Recovery and development of parasexual fusion products inEnteromorpha linza (L.) J. Ag. (Ulvales, Chlorophyta)

Abstract

Newly released zoospores fromEnteromorpha linza (L.) J. Ag. lack significant cellulose cell wall material and are suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca^{+ +}, PEG and centrifugation. Treated zoospores settled on glass cover slips within 3 h and were examined microscopically at 1000 ×. Presumptive fusion products were identified by their larger size and presence of twin chloroplasts and eyespots. Unfused zoospores adjacent to fusion cells were killed by 2–3 min exposure to blue light (410–490 nm) from a high pressure mercury illuminator. Unexposed fusion cells developed into uniseriate germlings within 10 days at which stage they could be readily identified at 60 × with a dissecting microscope and isolated by micropipette. Ten-day germlings from both unfused zoospores and fusion cells were stained with the DNA-localizing fluorochrome hydroethidine and relative nuclear DNA content determined with epi-(incident) UV illumination. All germlings were found to be uninucleate. Germlings from unfused zoospores had haploid nuclei with 1N = 10 and 1C and 2C levels of DNA, while germlings from fusion cells had diploid nuclei with 2N = 20 and 2C and 4C levels of DNA. These result are interpreted as evidence of karyogamy following parasexual zoospore fusions. Isolated diploid germlings, cultured for 10 weeks were found to conserve their 2N chromosome complements and elevated levels of nuclear DNA. Although most diploid germlings were morphologically similar to haploid control plants, some exhibited ‘gigas’ characteristics, including larger cells, chloroplasts, and nuclei. These results are discussed in terms of unique phenotypes that result when nuclear and organellar genes are combined in different ways.