A Simple and Sensitive DNA Assay for Plant Extracts - NCBI

Plant Physiol. (1982) 70, 999-1003 0032-0889/82/70/0999/05/$00.50/0

A Simple and Sensitive DNA Assay for Plant Extracts' Received for publication February 23, 1982 and in revised form April 13, 1982

GIANNI R. BAER2, STEVEN P. MEYERS, WILLIAM T. MOLIN, AND LARRY E. SCHRADER Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4',6'-diamidino-2phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCI to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.

Expressing enzyme activities per cell rather than per unit weight, Chl, or protein is extremely important in some areas of biologic research. The simplest and most accurate method of calculating the cell number in most higher plant tissues is by determining the amount of DNA per cell and per tissue preparation. Most available methods for the quantitation of DNA require considerable manipulation of the samples, which may result in a loss of DNA. Such methods are time-consuming and not very sensitive. The commonly used diphenylamine method (4) suffers from these limitations. The fluorescent dye DAPI3 complexes with DNA to give a product with fluorescence intensity several times greater than the dye alone. Using this property, Kapuscinski and Skoczylas (8) have shown that the specificity and sensitivity of this reaction could be used for a sensitive DNA assay. The method permitted the estimation of concentrations of DNA as low as 0.5 ng/ml, even in the presence of a 20-fold excess of RNA. They showed that the fluorescence intensity is highest when DAPI is complexed with native, highly polymerized DNA as compared to degraded or denatured DNA. Based on these findings, Brunk et al. (3) developed a simple and sensitive method for the quantitative assay of DNA in crude homogenates of microorganisms and animal tissues. This paper presents an adaptation of this method for plant extracts. Special emphasis is given to the optimum conditions for preparation of the extract. ' Supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison, and by USDA Competitive Research Grant 59010410-9-0361-0. 2 Supported I year by the Swiss National Science Foundation. 3Abbreviation: DAPI, 4',6'-diamidino-2-phenylindole.

MATERIALS AND METHODS Plant Material. Maize (Zea mays L.) plants (F1 hybrid W64A x W1 82E) were grown in a growth chamber or greenhouse in a mixture of peat moss and vermiculite (1:1, v/v) and irrigated with a nutrient solution (6). Alfalfa (Medicago sativa L.) genotypes were derived and grown as described (12). Reagents. DAPI was purchased from Accurate Chemical and Scientific Corporation, Hicksville, NY. Calf thymus DNA (type I), RNA, histones, DNase, and ribonuclease A were purchased from Sigma. Homogenization of Leaf Tissue. One g chopped leaves was homogenized in 10 or 25 ml homogenization buffer (0.1 or 2.0 M NaCl, 10 mm EDTA, 10 mm Tris [pH 7.0]) for I min at 48,000 rpm in a VirTis 60K homogenizer. Where indicated, the homogenization buffer contained 1% (w/v) sarkosyl or 5 mM MgCl2 and no EDTA. Extract of Protoplasts. The method for the preparation of alfalfa protoplasts is described elsewhere (11). An aliquot (0.2 ml) of suspension containing approximately one million protoplasts/ ml was centrifuged at 60g for 10 min. The supernatant was discarded, and the pellet was stored at - 15°C. The thawed pellet was suspended in 0.3 ml buffer (2.0 M NaCl, 10 mM EDTA, 10 mM Tris [pH 7.0]). Where indicated, sonication was performed for 15 s or 1% (w/v) sarkosyl was added to the suspension buffer. Chloroform Treatment. Chloroform (1.5 volumes) was added to the homogenate and vigorously mixed with a Vortex mixer. The phases were separated by centrifugation at l,000g for 10 min, and the aqueous supernatant was used for fluorimetric measurements. No loss of DNA resulted from chloroform treatment of homogenates or calf thymus DNA solution (data not shown). Fluorimetric Measurements. Fluorescence was measured with a Perkin-Elmer 650-1OS fluorescence spectrophotometer with the excitation and emission wavelengths set at 350 nm and 450 nm, respectively. A stock solution of DAPI (1 mg/100 ml H20) was prepared and could be stored at 4°C for several months. DAPI was used at final concentrations from 30 to 100 ng/ml buffer (0.1 M NaCl, 10 mm EDTA, 10 mm Tris [pH 7.0]). DNA in the homogenates was quantitated by either of two procedures, both using an internal standard. Calf thymus DNA (20-25 ,ig/ml buffer as above) was used as the internal standard. Procedure A after Brunk et al. (3) involved successive addition and mixing of four aliquots (usually 10 ,ul each) of homogenate and four aliquots of DNA standard solution into a single cuvette containing 3 ml of DAPI solution. Fluorescence was measured at the beginning of the assay and after addition of each aliquot. When fluorescence units were plotted against the cumulative volume of aliquots, two straight lines were obtained (one for homogenate and one for DNA standard) and their slopes were calculated (see Fig. 2). Procedure B consisted of two paired sets of measurements. One set contained 3 ml DAPI solution, 25 ,ul homogenate and 25 ul buffer (0.1 M NaCl, 10 mm EDTA, 10 mm Tris [pH 7.01). The other set contained 3 ml DAPI solution, 25 ,ul of the same homogenate and 25 ,ul of DNA standard solution. Assays were

