Plant Molecular Biology 5: 69-76, 1985 1985 Martinus Nijhoff Publishers, Dordrecht Printed in the Netherlands

Extraction of D N A from milligram amounts of fresh, herbarium and mummified plant tissues
Scott O. Rogers & Arnold J. Bendich l Departments of Botany and 1Botany and Genetics, University of Washington, Seattle, WA 98195, U.S.A.

Keywords: cetyltrimethylammonium bromide, CTAB

Summary

We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3-200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22-118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.

Introduction

Materials and methods

Most methods for extraction and purification of plant DNA have employed cesium chloride density gradients to eliminate enzyme-inhibiting polysaccharides [for example, 2, 14, 17]. These procedures are expensive and time-consuming since ultracentrifugation is required. Recent 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 [6, 18], do not provide details on yield [6] and are somewhat anecdotal [6]. We have developed a technique based on the CTAB (cetyltrimethylammonium bromide) nucleic acid extraction procedures of Murray and Thompson [14] and Taylor and Powell [17] which makes it possible to extract purified high molecular weight plant DNA without the use of expensive equipment a n d / o r time-consuming procedures. In most cases only three disposable microcentrifuge tubes are required for all operations from tissue homogenization to final restrictable DNA. 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.

Plant species used Various tissues from the following species were used: Allium cepa L., cv. White Globe (onion); Arabidopsis thaliana L. Schur.; Citrullus vulgaris L., cv. Dixie Queen (watermelon); Cucumis melo L., cv. Iroquois (muskmelon); Cucurbita pepo L., cv. Black Beauty (zucchini); Glycine max Merr., cv. Williams (soybean); Hordeum vulgare L., cv. Himalaya (barley); Linum usitatissimum L., cv. Tam F-201 (flax); Nicotiana tabacum L., cv. Burley 21 and cv. Xanthi NT-I (tobacco); Petunia hybrida Vilm.; Phaseolus vulgaris L., cv. Tender Green (bean); Pisum sativum L., cv. Alaska (pea); Triticum aestivum L., cv. CI13968 (wheat); Vicia faba L., cv. Broad Winsor (broad bean); Vicia villosa Roth. (hairy vetch); Vitis vinifera L. (grape); Zea mays L., leaves of cv. FR9 cms-CX FR37 and pollen of cv. Bantam (maize). Seeds were obtained commercially. Leaves, whole seedlings, pollen and suspension cultures were obtained locally. Samples of dried leaf tissue of Agropyronjunceum (Gramineae), Poa juncifolia (Gramineae), P. palustris, Triticum aestivum (Gramineae), and V. faba (Fabaceae) were obtained from the herbarium

70 collections of the University of Washington. The samples of Z. mays ssp. mays and the Teosinte/ Maize hybrid were obtained from the herbarium at the University of Wisconsin, Madison. Mummified seeds of Encelia virginensis (Asteraceae; 1210 +_ 80 years old), Eschscholtzia minutiflora (Papaveraceae; 500 + 60), Lycium shockleyi(Solanaceae; 1210 ___80), Juniperus osteosperma (Cupressaceae; 3460 + 150; 17 150 + 170 to 36 250_+ 910; >44 600), Opuntia ramosissirna (Cactaceae; 11 480 _+ 110) and Symphoricarpos sp. (Caprifolaceae; >44 600) were a gift from Dr W. Geoffrey Spaulding. They were obtained from packrat middens of 500 to greater than 44 600 years old (radiocarbon dated). (For a description of packrat middens and the process of mummification see [16].) MW 40 000] and 1X CTAB extraction buffer were added according to the proportions given in Table 1. The mixture was then heated in a 65 C water bath for 1 3 min. If a mortar and pestle was used, the mixture was transferred to a microfuge tube using a rubber policeman. If the tissue weighed less than about 50 mg, or the genome was small, 25-50 gg of yeast t R N A was added to aid in nucleic acid precipitation. An equal volume of chloroform/ isoamyl alcohol (24:1) was added. After gentle but thorough mixing, the tube was centrifuged at 11 000 g for 30 s. The top phase was transferred to a new microfuge tube and 1/ 10 volume of 65 C 10% CTAB [10% CTAB (w/v), 0.7 M NaC1] was added. A second chloroform/isoamyl alcohol extraction and centrifugation was performed. The top phase was transferred to a new tube. One volume of CTAB precipitation buffer [1% CTAB, 50 mM Tris (pH 8.0), 10 mM E D T A (pH 8.0)] was added, followed by gentle mixing. The tube was then centrifuged for 10 s (for fibrous precipitates) to 60 s (for fine precipitates). The pellet was rehydrated in high salt TE buffer [10 mM Tris (pH 8.0), 1 mM E D T A (pH 8.0), 1 M NaC1]. Heating to 65 C for5-10 min facilitated rehydration of some pellets. After complete rehydration, the nucleic acids were reprecipitated with two volumes of ethanol. The nucleic acids were then pelleted by 1 rain of centrifugation, washed with 80% ethanol and dried in a dessicator. Finally, the nucleic acids pellet was rehydrated in a

