Plant Molecular Biology Reporter

, Volume 17, Issue 3, pp 249–254 | Cite as

Modified CTAB Procedure for DNA Isolation from Epiphytic Cacti of the Genera Hylocereus and Selenicereus (Cactaceae)

  • N. Tel-zur
  • S. Abbo
  • D. Myslabodski
  • Y. Mizrahi
Article

Abstract

We present a simple protocol for DNA isolation from climbing cacti, genera Hylocereus and Selenicereus. The abundant polysaccharides present in Hylocereus and Selenicereus species interfere with DNA isolation, and DNA extracts, rich in polysaccharides, are poor templates for amplification using polymerase chain reaction (PCR). We used roots as the source tissue due to the lower viscosity of the extracts relative to that of other tissues. The extraction and isolation procedure we devised consists of the following steps: (1) three washes of ground tissue with the extraction buffer to remove the polysaccharides; (2) extraction with high-salt (4 M NaCl) cetyltrimethylammonium bromide (CTAB) buffer to remove the remaining polysaccharides; (3) removal of RNA by RNase; (4) phenol:chloroform extraction to remove proteins; (5) chloroform extraction to remove remaining phenols. The yields ranged from 10 to 20 μg DNA/g fresh roots. DNA samples prepared by our method were consistently amplifiable in the RAPD reaction and gave reproducible profiles.

