Elisabeth Matthys-Rochon

Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis

A multidisciplinary study was carried out to analyse the chromosome doubling process during the early stages of in vitro maize microspore embryogenesis. The main stages (microspore derivatives) that were formed in the course of the culture were analysed. Chromosome number was determined from squashed cells, and DNA content was measured by cytometry. In parallel, an ultrastructural analysis of the microspore derivatives demonstrated the occurrence of a nuclear fusion process. It seems likely that nuclear fusion ensures chromosome doubling at early stages of induced microspore embryogenesis. It occurs precisely at the 5/7xa0day stage in the embryonic domain and probably leads to polyploidy in the endosperm domain of the microspore derivatives. As a conclusion a scheme summarises the results and proposes an interpretation of the sequence of chromosome doubling events during early maize microspore embryogenesis. Understanding of this process will be important for future efforts to increase the percentage of homozygous plants for crop improvement.

High efficiency transformation of peppermint (Mentha × piperita L.) with Agrobacterium tumefaciens

Transgenic peppermint (Mentha × piperita L.) plants were obtained by using Agrobacterium tumefaciens-mediated gene transfer. The effects of the coculture period and of the Agrobacterium strain were tested. 10% transformed plants were regenerated by leaf disk culture after inoculation with strain EHA105MOG harbouring β-glucuronidase and neomycin phosphotransferase II genes, with a coculture period of 5 days. Rooting of regenerated plants was achieved on selective medium with 150 mg/l kanamycin. The presence of transgenes in DNA was shown through PCR and Southern blot hybridization and transgene product activity via histoenzymatic GUS test and leaf callus assay. Transgenic plants were successfully acclimatized in the greenhouse.

Nuclear DNA amounts in the egg and zygote of maize (Zea mays L.)

The nuclear DNA content of isolated eggs and zygotes of maize was estimated using 4′,6-diamidino-2-phenylindole (DAPI) staining and microspectrofluorometry. The data indicate that egg nuclei contain the 1C level of DNA (basic haploid amount) at the time of karyogamy, and that, by inference, the sperm nuclei are also at 1C. Fertilization occurred in most ovules by 24–28 h post-pollination (hpp), and DNA synthesis was well underway by 27–31 hpp. By 30–34 hpp, 80% of the zygotes were at the 3C DNA level or above, and many were undergoing mitosis. This study provides information that is pertinent to experiments on the microinjection of exogenous DNA into isolated zygotes of maize, and it will serve as a comparative base for future determinations of the DNA content of zygotes produced and cultured in vitro.

Isolation and microinjection of active sperm nuclei into egg cells and central cells of isolated maize embryo sacs

Artificial fertilisation was attempted in maize by microinjecting sperm nuclei into the egg cell or central cell of isolated embryo sacs. A protocol for isolation of nuclei from pollen grains was developed and a pure fraction of sperm nuclei was obtained after centrifugation on a Percoll gradient. The in vitro transcriptional activity of the nuclei was tested by incorporation of radioactive UTP into RNA. The level of labelled nucleotide incorporation increased and reached a maximum after between 30 and 40 min in the incubation medium. The embryo sacs were enzymatically isolated and their viability determined by observation of cytoplasmic streaming in the female cells. The embryo sacs were immobilised by embedding in low-melting-point agarose and a single male nucleus was injected with a bevelled microcapillary. The presence of the injected nucleus in the egg or central cell was demonstrated using a cytological approach. This paper presents an alternative method for studying the intimate processes of fertilisation in plants.

Deleterious effect of minimal enzymatic treatments on the development of isolated maize embryo sacs in culture

The long-term viability of isolated embryo sacs was studied in maize. Fertilised embryo sacs were digested in order to remove most of the nucellus cells present on their surfaces and then transferred to culture. Experiments on 161 embryo sacs showed that isolation treatments using even minimal enzymatic digestion affected the further development of the embryo sacs. Few embryo sacs survived in culture and those produced only abnormal embryos; they produced no plants. We concluded that embryo sacs isolated through enzymatic digestion may offer limited prospects for long-term studies where normal embryogenic development is required. Alternative strategies are discussed for maize.