Nucleic acid extraction

The tissue was weighed and ground into a fine powder in dry ice in the appropriate vessel (see Table 1). When grinding in microfuge tubes, 3 m m glass or stainless steel rods, filed at one end so they fit into the bottom of the tubes, were used as pestles. As grinding proceeded, the vessel was slowly warmed in order to evaporate the dry ice. I m m e diately after all of the dry ice had evaporated, 65 C 2X CTAB extraction buffer [2% CTAB (w/v), 100 mM Tris (pH 8.0), 20 mM E D T A (pH 8.0), 1.4M NaC1, 1% PVP (polyvinylpyrrolidone),
Table 1. Buffer/tissue proportions and vessel choices.

Weight of tissue 2X CTAB buffer 1X CTAB buffer

<10 mg 1 #1/mg bring to 20 t~l tot vol (incl tissue)

10--50 mg 1 #1/mg a) None b) 1 ~1/mg c) 2 ~zl/mg 0.5 ml or 1.5mlMT 0.5 ml MT

50-500 mg 1 #1/mg a) None b) 1 #1/mg c) 2/~1/mg 1.5 ml MT orM&P 0.5 ml or 1.5 ml MT

>500 mg 1 ~zl/mg a) None b) 1 tzl/mg c) 2 ~1/mg M &P

Grinding vessel

0.5 ml MT

Vessel for further manipulations

0.5 ml MT

CT

To use this Table find the appropriate column for the weight of the tissue. Note that the amount of I X CTAB buffer added depends on the tissue type: (a) leaves, roots, shoots, seedlings and tissue culture cells; (b) diploid cotyledons; (c) polyploid cotyledons (such as those found in legumes), embryos, endosperm, whole seeds, whole grains and pollen. For example, for a polyploid cotyledon weighing 30 rag, the tissue would be ground with dry ice in a 0.5 or 1.5 ml microfuge tube, followed by addition of 30 #1 of2X CTAB buffer and 60 #1 of 1X CTAB buffer. For a sample weighing 3 mg, the tissue would be ground in a 0.5 ml microfuge tube, followed by addition of 3 #1 of 2X CTAB buffer and 14 t~l of 1X CTAB buffer. For dried specimens, add 5 tzl of 1X CTAB buffer per mg of tissue and no 2X CTAB buffer. Vessel abbreviations: MT = microfuge tube; M & P = mortar and pestle; CT-= centrifuge tube.