cacti DNA extraction Hylocereus polysaccharides Selenicereus 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    An G: Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoters in transformed tobacco cells. Plant Physiol 81: 86–91 (1986).Google Scholar
  2. 2.
    Anderson MD, Chen Z, Klessig DF: Possible involvement of lipid peroxidation in SA-mediated induction of PR-1 gene expression. Phytochemistry 47: 555–566 (1998).Google Scholar
  3. 3.
    Bi Y-M, Kenton P, Mur L, Darby R, Draper J: Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression. Plant J 8: 235–245 (1995).Google Scholar
  4. 4.
    Bilha B, Kadish D, Levy Y., Cohen Y: Infectivity to potato, sporangial germination, and respiration of isolates of Phytophthora infestans from metalaxy-sensitive and metalaxyresistant populations. Phytopathology 79: 823–836 ( 1989).Google Scholar
  5. 5.
    Bradley DJ, Kjellbom P, Lamb C: Elicitor-and woundinduced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell 70: 21–30 (1992).Google Scholar
  6. 6.
    Burnett WV: Northern blotting of RNA denatured in glyoxal without buffer recirculation. BioTechniques 22: 668–671 (1997).Google Scholar
  7. 7.
    Cao H, Glazebrook J, Clarke JD, Volko S, Dong X: The Arabidopsis NPR 1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88: 57–63 (1997).Google Scholar
  8. 8.
    Chamnongpol S, Willekens H, Langebartels C, Van Montagu M, Inzé D, Van Camp W: Transgenic tobacco with a reduced catalase activity develop necrotic lesions and have constitutive pathogenesis-related gene expression under high light. Plant J 10: 491–503 (1996).Google Scholar
  9. 9.
    Chen Z, Klessig DF: Identification of a soluble salicylic acidbinding protein that may function in the signal transduction in the plant disease resistance response. Proc Natl Acad Sci USA 88: 8179–8183 (1991).Google Scholar
  10. 10.
    Chen Z, Lyer S, Caplan A, Klessig DF, Fan B: Differential accumulation of salicylic acid and salicylic acid-sensitive catalase in different rice tissues. Plant Physiol 114: 193–201 (1997).Google Scholar
  11. 11.
    Chen Z, Ricigliano JW, Klessig DF: Purification and characterization of a soluble salicylic acid-binding protein from tobacco. Proc Natl Acad Sci USA 90: 9533–9537 (1993).Google Scholar
  12. 12.
    Chen Z, Silva H, Klessig DF: Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 262: 1883–1886 (1993).Google Scholar
  13. 13.
    Conrath U, Chen Z, Ricigliano JW, Klessig DF: Two inducers of plant defence responses, 2,6-dichloroisonicotinic acid and salicylic acid, inhibit catalase activity in tobacco. Proc Natl Acad Sci USA 92: 7143–7147 (1995).Google Scholar
  14. 14.
    Coquoz JL, Buchala AJ, Meuwly PH, Metraux JP: Arachidonic acid induces local but not systemic synthesis of salicylic acid and confers systemic resistance to potato plants to Phytophthora infestans and Alternaria solani. Phytopathology 85: 1219–1224 (1995).Google Scholar
  15. 15.
    Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19–21 (1983).Google Scholar
  16. 16.
    Drory A, Woodson WR: Molecular cloning and nucleotide sequence of a cDNA encoding catalase from tomato. Plant Physiol 100: 1605–1606 (1992).Google Scholar
  17. 17.
    Du H, Klessig DF: Identification of a soluble, high affinity salicylic acid-binding protein in tobacco. Plant Physiol 113: 1319–1327 (1997).Google Scholar
  18. 18.
    Durner J, Klessig DF: Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses. Proc Natl Acad Sci USA 92: 11312–11316 (1995).Google Scholar
  19. 19.
    Durner J, Klessig DF: Salicylic acid is a modulator of tobacco and mammalian catalases. J Biol Chem 27: 28492–28501 (1996).Google Scholar
  20. 20.
    Enyedi A, Yalpani N, Silverman P, Rasin I: Localization, conjugation and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus. Proc Natl Acad Sci USA 89: 2480–2484 (1992).Google Scholar
  21. 21.
    Fauth M, Merten A, Hahn M, Jeblick W, Kauss H: Competence for elicitation of H2O2 in hypocotyls of cucumer is induced by breaching the cuticle and is enhanced by salicylic acid. Plant Physiol 110: 347–354 (1996).Google Scholar
  22. 22.
    Halliwell B, Gutteridge JMC: Free Radicals in Biology and Medicine. Oxford University Press, Oxford, UK (1989).Google Scholar
  23. 23.
    Kauss H, Jeblick W: Pretreatment of parsley suspension cultures with salicylic acid enhances spontaneous and elicited production of H2O2. Plant Physiol 108: 1171–1178 (1995).Google Scholar
  24. 24.
    Kauss H, Jeblick W: Influence of salicylic acid on the induction of competence for H2O2 elicitation. Plant Physiol 111: 755–763 (1996).Google Scholar
  25. 25.
    Kawano T, Sahashi N, Takahashi K, Uozumi N, Muto S: Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: the earliest events in salicylic acid signal transduction. Plant Cell Physiol 39: 721–730 (1998).Google Scholar
  26. 26.
    Leon J, Lawton MA, Raskin I: Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol 108: 1673–1678 ( 1995).Google Scholar
  27. 27.
    Levine A, Tenhaken R, Dixon R, Lamb C: H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79: 583–593 (1994).Google Scholar
  28. 28.
    Liu D., Raghothama KG, Hasegawa P, Bressan RA: Osmotin overexpression in potato delays development of disease symptoms. Proc Natl Acad Sci USA 91: 1881–1892 (1994).Google Scholar
  29. 29.
    Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNA from plant tissues. Anal Biochem 163: 16–20 (1987).Google Scholar
  30. 30.
    Malamy J, Carr J.P, Klessig DF, Raskin I: Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science 250: 1002–1004 (1990).Google Scholar
