Androgenesis-Based Doubled Haploidy: Past, Present, and Future Perspectives

Androgenesis, which entails cell fate redirection within the microgametophyte, is employed widely for genetic gain in plant breeding programs. Moreover, androgenesis-responsive species provide tractable systems for studying cell cycle regulation, meiotic recombination, and apozygotic embryogenesis within plant cells. Past research on androgenesis has focused on protocol development with emphasis on temperature pretreatments of donor plants or floral buds, and tissue culture optimization because androgenesis has different nutritional requirements than somatic embryogenesis. Protocol development for new species and genotypes within responsive species continues to the present day, but slowly. There is more focus presently on understanding how protocols work in order to extend them to additional genotypes and species. Transcriptomic and epigenetic analyses of induced microspores have revealed some of the cellular and molecular responses required for or associated with androgenesis. For example, microRNAs appear to regulate early microspore responses to external stimuli; trichostatin-A, a histone deacetylase inhibitor, acts as an epigenetic additive; ά-phytosulfokine, a five amino acid sulfated peptide, promotes androgenesis in some species. Additionally, present work on gene transfer and genome editing in microspores suggest that future endeavors will likely incorporate greater precision with the genetic composition of microspores used in doubled haploid breeding, thus likely to realize a greater impact on crop improvement. In this review, we evaluate basic breeding applications of androgenesis, explore the utility of genomics and gene editing technologies for protocol development, and provide considerations to overcome genotype specificity and morphogenic recalcitrance in non-model plant systems.

Genome Scale Constraint-Based Metabolic Reconstruction of Propionibacterium Freudenreichii DSM 20271

Propionibacterium is an anaerobic bacterium with a history of use in the production of Swiss cheese and, more recently, several industrial bioproducts. While the use of this strain for the production of organic acids and secondary metabolites has gained growing interest, the industrial application of the strain requires further improvement in the yield and productivity of the target products. Systems modeling and analysis of metabolic networks are widely leveraged to gain holistic insights into the metabolic features of biotechnologically important strains and to devise metabolic engineering and culture optimization strategies for economically viable bioprocess development. In the present study, a high-quality genome-scale metabolic model of P. freudenreichii ssp. freudenreichii strain DSM 20271 was developed based on the strain’s genome annotation and biochemical and physiological data. The model covers the functions of 23% of the strain’s ORFs and accounts for 711 metabolic reactions and 647 unique metabolites. Literature-based reconstruction of the central metabolism and rigorous refinement of annotation data for establishing gene-protein-reaction associations renders the model a curated omic-scale knowledge base of the organism. Validation of the model against experimental data indicates that the reconstruction can capture the key structural and functional features of P. freudenreichii metabolism, including the growth rate, the pattern of flux distribution, the strain’s aerotolerance behavior, and the change in the mode of metabolic activity during the transition from an anaerobic to an aerobic growth regime. The model also includes an accurately curated pathway of cobalamin biosynthesis, which was used to examine the capacity of the strain to produce vitamin B12 precursors. Constraint-based reconstruction and analysis of the P. freudenreichii metabolic network also provided novel insights into the complexity and robustness of P. freudenreichii energy metabolism. The developed reconstruction, hence, may be used as a platform for the development of P. freudenreichii-based microbial cell factories and bioprocesses.

Microbial Production of Bioactive Retinoic Acid Using Metabolically Engineered Escherichia coli

Microbial production of bioactive retinoids, including retinol and retinyl esters, has been successfully reported. Previously, there are no reports on the microbial biosynthesis of retinoic acid. Two genes (blhSR and raldhHS) encoding retinoic acid biosynthesis enzymes [β-carotene 15,15’-oxygenase (Blh) and retinaldehyde dehydrogenase2 (RALDH2)] were synthetically redesigned for modular expression. Co-expression of the blhSR and raldhHS genes on the plasmid system in an engineered β-carotene-producing Escherichia coli strain produced 0.59 ± 0.06 mg/L of retinoic acid after flask cultivation. Deletion of the ybbO gene encoding a promiscuous aldehyde reductase induced a 2.4-fold increase in retinoic acid production to 1.43 ± 0.06 mg/L. Engineering of the 5’-UTR sequence of the blhSR and raldhHS genes enhanced retinoic acid production to 3.46 ± 0.16 mg/L. A batch culture operated at 37 °C, pH 7.0, and 50% DO produced up to 8.20 ± 0.05 mg/L retinoic acid in a bioreactor. As the construction and culture of retinoic acid–producing bacterial strains are still at an early stage in the development, further optimization of the expression level of the retinoic acid pathway genes, protein engineering of Blh and RALDH2, and culture optimization should synergistically increase the current titer of retinoic acid in E. coli.

Production of Rice Based Alcoholic Beverages and their Quality Evaluation

Murcha is a traditional starter culture used for the production of alcoholic beverages in Nepal. The present study was conducted to compare and characterize rice-based alcoholic beverages prepared from varieties of rice as well as starter culture. Optimization of the production was performed using three criteria (pH, temperature, and Brix). Four different rice varieties (marsi/ red rice (RR), black rice, taichin, and khumal-4) and four different cultures (ATCC 18824, murcha, yeast isolated from murcha, and commercial wine yeast) were used to prepare rice-based alcoholic beverages under optimized condition. The highest acidity was found in black rice (BR) fermented by murcha (2.0±0.0 %). The highest alcohol was found in the taichin rice fermented by isolated yeast (23.38±0.00 %) and wine yeast (23.38±0.00 %). The highest antioxidant was found in BR fermented by wine yeast (WY) (68±0.002%). The highest phenolic content and reducing sugar was found in BR fermented by ATCC (103.2±0.02mg/l) and (1064±0.03µg/ml) respectively. The study showed different cultures and rice variation gives beverages with different characteristics. The results were subjected to statistical analysis. The result was found to be significant (p <0 .05).

Advances in Plant Regeneration: Shake, Rattle and Roll

Some plant cells are able to rebuild new organs after tissue damage or in response to definite stress treatments and/or exogenous hormone applications. Whole plants can develop through de novo organogenesis or somatic embryogenesis. Recent findings have enlarged our understanding of the molecular and cellular mechanisms required for tissue reprogramming during plant regeneration. Genetic analyses also suggest the key role of epigenetic regulation during de novo plant organogenesis. A deeper understanding of plant regeneration might help us to enhance tissue culture optimization, with multiple applications in plant micropropagation and green biotechnology. In this review, we will provide additional insights into the physiological and molecular framework of plant regeneration, including both direct and indirect de novo organ formation and somatic embryogenesis, and we will discuss the key role of intrinsic and extrinsic constraints for cell reprogramming during plant regeneration.