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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.