Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album
- Free Univ. of Brussels (Belgium); and others
The preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described. The principle has been to maintain transformed roots in vitro, synchronize and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes. Such in vitro material, unlike many cell suspensions, showed a stable karyotype. Factors critical to producing high-quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer. Agrobacterium rhizogenes transformed young growing root cultures were synchronized at G1/S with 50 {mu}M aphidicolin for 24 h and released to a mitotic block with 30 {mu}M oryzalin for 11 h. Protoplast preparations from such tissue routinely had metaphase indices reaching 15%. Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acid/disodium phosphate buffer. Except for the presence of clumps of autosomal chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4{prime},6-diamidino-2-phenylindole showed profiles similar to theoretical flow karyotypes. Two types of Y chromosomes, one full-length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21-fold enrichment of Y). These results are discussed in the context of sex determination and differentiation in higher plants. 45 refs., 6 figs., 2 tabs.
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 456824
- Journal Information:
- Cytometry, Journal Name: Cytometry Journal Issue: 4 Vol. 21; ISSN CYTODQ; ISSN 0196-4763
- Country of Publication:
- United States
- Language:
- English
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