Plant Cell Reports
Plant Cell Reports (1984) 3:237-239
© Springer-Verlag 1984
A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources Albert J. W i l s o n ~ and P r e m S. C h o u r e y 1, 2 USDA/ARS 1, Plant Pathology Department,University of Florida 2, Gainesville, Florida 32611, USA Received June 20, 1984 / Revised version received October I0, 1984- Communicated by J. Widholm
Abstract A simplified method for the isolation of mitochondrial DNA (mtDNA) of several plant species from either coleoptile or tissue cultured cells is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions (such as phenol, chloroform, ether, etc.). Protoplast isolation is not required for the release of organelles from cell suspension cultured cells. The entire procedure can he performed in a single day and employs differential low speed centrifugations for isolation of mitochondria and differential precipitations for the recovery of restrictable DNA. Introduction A great interest in mtDNA of higher plants has occurred over the past several years as investigators have documented the range of complexity in mitochondrial genomes, studied cytoplasmic male sterility in various plant systems, undertaken molecular mapping of mitochondrial genomes, isolated mitochondrial structural and non-structural genes and documented many molecular polymorphs by mtDNA restriction fragment profiles. An additional interest in the genome has stemmed from recent advances in plant tissue culture technology. Specifically, protoplast fusion leading to somatic hybridization provides a unique opportunity for organelle mixing and possibly genetic recombination in the organellar genomes (Belliard et al. 1979, Boeshore et al. 1983). In this regard mtDNA has also been analyzed to examine tissue culture mediated variation in regenerated plants or in cultured cell populations (Gengenbach et al. 1981, Belliard et al. 1979, Sisson et al. 1980, Sparks and Dale 1980, Ward et al. 1981, McNay et al. 1984). Because the higher plant mitochondrial genome is neither as immensely large and complex as the nuclear genome nor relatively simple or small as the chloroplast genome, it is readily analyzable subsequent to restriction endonuclease digestion, electrophoresis, and staining with ethidium bromide. In such analyses each sample displays a unique genetic fingerprint which is
Send inquiries
to:
P. S. Chourey
equivalent to many genetic markers at the molecular level. Paramount to all of these studies is the ability to isolate restrictable mtDNA. Many procedures have been developed over the years for isolation of plant mtDNA (Belliard et al. 1979, Sisson et al. 1980, Sparks and Dale 1980, Ward et al. 1981)o Most of these procedures are lengthy and sometimes yield mtDNA that is not restrictable with a particular restriction enzyme or with a battery of commonly used restriction endonucleases. The present report describes a procedure that has wide applicability for the isolation of mtDNA from several plant types as well as different plant tissues including coleoptile tissue, suspension cells, and plant callus tissue. The procedure is relatively fast, inexpensive and does not use toxic organic solvents. The procedure reproducibly yields undegraded, restrictable mtDNA. Material
and Methods
Suspension cultures of sorghum, maize, and tobacco were ~rown as described (Chourey and Zurawski 1981, Kao et al. 1974). Peanut suspension ceils were provided by Drs. P. Still and C. L. Niblett. For mtDNA isolation the growth media was removed by vacuum filtration, cells weighed, and the tissue ground in a prechilled mortar and pestle in chilled grinding buffer (Kemble and Bedbrook 1979) consisting of I0 mM n-tris (hydroxymethyl)methyl-2aminoethane-sulphonic acid (TES), pH 7.2, 0.5 M mannitol, i mM ethylene glycol-bis-(2-aminoethyL ~ther)-n, n'tetraacetic acid (EGTA), 0.2% wt/vol BSA, and 0.05% wt/vol