Isolation of Living Pollen Mother Cells in Brassica Species and Extraction of mRNA from PMCs.

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The first step of clonings of genes expressed in pollen mother cells (PMCs) is to isolate living PMCs. In the procedure of isolating PMCs from cabbage (Brassica oleracea var. capitata) and Chinese cabbage (B. campestris ssp. pekinensis), 1%∿30% sucrose, 2%∿10% mannitol, CPW medium (Frearson et al., 1973) and B5 medium (Gamborg et al., 1968) were used to maintain the cell vitality. The result showed that 7% sucrose was better in maintaining the cell vitality of PMCs. Percoll gradient density centrifugation was used to separate PMCs from tetra stage microspores and unicellular microspores by two steps. The first steps, 0% / 10% percoll, can separate unicellular microspores from PMCs and tetra microspores ; the second step, 0∿10% / 20% percoll, could isolate PMCs from tetra microspores. The isolation of PMCs of cabbage was effective than Chinese cabbage. The 0.7mg PMCs and 3.3mg tetra microspores were obtained from one gram buds. The total RNA was extracted from PMCs and tetra microspores. There were two major ribosomal RNA bands (18S rRNA and 28S rRNA) after total RNA agarose gel electrophoresis. The poly(A)+ RNA was purified from total RNA by the way of oligo(dT)-cellulose affinity column chromatography.