Download Flower petals allow simpler and better isolation of DNA for

Plant Molecular Biology Reporter
13 (3) 1995
pages 210-213
Commentary
Flower Petals Allow Simpler and Better
Isolation of D N A for Plant RAPD Analyses
Jing-Zhong Lin and Kermit Ritland
E-mail: [email protected]
Department of Botany, University of Toronto, 25 Willcocks St., Toronto,
Ontario, M5S 3B2, Canada
Key Words: DNA isolation, RAPD, flower petal, corolla
M
inipreps of plant genomic DNA for PCR and specifically
RAPD analyses usually use leaf tissue as the source of DNA
(e.g., Edwards et al., 1991; Stewart and Via, 1993). In some
cases, however, especially in small plants, the use of leaf tissue is not
feasible: it is often hard to collect sufficient fresh leaf tissue, particularly
in the later stages of the life cycle or at the end of the growing season when
most leaves are old. Additionally, in plant fitness studies, removal of
young leaves for DNA isolation will affect survival and fecundity, and
bias any estimate of fitness. Furthermore, in some species minipreps
often require that leaf tissue be ground with liquid nitrogen (Doyle and
Doyle, 1987), sterile sands or glass balls (Colosi and Schaal, 1993), or with
a special grinding device (Stewart and Via, 1993 ). Finally, there have been
enormous difficulties in extracting DNA from leaf tissue of some wild
species, e.g., Lythrum salicaria (below) and wild strawberries (Diane
Pavek, personal communication), probably because of the accumulation
of interfering secondary compounds. Often there is little or nothing
known about the biochemistry of these wild species.
The purpose of this note is to focus attention on another tissue, the
corolla, that we have found useful for isolating plant genomic DNA for
RAPD analysis. The major advantages are the ease of tissue grinding,
fewer steps in the isolation procedure, and the recovery of better quality
Abbreviations: CTAB,hexadecyltrimethylammonium bromide; PCR, polymerase
chain reaction; RAPD, random-polymorphic DNA.
210
Isolation of DNA fi'om Flozoer Petals
211
DNA compared to leaves, particularly older leaves. Also, removal of
corolla tissue has little effect on plant fitness, since flowering takes place
at the natural end of the life cycle or growing season, plants often produce
copious flowers to attract pollinators, and few actually produce seed
(Charlesworth, 1989).
To evaluate the general usefulness of corollas, we used a simplified
CTAB procedure to isolate DNA from five plant species, Mim ulus gu tta tus
(monkeyflower), Eichhornia paniculata, Aeschynanthus lobbianus (lipstick
plant), Lythrum salicaria (purple loosestrife), and Antirrhinum majus
(snapdragon). In each species, the isolation was replicated in at least four
individuals. The method is described below. For comparison, DNA from
the same plants was isolated using an equal amount of leaf tissue with
this procedure, except that an extra chloroform-isoamyl extraction was
required because of a visible protein precipitation at the interface after
only one extraction, followed by a 10-minute wash with 76 percent
ethanol and 10 mM ammonium acetate. Corollas of E. paniculata and A.
lobbianus also required a 10-minute wash to remove pigments. It was not
possible to isolate DNA from leaves of L. salicaria using this method since
the brei becomes extremely sticky, presumably from abundant polysaccharides.
Spectrophotometric measurements of the isolated DNA show that
corollas yield much more DNA than leaves in four of the five species. The
exception is E. paniculata in which corollas give slightly lower yield.
Except for A. lobbianus, DNA isolated from corollas shows a higher A260/
A280ratio (about 1.8) than that isolated from leaves (about 1.6).
The suitability of DNA isolated from corollas and leaves for RAPD
analyses was compared using two RAPD primers, OPLll and OPL17
(Operon Technologies, Alameda, USA). PCR was performed in a PerkinElmer Cetus DNA thermocycler (Perkin-Elmer Corp., Connecticut, USA)
using common RAPD PCR conditions (e.g., Williams et al., 1990).