cysteine. Enough buffer was added to the tissue to produce a thick slurry upon grinding (usually 0.5-0.8 ml/g tissue). After grinding for 3-5 min the suspension was filtered through 4 layers of cheeseclotb and one layer of miraclotb (Calbiochem) into prechilled tubes. The tissue was re-ground two more times in fresh buffer and the pooled filtrate was centrifuged at i000 x g for I0 min at 4 ° C. The supernatant was collected and centrifuged a second time in the same way. Mitochondria were collected from the supernatant by centrifugation at 16,000 × g for I0 min at 4 ° C. The mitochondrial
pellet was gently
238 resuspended with the aid of a small artists soft paint brush in 10 ml prechilled DNase buffer consisting of 0.3 M sucrose and 0.05 M Tris, pH 7.5 (McNay et al. 1984, Kemble et al. 1980). The resuspended mitochondria were centrifuged at I000 x g for i0 min at 4 ° C. The supernatant was collected and made 0 . 0 1 M MgCI 2 by the addition of i00 ul of 1 M MgCI 2 followed by addition of I00 ul DNase (2mg DNase/ml I mM MgCI 2 frozen stock solution). The suspension was incubated at 4 ° C for I hr. DNase was removed by carefully underlayin~ 20 ml shelf buffer (Kemble et al. 1980) consisting of 0.6 M sucrose, 0 . 0 1 M Tris, pH 7.2, and 0.02 M EDTA. The mitochondria were pelleted through the shelf buffer by centrifugation at 12,000 x ~ for 20 min at 4 ° C. The mitochondrial pellet was resnspended in I0 ml shelf buffer and repelleted at 14,000 x g for I0 min at 4 ° C. The pelleted mitochondria were lysed by the addition of 600 ul lysis buffer consisting of 0.i M Tris, pH 8.0, 0.05 M EDTA, 0 . 1 M NaCI, I% SDS, and 0 . 0 1 M mercaptoethanol and the solution was transferred to a 1.5 ml microcentrifuge tube and incubated 20 min at 65 ° C. Following lysis, the DNA isolation protocol is basically similar to the one used by yeast workers and has been described recently (Dellaporta et al. 1983). It consists of adding 200 ul 5 M potassium acetate, mixing the suspension and chilling on ice for a minimum of 15 min. Most proteins and polysaccharides were removed as a complex with the insoluble potassium dodecyl sulfate precipitate by a 3 min centrifugation in a microfuge at room temperature. The supernatant was collected and added to a microfuge tube containing 40 ul 5 M ammonium acetate and 400 ul isopropanol, The solution was mixed and incubated at -20 ° C for a minimum of 20 min. The precipitated DNA was pelleted for 5 min in a microfuge. The DNA pellet was washed with ice cold 70% ethanol and the tube centrifuged for 2 min to repellet the precipitate. The DNA pellet was dried in a vacuum dessicator and then rehydrated in 700 ul 50/10 buffer consisting of 50 mM Tris, pH 8.0, 10 mM EDTA. The resuspended DNA was made to 0.3 M sodium acetate by addition of 75 ul 3 M sodium acetate and precipitated by the addition of 500 ul isopropanol at room temperature. After mixing well, the DNA was pelleted in a microfuge for 30 s at room temperature. The DNA pellet was washed with 70% ethanol, dried and redissolved in a minimum volume of TE buffer consisting of 0 . 0 1 M Tris, (pH 8.0), 1.0 mM EDTA. Six to seven day old sorghum and maize coleoptiles were subjected to the same mtDNA isolation procedures used for suspension cell tissue. For mtDNA isolation from tobacco and peanut suspension cells, diethyldithiocarbamic acid (DIECA) was added to the buffers to a final concentration of I0 mM (McNay et al. 1984) for all buffers preceedin~ the lysis buffer. Maize callus tissue from agarose plates was scraped off the agarose surface, and ground as described for sorghum and maize coleoptile and suspension cell tissues. The yield of mtDNA per gm of tissue was variable. However, ideal extractions have yielded approximately 5 to 7 ug of DNA from 2.5 g of fresh weight of tissue or cultured cells. MtDNA extractions from less than 2.5 g of tissue were not attempted.