71 small volume of 0.1 T E buffer [1 mM Tris (pH 8.0), 0.1 mM E D T A (pH 8.0)]. of drying and the developmental stage of the plant at the time of drying. The D N A obtained from herbarium specimens (Table3) averaged about 400base pairs overall. However, sizes up to 20-30 kb were found in all of the samples. D N A from mummified seeds and embryos averaged from 0.2-7 kb, and ranged as high as 20-30 kb. There was a general trend toward D N A degradation with age of the mummified specimens, hut more data are needed to confirm this. After l h of DNase I treatment, all of the D N A in the treated samples was completely digested. Yields of D N A ranged from a low of 0.3 n g / m g of tissue to 200 ng/rag. The most consistently high yields were obtained from leaves, embryos and polyploid cotyledons (found in Pisum sativum [4] and Viciafaba [5]). The only exception to this was with the leaves of Vitis vinifera. These leaves were senescing, which may explain the low yield. The yield from Vicia faba pollen was by far the highest. F. faba has a large genome and its pollen grains are easily broken which probably accounts for the high yield. The yield from whole seeds and whole grains tends to be inconsistent from species to species. Of the 40 types of fresh tissues (from 19 species) and 17 types of dried and mummified tissues (from 13 species) none failed to yield measurable amounts of DNA. The D N A from endosperm and diploid cotyledons (both starch storage tissues) was difficult to extract. Also, some contamination of the D N A (possibly polysaccharides) was always seen with these tissues (as well as with D N A from the polyploid cotyledons of V. faba and Pisum sativum). While this caused some difficulties in rehydration of the DNA, it did not inhibit E c o R I digestion. Further purification can be obtained by rehydration in high salt TE (see Materials and methods), a 30 s centrifugation and reprecipitation of the supernatant. The cotyledons of zucchini (Cucurbita pepo) yielded D N A but it proved to be useless. Upon CTAB precipitation of the nucleic acids, another substance (polysaccharide a n d / o r lipid) also precipitated making rehydration of the D N A nearly impossible. Zucchini embryos, however, gave good yields of D N A as long as care was taken to exclude most of the cotyledons. The smallest amount of tissue that yielded a measurable a m o u n t of D N A was obtained from individual V. faba ovules, weighing as little as

Determination of DNA yields and susceptibility to restriction enzymes

All nucleic acid preparations from herbarium and mummified specimens (as well as some from fresh tissues) were treated with ribonucleases A ( 1 0 0 # g / m l ) and Yl (10 units/ml) for 30 min at 37 C. Parts of some preparations were also treated with DNase I (100 units/ml) for 1 h at 37 C. A sample of each nucleic acid solution was subjected to electrophoresis on 0.5 or 0.6% (for fresh tissue) or 1.0% or 1.5% (for herbarium and mummified specimens) agarose gels and compared to a standard a m o u n t of bacteriophage lambda D N A by densitometry of the fluorogram negative. D N A length was estimated by comparison to intact lambda D N A and a set of size markers ('1 kb ladder' from BRL); for some herbarium and mummified specimens a H a e I I I digest of lambda D N A was also used. For assay of the V.faba ovules, the extracted D N A was digested with EcoRI, subjected to electrophoresis on a 0.6% agarose gel and blotted onto Gene Screen Plus (NEN). Standard amounts of the unlabelled plasmid were also included on the blot. The blot was then hybridized to a 32p-labeled plasmid (pBD4) containing the yeast (Saccharomyces cerevisiae) ribosomal R N A genes [1] at 30 C below the thermal denaturation point. The autoradiogram of this blot was then analyzed by inspection. For E c o R I and B a m H I assays, 1 #g of the D N A was incubated overnight with 5 units of the enzyme in the appropriate buffer. The sample was then examined by gel electrophoresis.