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performed in triplicate. Fluorescence was measured against a blank that contained 3 ml of DAPI solution, 25 ,ul each of a 2.0 M NaCl and 0.1 M NaCl homogenization buffers. For both procedures, the concentration of DNA in the homogenate was calculated by multiplying the concentration of DNA in the standard solution by the ratio of the increase in fluorescence for the homogenate to the increase in fluorescence for the standard DNA solution. For procedure A, the ratio of the slopes was used

(Fig. 2).

Diphenylamine Assay. The assay described earlier (5) was used with slight modifications: 10 ml of methanol:ethanol:Triton X100:3 N HCI (40:60:1:1) were added to centrifuge tubes containing 2 ml of homogenate. After mixing with a Vortex mixer, tubes were centrifuged at l0,OOOg for 10 min. The supernatant was discarded and the procedure repeated three times. Nine ml of 0.3 N NaOH were added to each tube and the tubes were heated at 450C for 15 min. After cooling in ice-water, 1 ml of chilled HC104 (A.C.S., 60%1o) was added to each tube. Tube contents were mixed with a Vortex mixer and centrifuged at lO,OOOg for 10 min. Supernatants were decanted and 4 ml 1.05 M HC104 were added to each tube. To establish a standard curve, tubes containing 0 to 400 g calf thymus DNA in a total volume of 4 ml 1.05 M HC104 were prepared. All tubes were heated at 90°C to 950C for 20 min, then cooled and centrifuged at l0,OOOg for 10 min. Two ml of supernatant were pipetted into clean test tubes and 2 ml freshly prepared diphenylamine reagent (4) were added to each tube. After 24 h of color development, A at 600 nm was read against the blank. RESULTS AND DISCUSSION Preparation of the Homogenate. Fluorescence of the DAPIDNA complex depends on the structure of the DNA. Kapuscinski and Skoczylas (8) have shown that treatments that shear or degrade DNA (e.g. long sonication, boiling) reduce fluorescence intensity of the DAPI-DNA complex. However, we found no such effects when homogenates were sonicated for a 15-s period (data not shown). The same observation was reported earlier (3). High speed homogenization used to disrupt plant material may also degrade DNA, hence reducing fluorescence intensity of the DAPI-DNA complex. To test this possibility, a solution containing calf thymus DNA was homogenized under conditions identical to those described for the homogenization of leaf tissue. This treatment resulted in a loss of only 3.4% of the fluorescence enhancement capacity of the DNA. Complete recovery of a known quantity of calf thymus DNA added to the homogenization buffer was observed (data not shown). The same Chl to DNA ratio was found in leaves and protoplasts of alfalfa (10, 11). Because protoplast lysates were not homogenized, these data also suggest that shearing of DNA which may occur during homogenization did not significantly decrease the fluorescence intensity of the DAPI-DNA

