Plant Molecular Biology Reporter pages 106-109

12 (2) 1994

Commentary

A Modified CTAB D N A Extraction Procedure

for Plants Belonging to the Family Proteaceae
Key Words: DNA, Banksia,Dryandra,Leucadendron,Leucospermum,Protea,Mac-
adamia, Grevillea,Isopogon,Serruria,Hakea,Proteaceae, RAPD-PCR, digest
Abstract: This paper describes rapid and efficient DNA extraction methods for
mature leaves, resting buds and seedling leaves of genera in the familyProteaceae.
The procedures combine and modify previously published techniques. The
DNA can be digested by restriction endonucleases and is suitable for subsequent
PCR amplification.

he applications of current nucleic acid technologies to crop im-

T provement include gene mapping, genetic fingerprinting, popu-

lation studies, and phylogenetic analyses. These techniques have
application for the improvement of banksias, proteas and other Austra-
lian and African native plants for cut flower production. The develop-
ment of efficient DNA extraction techniques that yield DNA of a purity
adequate for restriction enzyme digest and PCR has proven difficult
from plant materials in these genera. After experimenting with a variety
of techniques, we have developed simple efficient procedures for the
extraction of DNA from Banksiaand other genera in the family Proteaceae.
The difficulties associated with DNA extraction from plants high in
polysaccharides and phenolics, such as occurs in Banksia, have been
recognised previously (Couch and Fritz, 1990; John, 1992; Do and Ad-
ams, 1991). Our combined techniques will be useful to others who also
experience similar difficulties isolating DNA from plant material using
the CTAB method.

Materials and Methods

DNA was extracted from plant material of Banksia, Dryandra,
Leucadendron, Isopogon,Protea,Macadamia,Leucospermum,Grevillea,Hakea
and Serruria (Family Proteaceae) using the following modifications of the
methods of Doyle and Doyle (1988) and Fang et al. (1992).

106
Extraction of DNA from the Proteaceae 107

Fig. 1. Restriction of DNA extracted by CTAB procedure. The extracted DNA

is suitable for digestion by the restriction enzymes Hae III and Dra I(lanes 2-4).
The DNA is also able to be used in RAPD PCR (lanes 5-7).

Mature leaves
The m e t h o d of Doyle and Doyle (1988) was used with the following
modifications:

9 70% ethanol instead of 76% ethanol plus 10 m M a m m o n i u m acetate

w a s h buffer,
9 D N A r e s u s p e n d e d in 400 ~tL rather than I m L TE buffer.
9 RNA r e m o v e d using 2 ~tL RNase A at 40 ~tg/mL at 5~ overnight.
9 N o second D N A precipitation.

Resting buds
The m e t h o d for m a t u r e leaves was used with the following modifica-
tions:

9 Incubate 45 min rather than 30 min in CTAB extraction buffer.

9 Precipitate D N A with isopropanol for 30 min at -20~
108 Maguire, Collins, and Sedgley

Seedling leaves
The method for resting buds was used with the following modifications:
9 Extraction buffer comprised 3% rather than 2% CTAB, 0.4% rather
than 0.2% 2-mercaptoethanol, plus 2% PVP.
9 Two chloroform-isoamyl extractions rather than one.
9 DNA precipitated with isopropanol containing 50 ~tL sodium
acetate (pH 4.8) for 30 rain at -20~
Polysaccharides co-precipitate with the DNA forming a viscous layer
and pellet. The following protocol was developed to separate the DNA
from the polysaccharide impurities:
9 The viscous layer and pellet were precipitated with 95% ethanol
and centrifuged for 10 rains at 4000 rpm
9 The pellet was dissolved in 400 ~tLTE buffer with a Pasteur pipette.
1.6 mL of 2.5 M NaC1 was added to give a total concentration of 2
M NaC1, and the DNA precipitated with 95% ethanol and centri-
fuged for 10 min at 4000 rpm. At this concentration of NaC1,
contaminating material remained in solution (Fang et al. 1992)
9 Final DNA pellet resuspended in 100 ~tL of TE buffer
DNA extracted using the seedling leaves method was restricted using
Hae III or Dra I endonucleases, or subjected to RAPD PCR (Hu and
Quiros, 1991; Klein-Lankhorst et al., 1991).

Results and Discussion

Using this technique we obtained DNA yields of 8-12 ~tg/g fresh wt
leaf tissue from all 10 genera tested. The best results for extraction of
DNA from Banksia were with fresh leaf tissue harvested from mature
plants. Seedling material was more difficult due to higher levels of
interfering compounds, hence development of the technique suitable for
this tissue. The DNA samples were completely digested with Hae III and
Dra I restriction enzymes (Fig. 1), indicating low levels of DNA methy-
lation (Nelson & McClelland 1991). The DNA was also suitable for RAPD
PCR (Fig. 1).
Acknowledgments. Thanks to Peter Langridge for advice, Michelle Sierp for
assistance, and Jenny Groom for photography. TLM was supported by an
Australian Postgraduate Research Award.

Tina L. Maguire, Graham G. Collins and Margaret Sedgley

Department of Horticulture, Viticulture and Oenology, Waite Campus,
University of Adelaide, Glen Osmond, SA, 5064, Australia
Extraction of DNA from the Proteaceae 109

References

Couch, J.A. and P.J. Fritz. 1990. Isolation of DNA from plants high in polyphenolics. Plant
Mol. Biol. Reptr. 8:8-12.
Do, N. and R.P. Adams. 1991. A simple technique for removing plant polysaccharide
contaminants from DNA. Biotechniques 10:163-166.
Doyle, J.J. and J,L. Doyle. 1988. Isolation of plant DNA from fresh tissue. Focus 12:13-15.
Fang, G., S. Hammar and R. Grumet. 1992. A quick inexpensive method for removing
polysaccharides from plant genomic DNA. Biotechniques 13:52-55.
Hu, J., and C.F. Quiros. 1991. Identificationof broccoli and cauliflower cultivars with RAPD
markers. Plant Cell Reports. 10:505-511.
John, M.E. 1992. An efficient method for isolation of RNA and DNA from plants containing
polyphenolics. Nucleic Acids Res. 20:2381.
Klein-Lankhorst, R.M., A, Vermunt, R. Weide, T. Liharska, and P. Zabel. 1991. Isolation of
molecular markers from tomato (L. esculentum)using random amplified polymorphic
DNA (RAPD). Theor. Appl. Genet. 83:108-114.
Nelson, M. and M. M~Clelland. 1991. Site specific methylation: effect on DNA modification
methyltransferases and restriction endonucleases. Nucleic Acids Res. 19: 2045-2070.