Results

The results are summarized in Tables 2 and 3. Prominent 25S and 18S ribosomal R N A bands were seen in some of the preparations. Most of the D N A for each species was 40-70 kb in length. Exceptions to this were seen in the D N A extracted from the herbarium and mummified samples. The extent of D N A degradation for the herbarium specimens appeared to be related to the condition of the leaf rather than the year in which it was dried. The condition of the leaf would depend on the method

72
Table 2. DNA yields from fresh plant tissues. Tissue Species Genome (pg/1 C) 3.9 2.0 15.7 15.7 15.7 13.2 4.0 4.0 0.08 3.9 16.5 1.1 0.9 5.5 0.7 1.8 5.0 15.7 4.0 1.1 1.0 1.3 0.9 5.5 1.8 5.0 15.7 13.2 2.0 4.0 1.1 5.0 13.2 5.5 15.7 4.0 Weight (mg) 500 138 86 74 52 500 500 500 39.0 28.5 600 6.0 1O0 400 68.0 5.0 950 450 63.0 340 3.8 1.5 6.0 11.2 3.5 10.0 10.4 4.5 30.0 1.0 88.0 61.5 258 180 57.0 74.5 125 100 250 24.0 DNA (ng) 19 000 1 900 3 900 3 600 2 650 20 000 210 20 000 1 900 230 6 100 330 1O0 2 200 650 140 2 I00 8 200 1 400 6 100 160 20 83 620 85 330 600 67 2 300 40 2 900 20 11 000 7 300 240 340 130 2 000 50 000 59 Yield (ng/mg) 38 14 45 48 52 40 0.4 40 49 8.0 10 55 1.0 5.5 9.6 28 2.2 18 22 18 42 13 14 55 24 33 58 15 77 40 33 0.3 43 41 4.6 4.2 1.0 20 200 2.5 Restrictability a E

Leaves

Nicotiana tabacurn (young) Petunia hybrida (young) Triticum aestivum (flag) Triticum aestivum (mature) Triticum aestivum (young) Viciafaba (young) Vitis vinifera (senescing) Zea mays (mature) Zea mays (seedling) Arabidopsis thaliana Nicotiana tabacurn (NT-I) A llium cepa CitruHus vulgaris Glycine max Hordeurn vulgate Linum usitatissimum Phaseolus vulgaris Pisum sativum Triticum aestivum Zea mays Citrullus vulgaris Cucumis melo Cucurbita pepo Glycine max Hordeum vulgare Phaseolus vulgaris Pisum sativum Triticum aestivum Viciafaba Vicia villosa Zea mays Citrullus vulgaris Pisum sativum Viciafaba Hordeurn vulgare Triticum aestivum Zea mays Cedrus deodara Viciafaba Zea mays

E E, B E E

Whole seedling Suspension culture Whole seeds/grains

E E

E E

E E E

Embryos and embryo axes

E E E E E E E, B E

Cotyledons

E E, B E E E

Endosperm

Pollen

13.2 4.0

Yields are given in nanograms of DNA per milligram of starting tissue. All tissues were measured as fresh weights. Extractions were performed on individual plants or parts of individual plants, except in the case of pollen and suspension cells. For example, the data given for embryos are all for single embryos. The data for cotyledons are for single cotyledons or portions of a single cotyledon. Haploid nuclear genome size given for Arabidopsis thaliana is from [12] and for Glycine max is from [7]. The value given for Citrullus vulgaris represents the mean of the range of 0.7-1.5 pg for seven cucurbit species [ 10]. The remaining values are from [3]. No data are available for Vitis vinifera or Cedrus deodara, but the latter probably has a large genome since the range for closely related gymnosperms is from 60-140 pg [15]. a Samples were tested with EcoRl (E) or BamHl (B). No inhibition of either enzyme was evident.

73
Table 3. DNA yields from herbarium and mummified plant tissues.
Tissue Species Agea (yrs) 118 93 93 24 69 22 65 27 23 1 200 500 1 200 3 500 27 000 >45 000 11 000 >45 000 Weight (mg) 62 100 53 64 31 38 25 5.6 57 36 8.3 5.0 7.0 8.0 8.5 2.2 5.6 DNA (ng) 1 350 I 660 1 200 560 1 500 3 200 225 145 1 460 I 200 316 240 40 20 10 15 8 Yield (ng/mg) 22 17 23 9 49 84 9 26 26 33 38 48 6 3 1 7 1 Length (kb) max avg 20 30 30 30 20 20 20 30 30 1.5 20 30 10 10 10 20 4 0.1 0.2 0.3 0.4 0.2 0.7 0.5 l 0.3 0.2 7 4 7 5 3 0.5 0.5 Restrictability b