complex. Effects of filtration, centrifugation, NaCl concentration, and addition of detergent on extraction of DNA are shown in Table I. The presence of 2.0 M NaCl in the homogenization buffer was necessary to obtain a maximal yield of DNA. Yield of DNA was not increased when NaCl concentration was 3 M rather than 2 M (data not shown). Homogenizing the leaves in a buffer containing 0.1 M NaCl rather than 2.0 M resulted in variable and lower yield of DNA. Post homogenization adjustment of the NaCl concentration from 0.1 to 2.0 M resulted in a yield of DNA nearly equivalent to that obtained with 2.0 M NaCl in the homogenization medium. The concentration of NaCl in calf thymus DNA containing buffer did not affect fluorescence enhancement of DAPI (Table III). This is because DNA containing solutions were diluted over 50-fold when reacted with DAPI solution. If high salt concentration affected DAPI-DNA complex fluorescence, one should observe an inhibition rather than an increase of fluorescence (Table IV) (8). This indicates that the presence of high salt concentration was

Table I. Conditionsfor Maximal Extraction of DNA from Leaf Tissue One g alfalfa or maize leaves was homogenized in 10 ml homogenization buffer. To obtain the desired concentrations of NaCl and sarkosyl, 1.5 volumes of buffer (10 mm EDTA, 10 mm Tris [pH 7.01) containing appropriate amounts of NaCl and sarkosyl were added to the homogenates. The diluted homogenates were sonicated for 15 s, treated with chloroform as described in "Materials and Methods," and reacted with DAPI using procedure B. The additional treatments specified in the table were performed as follows: filtration through two layers of cheesecloth and centrifugation at 20,000g for 10 mi. NaCl Concn. Relative

In homogem- After dization buffer lution

Additional Treatments

DNA Cnn Concn.

M

2.0 0.1 0.1 2.0 0.1 2.0 0.1 2.0 a Mean ± SD.

2.0 0.1 2.0 2.0 2.0 2.0 2.0 2.0

Filtration after dilution Filtration prior to dilution Centrifugation after dilution Centrifugation prior to dilution +1% (w/v) sarkosyl

100 ± 5a 23±20 95 ± 4 98 ± 8 100 ± 6 100 ± 4 26 ± 3 113 ± 8

Table II. Effects of Sonication and Sarkosyl on the Extraction of DNA from Alfalfa Protoplasts A suspension containing 3.27 x 106 protoplasts/ml was lysed and treated with chloroform as described in "Materials and Methods." Relative DNA Concn. in the Homogenatea 100 ± 2b Sonication 87 ± 8 Sarkosyl 1% (w/v) 100 ± 5 Sarkosyl 1% (w/v) + sonication a The actual DNA concentration was 4.66 ,ug/300 ,il or/6.54 x 105 protoplasts. b Mean ± SD. Table III. Effects of Histones on Fluorescence Enhancement of DAPI by DNA

Solutions of DNA and histones were mixed, and 0.1 ml of the mixture was reacted with 4.9 ml DAPI solution. The buffer was 10 mM EDTA, 10 mm Tris [pH 7.0] and NaCl as indicated. Relative Fluorescence NaCl Concn. Addition to Calf in Buffer Enhancement Thymus DNA M

0.1 None 2.0 None 0.1 Histones (1:2.5)b 2.0 Histones (1:2.5) 0.1 Histones (1:1) 2.0 Histones (1:1) a Mean ± SD. b Ratio of calf thymus DNA:histones (w/w).

