Plant Molecular Biology Reporter 19: 273af, 2001 2001 International Society for Plant Molecular Biology.

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DNA Isolation Methods for Medicinal and Aromatic Plants

ANNA MARIA PIRTTIL, MERJA HIRSIKORPI, TERTTU KMRINEN*, LAURA JAAKOLA and ANJA HOHTOLA
Department of Biology/Botany, University of Oulu, POB 3000, FIN-90014 Oulu, Finland Abstract. Several protocols described for plant DNA isolation fail to produce good quality DNA from medicinal herbs and aromatic plants. These plants contain exceptionally high amounts of secondary metabolites that interfere with DNA isolation. To address this problem, we developed 2 DNA isolation methods for sundew and tarragon that produce DNA suitable for molecular biological applications. One of the methods also is applicable for milfoil and Siberian ginseng. Key words: Achillea millefolium, Artemisia dracunculus, DNA isolation, Drosera rotundifolia, Eleutherococcus senticosus, herbs, secondary metabolites

Introduction Herbal and aromatic plants are attracting more attention among contemporary plant researchers because some human diseases resulting from bacterial antibiotic resistances have gained worldwide concern. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that have medicinal properties. Application of molecular technology would increase and facilitate production of these substances (Stckigt et al., 1995) and help save natural resources. However, the same substances that make the herbal and aromatic plants worthy of such intensive study also may hinder molecular approaches with these plants. Polyphenols and polysaccharides bind firmly to nucleic acids during DNA isolation and interfere with subsequent reactions. A number of methods are available and are being developed for the isolation of nucleic acids from plants. Because plants contain high amounts of many different substances, it is unlikely that just one nucleic acid isolation method suitable for all plants can ever exist (Loomis, 1974). We tested several DNA isolation methods on round-leaved sundew (Drosera rotundifolia L.) and tarragon (Artemisia dracunculus L.) (Bekesiova et al., 1999; Maass and Dalhoff, 1994; Mercado et al., 1999; Lodhi et al., 1994; Rogers and
*Author for correspondence. Present address: Botanical Gardens, POB 3000, FIN-90014 Oulu, Finland; e-mail: Terttu.Kamarainen@oulu.fi; fax: +358-8-553 1584; ph: +358-8-553 1544.

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Bendich, 1994; Sul and Korban, 1996). All these methods resulted in brown or yellow DNA precipitate that could not be reliably amplified by PCR. Therefore, we developed 2 new methods that produced good quality DNA from these sources. One of the methods also was applied successfully to Siberian ginseng (Eleutherococcus senticosus [Maxim.] Maxim.) and milfoil (Achillea millefolium L.), which are considered even more recalcitrant to DNA isolation than sundew and tarragon. Materials and Methods Plant material Leaves of greenhouse-grown Siberian ginseng (Eleutherococcus senticosus [Maxim.] Maxim.) and field-grown milfoil (Achillea millefolium L.) were harvested fresh before DNA isolation. Seedlings of sundew (Drosera rotundifolia L.) and tarragon (Artemisia dracunculus L.) were tissue cultured for 6 months on a half strength MS medium (Murashige and Skoog, 1962) supplemented with BAP (6-benzylamino purine) and NAA (-naphthaleneacetic acid). The seedlings were detached from the medium and used for DNA isolation. Reagents and solutions

1. 2.

Extraction buffer: 2% hexadecyltrimethyl-ammonium bromide (CTAB) (w/v), 100 mM Tris-HCl (pH 8), 20 mM EDTA (pH 8), 1.4 M NaCl; autoclave and add 2% polyvinylpyrrolidone (PVP) Mr 750,000 (Serva) (w/v) and 2% mercaptoethanol (v/v) immediately before use 8 M LiCl, autoclave Chloroform-isoamyl alcohol (IAA) (24:1 v/v) 3 M potassium acetate (pH 4.8) Isopropanol, -20C Absolute ethanol, -20C 70% ethanol -20C Distilled autoclaved water

Protocol 1 Mix 350 L of extraction buffer with 350 L of 8 M LiCl and prewarm to 65C. Grind the fresh plant tissue into a fine powder in liquid nitrogen using a mortar and pestle. Weigh 0.1 g of the powder in a microcentrifuge tube using a precooled spattle and add the prewarmed isolation buffer1. Vortex thoroughly and place the tube at 65C. Hold the tube at 65C for 10 min and vortex 3-4 times during the incubation. Add 700 L chloroform-IAA (24:1) and mix thoroughly. Centrifuge the tube at 13,000 rpm (13,793 g-force) for 5 min at room temperature. Transfer the upper phase into a new tube and extract again with 700 L chloroform-IAA, centrifuge.