Herbarium leaves

Agropyronjunceum (1867) Poa juncifolia (1892) Poa palustris (1892)

Teosinte/Maize hybrid ( 1961 ) Triticum aestivum (1916) Triticum aestivum (1963) Viciafaba (1920) Viciafaba (1958) Zea mt~vs (1962) c

Mummified seeds/embryos

Encelia virginensis Eschscholtzia minutiflora" Lycium shockleyi c Juniperus osteosperma Juniperus osteosperma Juniperus osteosperma Opuntia ramosissima Symphoricarpos sp.

Yields are in nanograms of DNA per milligram of tissue. Dates of mounting of the herbarium specimens are in parentheses. The maximum and average lengths in kb are given. The amounts of tissue used were: l seed of E. virginensis, l0 seeds of E. minutiflora, 1 embryo of L. shockleyi, 2 embryos of O. ramosissima, 4 embryos each of the three samples of J. osteosperma, and 5 embryos of

Symphoricarpos.
a Ages for the mummified specimens are based on radiocarbon analyses. Precise data on age is given in Materials and methods section. b Samples were tested with EcoRI (E). No inhibition of this enzyme was evident. c A portion of this DNA was completely digested with DNase I.

0.2 mg. Yield in this case was estimated by blotting and hybridization with a ribosomal D N A probe ( r D N A makes up about 0.1% of the genome of V. faba [13]; genome size = 13.2 p g / I C ) . About 10 ng of D N A was obtained from these single ovules, with an average yield of 35 ng/mg. With such small pieces of tissue, carrier t R N A was a necessity in order to aid in precipitation of the small amounts of DNA. In general, if the tissue weighed less than 50 rag, 25-50 #g of carrier t R N A was added. Overnight soaking in water facilitated the grinding of dry and hard tissues (although dry tissues were used occasionally). For large pieces of hard tissue (such as Viciafaba cotyledons) grinding with dry ice in a small coffee mill achieved better breakage than in a mortar and pestle. More complete breakage was evident with a mortar and pestle than a microfuge tube, but transfer of the homogenate from the mortar was difficult for volumes belbw 50 #1. A rubber policeman proved useful for this transfer. In general, tissues weighing less than 50 mg (except pollen or other tough tissues) should be ground in microfuge tubes for highest yields.

Before the 10% CTAB or Precipitation Buffer solutions are added, the volume of the extract must be measured accurately. This can be accomplished by using a micropipetter (such as a Pipetman). If care is not taken in this measurement, yield may be adversely affected. By overestimation or underestimation either incomplete precipitation may occur or contaminants will be precipitated with the DNA. The 10% CTAB solution is quite viscous at room temperature, making pipetting difficult. Warming to 65 C will decrease the viscosity somewhat, but measurements below 50 #1 are difficult. Positive displacement pipetters (such as a Microman) are useful for this purpose. An alternative is to use one-fifth volume of a 5% CTAB solution (again at 65 C) instead of the one-tenth volume of 10% CTAB. P V P in the extraction buffer was necessary only for some leaf tissue (such as V. faba) to inhibit browning of the extract. When PVP was not used, the D N A precipitate was occasionally brown to black. The coloration did not seem to inhibit digestion by EcoRI, but other enzymes were not tested.

