Vital fluorescent staining technique for microspores of Brassica napus.

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus. It provided high contrast when used in combination with Tinapol 5 BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.

The transfer of 'Polima' cytoplasmic male sterility from oilseed rape (Brassica napus) to broccoli (B. oleracea) by protoplast fusion.

Protoplast fusion was utilised to transfer Polima type cytoplasmic male sterility (CMS) from Brassica napus, canola cv. Polima Karat (Pol-Karat) to B. oleracea, broccoli, var. "Green Comet". Southern and RFLP analysis confirmed that four cybrids possessed nuclear genomes of broccoli with Polima mitochondria and chloroplasts. A fifth cybrid was a nuclear hybrid between broccoli and Pol-Karat, with Polima mitochondria and chloroplasts of broccoli. The broccoli type cybrids were morphologically similar to "Green Comet", while the hybrid type was an intermediate of the two fusion parents. Flowers on the cybrids were distinctive in that although they possessed a morphology typical of Polima, they had very reduced petals. The broccoli type cybrids exhibited some female fertility, albeit low, establishing potential for F1 hybrid production.

The combination of polima cms and cytoplasmic triazine resistance in Brassica napus.

Protoplast fusion was used to combine cytoplasmic triazine resistance (ctr) and Polima type cytoplasmic male sterility (cms) in Brassica napus. The cybrids produced constitute the major biological input required for the production of commercial single-cross hybrid rapeseed bearing cytoplasmic triazine resistance. The results also indicate that Polima cms is associated with the mitochondrial genome.

Plant regeneration from cultured cell-derived protoplasts of Pelargonium aridum, P. x hortorum and P. peltatum.

Cultured protoplasts from cell suspensions of Pelargonium aridum, P.x hortorum and P. peltatum divided and formed callus. On agar-solidified regenerative medium, such protoplast-derived calli (p-calli) underwent plant regeneration at frequencies approaching 100% for P. aridum and 10% for P.x hortorum. Under similar conditions shoot primordia arose in 5% of P. peltatum p-calli, but these never developed into normal shoots. However, following a liquid-shake culture regime, whole plants were induced in 20% of P. peltatum p-calli. This approach also improved regeneration of P.x hortorum to 60%.

A rapid and efficient alternative procedure for the regeneration of plants from hypocotyl protoplasts of Brassica napus.

Protoplasts of several spring and winter varieties of Brassica napus were isolated from hypocotyl tissue. Protoplasts divided and formed cell colonies at high frequency, without browning when cultured in modified Shepards' medium. This high efficiency of proliferation was sustained through to plant regeneration with all varieties cultured. This has been attributed to the incorporation of a reservoir medium, the presence of 2,4-D in the proliferation medium, and the presence of kinetin in conjunction with lowering of the sucrose concentration in the regeneration medium.

The introduction of CMS mitochondria to triazine tolerant Brassica napus L., var. "Regent", by micromanipulation of individual heterokaryons.

Cytoplasmic triazine tolerance and cytoplasmic male sterility traits were combined in the nuclear genomic background of the Brassica napus variety 'Regent', following protoplast fusion, selection of fusion products by manual micro-manipulation, and culture in a Nicotiana tabacum nurse system. Whole plant cybrid regenerants were morphologically normal and produced seed on pollination, demonstrating their potential for incorporation into a breeding program.

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts.

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.