Induction Of Microspore Embryogenesis Research Articles

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.

Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis

Culture temperature determines the developmental fate of isolated microspores from Brassica napus L. At 18°C, tricellular pollen develops, whereas culture at 32°C for 8 h leads to the quantitative and synchronous induction of embryogenesis, and ultimately to the formation of embryos. We investigated the changes in protein synthesis that are associated with this 8-h inductive period by using in-situ [35S]methionine labeling, followed by two-dimensional (2-D) gel electrophoretic analysis of the radiolabeled proteins. Qualitative and quantitative computer analyses of 2-D [35S]methionine protein patterns showed six polypeptides specifically labeled under embryogenic culture conditions. Eighteen polypeptides incorporated [35S]methionine at a statistically significant higher rate under embryogenic culture conditions (32°C) than in the controls (18°C), whereas one protein was preferentially labeled under non-embryogenic culture conditions (18°C). These results indicate that only a limited number of proteins detectable in the 2-D gels of microspore extracts are associated with the early induction of embryogenesis. The reproducible identification of the differentially radiolabeled proteins in the 2-D gels allow the sequencing of representative peptides and the isolation of the corresponding cDNAs. This may lead to the identification and characterization of proteins associated with the very first stages of plant embryogenesis.

Induction of microspore embryogenesis in Camellia japonica cv. Elegans

Three methods of microspore culture were tested for the induction of microspore embryogenesis in Camellia japonica L. cv. Elegans. Culture was performed on 17 different media consisting of Murashige and Skoog (MS) and N6 basal media with different combinations of carbon, growth regulators, serine and glutamine. Microspore suspensions plated over solid MS medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 0.5 μM kinetin, with sucrose (MS6) or glucose (MS9) were seen as the best culture conditions for induction of embryogenesis. The development of microspore derived proembryos was obtained in MS medium supplemented with 2.2 μM N6-benzyladenine (MS10) and reached the highest level when the microspores were cultured in MS6 inducing medium. The development of microspore-derived embryos ceased at the maturation stage.

Messenger-RNA and protein changes associated with induction of Brassica microspore embryogenesis

Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.

Induction of microspore embryogenesis in cultured anthers of hordeum vulgare. The effects of ammonium nitrate, glutamine and asparagine as nitrogen sources

Improvement of the yield of green plants was obtained by decreasing the concentration of ammonium nitrate in the culture medium and using glutamine as a nontoxic nitrogen source. Embryoids developed directly from the microspores on such media, and frequently green plants were obtained by germination of the embryoids on the induction medium, whereas formation of calli was greatly reduced.Ficoll was tested as a carrier agent in liquid barley anther culture. It gave a higher total yield of embryoids resulting from an increased number of responding anthers and an increased number of developed structures. When anthers were floated on a Ficoll medium surface the microspores developed inside the anther. After several mitotic divisions the anther wall burst and the proembryoids were expelled onto the surface of the medium. Here their development continued by germination. The substitution of ammonium nitrate by glutamine or asparagine in the liquid medium containing Ficoll was found to be beneficial for culture development.This report is based on the regeneration of 6,827 green plants, with an optimal average ratio of green to albino plants of 1.8. A frequency of 4.6 green plants per plated anther was obtained.

Relationships Between Anther Browning and Plantlet Formation in Anther Culture of Nicotiana tabacum L.

Summary Anthers of Nicotiana tabacum cv. Wisconsin 38 contammg pollen grains in various developmental stages were cultured for 55 days at 23°C to investigate the relationship between the formation of plantlets from pollen and browning of the anther. Browning took place in anthers containing binucleate pollen grains during the first 10 days after inoculation onto Nitsch's basal medium (1969) , whereas it was continuously observed for as long as a month in anthers containing micros pores in the uninucleate stage and in those undergoing pollen mitosis. Plantlet formation in the first instance was observed during the first 3 to 5 weeks of culture and was never detected thereafter. There was no positive correlation between anther browning and plantlet formation. Plantlet induction was delayed for one week, but it was observed to be continuously effective until the cultures were 50 days old in the anthers containing uninucleate and mitotic microspores. The protraction of senescence in anthers appears to be of great importance for the induction of embryogenesis in microspores at this stage of development. There was a positive correlation between the duration of anther browning and the time required for plantlet production. That is, the percentage of plantlet formation in anthers having browned during the first 2 weeks was low, whereas the frequency of plantlet formation was high in long-lived anthers. These results suggest that there is a critical period for the induction of pollen embryogenesis in the early binucleate stage. Most microspores not yet having undergone pollen mitosis can attain to this stage with the nutritional or hormonal aid of living anther tissue; the embryogenesis can never proceed if anther browning has occurred before this stage has been attained. In a more advanced stage the duration of such a situation seems to be shorter.