100.0 ± 1.9a 100.0 ± 1.9 50.0 ± 0.8 82.7 ± 0.6 82.8 ± 2.3 94.8 ± 0.8

important during homogenization rather than during the dye binding reaction. Filtration of the homogenate through cheesecloth did not affect the yield of DNA. No effect of centrifugation of homogenates containing 2.0 M NaCl after dilution was observed. However, centrifugation of homogenates containing 0.1 M NaCl prior to dilution reduced the yield of DNA to 26% of the maxi-

Addition of sarkosyl slightly enhanced the yield of DNA. Sonication was more efficient than addition of sarkosyl for

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Volume Added (,ul) FIG. 1. Quenching of fluorescence of the DAPI-DNA complex by leaf homogenate. Procedure A was used. The homogenate was obtained by homogenizing I g alfalfa leaves in 25 ml buffer (2.0 M NaCl, 10 mm EDTA, 10 mM Tris [pH 7.01). Chloroform treatment was omitted. (0), Addition of homogenate. (0), Addition of standard DNA solution (24 ,sg/ ml) following the addition of variable volumes of homogenate as indicated by the thick arrows. The fluorescence observed without homogenate or DNA standard (Blank) was due to fluorescence of DAPI.

Table IV. Quenching of Fluorescence of the DAPI-DNA Complex by Homogenates of Alfalfa Leaves Procedure A was used with addition of 0.1 ml homogenization buffer or homogenate prior to the addition of standard DNA solution. A standard curve was established using DNA standard without prior additions. Homogenates were prepared in 2.0 M NaCl homogenization buffer. Addition Quenchinga None 1.00 0.1 M NaCl homogenization buffer 0.97 ± 0.01b 2.0 M NaCI homogenization buffer 0.93 ± 0.02 Leaf homogenate (1:25)c 0.34 ± 0.02 Leaf homogenate (1:25) + chloroform treatment 0.63 ± 0.01 Leaf homogenate (1:50) + chloroform treatment 0.74 ± 0.01 a Quenching reflects the relative fluorescence enhancement when additions were made to the DNA standards, and is expressed as the ratio of the slope for DNA standard in presence of homogenization buffer or homogenate to the slope for DNA standard without any addition. b Mean ± SD. c Final dilution ratio (g tissue/ml buffer).

extracting DNA from protoplasts (Table II). Sarkosyl plus sonication was no better than sonication alone. Histones limit the accessibility to DNA of several compounds which bind to DNA (e.g. ethidium bromide, DABA, mithramycin) (1, 7, 9). High salt concentrations are known to dissociate histones from DNA (2). Therefore, the higher estimation of DNA in the presence of high NaCl is possibly due to an increased accessibility of DAPI to DNA, through dissociation of histones from DNA. In order to test this possibility, histones and calf thymus DNA were mixed prior to addition to DAPI solution. Presence of histones reduced fluorescence of the DAPI-DNA complex (Table III). The higher the proportion of histones relative to DNA, the higher was the inhibition of fluorescence. A 2.0 M NaCl concentration in the buffer prevented part of the inhibition of fluorescence due to the presence of histones, but did not eliminate it totally. Quenching of Fluorescence by Leaf Homogenate. Fluorescence of the DAPI-DNA complex increases linearly with increasing amounts of DNA until DNA concentration exceeds that of DAPI by 10-fold (w/w) (3). In the presence of compounds that quench

0

40 80 Volume Added (,ul)

