Occurrence of albinism during wheat androgenesis is correlated with repression of the key genes required for proper chloroplast biogenesis.

MAIN CONCLUSION: The phenomenon of albinism in wheat androgenesis is linked to the transcriptional repression of specific genes involved in chloroplast biogenesis during the first weeks of in vitro culture. Isolated microspore culture is widely used to accelerate breeding programs and produce new cultivars. However, in cereals and particularly in wheat, the use of this technique is limited due to the high proportion of regenerated albino plantlets. The causes and mechanisms leading to the formation of albino plantlets in wheat remain largely unknown and, to date, no concrete solution has been found to make it possible to overcome this barrier. We performed a molecular study of proplastid-to-chloroplast differentiation within wheat microspore cultures by analyzing the expression of 20 genes specifically involved in chloroplast biogenesis. Their expression levels were compared between two wheat genotypes that exhibit differential capacities to regenerate green plantlets, i.e., Pavon and Paledor, which produce high and low rates of green plants, respectively. We observed that chloroplast biogenesis within wheat microspores was affected as of the very early stages of the androgenesis process. A successful transition from a NEP- to a PEP-dependent transcription during early plastid development was found to be strongly correlated with the formation of green plantlets, while failure of this transition was strongly correlated with the regeneration of albino plantlets. The very low expression of plastid-encoded 16S and 23S rRNAs within plastids of the recalcitrant genotype Paledor suggests a low translation activity in albino plastids. Furthermore, a delay in the activation of the transcription of nuclear encoded key genes like GLK1 related to chloroplast biogenesis was observed in multicellular structures and pro-embryos of the genotype Paledor. These data help to understand the phenomenon of albinism in wheat androgenesis, which appears to be linked to the transcriptional activation of specific genes involved in the initial steps of chloroplast biogenesis that occurs between days 7 and 21 of in vitro culture.

Impedance flow cytometry allows the early prediction of embryo yields in wheat (Triticum aestivum L.) microspore cultures.

Haplomethods are key biotechnological tools that make it possible to rapidly produce perfectly homozygous lines, speeding up plant breeding programs. Under specific stress conditions, microspores are reprogrammed toward sporophytic pathways, leading to embryo formation. Various endogenous and exogenous factors affect embryo yield in androgenesis, so the improvement of androgenesis efficiency requires the development of early, reliable and robust reactivity markers. During the last decade, numerous cytological, cellular and biochemical approaches were carried out to finely characterize microspore development and fate during androgenesis. However, the different available markers are often species-dependent, and their development and application are time-consuming and cumbersome. In this study, we show the suitable use of impedance flow cytometry (IFC) to develop new robust, reliable and strong markers of androgenesis reactivity in wheat, leading to: (i) routine monitoring of the viability of heterogeneous cell cultures; (ii) quick and simple evaluation of stress treatment efficiency; and (iii) early prediction of embryo yields from microspore suspensions. IFC can therefore provide the fine characterization of all of the microspore developmental pathways that occur in a cell suspension, for embryogenic microspores as well as pollen-like microspores. IFC technology has become a very useful tool to track and characterize wheat microspores in androgenesis, but can also be adapted to other species and other in vitro cell culture systems.

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis - Mass spectrometry: Assessment and method validation.

Characterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.