Single-cell cloning of a crown gall from protoplasts regenerated in hormone-free medium. Establishment of pure transformed cell-lines of Catharanthus roseus |
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Authors: | J Trémouillaux-Guiller H Kodja J C Chénieux |
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Institution: | (1) Biologie Cellulaire et Biochimie Végétale EA 1370 DRED-Faculté de Pharmacie, 31 Avenue Monge, 37200 Tours Cédex, France |
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Abstract: | Protoplasts enzymatically isolated from cell line of Catharanthus roseus G. Don crown gall, were cultured at high density (105 P ml-1) in modified B5 liquid medium (Gamborg et al. 1976). In the absence of growth regulators C. roseus protoplasts were able to regenerate a cell-wall, divide and, subsequently, yield very numerous clones in the absence of growth regulators. After two weeks, the cultures were greatly diluted in order to obtain clones of single-cell origin. Most of the clones individually transferred onto solid medium can proliferate indefinitely, without growth regulators. Among analyzed clones, 90% were nopaline positive. Their ajmalicine and serpentine content was compared with that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting model for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or for the delicate culture of tree protoplasts.Abbreviations B5
Gamborg et al. (1976) medium
- 2,4-d
2,4-dichlorophenoxyacetic acid
- Kin
Kinetin
- NAA
naphthalene acetic acid
- BA
N6 (benzyl) adenine |
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Keywords: | Catharanthus roseus crown gall tumor heterokaryons protoplasts |
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