74 As stated above, we routinely obtained D N A 40 70 kb with little precaution to prevent shearing. If longer D N A is sought, however, some precautions must be taken. When D N A is in solution, it is vulnerable to shearing. Use of wide bore pipettes and minimum agitation will cut down on this breakage (see Freifelder [8] for a more thorough discussion). Because CTAB is a strong detergent it releases D N A from membranes and proteins. Upon reduction of the salt concentration to below about 0.5 M (at room temperature) CTAB forms insoluble complexes with nucleic acids [17]. Enzyme-inhibiting polysaccharides do not precipitate at this point and are discarded in the supernatant. The CTAB must subsequently be removed, since the D N A / C T A B complex is only soluble in high salt. This is the reason for the rehydration in high salt TE and the subsequent precipitation with ethanol. The CTAB is soluble in the 80% ethanol and is easily removed. After drying and rehydration in 0.1X TE the D N A is free of nucleases, inhibiting polysaccharides and CTAB. In no case tested was inhibition observed for restriction enzymes EcoRI or B a m H I (see Tables 2 and 3).

Discussion

The major aim of this study was to develop a D N A isolation method for milligram amounts of plant tissue. N a n o g r a m and microgram amounts of D N A were thus obtained. Such quantities of D N A are generally sufficient to analyze picogram amounts of gene sequences given the sensitivity of contemporary nucleic acid hybridization assays. Figure l allows determination of the minimum amount of D N A (Fig. la) and tissue (Fig. lb) necessary to be able to detect a gene. Several pieces of information are needed for a prediction: genome size, gene length, copy number, detection limit and yield. In most cases milligram (and sometimes microgram amounts of tissue are required.

-3

06

-3

'

-7 I8
-2

I
0 1 2 3 4 -8

-1

-2
Io K Nanograms DNA Needed

I -1

I 0

I 1

Io s Milligrams Tissue Needed (1 PK detection limit)

Fig. lb. A m o u n t of tissue needed based on yield and fraction of the g e n o m e t h a t the gene (or gene set) comprises. [The g r a p h is set at the 1 pg limit of detection. F o r use at other detection limits, divide the tissue yield by the d e t e c t i o n limit and find that yield n u m b e r on the graph.] The a m o u n t of tissue needed = ( a m o u n t of D N A required)/(yield). To e x t e n t the e x a m p l e in Fig. la: for y o u n g maize leaves (yield - 49 n g / m g ) , a m i n i m u m of 55 mg of tissue w o u l d be required to o b t a i n the 2.7/.tg of D N A necessary to detect the single copy gene.

Fig. la. A m o u n t of D N A needed based on d e t e c t i o n limit a n d the f r a c t i o n of the g e n o m e t h a t the gene (or gene set) comprises. F r a c t i o n of g e n o m e ~ (gene length) X ( n u m b e r of c o p i e s ) / ( g e n o m e size). F o r e x a m p l e , a single c o p y gene of length 1.5 k b in Zea rnays ( g e n o m e size = 4.0 pg, e q u i v a l e n t to 4 x 106 kb) w o u l d c o m p r i s e 3.75 X 10 7 of the genome. The a m o u n t of D N A req u i r e d = ( d e t e c t i o n l i m i t ) / ( f r a c t i o n of genome). Therefore, at a d e t e c t i o n limit of 1 pg, a m i n i m u m of 2.7 # g of D N A w o u l d be required to detect the gene.