  31. 31.
    Malamy J, Hennig J, Klessig DF: Temperature-dependent induction of salicylic acid and its conjugates during the resistance response to tobacco mosaic virus infection. Plant Cell 4: 359–366 (1992).Google Scholar
  32. 32.
    Metraux JP, Signer H, Ryals JA, Ward E, Wyss-Benz M, Gaudin J, Raschdorf K, Schmid E, Blum W, Inveradi B: Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250: 1004–1006 (1990).Google Scholar
  33. 33.
    Mur LA, Naylor G, Warner SAJ, Sugars JM, White RF, Draper J: Salicylic acid potentiates defense gene expression in leaf tissue exhibiting acquired resistance to pathogen attack. Plant J 9: 559–571 (1996).Google Scholar
  34. 34.
    Neuenschwander U., Vernooij B., Friedrich L., Uknes S., Kessmann H, Ryals J: Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance. Plant J 8: 227–233 (1995).Google Scholar
  35. 35.
    Niebel A., Heungens K., Barthels N., Inzé D., Van Montagu M, Gheysen G: Characterization of a pathogen-induced potato catalase and its systemic expression upon nematode and bacterial infection. Mol Plant-Microbe Interact 8: 371–378 (1995).Google Scholar
  36. 36.
    Raskin I, Skubatz H, Tang W, Meeuse BJD: Salicylic acid levels in thermogenic and nonthermogenic plants. Ann Bot 66: 369–373 (1990).Google Scholar
  37. 37.
    Rasmussen JB, Hammerschmidt R, Zook MN: Systemic induction of salicylic acid accumulation in cucumber after inoculation with Psedomonas syringae pv. syringae. Plant Physiol 97: 1342–1347 (1991).Google Scholar
  38. 38.
    Ruffer M, Steipe B, Zenk MH: Evidence against specific binding of salicylic acid to plant catalase. FEBS Lett 377: 175–180 (1995).Google Scholar
  39. 39.
    Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD: Systemic acquired resistance. Plant Cell 8: 1809–1819 (1996).Google Scholar
  40. 40.
    Ryals J, Weymann K, Lawton K, Friedrich L, Ellis D, Steiner HY, Johnson J, Delaney TP, Jesse T, Vos P, Uknes S: The Arabidopsis thaliana NIM1 protein shows homology to the mammalian transcription factor inhibitor I_B. Plant Cell 9: 425–439 (1997).Google Scholar
  41. 41.
    Shah J., Tsui F, Klessig DF: Characterization of a salicylic acid-insensitive mutant (sai1) of Arabidopsis thaliana identi-fied in a selective screening utilizing the SA-inducible expression of the tms2 gene. Mol Plant-Microbe Interact 10: 69–76 (1997).Google Scholar
  42. 42.
    Shirasu K, Nakajima H, Rajasekhar VY, Dixon RA, Lamb C: Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms. Plant Cell 9: 261–270 (1996).Google Scholar
  43. 43.
    Takahashi H, Chen Z, Du H, Liu Y, Klessig D.F: Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels. Plant J 11: 993–1005 (1997).Google Scholar
  44. 44.
    Timmermans MCP, Maliga P, Veira J, Messing, J: The pFF plasmids: cassettes utilising CaMV sequences for expression of foreign genes in plants. J Biotechnol 14: 333–344 (1990).Google Scholar
  45. 45.
    Tooley PW, Swelgard JA, Fry WE: Fitness and virulence of Phytophthora infestans isolates from sexual and asexual populations. Phytopathology 76: 1209–1212 (1980).Google Scholar
  46. 46.
    Uknes S, Winter A, Delaney T, Vernooij B, Morse A, Friedrich L, Potter S, Slusarenko A, Ward E, Ryals J: Biological in488 duction of systemic acquired resistance in Arabidopsis. Mol Plant-Microbe Interact 6: 680–685 (1993).Google Scholar
  47. 47.
    Vernooij B, Friedrich L, Morse A, Reist R, Koldttz-Jawhar R, Ward E, Uknes S, Kessmann H, Ryals J: Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. Plant Cell 6: 959–969 (1994).Google Scholar
  48. 48.
    Wendehenne D, Durner J, Chen Z, Klessig DF: Benzothiadiazole, an inducer of plant defenses, inhibits catalases and ascorbate peroxidase. Phytochemistry 47: 651–657 (1998).Google Scholar
  49. 49.
    Wenzler HC, Mignery G, May G, Park WD: A rapid and efficient transformation method for the production of large numbers of transgenic potato plants. Plant Sci 63: 79–85 (1989).Google Scholar
  50. 50.
    Weyman K, Hunt M, Uknes S, Neuenschwander U, Lawton K, Steiner H, Ryals J: Suppression and restoration of lesion formation in Arabidopsis Isd mutants. Plant Cell 7: 2013–2022 (1995).Google Scholar
  51. 51.
    Willekens H, Villarroel, Van Camp W, Van Montagu M, Inzé D: Molecular identification of catalases from Nicotinana plumbaginfolia (L). FEBS Lett 352: 79–83 (1994).Google Scholar
  52. 52.
    Willekens H, Langebartels C, Tire C, Van Montagu M, Inzé D, Van Camp W: Differential expression of catalase genes in Nicotinana plumbagiifolia. Proc Natl Acad Sci USA 91: 10450–10454 (1994).Google Scholar
  53. 53.
    Wu G, Shortt BJ, Lawrence EB, Levine EB, Fitzsimmons KC, Shah DM: Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants. Plant Cell 7: 1357–1368 (1995).Google Scholar
  54. 54.
    Yu D, Liu Fan B, Klessig DF, Chen Z: Is the high basal level of salicylic acid important for disease resistance in potato? Plant Physiol 115: 343–349 (1997).Google Scholar
  55. 55.
    Zhu B, Chen THH, Li PH: Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein. Planta 198: 70–77 (1996).Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • N. Tel-zur
    • 1
  • S. Abbo
    • 2
  • D. Myslabodski
    • 3
  • Y. Mizrahi
    • 1
  1. 1.Department of Life SciencesBen-Gurion University of the NegevBeer-ShevaIsrael
  2. 2.Faculty of Agriculture, Food and Environmental Quality SciencesThe Hebrew UniversityRehovotIsrael
  3. 3.Israel Oceanographic and Limnological Research, Ltd. Tel ShikmonaHaifaIsrael

Personalised recommendations