Planta

, Volume 190, Issue 2, pp 190–198 | Cite as

Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes

  • K. S. Ramulu
  • P. Dijkhuis
  • I. Famelaer
  • T. Cardi
  • H. A. Verhoeven
Article
Received:
Accepted:

Abstract

Results on the enhancement of the frequency of protoplasts with micronuclei, and on the isolation and enrichment of smaller sub-diploid microprotoplasts in transformed Nicotiana plumbaginifolia Viv. are reported. Suspension cells were treated with the spindle toxin amiprophos-methyl (APM) for 48 h, and subsequently incubated in a mixture of cell-wall-digesting enzymes in the presence of APM and cytochalasin-B. During enzyme incubation, the frequency of micronucleated protoplasts increased by a factor of 2–6. A shorter period (3 h) of incubation with a higher concentration of enzymes as well as a longer period (16 h) of incubation with a lower concentration of enzymes gave similar frequencies of micronucleated protoplasts and yields of micronuclei. Further, synchronization by sequential treatment with the DNA-synthesis inhibitor hydroxy urea, or aphidicolin, followed by APM and enzyme incubation, significantly increased the frequency of micronucleated protoplasts and the number of micronuclei. The suspension of protoplasts (mono- and micronucleated) obtained after enzyme incubation was fractionated throuh a continuous iso-osmotic gradient of Percoll, using high-speed centrifugation. This resulted in one large and a few small bands, which contained a heterogeneous population of microprotoplasts, protoplasts and cytoplasts. In contrast to the large band, the small bands contained a relatively higher frequency of small sub-diploid microprotoplasts. To separate the small sub-diploid microprotoplasts from the large microprotoplasts and protoplasts of the bands, discontinuous Percoll gradients and sequential filtration through nylon sieves of decreasing pore size (48-20-15-10-5 μm) were investigated. Compared with the former method, the latter gave a highly enriched fraction containing predominantly (≈80%) small subdiploid microprotoplasts with DNA contents equivalent to that of one to four chromosomes, as revealed by microdensitometric and flow-cytometric analyses. The application of this technique for partial genome and limited gene transfer is discussed.

Key words

Amiprophos-methyl Cytochalasin-B Genome (partial, transfer) Microprotoplast Nicotiana Synchronization 