120

FIG. 2. Quantitation of DNA in leaf homogenates using procedure A. One g alfalfa leaves was homogenized in 25 ml 2.0 M NaCl homogenization buffer. A 1-ml aliquot was diluted 1:1 with the same buffer. Chloroform treatment and quantitation of DNA (procedure A) were performed as described in "Materials and Methods." (0), Addition of homogenate. (0), Addition of four aliquots (25 ,tl each) of standard DNA solution following the addition of four aliquots (10 1l each) of homogenate. (U), DNA standard curve in the absence of homogenate. (S), Slope of the curve. The fluorescence observed without homogenate or DNA standard (Blank) was due to fluorescence of DAPI. The ratio of the slope of fluorescence enhancement for the homogenate to the slope for the DNA standard (in the presence of homogenate) is 0.820. This value times the DNA standard concentration (24 ILg/ml) yields 19.7 ug/ml, the DNA concentration of the homogenate. Comparing the slope for the DNA standard in the presence of homogenate with the slope for the DNA standard in the absence of homogenate, the ratio is 0.89. This ratio represents the quenching of fluorescence of the DAPI-DNA complex by the homogenate.

fluorescence, this linearity is no longer observed. Quenching of fluorescence of the DAPI-DNA complex by leaf homogenate was observed using procedure A (Fig. 1). Prior addition of leaf homogenate decreased the slope of the standard curve proportionally to the volume of leaf homogenate added prior to calf thymus DNA. Thus, the leaf homogenate contained substances that were inhibitory to fluorescence of the DAPI-DNA complex. Some factors affecting quenching were measured using procedure A (Table IV). The presence of 2.0 M NaCl in the buffer quenched slightly as was reported earlier (8). Quenching by leaf homogenate was reduced by dilution of homogenate and by chloroform treatment of homogenate. The latter observation suggests that pigments and possibly proteins caused part of the quenching. It should be noted that the data of Table IV were obtained by adding 0.1 ml of homogenate to 3 ml of DAPI solution, whereas only 25 to 40 ,ul homogenate were normally used in DNA assays. These smaller aliquots reduced the quenching to 0.85 to 0.90 (i.e. 15-10%o inhibition of fluorescence intensity of the DAPI-DNA complex). An illustration of procedure A with a diluted, chloroform treated homogenate is presented in Figure 2. Temperature Sensitivity of the Fluorescence of DAPI and of the DAPI-DNA Complex. Fluorescence of both DAPI and the DAPI-DNA complex decreased linearly with increasing temperature (Fig. 3). For both solutions, as the temperature increased I °C, the fluorescence decreased by 1%. Therefore, it was necessary to either keep the temperature constant or adjust the blank frequently when long series of fluorescence measurements were made. Specificity and Interferences. The specificity of the fluorescence

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Deoxyribonuclease digestion of DNA is another means ofshowing the specificity of the DNA-enhanced DAPI fluorescence. Treatment of calf thymus DNA with DNase reduced the fluoresDAPI - DNA cence enhancement to 2.2% of the nontreated control (Table VI). 80 DNase treatment of alfalfa and maize leaf homogenates reduced the fluorescence enhancement to 3.5% and 20.2% of the controls, respectively. No further decrease of the fluorescence enhancement U, 60 was observed when DNase-treated homogenates were treated with c RNase (data not shown). The DNase-resistant fluorescence enD hancement of DAPI by maize leaf homogenates cannot therefore be attributed to RNA, but possibly to the presence of DNaseU1) 40 resistant DNA. It has been shown that DAPI binding to DNA is specific for U1) DAPI adenine-thymine base pairs (3, 14). As a consequence, DNA 0 differing widely in their mole percent adenine plus thymine (A 20 + T) may show different fluorescence intensity upon binding of DAPI. Therefore, quantitative comparisons of DNA between , , , species differing in their mole percent A + T should be done with 0 caution. The use of a DNA standard with similar base composition 5 10 c 15 20 25 30 to the DNA from the species studied will give the most accurate Temperature (Ca) results. Comparison with the Diphenylamine Method. The DAPI FIG. 3.'Temperature dependence of the fluorescence of DAPI and of the DAPI-I)NA complex. Cuvettes containing solutions of DAPI or DAPI method was compared with the diphenylamine method for quanand calf th3ymus DNA were incubated at the indicated temperatures, and tifying DNA. The concentration of DNA in alfalfa leaves, estimated by the diphenylamine method, was only 59% of the conthe fluoresc:ence was determined immediately. centration of DNA found using the DAPI method (average of 13 Tabile V. Spec(ficity of Fluorescence Enhancement of DAPI independent experiments). The DAPI method was highly reproOne hunidred ul of sample were reacted with 4.9 ml DAPI solution. ducible; a coefficient of variation of 3.7% was found when seven DNA, RN)A, and histones solutions contained 21.5 ug/ml buffer (2.0 M independent determinations were performed on a single suspenNaCl, 5 mi d MgCl2, 10 mM Tris [pH 7.0]). BSA solution contained 30 mg/ sion of alfalfa protoplasts. Comparable coefficients of variation ml buffer ((2.0 M NaCl, 10 mM EDTA, 10 mM Tris [pH 7.0]). RNA and were observed for determinations of DNA from leaf tissue. histones we-re treated with DNase (I mg/ml) for 6 h at room temperature.

