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.
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Abbreviations
- APH:
-
aphidicolin
- APM:
-
amiprophos-methyl
- Au:
-
arbitrary units
- CB:
-
cytochalasin B
- DAPI:
-
4,6-diamidino-2-phenylindole
- FD:
-
fluorescein diacetate
- HU:
-
hydroxy urea
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Our thanks are due to Professor J. Sybenga, department of Genetics, Agricultural University, Wageningen, The Netherlands and Dr. C.M. Colijn-Hooymans, CPRO-DLO, Wageningen, for critically reading the manuscript and to Ms. Gea Jonker and Ms. Tineke Lubout for typing the manuscript.
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Ramulu, K.S., Dijkhuis, P., Famelaer, I. et al. Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes. Planta 190, 190–198 (1993). https://doi.org/10.1007/BF00196611
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DOI: https://doi.org/10.1007/BF00196611