris 1.8 2.2
Pisum sativum 5.0 18
Triticum aestivum 15.7 22
Zea mays 4.0 18
Embryos and embryo axes Citrullus vulgaris 1.1 42
Cucumis melo 1.0 13
Cucurbita pepo 1.3 14
Glycine max 0.9 55
Hordeum vulgare 5.5 24

PMAN-A6/2