Calf thymus DNA Wheat germ tRNA (type V) Bakers yeast RNA (type III) Calf liver RNA (type IV) Histones (type II-S) BSA (3% w/v) a Mean ± SD.

Relative Fluorescence Enhancement 100.0 + 1.8a 1.2 ± 0.1 1.9 ± 0.1 1.0 ± 0.1 0.3 ± 0.4 15.3 ± 2.1

Table VI. Effect of DNase Treatment on the Fluorescence Enhancement by Leaf Homogenates and Calf Thymus DNA One mg DNase I (DN 25, Sigma) was added to I ml calf thymus DNA solution or leaf homogenates, both in 2.0 M NaCl, 5 mM MgC12, 10 mM Tris (pH 7.0). Solutions were incubated at room temperature until completion of digestion. Controls were incubated without DNase. Fluorescence Enhancement after Sample DNase Treatment % controls ± SD Calf thymus DNA 2.2 ± 1.8 Alfalfa leaves homogenate 3.5 ± 0.7 Maize leaves homogenate 20.2 ± 3.6

enhancement of DAPI by DNA was demonstrated earlier (3, 8). Our results show a fluorescence enhancement of DAPI by RNA between 1% and 2% of an equivalent concentration of calf thymus DNA (Table V). Histones did not affect the fluorescence of DAPI alone. To check the effect of homogenization in buffer containing 3% (w/v) BSA, an aliquot of this buffer equivalent to that used for calf thymus DNA was added to DAPI; fluorescence of the BSA was 15% of that observed with DNA. When homogenates containing 3% BSA were treated with chloroform, the effects of BSA were essentially eliminated.

CONCLUSIONS This assay can be used to quantitate DNA in homogenates in parallel with enzyme assays or determinations of other cellular components. The following procedure is followed: Aliquots of the homogenate, taken before any centrifugation is performed, are diluted with a suitable buffer to adjust the concentration of NaCl and EDTA to 2.0 and 0.01 M, respectively. A final dilution factor of the homogenate of 1 g fresh weight tissue to 25 or 50 ml buffer is suitable. Chloroform (1.5 volumes) is added to the homogenate and the phases combined using a Vortex mixer. The phases are separated by centrifugation at 1,000g for 10 min. The clear, aqueous, supernatant is used for fluorimetric measurements. In some instances, clarification of the supernatant by centrifuging at 20,000g for 10 min is necessary. Fluorimetric measurements can be done by either procedure A or B as described in "Materials and Methods." No significant differences were observed when the two procedures were compared (data not shown). Although reproducibility was higher with procedure A, it was difficult to work with procedure A when dust was present in the air surrounding the spectrofluorimeter. Procedure B was much less susceptible to this problem. In order to help keep dust out of the solutions, glassware should be rinsed with distilled H20 before use, and all solutions (except those containing DNA) filtered through a 0.45 MLm Millipore filter. Because of the quenching of fluorescence of the DAPI-DNA complex by leaf homogenates (Figs. I and 2; Table IV), a standard curve established with purified DNA is not satisfactory for this assay. An internal standard is therefore required to correct for quenching. A quenched standard curve (ie. established in the presence of leaf homogenate) could be used for this purpose. However, variations of quenching between samples would lead to