Restriction enzyme digests were performed as described by the manufacturer of the enzymes. RNase (DNase free) was added to all restriction digestion reactions (25 ng/ul final concentration). Electrophoresis was performed using 0.8% agarose gels at 2.5 volts/cm. Gels were stained with ethidium bromide (0.5 ug/ml final concentration) for 1 hr, destained with water, and photographed under UV light using POLAROID type 55 land film. Results and Discussion The procedure described in this report has been used sucessfully for the isolation of restrictable mtDNA from maize suspension cells, maize coleoptiles, maize callus, sorghum coleoptiles, sorghum suspension cells, peanut suspension cells, and tobacco suspension cells (Fig. I). RNase was added to all restriction digestion reactions to help minimize the "smiling effect" that large amounts of RNA have on DNA banding of restriction fragments in agarose gels. DNase treatment of isolated mitochondria was performed routinely although this step may be optional for some isolations. For example, a degraded nuclear smear background was only slightly detectable in non-DNase treated isolations from sorghum suspension cells. DNase treatment in such isolations reduced the degraded nuclear background only slightly. This nuclear DNA background was generally more substantial for isolations from coleoptile tissue where DNase treatment was considered essential. Incorporation of DIECA in isolation buffers for the two dicotyledonous species (tobacco and peanut) and mature plants of sorghum was found to be essential for the isolation of undegraded mtDNA. Without DIECA the mtDNA's were almost totally degraded. The mtDNA isolated from callus tissue grown on agar was nonrestrictable. The problem was unrelated to this method of DNA isolation since the mtDNA's isolated by other methods have similarly been non-digestible. Total genomic DNA from agargrown maize calli isolated by various methods was similarly non-restrictable (Chourey and Still, unpublished). In fact a minimum of four weeks of subculture on agarose or in liquid medium, upon transfer from agar, seems to be an essential step before any restrictable DNA is obtained. Sometimes addition of spermidine (4.0 mM final concentration) (Bouche 1981) to the restriction digest has yielded digestible DNA in those samples which have otherwise been nonrestrictable. The entire isolation procedure can be done in less than 3 hr if the DNase treatment is omitted and in less than 5 hr when the DNase treatment is included. The procedure has the marked advantage over other procedures in that no organic solvent extractions of the mtDNA with phenol~ chloroform, or ether is necessary. Another advantage of this method is that ultracentrifugation for either the isolation of mitochondrial organelles on sucrose or pereoll gradients or separation of mtDNA from plastid and nuclear DNA's on CsCI density gradients is not needed. A disadvantage of this method, however, is that the mtDNA thus purified has
239
1
2
3
4
5
6
7
8
9
10
amenable to a wide variety of other plants even though we have tested only a few. For researchers interested in using this procedure for isolating mtDNA from plants that contain mtDNA episomes as in certain cytoplasmic male sterile lines of maize (Pring et al. 1977), we suggest the incorporation of a protease step prior to the potassium acetate-SDS precipitation step in the isolation procedure. Protease digestion removes the protein bound to the ends of the mtDNA episomes so that the episomal DNA is not precipitated in the protein-potassium acetate-SDS complex (Chris Chase, personal communication). Acknowledgements: We thank Dr. D. R. Pring for the critical review of the manuscript and Ms. Diana Z. Sharpe and P. Still for technical assistance in this work. Cooperative Investigation, U. S. Department of Agriculture, ARS and I.F.A.S. University of Florida, Florida Agricultural Experiment Station Journal Series No. 5806. References
Fig. 1.Eco RI digests of mtDNA from: 2) maize Black Mexican Sweet suspension culture cells, 3) maize coleoptile tissue (aDs stock), 4) agarose grown maize callus tissue (aMu stock), 5) sorghum NK300 suspension culture cells, 6) sorghum NK300 cleoptile tissue, 7) sorghum M35-I suspension culture cells, 8) sorghum M35-I coleoptile tissue, 9) peanut suspension culture cells, 10) tobacco suspension culture cells. In lane i is shown lambda-Hind III molecular size markers. more chloroplast DNA than some of the other methods which require an ultracentrifugation step to separate the organelles. This isolation procedure reproducibly yields restrictable mtDNA from sorghum which is frequently difficult to obtain by existing procedures (Chase and Pring, personal communication). This procedure should also be
Belliard G, Vedel J, Pelletier G (1979) Nature 281:401-403 Boeshore M L, Lifshitz I, Hanson M R, Izhar S (1983) Molec Gen Genet 190:459-467 Boucbe J P (1981) Anal Biochem 115:42-45 Chourey P S, Zurawski D B (1981) Tbeor Appl Genet 59:341-344 Dellaporta S L, Wood J, Hicks J B (1983) Maize Genet Coop News Letter 57:26-29 Gengenbach B G, Connelly J A, Pring D R, Conde M F (1981) Theor Appl Genet 59:161-167 Kao K N, Constable F~ Michayluk M R, Gamborg O L (1974) Planta 120:215-227 Kemble R J, Bedbrook J R (1979) Maydica XXIV:175-180 Kemble R J, Gunn R E, Flavell R B (1980) Genetics 95:451-458 McNay J W, Chourey P 8, Pring D R (1984) Theor Appl Genet 67:433-437 Nagy F, Lazar G, Menczel L, Maliga P, (1983) Theor Appl Genet 66:203-207 Pring D R, Levings III C S, Hu W W L, Timothy D. H (1977) Proc Natl Acad Sci USA 74:2904-2908 Sisson V A, Brim C A, Levings III C S (1978) Crop Science 18:991-996 Sparks Jr R B, Dale R M K (1980) Molec Gen Genet 180:351-355 Ward B L, Anderson R S, Bendicb A J (1981) Cell 25:793-803.
