Transformation of tef (Eragrostis tef) by Agrobacterium through immature embryo regeneration system for inducing semi-dwarfism

Successful application of genetic transformation for integration of a transgene is much dependent upon availability of an efficient in vitro plant regeneration procedure and detection of transgene insertion and expression. Isolated immature embryos (IEs) of E. tef cultivar DZ-01-196 were used for embryogenic callus formation and the callus was transformed with GA inactivating gene PcGA2ox under the control of a triple CaMV 35S promoter using Agrobacterium transformation procedure. Embryogenic callus was induced from immature embryos in a medium containing KBP minerals in the presence of 2,4- dichlorophenoxiyacetic acid. The embryogenic calli were further inoculated with Agrobacterium and the calli were grown in co-cultivation medium (CCM) followed by selection in KBP and regeneration (K4NB) media. Putatively transformed E. tef embryogenic calli were tolerant to treatment with the selectable marker kanamycin, while 75mg l-1 geneticin inhibited growth of non-transformed shoots derived from matured embryos completely after 12 days. A total of 55 plants were regenerated from all the embryogenic calli to fully viable plants setting seeds at maturity. Eight putatively transformed T0 plants were produced carrying the transgene in their genome which was detected by PCR. Sequence analysis confirmed amplified PCR products to have 97.2 and 99.8% sequence identity to PcGA2ox and nptII, respectively. However, detection of the transgene, PcGA2ox or nptII, in T1 plants was inconsistent although phenotypic analysis of T1 plants showed changes in pheno-morphic and agronomic characters such as plant height, number of internodes, tillering, panicle length, biomass, yield as well as GA content. Culm reduction was due to absence of elongation of the upper-most internodes. Panicle length in semi-dwarfed plants showed no relation with culm length. GA analysis showed plants with semi-dwarf phenotype to be associated with a low level of bioactive GA1 and its immediate precursors. Up to 3.7 fold increase in grain yield per plant was found in some semi-dwarfed plants.Ethiopian Institute of Agricultural Research, the University of Pretoria (FABI in Faculty of Plant Science) and the Rothamsted International.http://www.elsevier.com/locate/sajbhb201

Identification and Application of Phenotypic and Molecular Markers for Abiotic Stress Tolerance in Soybean

Neurology & clinical neurophysiolog

Proteolysis of recombinant proteins in bioengineered plant cells

Plants are increasingly used as alternative expression hosts for the production of recombinant proteins offering many advantages including higher biomass and the ability to perform posttranslational modifications on complex proteins. Key challenges for optimized accumulation of recombinant proteins in a plant system still remain, including endogenous plant proteolytic activity, which may severely compromise recombinant protein stability. Several strategies have recently been applied to improve protein stability by limiting protease action such as recombinant protein production in various sub-cellular compartments or application of protease inhibitors to limit protease action. A short update on the current strategies applied is provided here, with particular focus on sub-cellular sites previously selected for recombinant protein production and the co-expression of protease inhibitors to limit protease activity.http://www.tandfonline.com/loi/kbie20hb201

Review : The future of cystatin engineering

Plant cystatins are naturally occurring protease inhibitors that prevent proteolysis by papain-like cysteine proteases. Their protective action against environmental stresses has been relatively well characterised. Still, there is a need to greatly improve both potency and specificity based on the current rather poor performance of cystatins in biotechnological applications. Research in creating more potent and specific cystatins, including amino acid substitutions in either conserved cystatin motifs and/or at variable amino acid sites, is reviewed. Existing gaps for better understanding of cystatin-protease interactions are further explored. Current knowledge on multi-cystatins or hybrid protease inhibitors involving cystatins as an additional option for cystatin engineering is further outlined along with the nuances of how cystatins with rather unusual amino acid sequences might actually help in cystatin engineering. Finally, future opportunities for application of cystatins are highlighted which include applications in genetically modified transgenic plants for environmental stress protection and also as nutraceuticals, as part of more nutritious food. Further opportunities might also include the possible management of diseases and disorders, often associated with lifestyle changes, and the most immediate and promising application which is inclusion into plant-based recombinant protein production platforms.International Foundation of Science (IFS grant C/5151-1), the NRF Incentive funding for rated researchers (90779) and the NRF National Bioinformatics Functional Genomics program(86947). Funding received from the Genomic Research Institute (GRI), University of Pretoria.http://www.elsevier.com/locate/plantsci2017-05-31hb2016Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil SciencePlant Scienc

Use of transgenic Oryzacystatin-I expressing plants enhances recombinant protein production

Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter–GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein which is very likely due to the reduced activity of endogenous cysteine protease.This work was supported by a grant from the National Research Foundation in South Africa.http://link.springer.com/journal/12010hb201

Whole genome sequencing and assembly data of Moricandia moricandioides and M. arvensis

Moricandia is a genus belonging to the family Brassicaceae. C-3 and C-3-C-4 photosynthesis Moricandia species exist in a close phylogenetic proximity, as well as to Brassica crops. Here, we performed PacBio genome sequencing on M. moricandioides and M. arvensis. The genomes were assembled using Flye assembler, then polished with Illumina reads and reduced duplication with Purge Haplotigs. The total length of genome assemblies of M. moricandioides and M. arvensis was 498 Mbp and 759 Mbp, respectively. These data will be useful for studies of the genetic control of C-3-C-4 characteristics, therefore gaining new insights into the early evolutionary steps of C-4 photosynthesis. Further, it can be integrated into Brassica crop breeding. The data can be accessed at ENA under the project number PRJEB39764. (C) 2021 The Author(s). Published by Elsevier Inc

Effect of water-deficit stress on cotton plants expressing the Bacillus thuringiensis toxin

Bacillus thuringiensis (Bt) crops require a high dosage of Bt toxin to delay development of insect resistance, in particular, when the refuge strategy is applied. This strategy is threatened by plant developmental and environmental factors that might reduce Bt toxin concentration and Bt efficacy in Bt crops. Growth of Bt (Cry1Ac) cotton under prolonged, moderate water deficit as a single stress factor was evaluated. Bt cotton plants were analysed for physiological performance, Bt toxin concentration and Bt efficacy. For performance analysis, leaf and total plant dry weight and leaf area were measured. Bt toxin concentration was determined by an immuno-assay. Effects of Bt toxin on growth and mortality of African cotton bollworm, Helicoverpa armigera, larvae were measured in different plant organs. Leaves from young plants exposed for 30 days to moderate water deficit had both higher Bt toxin concentrations and were more effective against larvae than leaves, flowers or bolls from mature flowering plants exposed to 60 days of moderate water deficit. Although growth of Bt cotton plants under moderate water-deficit conditions decreased Bt concentrations in leaves, flowers and bolls, this had no effect on efficacy against first-instar cotton bollworm larvae. No significant evidence was found that moderate water deficit, as a single stress factor, decreases Bt efficacy in Bt cotton

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of active cysteine proteases following inhibitor ingestion. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were first conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet bioassay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of young weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg- MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the diagnostic cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with these broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatininfiltrated banana stem disks. These promising results, which illustrate the natural susceptibility of C. sordidus to plant cystatins, are discussed in the light of current genomic data on coleopteran cysteine cathepsins and recent hypotheses suggesting a key role for digestive cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.Research Foundation of South Africa; Natural Science and Engineering Research Council of Canada; Rockefeller Foundation