results. Two modifications of the DAPI method described by Brunk et al (3) were made to adapt this method to plant extracts. Addition of high salt concentration to homogenates maximized dissociation

erroneous

DNA ASSAY of histones from DNA and DNA recovery. Treatment of homogenates with chloroform extracted pigments and proteins, thereby reducing quenching. No loss of DNA resulted from this treatment. Chloroform treatment is important when the homogenization buffer contains high concentrations of inert protein (e.g. BSA or casein) added for extraction of unstable enzymes (13). The range of DNA concentrations that was found in protoplast suspensions (1-7 jig in 0.3 ml) could not be determined using the diphenylamine assay. However, much less time and effort was spent trying to improve the diphenylamine than the DAPI method. The lower limit of sensitivity of the DAPI method was not reached in the determination of DNA in our protoplast suspensions. Sensitivity in the nanogram range has been reported by others (3, 8). We have not used the fluorimetric technique with organelles, but based on these reports, DNA determinations in the nanogram range on organelle preparations should be possible. LITERATURE CITED 1. BARTH CA, BS WILLERSHAUSEN 1978 Improvement of DNA quantitation following proteolytic extraction. Anal Biochem 90: 167-173 2. BONNER J 1976 The Nucleus. In J Bonner, J Varner, eds, Plant Biochemistry, Ed 3. Academic Press, New York, pp 37-64 3. BRUNK CF, KL JONES, TW JAMES 1979 Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem 92: 497-500 4. BURTON K 1956 A study of the conditions and mechanism of the diphenylamine

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reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62: 315-323 CHEVALIER MM 1978 Biochemical and genetic variation of sucrose phosphate synthetase from maize leaf blades. PhD thesis. University of Wisconsin, Madison CHEVALIER MM, LE SCHRADER 1977 Genotypic differences in nitrate absorption and partitioning of N among plant parts in maize. Crop Sci 17: 897-901 GROYER A, P ROBEL 1980 DNA measurement by mithramycin fluorescence in chromatin solubilized by heparin. Anal Biochem 106: 262-268 KAPUSCINSKI J, B SKOCZYLAS 1977 Simple and rapid fluorimetric method for DNA microassay. Anal Biochem 83: 252-257 KARSTEN U, A WOLLENBERGER 1977 Improvements in the ethidium bromide method for direct fluorimetric estimation of DNA and RNA in cell and tissue homogenates. Anal Biochem 77: 464 470 MEYERS SP, SL NICHOLs, GR BAER, WT MOLIN, LE SCHRADER 1982 Ploidy effects in isogenic populations of alfalfa (Medicago Sativa L.). I. Ribulose- 1,5bisphosphate carboxylase, soluble protein, chlorophyll and DNA in leaves. Plant Physiol. In press MOLIN WT, SP MEYERS, GR BAER, LE SCHRADER 1982 Ploidy effects in isogenic populations of alfalfa (Medicago sativa L.). II. Photosynthesis, chloroplast number, ribulose-1,5-bisphosphate carboxylase, chlorophyll and DNA in protoplasts. Plant Physiol. In press PFEIFFER T, LE SCHRADER, ET BINGHAM 1980 Physiological comparisons of isogenic diploid-tetraploid, tetraploid-octoploid alfalfa populations. Crop Sci 20: 299-303 SCHRADER LE, DA CATALDO, DM PETERSON 1974 Use of protein in extraction and stabilization of nitrate reductase. Plant Physiol 53: 688-690 WILLIAMSON DH, DJ FENNEL 1974 The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Methods Cell Biol 12: 335-351