Plant Cell Reports (1984) 3:237-239
© Springer-Verlag 1984
A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources Albert J. W i l s o n ~ and P r e m S. C h o u r e y 1, 2 USDA/ARS 1, Plant Pathology Department,University of Florida 2, Gainesville, Florida 32611, USA Received June 20, 1984 / Revised version received October I0, 1984- Communicated by J. Widholm
Abstract A simplified method for the isolation of mitochondrial DNA (mtDNA) of several plant species from either coleoptile or tissue cultured cells is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions (such as phenol, chloroform, ether, etc.). Protoplast isolation is not required for the release of organelles from cell suspension cultured cells. The entire procedure can he performed in a single day and employs differential low speed centrifugations for isolation of mitochondria and differential precipitations for the recovery of restrictable DNA. Introduction A great interest in mtDNA of higher plants has occurred over the past several years as investigators have documented the range of complexity in mitochondrial genomes, studied cytoplasmic male sterility in various plant systems, undertaken molecular mapping of mitochondrial genomes, isolated mitochondrial structural and non-structural genes and documented many molecular polymorphs by mtDNA restriction fragment profiles. An additional interest in the genome has stemmed from recent advances in plant tissue culture technology. Specifically, protoplast fusion leading to somatic hybridization provides a unique opportunity for organelle mixing and possibly genetic recombination in the organellar genomes (Belliard et al. 1979, Boeshore et al. 1983). In this regard mtDNA has also been analyzed to examine tissue culture mediated variation in regenerated plants or in cultured cell populations (Gengenbach et al. 1981, Belliard et al. 1979, Sisson et al. 1980, Sparks and Dale 1980, Ward et al. 1981, McNay et al. 1984). Because the higher plant mitochondrial genome is neither as immensely large and complex as the nuclear genome nor relatively simple or small as the chloroplast genome, it is readily analyzable subsequent to restriction endonuclease digestion, electrophoresis, and staining with ethidium bromide. In such analyses each sample displays a unique genetic fingerprint which is
Send inquiries
to:
P. S. Chourey
equivalent to many genetic markers at the molecular level. Paramount to all of these studies is the ability to isolate restrictable mtDNA. Many procedures have been developed over the years for isolation of plant mtDNA (Belliard et al. 1979, Sisson et al. 1980, Sparks and Dale 1980, Ward et al. 1981)o Most of these procedures are lengthy and sometimes yield mtDNA that is not restrictable with a particular restriction enzyme or with a battery of commonly used restriction endonucleases. The present report describes a procedure that has wide applicability for the isolation of mtDNA from several plant types as well as different plant tissues including coleoptile tissue, suspension cells, and plant callus tissue. The procedure is relatively fast, inexpensive and does not use toxic organic solvents. The procedure reproducibly yields undegraded, restrictable mtDNA. Material
and Methods
Suspension cultures of sorghum, maize, and tobacco were ~rown as described (Chourey and Zurawski 1981, Kao et al. 1974). Peanut suspension ceils were provided by Drs. P. Still and C. L. Niblett. For mtDNA isolation the growth media was removed by vacuum filtration, cells weighed, and the tissue ground in a prechilled mortar and pestle in chilled grinding buffer (Kemble and Bedbrook 1979) consisting of I0 mM n-tris (hydroxymethyl)methyl-2aminoethane-sulphonic acid (TES), pH 7.2, 0.5 M mannitol, i mM ethylene glycol-bis-(2-aminoethyL ~ther)-n, n'tetraacetic acid (EGTA), 0.2% wt/vol BSA, and 0.05% wt/vol cysteine. Enough buffer was added to the tissue to produce a thick slurry upon grinding (usually 0.5-0.8 ml/g tissue). After grinding for 3-5 min the suspension was filtered through 4 layers of cheeseclotb and one layer of miraclotb (Calbiochem) into prechilled tubes. The tissue was re-ground two more times in fresh buffer and the pooled filtrate was centrifuged at i000 x g for I0 min at 4 ° C. The supernatant was collected and centrifuged a second time in the same way. Mitochondria were collected from the supernatant by centrifugation at 16,000 × g for I0 min at 4 ° C. The mitochondrial