Fig. 1 shows the RAPD banding patterns amplified with primers
OPLll and OPL17 using DNA isolated from corollas. This DNA amplified consistently well for all species tested. In contrast, DNA from leaves
of the same plants produced variable RAPD results (not shown). It
amplified equally well in A. lobbian us, amplified erratically inM. gu ttatus,
and did not amplify at all in A. majus and E. paniculata.
Although more elaborate procedures (e.g., Doyle and Doyle, 1987)
may be employed to isolate useful DNA from leaves, we stress the
efficiency and utility of corollas. Because flower petals presumably
contain fewer carbohydrates and polysaccharides, and are softer in
212
Lin & Ritland
M. guttatus
E. paniculata
A. rnajus A. lobbianus
L. salicaria
M. guttatus
A. majus
q
E. paniculata
A. lobbianus
L. salicaria
W
g
-'"-2-
aJ
.i
-
Fig. 1. RAPD results with corolla DNA. DNA isolated from corollas of five plant
species was amplified using primers OPLll (A, upper) and OPL17 (B, lower).The
XDNA was digested with Hind III-Eco RI.
texture than leaves, they are easier to grind, involve fewer steps in the
isolation procedure (e.g., omission of grinding in liquid nitrogen and the
extra chloroform-isoamyl extraction), and produce a higher yield of
purer DNA. Because isolation is carried out in microfuge tubes, multiple
samples can be handled readily. In our laboratory, we can obtain enough
DNA for at least 100 RAPD analyses with only a minute amount of
corollas (less than 200 mg fresh weight), and we can isolate at least 100
samples a day. Corollas are especially appropriate for DNA isolation in
Isolation of DNA f~'om Flower Petals
213
species that h a v e t o u g h leaf tissue (e.g., A. majus) or h a v e interfering
chemical c o m p o u n d s in leaves (e.g., L. salicaria), a n d in s t u d i e s w h e r e
p l a n t fitness is assessed.
Methods and Materials
9 To a 1.5-mL m i c r o f u g e tube, a d d a b o u t 150 m g of corolla tissue a n d
20 ~tL CTAB b u f f e r 1 ( w i t h o u t 2 - m e r c a p t o e t h a n o l ) .
9 M a c e r a t e tissue w i t h a d i s p o s a b l e pellet pestle (VWR Scientific) for
a b o u t I min.
9 A d d 2 - m e r c a p t o e t h a n o l (0.2 % final) a n d m o r e CTAB b u f f e r to
m a k e a total v o l u m e of 500 p.L, t h e n
9 p u t the tube in w a t e r b a t h at 60 ~ for 15-30 min.
9 Extract w i t h c h l o r o f o r m - i s o a m y l (24:1) for 5 min, t h e n centrifuge at
13,000 r p m for 5 m i n either at 4 ~ or r o o m t e m p e r a t u r e .
9 T r a n s f e r s u p e r n a t a n t to a n e w m i c r o f u g e tube, a n d precipitate w i t h
300 ~L cold i s o p r o p a n o l , t h e n centrifuge at 13,000 r p m for 5
minutes.
9 D r y pellet in v a c u u m a n d r e s u s p e n d in 150 ~tL TE buffer. 2
Notes
1. CTAB buffer: 100 mM tris-HC1, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2 %
hexadecyltrirnethylammonium bromide (CTAB).
2. For long-term use, further cleaning of this DNA with RNAse A will reduce
degradation.
References
Charlesworth, D. 1989. Why do plants produce so many more ovules than seeds? Nature
338:21-22.
Colosi, J.C. and B.A. Schaal. 1993. Tissue grinding with ball bearings and votex mixer for
DNA extraction. Nucleic Acids Res. 21:1051-1052.
Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of
fresh leaf tissue. Phytochem. Bull. 19:11-15.
Edwards, K., C. Johnstone and C. Thompson. 1991. A simple and rapid method for the
preparation of genomic plant DNA for PCR analysis. Nucleic Acids Res. 19:1349.
Stewart, Jr.C.N. and L.E. Via. 1993. A rapid CTAB DNA isolation technique useful for
RAPD fingerprinting and other PCR applications. BioTech. 14:748-750.
Williams, J.G.K., A.K. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey. 1990. DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic
Acids Res. 18:6531-6535.