Analysis of ZmAE3 upstream sequences in maize endosperm and androgenic embryos

The single copy gene ZmAE3 (androgenic embryo) has been isolated from early androgenic embryos of maize. It codes for a small hydrophilic protein with partial similarity to basal layer antifungal proteins. During normal reproductive development ZmAE3 is specifically expressed in the embryo-surrounding region, a specialised region of the endosperm. Here we report the cloning, sequencing and functional analysis of upstream sequences of ZmAE3. The fusion of a 1.1xa0kb potential promoter fragment of ZmAE3 to a uidA reporter gene resulted in β-glucuronidase activity in transiently transformed androgenic embryos and in Black Mexican Sweet maize suspension cells. The AE box, an 18xa0bp sequence present in the ZmAE3 promoter and in other endosperm-specific promoters, was shown to bind proteins present specifically in nuclear extracts of the lower half of the maize kernel. The potential role of this and other cis elements in the binding of trans-acting factors will be discussed.

In vitro pollination and fertilisation in maize (Zea mays L.): technical procedures and prospects for the dissection of the double fertilisation process

Abstract In this review, we report the considerable worldwide progress accomplished in Angiosperm fertilisation, especially in Zea mays L. In this species, the precise sequence of events during the process of double fertilisation has been determined. A method of in vitro pollination which allows definition of the precise nutritional and environmental conditions appears to be a suitable technique to study seed set, female receptivity and the effects of environmental stress on the fertilisation process. A protocol for sperm cells and female gamete isolation has been described and offers a unique opportunity for studying gamete interactions. Different systems of in vitro fertilisation have been developed including electrofusion, calcium-induced fusion of isolated gametes and sperm nuclei microinjection into the female cells. Regeneration of electrofused zygotes into fertile hybrid plants and culture of isolated fertilised embryo sacs provide new possibilities for investigating the early steps of fertilisation and embryogenesis at the cellular and molecular level.

In situ detection of Esr proteins secretion during maize microspore embryogenesis and their secretion blockage show effects on the culture progression

In vitro plant cells in culture release proteins and carbohydrates, but the active molecules responsible for sustaining the switch in embryogenic development and progression have not yet been identified. In maize (Zea mays L.), the Esr genes encode for small hydrophilic proteins and are expressed in the restricted region of maize endosperm surrounding the embryo: the embryo surrounding region (ESR). In the present work, the possible influence of secreted molecules in the liquid medium during microspore-derived embryo development and specifically, the presence of Esr proteins, has been analysed in maize microspore cultures. The study has been conducted with in situ monitoring of the structural and cellular organisation of developing embryos and the subcellular localisation of the Esr proteins by immunofluorescence and immunogold labelling. The results obtained using confocal and electron microscopy revealed that Esr proteins were localised in elements of the secretory pathway and cell walls in microspore-derived embryo cells during early embryogenesis. Esr proteins were also detected in the liquid medium of maize microspore cultures and accumulated at 20 days of culture. Tunicamycin treatment to block protein glycosilation and, therefore, secretion inhibited microspore-derived embryo development, which was subsequently recovered by supplementation with medium containing all the secreted factors from a well developed microspore culture. Esr labelling was not present in non-developing microspore embryos of cultures treated with tunicamycin, whereas labelling was present again in the Golgi elements and secretory vesicles of embryo cells when development was restored. The results indicate that Esr proteins are part of the secreted proteins, which show a nursing or signalling role during in vitro embryo development in maize microspore embryogenesis cultures and provide new evidence for an endosperm-like function of microspore-derived embryo structures during the early stages.

In vitro culture of digested maize embryo sacs leads to abnormal embryogenesis

Angiosperms present a megagnmetophytc generation of a reduced srze: The embryo sac. It contains the two female gametes and is the site where the double fertilisation characteristic of the flowering plants takes place. This embryo sacs is deeply inserted in the ovular tissues and this made the studv of fertilisation events and of embryogenesis in flowering plants particularly difficult. Isolation techniques were developed to gain access to the embrvo sacs in several species. Microdissection -and enzymatic treatments have allowed the the isolation of living embryo sacs in some and these have been considered as useful tools for the study ofsexual reproduction events as well as for genetic manipulations. So far viability of isolated embryo sacs was only assessed by FCR test or through the observations of cytoplasmic streaming. In these cases, the viability only reported for few hours or days after isolation. If isolated embryo sacs are to be used for long term experiments such as for the study of embryogenesis, or the recovery of transformed embryos, these should remain viable and undergo normal development until maturity. No report to-date approached this question.