pellet was gently
238 resuspended with the aid of a small artists soft paint brush in 10 ml prechilled DNase buffer consisting of 0.3 M sucrose and 0.05 M Tris, pH 7.5 (McNay et al. 1984, Kemble et al. 1980). The resuspended mitochondria were centrifuged at I000 x g for i0 min at 4 ° C. The supernatant was collected and made 0 . 0 1 M MgCI 2 by the addition of i00 ul of 1 M MgCI 2 followed by addition of I00 ul DNase (2mg DNase/ml I mM MgCI 2 frozen stock solution). The suspension was incubated at 4 ° C for I hr. DNase was removed by carefully underlayin~ 20 ml shelf buffer (Kemble et al. 1980) consisting of 0.6 M sucrose, 0 . 0 1 M Tris, pH 7.2, and 0.02 M EDTA. The mitochondria were pelleted through the shelf buffer by centrifugation at 12,000 x ~ for 20 min at 4 ° C. The mitochondrial pellet was resnspended in I0 ml shelf buffer and repelleted at 14,000 x g for I0 min at 4 ° C. The pelleted mitochondria were lysed by the addition of 600 ul lysis buffer consisting of 0.i M Tris, pH 8.0, 0.05 M EDTA, 0 . 1 M NaCI, I% SDS, and 0 . 0 1 M mercaptoethanol and the solution was transferred to a 1.5 ml microcentrifuge tube and incubated 20 min at 65 ° C. Following lysis, the DNA isolation protocol is basically similar to the one used by yeast workers and has been described recently (Dellaporta et al. 1983). It consists of adding 200 ul 5 M potassium acetate, mixing the suspension and chilling on ice for a minimum of 15 min. Most proteins and polysaccharides were removed as a complex with the insoluble potassium dodecyl sulfate precipitate by a 3 min centrifugation in a microfuge at room temperature. The supernatant was collected and added to a microfuge tube containing 40 ul 5 M ammonium acetate and 400 ul isopropanol, The solution was mixed and incubated at -20 ° C for a minimum of 20 min. The precipitated DNA was pelleted for 5 min in a microfuge. The DNA pellet was washed with ice cold 70% ethanol and the tube centrifuged for 2 min to repellet the precipitate. The DNA pellet was dried in a vacuum dessicator and then rehydrated in 700 ul 50/10 buffer consisting of 50 mM Tris, pH 8.0, 10 mM EDTA. The resuspended DNA was made to 0.3 M sodium acetate by addition of 75 ul 3 M sodium acetate and precipitated by the addition of 500 ul isopropanol at room temperature. After mixing well, the DNA was pelleted in a microfuge for 30 s at room temperature. The DNA pellet was washed with 70% ethanol, dried and redissolved in a minimum volume of TE buffer consisting of 0 . 0 1 M Tris, (pH 8.0), 1.0 mM EDTA. Six to seven day old sorghum and maize coleoptiles were subjected to the same mtDNA isolation procedures used for suspension cell tissue. For mtDNA isolation from tobacco and peanut suspension cells, diethyldithiocarbamic acid (DIECA) was added to the buffers to a final concentration of I0 mM (McNay et al. 1984) for all buffers preceedin~ the lysis buffer. Maize callus tissue from agarose plates was scraped off the agarose surface, and ground as described for sorghum and maize coleoptile and suspension cell tissues. The yield of mtDNA per gm of tissue was variable. However, ideal extractions have yielded approximately 5 to 7 ug of DNA from 2.5 g of fresh weight of tissue or cultured cells. MtDNA extractions from less than 2.5 g of tissue were not attempted.