3. 4. 5. 6.

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18.

Pipet the upper phase into a new tube. Add 0.5 vol (300 L) 3 M potassium acetate (pH 4.8), mix by inverting the tube carefully. Place the tube at -20C for 30 min. Centrifuge the tube at 13,000 rpm (13,793 g) for 10 min at 4C and pipet the supernatant into a new tube2. Add 0.6 vol (600 L) cold isopropanol and invert the tube carefully several times to mix the 2 layers3. Place the tube at -20C for 30 min. Centrifuge the tube at 13,000 rpm (13,793 g) for 10 min at 4C. Discard the supernatant and dry the pellet either at 60C or using a SpeedVac (Savant)4. Dissolve the pellet in 300 L distilled autoclaved water. Add 2 vol (600 L) cold absolute ethanol and place the tube at -20C for 1 h or -70C for 30 min. Centrifuge the tube at 13,000 rpm (13,793 g) for 10 min at 4C and wash the DNA pellet with cold 70% ethanol (300 L), centrifuge at 13,000 rpm (13,793 g) for 5 min at 4C. Dry the DNA pellet and dissolve in 10-50 L sterile distilled water. Check the quality and concentration of the DNA with a spectrometer and on a 1% agarose gel.

Protocol 2 1. 2. 3. Perform steps 1-6 of protocol 1. Pipet the upper phase into a new tube. Slowly add 1000 L of -20C ethanol on top of the liquid and place the tube at -20C for 10-20 min. Invert the tube carefully until a white DNA fiber appears between the 2 layers5. Transfer the lower colored layer carefully to a new tube using a narrow-tip pipet6. Add 500 L of sterile water into the ethanol-containing tube, mix carefully3, and leave at -20C for 1 h or at -70C for 30 min. Extract the colored layer again with ethanol as described in steps 2-3. Then remove the colored layer and discard. Centrifuge the ethanol-containing tubes at 13,000 rpm (13,793 g) for 10 min at 4C. Discard the supernatant and wash the DNA pellet with (-20C) 70% ethanol. Centrifuge the tubes at 13,000 rpm (13,793 g) for 5 min at 4C. Dry the DNA pellet and dissolve in 10-20 L sterile distilled water. Check the quality and concentration of the DNA with a spectrometer and on a 1% agarose gel.

4. 5.

6. 7. 8.

Notes: 1. The powder should not be allowed to thaw at any point before adding the isolation buffer. By keeping the temperature below 0C, the oxidizing enzymes are inactivated during this step. If larger amounts of plants are processed simultaneously, the powder can be stored in a -20C freezer. If precipitation is not observed, the centrifugation may be omitted.

2.

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Figure 1. Digestion and amplification of isolated DNA from herbs investigated. (A) DNA undigested (lanes 3, 5, 7, 9) and digested with BamH I (lanes 2, 4, 6, 8). (B) DNA amplified with universal primers that produce a fragment of chloroplast and mitochondrial 16S rDNA. (A and B) Lane 1: Lambda DNA/EcoR I + Hind III Marker 3 (MBI Fermentas); lanes 2, 3, and 11: tarragon; lanes 4, 5, and 12: Siberian ginseng; lanes 6, 7, and 13: milfoil; lanes 8, 9, and 14: sundew; lane 10: negative PCR control (no template).

3. 4. 5.

6.

The tube should not be shaken vigorously because the DNA is very vulnerable to fragmentation at this step. Care should be taken so that the pellet is not dried excessively, making it difficult to dissolve. Precipitation can be aided by stirring the liquid carefully with the narrow-tip pipet. Even if DNA fiber is not observed at this point, the procedure should be continued. The colored liquid should be removed totally. If any color is left in the bottom of the tube, the DNA will be colored during the subsequent centrifuging. Care should be taken not to remove the DNA from the tube along with the colored liquid.

