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Liquid Culture Systems for in vitro Plant Propagation

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Potential of flow cytometry 343<br />

that even though regeneration decreases, aberrant plants might still be<br />

regenerated. Screen<strong>in</strong>g <strong>for</strong> off-types is there<strong>for</strong>e necessary at an early stage<br />

of the somatic embryogenesis process.<br />

Flow cytometry could also be used to analyse the cell cycle k<strong>in</strong>etics by<br />

calculat<strong>in</strong>g the ratio of cells <strong>in</strong> the G2 and G1 phase of the cell cycle. The<br />

majority of cells are <strong>in</strong> the G1 phase dur<strong>in</strong>g the first 3 days after subculture.<br />

This is important to determ<strong>in</strong>e the most appropriate moment <strong>for</strong> mutagenic<br />

treatment or <strong>for</strong> genetic trans<strong>for</strong>mation but also as an <strong>in</strong>dicator on the<br />

proportion of cycl<strong>in</strong>g cells. In addition, the unicellular orig<strong>in</strong> of somatic<br />

embryos was verified by treat<strong>in</strong>g embryogenic cell suspensions with<br />

colchic<strong>in</strong>e and by determ<strong>in</strong><strong>in</strong>g the ploidy of regenerated plants by flow<br />

cytometric analysis.<br />

To conclude, our results prove that ploidy levels of Musa embryogenic<br />

cell suspension cultures can easily be determ<strong>in</strong>ed us<strong>in</strong>g flow cytometry. The<br />

results of this study also <strong>in</strong>dicate that <strong>in</strong> <strong>vitro</strong> cultures may become<br />

mixoploid <strong>in</strong> a relatively short period and that abnormal ploidy levels<br />

co<strong>in</strong>cide with poor regeneration abilities. To develop protocols, which avoid<br />

variation at least <strong>in</strong> DNA content, we have now a simple and highthroughput<br />

assay at hand that can be applied immediately after tissue culture<br />

<strong>in</strong>itiation and that gives representative data on ploidy level of cultured cells.<br />

Acknowledgements<br />

This work was supported by a Jo<strong>in</strong>t FAO/IAEA/DGDC (Belgian General<br />

Direction <strong>for</strong> International Cooperation) Coord<strong>in</strong>ated Research Project. The<br />

study was undertaken as part of the Global Programme <strong>for</strong> Musa<br />

Improvement (PROMUSA).<br />

References<br />

Dhed’a D, Dumortier F, Panis B, Vuylsteke D & De Langhe E (1991) <strong>Plant</strong> regeneration <strong>in</strong><br />

cell suspension cultures of the cook<strong>in</strong>g banana cv. ‘Bluggoe’ (Musa spp. ABB group).<br />

Fruits 46: 125-135<br />

Doležel J (1997) Cytological evidence of spontaneous and <strong>in</strong>duced mutations and<br />

cytogenetics techniques. 15th IAEA/FAO Interregional tra<strong>in</strong><strong>in</strong>g course on advances <strong>in</strong><br />

technologies <strong>for</strong> <strong>in</strong>duced mutations <strong>in</strong> crops. IAEA, Vienna<br />

Kubalakova M, Dolezel J & Lebeda A (1996) Ploidy <strong>in</strong>stability of embryogenic cucumber<br />

(Cucumis sativus L.) callus culture. Biol. <strong>Plant</strong>. 38: 475-480<br />

Panis B, Totte N, Van Nimmen K, Withers LA & Swennen R (1996) Cryopreservation of<br />

banana (Musa spp.) meristems cultures after preculture on sucrose. <strong>Plant</strong> Sciences<br />

21: 95-106

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