75 This D N A extraction method offers two advantages over previous methods. First, as little as a few milligrams of tissue (or less in some cases) can be used. This makes practical the non-destructive sampling of individual plants, including individual embryos. For example, we have extracted D N A from the leaves, shoots, roots, whole flowers, petals, ovaries, anthers, ovules, pods, embryos and cotyledons from individual Vicia faba plants with a minimum of damage to the plants. The D N A from 0.2-25 mg of each tissue was sufficient to assay the EcoRI ribosomal D N A pattern of blot hybridization (our unpublished results). In addition, parts of embryos and seeds, such as cotyledons, plumules, scutella, endosperm and coleoptiles may be excised and used without sacrificing the entire embryo, seed or grain. The second advantage is that the method is inexpensive, both in terms of equipment and time. Methods that employ CsC1 density gradients require ultracentrifuges and 1 or 2 days ofcentrifugation. In addition, both detection and recovery of small amounts of D N A are difficult with such gradients. One microgram of bacterial D N A is barely detectable as a fluorescent band in CsC1 gradients containing ethidium bromide (our unpublished observations). Thus, for n a n o g r a m amounts of D N A the CsCI method is not convenient. While the minipreparation method of Dellaporta et al. [6] does not require CsC1 gradients, it employs much more tissue and 30 ml glass centrifuge tubes. With our technique 2-3 dozen samples can be processed in 1 day by one person using microfuge tubes exclusively. We attempted to identify the factors that determine D N A yield. Genome size and ploidy are obviously important, but yield could not always be predicted by these two factors alone. We next tried various combinations of genome size, ploidy, estimated cell size and tissue weight, but the correlations were weak. A general pattern did emerge, however. Embryos and embryo axes (excluding cotyledons) gave the highest D N A yields, followed by leaves. Starch storage tissues (cotyledons and endosperm) gave the lowest yields, with the exception of polyploid cotyledons. Whole seeds and grains were, as expected, intermediate between storage organs and embryos. Species-to-species variability in cell size for corresponding tissues would alter the n u m b e r of genome copies per milligram of tissue, and thus affect the yield. Additional factors could also affect the yield. In tissues with a high starch content, some trapping of the D N A may occur. This appeared to be the case in cotyledons and endosperm. In certain species the CTAB and E D T A may not be sufficient to completely inactivate nucleases. An EDTA-stimulated DNase exists in the shoots of wheat [11]. When compared to maize, the yield for wheat was greater in endosperm, about equal in whole grains and lower in embryos (Table 2). The genome of wheat is four times that of maize. Another example of unexplained yield variability occurred with cucurbit embryos, which all have approximately equal haploid genome sizes (Table 2). The embryos of two of them (muskmelon and zucchini) gave very different yields from that of a third (watermelon). While genome size, ploidy and cell size are all undoubtably factors that influence D N A yields, other as yet unexplained factors are evidently important. The extraction, cloning and sequencing of D N A from a 140 year old sample of dried muscle tissue from the extinct quagga, a zebra-like animal [9], prompted our attempts with herbarium specimens and mummified tissue. The mean size of quagga D N A was about 500 base pairs. The D N A obtained from herbarium specimens (Table3) averaged about 400 base pairs. However, sizes up to 20-30 kb were found in all of the samples. D N A was also obtained from mummified embryos contained in packrat middens 500-45 000 years old (see [16] for a description of packrat middens). While the D N A from all of the samples was degraded, some of the samples contained high molecular weight DNA. Thus, it may be possible to digest these DNAs with restriction enzymes and clone the fragments for further study. Small pieces of herbarium and mummified specimens can be used to obtain D N A for evolutionary studies. It is also possible that pieces of plant material recovered from archaeological excavation sites would prove useful in assessing the effects of agricultural practices on the evolution of certain genes in crop plants such as wheat and maize. We have shown that this micropreparation method for D N A is fast, reliable, easy to perform and can be used with a broad range of monocot and dicot species, as well as with gymnosperms. It should prove useful whenever D N A is needed from a small amount of tissue, from a large number of individuals, from various parts of the same plant, or from dried and mummified plants.

76

Acknowledgement
This work was supported by a grant from the National Science Foundation. SOR was supported by an ARCO Fellowship.

