Theoretical and Applied Genetics

, Volume 83, Issue 6–7, pp 799–806 | Cite as

Mitochondrial genome variability in Sorghum cell culture protoclones

  • E. J. Kane
  • A. J. Wilson
  • P. S. Chourey


Sorghum bicolor cv NK300 seedlings, a cell suspension culture, and five protoclone suspension cultures were compared for the occurrence of somaclonal variation by analysis of their mitochondrial DNA (mtDNA). Restriction digests of the mtDNA showed qualitative and quantitative variation of restriction fragments. Southern analyses were performed using a 14.7-kb EcoRI mitochondrial genome fragment and regions carrying mitochondrial protein coding genes, atpA, atp6, cob, and coxI as probes. These analyses revealed part of the 14.7-kb EcoRI region to be present as a repeat in planta, and to be hypervariable when cells were subjected to protoplast culture. All protoclones differed from each other, from the parental cell suspension culture, and from the seedlings in their mitochondrial genome arrangement. Seedlings of five independent sorghum accessions, unrelated to cv NK300, of diverse geographic origin showed conservation of this mitochondrial fragment. Southern analyses of the mtDNA showed no variation for genomic organization of the region carrying coxI, and atpA was identical in all the tissue culture lines. The atp6 gene was present as two copies in the seedlings, and one copy was rearranged upon tissue culture. The region carrying the cob gene was also found to be variant between tissue culture and seedling mtDNA. A substoichiometric 3.3-kb EcoRI cob fragment present in seedlings was amplified in the tissue culture lines. Protoclone S63 differed from the original suspension culture and remaining protoclones in that it had lost the 3.0-kb EcoRI band, the most abundant fragment in seedlings. A new set of fragments was detected in this protoclone. Northern analysis for the cob gene demonstrated altered transcript size in protoclone S63.