Abbreviations

APH

aphidicolin

APM

amiprophos-methyl

Au

arbitrary units

CB

cytochalasin B

DAPI

4,6-diamidino-2-phenylindole

FD

fluorescein diacetate

HU

hydroxy urea

This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Athwal, R.S., Dhar, V. (1984) Transfer of specific chromosomes by microcell fusion and gene mapping in mammalian cells. In: Gene transfer and cancer, pp. 21–29, Pearson, M.L., Sternberg, N.L., eds. Raven Press, New YorkGoogle Scholar
  2. Barfield, D.G., Robinson, S.J., Shields, R. (1985) Plant regeneration from protoplasts of long term haploid suspension cultures of N. plumbaginifolia. Plant Cell Rep. 4, 104–107Google Scholar
  3. Bokelmann, G.S., Roest, S. (1983) Plant regeneration from protoplasts of potato (Solanum tuberosum cv. Bintje). Z. Pflanzenphysiol. 109, 259–265Google Scholar
  4. De Laat, A.M.M., Blaas, J. (1987) An improved method for protoplast microinjection suitable for transfer of entire plant chromosomes. Plant Sci. 50, 161–169Google Scholar
  5. De Laat, A.M.M., Verhoeven H.A., Ramulu, K.S., Dijkhuis, P. (1987) Efficient induction by amiprophos-methyl and flow cytometric sorting of micronuclei in Nicotiana plumbaginifolia. Planta 172, 473–478Google Scholar
  6. Ege, T., Ringertz, N.R., Hamber, H., Sidebottom, E. (1977) Preparation of microcells. In: Methods in cell biology, 15, pp. 339–358, Prescott, D.M., ed. Academic Press, New YorkGoogle Scholar
  7. Famelaer, I., Negrutiu, I., Mouras, A., Vaucheret, H., Jacobs, M. (1990) Asymmetric hybridization in Nicotiana by “gamma fusion” and progeny analysis of self-fertile hybrids. Theor. Appl. Genet. 79, 513–520Google Scholar
  8. Fournier, R.E.K. (1982) Microcell-mediated chromosome transfer. In: Techniques in somatic cell genetics, pp. 309–327, Shay, J.W., ed. Plenum Press, New York, LondonGoogle Scholar
  9. Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., Firoozabady, E. (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051Google Scholar
  10. Gasser, G.S., Fraley, R.T. (1989) Genetically engineered plants for crop improvement. Science 244, 1293–1299Google Scholar
  11. Gilissen, L.J.W., Van Staveren, M.J., Verhoeven, H.A., Ramulu, K.S. (1992) Somatic hybridization between potato and Nicotiana plumbaginifolia. 1. Spontaneous biparental chromosome elimination and production of asymmetric hybrids. Theor. Appl. Genet. 84, 73–80Google Scholar
  12. Gleba, Y.Y., Sytnik, K.M. (1984) Protoplast fusion — Genetic engineering in higher plants. In: Monographs on theoretical and applied genetics, vol. 8, pp. 1–220, Shoeman, R., ed. Springer, Berlin, HeidelbergGoogle Scholar
  13. Landolph, J.R., Fournier, R.E.K. (1983) Microcell-mediated transfer of carcinogen-induced ouabain resistance from C3H/10 T1/2C1 8 mouse fibroblasts to human cells. Mut. Res. 107, 447–463Google Scholar
  14. Lugo, T.G., Fournier, R.E.K. (1986) Microcell fusion and mammalian gene transfer. In: Gene transfer, pp. 79–93, Kucherlapati, R., ed. Plenum publishing Corp., New YorkGoogle Scholar
  15. Negrutiu, I., Mouras, A., Horth, M., Jacobs, M. (1987) Direct gene transfer to plants: present developments and some future prospectives. Plant Physiol. Biochem. 25, 493–503Google Scholar
  16. Piastuch, W.G., Bates, G.W. (1990) Chromosomal analysis of Nicotiana asymmetric somatic hybrids by dot blotting and in situ hybridization. Mol. Gen. Genet. 222, 97–103Google Scholar
  17. Potrykus, I. (1990) Gene transfer to cereals: an assessment. Bio Technology 8, 535–542Google Scholar
  18. Ramulu, K.S., Dijkhuis, P., Roest, S., Bokelmann, G.S., De Groot, B. (1984) Early occurrence of genetic instability in protoplast cultures of potato. Plant Sci Lett. 36, 79–86Google Scholar
  19. Ramulu, K.S., Verhoeven, H.A., Dijkhuis, P. (1988) Mitotic dynamics of micronuclei induced by amiprophos-methyl and prospects for chromosome-mediated gene transfer in plants. Theor. Appl. Genet. 75, 575–584Google Scholar
  20. Ramulu, K.S., Verhoeven, H.A., Dijkhuis, P., Gilissen, L.J.W. (1990) A comparison of APM-induced micronucleation and influence of some factors in various genotypes of potato and Nicotiana. Plant Sci. 69, 123–133Google Scholar
  21. Ramulu, K.S., Verhoeven, H.A., Dijkhuis, P. (1991) Mitotic blocking, micronucleation and chromosome doubling by oryzalin, amiprophos-methyl and colchicine in potato. Protoplasma 160, 65–71Google Scholar
  22. Ramulu, K.S., Dijkhuis, P., Verhoeven, H.A., Famelaer, I., Blaas, J. (1992) Microprotoplast isolation, enrichment and fusion for partial genome transfer in plants. Physiol. Plant. 85, 315–318Google Scholar
  23. Simmonds, D.H. (1991) Microtubules in cultured plant protoplasts. Acta Bot. Neerl. 40, 183–195Google Scholar
  24. Sybenga, J. (1989) Genetic manipulation. Generative versus somatic. In: Biotechnology in agriculture and forestry, 9: Plant protplasts and genetic engineering II, pp. 26–53, Bajaj, Y.P.S. ed. Springer, HeidelbergGoogle Scholar
  25. Tempelaar, M.J., Drenth-Diephuis, L.J., Saat, T.A.W.M., Jacobsen, E. (1991) Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia. Euphytica 56, 287–296Google Scholar
  26. Traas, J.A. (1990) The plasma membrane-associated cytoskeleton. In: The plant plasma membrane, pp. 269–292, Larsson, C., Moller, I.M., eds. Springer, Berlin, HeidelbergGoogle Scholar
  27. Verhoeven, H.A., Ramulu, K.S. (1991) Isolation and characterization of microprotoplasts from APM treated suspension cells of Nicotiana plumbaginifolia. Theor. Appl. Genet. 82, 346–352Google Scholar
  28. Verhoeven, H.A., Ramulu, K.S., Dijkhuis, P. (1990) Comparison of the effects of various spindle toxins on metaphase arrest and formation of micronuclei in cell-suspension cultures of Nicotiana plumbaginifolia. Planta 182, 408–414Google Scholar
  29. Verhoeven, H.A. Ramulu, K.S., Gilissen, L.J.W., Famelaer, I., Dijkhuis, P., Blaas, J. (1991a) Partial genome transfer through micronuclei in plants. Acta Bot. Neerl. 40, 97–113Google Scholar
  30. Verhoeven, H.A., Ramulu, K.S., Blaas, J., Dijkhuis, P. (1991b) Control of cell cycle progression. In: A laboratory guide for cellular and molecular plant biology, pp. 346–355, Negrutiu, I., Ghatri-Chhetri, G.B., eds. Birkhäuser, BaselGoogle Scholar
  31. Wallin, A., Waara, S., Eriksson, T. (1989) Miniprotoplasts and their use in genetic engineering. In: Biotechnology in agriculture and forestry, 9: Plant protoplasts and genetic engineering II, pp. 480–493, Bajaj, Y.P.S., ed. Springer, Berlin, HeidelbergGoogle Scholar
  32. Wijbrandi, J., Zabel, P., Koornneef, M. (1990) Restriction fragment length polymorphism analysis of somatic hybrids between Lycopersicon esculentum and irradiated L. peruvianum: Evidence for limited donor genome elimination and extensive chromosome rearrangements. Mol. Gen. Genet. 222, 270–277Google Scholar
  33. Wolters, A.M.A., Schoenmakers, H.C.H., Van der Meulen-Muisers, J.J.M., Van der Knaap, E., Derks, F.H.M., Koornneef, M., Zelcer, A. (1991) Limitied DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum tuberosum. Theor. Appl. Genet. 83, 225–232Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • K. S. Ramulu
    • 1
  • P. Dijkhuis
    • 1
  • I. Famelaer
    • 1
  • T. Cardi
    • 2
  • H. A. Verhoeven
    • 1
  1. 1.Department of Cell BiologyDLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO)AA WageningenThe Netherlands
  2. 2.Research Centre for Vegetable BreedingNational Research Council (CNR)PorticiItaly

Personalised recommendations