Restriction enzyme digests were performed as described by the manufacturer of the enzymes. RNase (DNase free) was added to all restriction digestion reactions (25 ng/ul final concentration). Electrophoresis was performed using 0.8% agarose gels at 2.5 volts/cm. Gels were stained with ethidium bromide (0.5 ug/ml final concentration) for 1 hr, destained with water, and photographed under UV light using POLAROID type 55 land film. Results and Discussion The procedure described in this report has been used sucessfully for the isolation of restrictable mtDNA from maize suspension cells, maize coleoptiles, maize callus, sorghum coleoptiles, sorghum suspension cells, peanut suspension cells, and tobacco suspension cells (Fig. I). RNase was added to all restriction digestion reactions to help minimize the "smiling effect" that large amounts of RNA have on DNA banding of restriction fragments in agarose gels. DNase treatment of isolated mitochondria was performed routinely although this step may be optional for some isolations. For example, a degraded nuclear smear background was only slightly detectable in non-DNase treated isolations from sorghum suspension cells. DNase treatment in such isolations reduced the degraded nuclear background only slightly. This nuclear DNA background was generally more substantial for isolations from coleoptile tissue where DNase treatment was considered essential. Incorporation of DIECA in isolation buffers for the two dicotyledonous species (tobacco and peanut) and mature plants of sorghum was found to be essential for the isolation of undegraded mtDNA. Without DIECA the mtDNA's were almost totally degraded. The mtDNA isolated from callus tissue grown on agar was nonrestrictable. The problem was unrelated to this method of DNA isolation since the mtDNA's isolated by other methods have similarly been non-digestible. Total genomic DNA from agargrown maize calli isolated by various methods was similarly non-restrictable (Chourey and Still, unpublished). In fact a minimum of four weeks of subculture on agarose or in liquid medium, upon transfer from agar, seems to be an essential step before any restrictable DNA is obtained. Sometimes addition of spermidine (4.0 mM final concentration) (Bouche 1981) to the restriction digest has yielded digestible DNA in those samples which have otherwise been nonrestrictable. The entire isolation procedure can be done in less than 3 hr if the DNase treatment is omitted and in less than 5 hr when the DNase treatment is included. The procedure has the marked advantage over other procedures in that no organic solvent extractions of the mtDNA with phenol~ chloroform, or ether is necessary. Another advantage of this method is that ultracentrifugation for either the isolation of mitochondrial organelles on sucrose or pereoll gradients or separation of mtDNA from plastid and nuclear DNA's on CsCI density gradients is not needed. A disadvantage of this method, however, is that the mtDNA thus purified has
239
1
2
3
4
5
6
7
8
9
10
amenable to a wide variety of other plants even though we have tested only a few. For researchers interested in using this procedure for isolating mtDNA from plants that contain mtDNA episomes as in certain cytoplasmic male sterile lines of maize (Pring et al. 1977), we suggest the incorporation of a protease step prior to the potassium acetate-SDS precipitation step in the isolation procedure. Protease digestion removes the protein bound to the ends of the mtDNA episomes so that the episomal DNA is not precipitated in the protein-potassium acetate-SDS complex (Chris Chase, personal communication). Acknowledgements: We thank Dr. D. R. Pring for the critical review of the manuscript and Ms. Diana Z. Sharpe and P. Still for technical assistance in this work. Cooperative Investigation, U. S. Department of Agriculture, ARS and I.F.A.S. University of Florida, Florida Agricultural Experiment Station Journal Series No. 5806. References
Fig. 1.Eco RI digests of mtDNA from: 2) maize Black Mexican Sweet suspension culture cells, 3) maize coleoptile tissue (aDs stock), 4) agarose grown maize callus tissue (aMu stock), 5) sorghum NK300 suspension culture cells, 6) sorghum NK300 cleoptile tissue, 7) sorghum M35-I suspension culture cells, 8) sorghum M35-I coleoptile tissue, 9) peanut suspension culture cells, 10) tobacco suspension culture cells. In lane i is shown lambda-Hind III molecular size markers. more chloroplast DNA than some of the other methods which require an ultracentrifugation step to separate the organelles. This isolation procedure reproducibly yields restrictable mtDNA from sorghum which is frequently difficult to obtain by existing procedures (Chase and Pring, personal communication). This procedure should also be
Belliard G, Vedel J, Pelletier G (1979) Nature 281:401-403 Boeshore M L, Lifshitz I, Hanson M R, Izhar S (1983) Molec Gen Genet 190:459-467 Boucbe J P (1981) Anal Biochem 115:42-45 Chourey P S, Zurawski D B (1981) Tbeor Appl Genet 59:341-344 Dellaporta S L, Wood J, Hicks J B (1983) Maize Genet Coop News Letter 57:26-29 Gengenbach B G, Connelly J A, Pring D R, Conde M F (1981) Theor Appl Genet 59:161-167 Kao K N, Constable F~ Michayluk M R, Gamborg O L (1974) Planta 120:215-227 Kemble R J, Bedbrook J R (1979) Maydica XXIV:175-180 Kemble R J, Gunn R E, Flavell R B (1980) Genetics 95:451-458 McNay J W, Chourey P 8, Pring D R (1984) Theor Appl Genet 67:433-437 Nagy F, Lazar G, Menczel L, Maliga P, (1983) Theor Appl Genet 66:203-207 Pring D R, Levings III C S, Hu W W L, Timothy D. H (1977) Proc Natl Acad Sci USA 74:2904-2908 Sisson V A, Brim C A, Levings III C S (1978) Crop Science 18:991-996 Sparks Jr R B, Dale R M K (1980) Molec Gen Genet 180:351-355 Ward B L, Anderson R S, Bendicb A J (1981) Cell 25:793-803.