Results and Discussion We successfully isolated DNA from sundew, tarragon, milfoil, and Siberian ginseng using the 2 protocols described above. High quality DNA was isolated from milfoil and Siberian ginseng using protocol 1, while protocol 2 failed to produce usable DNA from these sources. Protocol 2 was considered preferable for sundew, but the DNA yield was low. Both protocols worked well for tarragon. These 2 methods produced RNA-free DNA (Figure 1A) from 10-250 g per gram of fresh tissue from the plant sources. The highest amount of DNA was obtained from tarragon using protocol 1 (data not shown). When protocol 2 was used, some DNA likely remained in the colored layer, which was discarded after 2 ethanol extractions. Although we considered 2 extractions adequate, the extractions may be continued if a larger amount of DNA is required in protocol 2; however, in our experience, the quality of DNA decreases with the number of ethanol extractions performed. When the isolated DNA was digested with BamH I, a characteristic smear was observed on the agarose gel for all 4 species (Figure 1A). We also tested the isolated DNA by amplification in PCR and obtained the expected product of 1.5 kbp (Figure 1B). The isolated DNA had normal spectra in which the A260/A280 ratios were 1.6-1.7 (data not shown; Pich and Schubert, 1993).

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Using either of our 2 methods, colorless DNA from the 4 plant sources was obtained. With all other methods, the isolated DNA was dark brown or yellow and, for the most part, unusable. Our methods do not require complicated and long ultracentrifugations but are straightforward and can be performed within 5 hours. The methods are based on protocols described by Sul and Korban (1996) and Jobes et al. (1995). In both methods, the extraction buffer contains high amounts of PVP and -mercaptoethanol, which prevent oxidation of the secondary metabolites in the disrupted plant material. CTAB is used as a detergent in the extraction buffer to separate polysaccharides from DNA. Addition of lithium chloride in the extraction buffer eliminates the isolation of RNA during both procedures. In protocol 1, we found the combined use of potassium acetate and isopropanol during the precipitation of DNA efficient in removing most of the secondary metabolites and polysaccharides from the DNA. However, this resulted in precipitation of large quantities of potassium salt along with the DNA. Therefore, the pellet was resuspended in water and precipitated again with ethanol. Although we found isopropanol to work better in combination with potassium acetate, ethanol may be used also. Protocol 2 is based on an ethanol extraction where DNA precipitates between the poorly dissolved ethanol and water phases. The ethanol extraction is feasible because the high levels of polyphenols and polysaccharides remaining in the isolation buffer make the water phase heavy and less soluble in ethanol. As we discovered, this procedure may work better for particular plants. To our knowledge, this work represents a novel method that does not require ultracentrifugation to isolate DNA from a variety of herbal or aromatic plants. Because we found the methods described in this paper functional for plants that were otherwise recalcitrant to DNA isolation, we believe that these methods will be of help for molecular biological studies of many other aromatic and herbal plants. Acknowledgements This work was supported by Kone Foundation and the Foundation for Research of Natural Resources in Finland. References
Bekesiova I, Nap JP and Mlynarova L (1999) Isolation of high quality DNA and RNA from leaves of the carnivorous plant Drosera rotundifolia. Plant Mol Biol Reptr 17: 269-277. Jobes DV, Hurley DL and Thien LB (1995) Plant DNA isolation: a method to efficiently remove polyphenolics, polysaccharides, and RNA. Taxon 44: 379-386. Lodhi MA, Ye GN, Weeden NF and Reisch BI (1994) A simple and efficient method for DNA extraction from grapevine cultivars and vitis species. Plant Mol Biol Reptr 12: 613. Loomis WD (1974) Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Meth Enzymol 31: 528-545. Maass M and Dalhoff K (1994) Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR. J Clin Microbiol 32: 2616-2619.

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Mercado JA, el Mansouri I, Jimenez-Bermudez S, Pliego-Alfaro F and Quesada MA (1999) A convenient protocol for extraction and purification of DNA from Fragaria. In Vitro Cell Dev Biol Plant 35: 152-153. Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497. Pich U and Schubert I (1993) Miniprep method for isolation of DNA from plants with a high content of polyphenols. Nucleic Acids Res 21: 3328. Rogers SO and Bendich AJ (1994) Extraction of total cellular DNA from plants, algae and fungi. In: Gelvin SB and Schilperoort RA (eds), Plant Molecular Biology Manual. Second Edition. pp D1: 1-8, Kluwer Academic Publishers, Dordrect, The Netherlands. Stckigt J, Obitz P, Falkenhagen H, Lutterbach R and Endress S (1995) Natural products and enzymes from plant cell cultures. Plant Cell Tissue Organ Culture 43: 97-109. Sul IW and Korban SS (1996) A highly efficient method for isolating genomic DNA from plant tissues. Plant Tissue Culture Biotechnol 2: 113-116.