References
1. Bell GI, DeGennaro LJ, Gelfand DH, Bishop R J, Valenzuela P, Rutter W J: Ribosomal RNA genes of Saccharomyces cerevisiae. J Biol Chem 252:8118 8125, 1977. 2. Bendich A J, Anderson RS, Ward BL: Plant DNA: long, pure and simple. In: Leaver CJ (ed) Genome organization and expression. Plenum Press, New York, 1980, pp 31-33. 3. Bennett MD, Smith JB: Nuclear DNA amounts in angiosperms. Phil Trans Royal Soc London, ser B, 274:227-274, 1976. 4. Cullis CA; Chromatin-bound DNA-dependent RNA polymerase in developing pea cotyledons. Planta (Berl) 131: 293-298, 1976. 5. Davies RD: DNA contents and cell number in relation to seed size in the genus Vicia. Heredity 39:153-163, 1977. 6. Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version 11. Plant Mol Biol Rep 1:19 21, 1983. 7. Doerschug EB, Miksche JP, Palmer RG: DNA content, ribosomal-RNA gene number, and protein content in soybeans. Can J Genet Cytol 20:531 538, 1978. 8. Freifelder D: Physical biochemistry. Applications to biochemistry and molecular biology (2nd edn). W. H. Freeman and Company, San Francisco, 1982, p 686. 9. Higuchi R, Bowman B, Freiberger M, Ryder OA, Wilson AC: DNA sequences from the quagga, an extinct member of the horse family. Nature 312:282-284, 1984.

10. Ingle J, Timmis J, Sinclair J: The relationship between satellite DNA, rRNA, gene redundancy and genome size in plants. Plant Physiol 55:496-501, 1975. 11. Jones MC, Boffey SA: Deoxyribonuclease activities of wheat seedlings. FEBS Lett 174:215-218, 1984. 12. Leutweiter LS, Hough-Evans BR, Meyerowitz EM: The DNA of Arabidopsis thaliana. Mol Gen Genet 194:15 23, 1984. 13. Maher EP, Fox DP: Multiplicity of ribosomal RNA genes in Vicia species with different nuclear DNA contents. Nature New Biol 245:170 172, 1973. 14. Murray HG, Thompson WF: Rapid isolation of high molecular weight DNA. Nucleic Acids Res 8:4321-4325, 1980. 15. Sparrow AH, Price H J, Underbrink AG: A survey of DNA content per cell and per chromosome of prokaryotic and eukaryotic organisms: some evolutionary considerations. In: Smith HH (ed) Evolution of genetic systems. Gordon and Breach, New York, 1972, pp 451 494. 16. Spaulding WG, Leopold EB, Van Devender TR: Late Wisconsin paleoecology of the American Southwest. In: Wright HE Jr (ed) Late-quaternary environments of the United States, Vol l, The late pleistocene, Stephen Porter (ed) University of Minnesota Press, Minneapolis, 1983, pp 263 265, 266 276. 17. Taylor B, Powell A: Isolation of plant DNA and RNA. Focus 4:4-6, 1982. 18. Zimmer EA, Newton KJ: A simple method for the isolation of high molecular weight DNA from individual maize seedlings and tissues. In: Sheridan WF (ed) Maize for biological research. University Press, University of North Dakota, Grand Forks, ND, 1982.

Received 5 February 1985; in revised form6 May 1985; accepted 13 May 1985.

Note added in proof

F o r s o m e t i s s u e s m o r e e x t r a c t i o n b u f f e r m a y b e n e e d e d t h a n is s t a t e d in T a b l e 1. F o r e x a m p l e , s o m e g r a s s l e a v e s a r e q u i t e d r y a n d r e q u i r e a d d i t i o n a l 1 X C T A B b u f f e r f o r e f f e c t i v e D N A e x t r a c t i o n . If less l i q u i d is r e c o v e r e d i n t h e f i r s t c h l o r o f o r m s u p e r n a t a n t t h a n w a s a d d e d as e x t r a c t i o n b u f f e r , m o r e 1 X C T A B b u f f e r should be added, the tube remixed and recentrifuged. M o r e t h o r o u g h t i s s u e g r i n d i n g m a y b e a c h i e v e d w i t h a m o r t a r a n d p e s t l e t h a n in a m i c r o f u g e t u b e . S m a l l a m o u n t s o f t i s s u e ( 1 0 m g o r less) m a y b e g r o u n d i n a c h i l l e d m o r t a r a n d e f f i c i e n t l y t r a n s f e r r e d t o a m i c r o f u g e t u b e u s i n g p o w d e r e d d r y ice as c a r r i e r f o r t h e p o w d e r e d tissue.