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Cryopreservation of Embryogenic Culture of Pinus roxburghii

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Biologia Plantarum

Abstract

Embryogenic cultures of chir pine (Pinus roxburghii Sarg.) were cryopreserved successfully in liquid nitrogen. It was found that using sorbitol and dimethyl sulfoxide (DMSO) as cryoprotectants was essential for the survival of the tissue. Among the different concentrations of the cryoprotectants used, the most effective treatment was observed to be 0.3 M sorbitol and 5 % DMSO. On staining the cryopreserved tissue with fluorescein diacetate, it was observed that only a few meristematic embryo heads survived and resumed growth after a very short initial lag phase. The recovered cultures showed normal regrowth on proliferation medium and, it was also observed that washing off the cryoprotectants was necessary for the cultures to survive. The results indicate that cryopreservation can be used for conserving the germplasm, and in maintaining the embryogenic capacity of the tissue.

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References

  • Aronen, T.S., Krajnakova, J., Häggman, H.M., Ryynänen, L.A.: Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica.-Plant Sci. 142: 163–172, 1999.

    Article  CAS  Google Scholar 

  • Arya, S., Kalia, R.K., Arya, I.D.: Induction of somatic embryogenesis in Pinus roxburghii Sarg.-Plant Cell Rep. 19: 775–780, 2000.

    Article  CAS  Google Scholar 

  • Attree, S.M., Fowke, L.C.: Embryogeny of gymnosperms: advances in synthetic seed technology of conifers.-Plant Cell Tissue Organ Cult. 35: 1–35, 1993.

    Article  CAS  Google Scholar 

  • Cyr, D.R., Lazaroff, W.R., Grimes, S.M.A., Quan, G., Bethune, T.D., Dunstan, D.I., Roberts, D.R.: Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures.-Plant Cell Rep. 13: 574–577, 1994.

    Article  Google Scholar 

  • DeVerno, L.L., Park, Y.S., Bonga, J.M., Barrett, J.D.: Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.].-Plant Cell Rep. 18: 948–953, 1999.

    Article  CAS  Google Scholar 

  • Eastman, P.A.K., Webster, F.B., Pitel, J.A., Roberts, D.R.: Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea engelmannii complex) using culture morphology and isozyme analysis.-Plant Cell Rep. 10: 425–430, 1991.

    Article  Google Scholar 

  • Find, J.I., Floto, F., Krogstrup, P., Moller, J.D., Norgaard, J.V., Kristensen, M.M.H.: Cryopreservation of an embryogenic suspension culture of Picea sitchensis and subsequent plant regeneration.-Scand. J. Forest Res. 8: 156–162, 1993.

    Article  Google Scholar 

  • Ford, C.S., Jones, N.B., Van Staden, J.: Cryopreservation and plant regeneration from somatic embryos of Pinus patula.-Plant Cell Rep. 19: 610–615, 2000.

    Article  CAS  Google Scholar 

  • Fourré, J.L., Berger, P., Niquet, L., André, P.: Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenesis, cytogenetic and molecular approaches.-Theor. appl. Genet. 94: 159–169, 1997.

    Article  Google Scholar 

  • Gupta, P. K., Durzan, D.J.: Shoot multiplication from mature trees of Douglas fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana).-Plant Cell Rep. 4: 177–179, 1985.

    Article  CAS  Google Scholar 

  • Gupta, P.K., Durzan, D.J., Finkle, B.J.: Somatic embryogenesis in embryogenic cell masses of Picea abies (Norway spruce) and Pinus taeda (loblolly pine) after thawing from liquid nitrogen.-Can. J. Forest Res. 17: 1130–1134, 1987.

    Google Scholar 

  • Gupta, P.K., Grob, J.A.: Somatic embryogenesis is conifers.-In: Jain, S.M., Gupta, P.K., Newton, R.J. (ed.): Somatic Embryogenesis in Woody Plants. Vol. 1. Pp. 81–98. Kluwer Academic Publishers, Dordrecht 1995.

    Google Scholar 

  • Häggman, H.M., Ryynänen, L.A., Aronen, T.S., Krajnakova, J.: Cryopreservation of embryogenic cultures of Scots pine.-Plant Cell Tissue Organ Cult. 54: 45–53, 1998.

    Article  Google Scholar 

  • Isabel, N., Tremblay, L., Michaud, M., Tremblay, F.M., Bosquet, J.: RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana.-Theor. appl. Genet. 86: 81–87, 1993.

    Article  CAS  Google Scholar 

  • Kartha, K.K., Fowke, L.C., Leung, N.L., Caswell, K.L., Hakman, I.: Induction of somatic embryos and plantlets from cryopreserved cell cultures of white spruce (Picea glauca).-Plant Physiol. 132: 529–539, 1988.

    CAS  Google Scholar 

  • Klimaszewska, K., Ward, C., Cheliak, W.M.: Cryopreservation and plant regeneration from embryogenic cultures of larch (Larix × eurolepis) and black spruce (Picea mariana).-J. exp. Bot. 43: 73–79, 1992.

    CAS  Google Scholar 

  • Laine, E., Bade, P., David, A.: Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea.-Plant Cell Rep. 11: 295–298, 1992.

    Article  Google Scholar 

  • Mathur, G., Von Arnold, S., Nadgauda, R.S.: Studies on somatic embryogenesis from immature zygotic embryos of chir pine (Pinus roxburghii Sarg.).-Curr. Sci. 79: 999–1004, 2000.

    Google Scholar 

  • Nørgaard, J.V., Baldursson, S., Krogstrup, P.: Genotypic differences in the ability of embryogenic Abies normanniana cultures to survive cryopreservation.-Silvae Genet. 42: 93–97, 1993a.

    Google Scholar 

  • Nørgaard, J.V., Duran, V., Johnsen, Ø., Krogstrup, P., Baldursson, S., Von Arnold, S.: Variations in cryotolerance of embryogenic Picea abies cell lines and the association to genetic, morphological, and physiological factors.-Can. J. Forest Res. 23: 2560–2567, 1993b.

    Google Scholar 

  • Tewari, D.N.: A Monograph on Chir Pine (Pinus roxburghii Sarg.).-Vedam Books International, New Delhi 1994.

    Google Scholar 

  • Timmis, R.: Bioprocessing for the tree production in the forest industry: conifer somatic embryogenesis.-Biotechnol. Progr. 14: 156–166, 1998.

    Article  CAS  Google Scholar 

  • Widholm, J.M.: The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells.-Stain Technol. 47: 189–194, 1972.

    PubMed  CAS  Google Scholar 

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Mathur, G., Alkutkar, V. & Nadgauda, R. Cryopreservation of Embryogenic Culture of Pinus roxburghii . Biologia Plantarum 46, 205–210 (2003). https://doi.org/10.1023/A:1022894409408

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