Key words

Sorghum bicolor Protoplast Mitochondri al DNA Somaclonal variation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bailey-Serres J, Dixon LK, Liddel AD, Leaver CJ (1986a) Nuclear-mitochondrial interactions in cytoplasmic male-sterile sorghum. Theor Appl Genet 73:252–260Google Scholar
  2. Bailey-Serres J, Hanson DK, Fox T, Leaver CJ (1986b) Mitochondrial genome rearrangement leads to extension and relocation of the cytochrome c oxidase subunit I gene in sorghum. Cell 47:567–576Google Scholar
  3. Bhaskaran S, Smith RH, Paliwal S, Schertz KF (1987) Somaclonal variation from Sorghum bicolor (L.) Moench cell culture. Plant Cell Tiss Org Cult 9:189–196Google Scholar
  4. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HC, Boyer HW (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113Google Scholar
  5. Bonen L, Bird S (1988) Sequence analysis of the wheat mitochondrial atp6 gene reveals a fused upstream reading frame and markedly divergent N termini among plant ATP6 proteins. Gene 73:47–56Google Scholar
  6. Braun CJ, Levings CS III (1985) Nucleotide sequence of the F1-ATPase subunit gene from maize mitochondria. Plant Physiol 79:571–577Google Scholar
  7. Brears T, Curtis GJ, Lonsdale DM (1989) A specific rearrangement of mitochondrial DNA induced by tissue culture. Theor Appl Genet 77:620–624Google Scholar
  8. Cai T, Ejeta G, Axtell JD, Butler LG (1990) Somaclonal variation in high tannin sorghums. Theor Appl Genet 79:737–747Google Scholar
  9. Chourey PS, Sharpe DZ (1985) Callus formation from protoplasts of Sorghum cell suspension cultures. Plant Sci 39:171–175Google Scholar
  10. Chourey PS, Lloyd RE, Sharpe DZ, Isola NR (1986) Molecular analysis of hypervariability in the mitochondrial genome of tissue cultured cells of maize and sorghum. In: Mantell SH, Chapman GP, Street PFS (eds) The chondriome — chloroplast and mitochondrial genomes. Wiley and Sons, New York, pp 177–191Google Scholar
  11. Chowdhury MKU, Schaeffer GW, Smith RL, Matthews BF (1988) Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice. Theor Appl Genet 76:533–539Google Scholar
  12. Chowdhury MKU, Schaeffer GW, Smith RL, DeBonte LR, Matthews BF (1990) Mitochondrial DNA variation in longterm tissue cultured rice lines. Theor Appl Genet 80:81–87Google Scholar
  13. Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13Google Scholar
  14. Gengenbach BG, Connelly JA, Pring DR, Conde MF (1981) Mitochondrial DNA variation in maize plants regenerated during tissue culture selection. Theor Appl Genet 59:161–167Google Scholar
  15. Grayburn WS, Bendich AJ (1987) Variable abundance of a mitochondrial DNA fragment in cultured tobacco cells. Curr Genet 12:257–261Google Scholar
  16. Hakansson G, Bonnett HT, Glimelius K (1990) Extensive nuclear influence on mitochondrial transcription and genome structure in male-fertile and male-sterile alloplasmic Nicotiana materials. In: Proc 4th Int Workshop plant Mitochondria, Sept. 23–27, Ithaca, NY, 1990, pp 58Google Scholar
  17. Hartmann C, Henry Y, DeBuyser J, Aubry C, Rode A (1989) Identification of new mitochondrial genome organizations in wheat plants regenerated from somatic tissue cultures. Theor Appl Genet 77:169–175Google Scholar
  18. Johnson DA, Gautsch JW, Sportsman JR, Elder JH (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Techn 1:3–8Google Scholar
  19. Kemble RJ, Shepard JF (1984) Cytoplasmic DNA variation in a potato protoclonal population. Theor Appl Genet 69:211–216Google Scholar
  20. Larkin PJ, Scowcroft WR (1981) Somaclonal variation — a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214Google Scholar
  21. Laughnan JR, Gabay-Laughnan S, Carlson JE (1981) Characteristics of cms-S reversion to male fertility in maize. Stadler Gen Symp 13:93–114Google Scholar
  22. Leaver CJ, Isaac PG, Bailey-Serres J, Small ID, Hanson DK, Fox TD (1985) Recombination events associated with the cytochrome c oxidase subunit I gene in fertile and cytoplasmic male sterile maize and sorghum. In: Quagliariello E, Slater EC, Palmieri F, Saccone C, Kroon AM (eds) Achievements and perspectives of mitochondrial research, Vol. II. Biogenesis. Elsevier, New York, pp 111–122Google Scholar
  23. Lonsdale DM, Brears T, Hodge TP, Melville SE, Rottmann WH (1988) The plant mitochondrial genome: homologous recombination as a mechanism for generating heterogeneity. Philos Trans R Soc Lond Ser B319:149–163Google Scholar
  24. Mackenzie SA, Chase CD (1990) Fertility restoration is associated with loss of a portion of the mitochondrial genome in cytoplasmic male-sterile common bean. The Plant Cell 2:905–912Google Scholar
  25. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor/NYGoogle Scholar
  26. McNay JW, Chourey PS, Pring DR (1984) Molecular analysis of genomic stability of mitochondrial DNA in tissue cultured cells of maize. Theor Appl Genet 67:433–437Google Scholar
  27. Pring DR, Conde MF, Schertz KF (1982) Organelle genome diversity in sorghum: male sterile cytoplasms. Crop Sci 22:414–421Google Scholar
  28. Pring DR, Gengenbach BG, Wise RP (1988) Recombination is associated with polymorphism of the mitochondrial genomes of maize and sorghum. Phils Trans R Soc Lond Ser B 319: 187–198Google Scholar
  29. Saleh NM, Gupta HS, Finch RP, Cocking EC, Mulligan BJ (1990) Stability of mitochondrial DNA in tissue-cultured cells of rice. Theor Appl Genet 79:342–346Google Scholar
  30. Shirzadegan M, Christey M, Earle ED, Palmer JD (1989) Rearrangement, amplification, and assortment of mitochondrial DNA molecules in cultured cells of Brassica campestris. Theor Appl Genet 77:17–25Google Scholar
  31. Shirzadegan M, Christey M, Earle ED, Palmer JD (1991) Patterns of mitochondrial DNA instability in Brassica campestris cultured cells. Plant Mol Biol 16:21–37Google Scholar
  32. Small ID, Isaac PG, Leaver CJ (1987) Stoichiometric differences in DNA molecules containing the atpA gene suggest mechanisms for the generation of mitochondrial genome diversity in maize. EMBO J 6:865–869Google Scholar
  33. Small ID, Earle ED, Escote-Carlson LJ, Gabay-Laughnan S, Laughnan JR, Leaver CJ (1988) A comparison of cytoplasmic revertants to fertility from different CMS-S maize sources. Theor Appl Genet 76:609–618Google Scholar
  34. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  35. Wilson AJ, Chourey PS (1984) A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources. Plant Cell Rep 3:237–239Google Scholar
  36. Wilson AJ, Chourey PS, Sharpe DZ (1985) Protoclones of Sorghum bicolor with unusually high mitochondrial DNA variation. In: Henke RR, Hughes KW, Constantin MJ, Hollaender A (eds) Tissue culture in forestry and agriculture. Plenum, New York, pp 368–369Google Scholar
  37. Zack CD, Chourey PS (1985) Molecular characterization of a region of mitochondrial DNA which is hypervariable in cultured cells of Sorghum bicolor cv NK300. In: Abstr 1st Int Congr Plant Mol Biol Oct 27–Nov 2, Savannah/GA, pp 105Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • E. J. Kane
    • 1
  • A. J. Wilson
    • 1
  • P. S. Chourey
    • 1
    • 2
    • 3
  1. 1.Department of Plant PathologyGainesvilleUSA
  2. 2.Department of AgronomyUniversity of FloridaGainesvilleUSA
  3. 3.USDA/ARSGainesvilleUSA

Personalised recommendations