Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos

The impact of cassava transformation technologies for agricultural development in Africa will depend largely on how successfully these capabilities are transferred and adapted to the African environmentand local needs. Here we report on the first successful establishment of cassava regeneration and transformation capacity in Africa via organogenesis, somatic embryogenesis and friable embryogeniccallus (FEC). As a prerequisite for genetic engineering, we evaluated six African cassava genotypes for the ability of a) induction of FEC b) hygromycin sensitivity and c) T-DNA integration potential bydifferent Agrobacterium strains. FEC was induced in genotypes TMS 60444, TME 1 and TMS 91/02327. Potential tissues for FEC formation were induced in TMS 91/02324, TME 12 and TME 13. Pure andproliferating FEC was obtained and maintained only in TMS 60444. FEC growth and shoot organogenesis were completely suppressed when hygromycin was used at a concentration of 20 mg/l in all tissue types and genotypes. With somatic cotyledons, statistically significant differences (p0.05) were observed between Agrobacterium strains and genotypes with respect to T-DNA transfer efficiency.Using somatic cotyledons, TME 8 was found to be the most amenable to transformation with maximum blue spots per GUS-positive explants, and Agrobacterium GV3101 proved to be superior to EHA105,LBA4404, and AGl-1 for T-DNA transfer based on transient assays with a reporter gene (GUS). With FEC, Agrobacterium LBA4404 was superior to other strains. This study also identified EHA105 as a newvir helper strain to recover transgenic cassava plants. PCR and Southern hybridization of genomic DNA of the hygromycin-resistant cassava plants to a hpt probe confirmed the integration of hpt withintegration events varying between 1 and 2 insertions. The benefit of combining the FEC and shoot organogenesis systems for recovering transgenic cassava plants is described. The contributions ofthis report to enhancing the development and deployment of genetic engineering of cassava for agricultural biotechnology development in Africa are discussed

Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus

Background: Secondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression. Results: We overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas, AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants. Conclusions: Overall, saccharification efficiency was increased by 35–50%, 21–42%, and 8–39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance

Reducing Biomass Recalcitrance by Heterologous Expression of a Bacterial Peroxidase in Tobacco (\u3cem\u3eNicotiana benthamiana\u3c/em\u3e)

Commercial scale production of biofuels from lignocellulosic feed stocks has been hampered by the resistance of plant cell walls to enzymatic conversion, primarily owing to lignin. This study investigated whether DypB, the lignin-degrading peroxidase from Rodococcus jostii, depolymerizes lignin and reduces recalcitrance in transgenic tobacco (Nicotiana benthamiana). The protein was targeted to the cytosol or the ER using ER-targeting and retention signal peptides. For each construct, five independent transgenic lines were characterized phenotypically and genotypically. Our findings reveal that expression of DypB in the cytosol and ER does not affect plant development. ER-targeting increased protein accumulation, and extracts from transgenic leaves showed higher activity on classic peroxidase substrates than the control. Intriguingly, in situ DypB activation and subsequent saccharification released nearly 200% more fermentable sugars from transgenic lines than controls, which were not explained by variation in initial structural and non-structural carbohydrates and lignin content. Pyrolysis-GC-MS analysis showed more reduction in the level of lignin associated pyrolysates in the transgenic lines than the control primarily when the enzyme is activated prior to pyrolysis, consistent with increased lignin degradation and improved saccharification. The findings reveal for the first time that accumulation and in situ activation of a peroxidase improves biomass digestibility

CALLUS INDUCTION AND MORPHOGENESIS IN DIOSCOREA DUMETORUM FOR STEROID PRODUCTION

Diosgenin is characteristic of the genus Dioscorea (Dioscoreaceae), which is used in partial synthesis of steroid drugs. It was not detected in the crude sapogenin extract of Dioscorea dumetorum and in the calluses. Callus was induced from the meristem of the two genotypes and maintained successfully on Murashige and Skoog's medium (MS) supplemented with 0.1mg/l of 2, 4-dichlorophenoxyacetic acid (2,4-D). Shoots induced from nodal cuttings of the white and yellow genotypes regenerated plantlets, which were subsequently maintained on MS medium, and Linsmaier and Skoog's medium (LS) supplemented with 0.5mg/l kinetin. Control of microbial contamination was achieved with tetracycline. Microtubers were also obtained from the yellow genotype plantlet growing on the MS medium. Steroids, especially the Δ5, 3-hydroxy steroids were detected only in the yellow genotype callus. Keywords: Dioscorea dumetorum; diosgenin; 3-hydroxy steroids; callus; shoot culture; meristem culture; yellow genotype Nigerian Journal of Natural Products and Medicine Vol.7 2003: 5-

The Maize Corngrass1 miRNA-Regulated Developmental Alterations Are Restored by a Bacterial ADP-Glucose Pyrophosphorylase in Transgenic Tobacco

Crop-based bioethanol has raised concerns about competition with food and feed supplies, and technologies for second- and third-generation biofuels are still under development. Alternative feedstocks could fill this gap if they can be converted to biofuels using current sugar- or starch-to-ethanol technologies. The aim of this study was to enhance carbohydrate accumulation in transgenic Nicotiana benthamiana by simultaneously expressing the maize Corngrass1 miRNA (Cg1) and E. coli ADP-glucose pyrophosphorylase (glgC), both of which have been reported to enhance carbohydrate accumulation in planta. Our findings revealed that expression of Cg1 alone increased shoot branching, delayed flowering, reduced flower organ size, and induced loss of fertility. These changes were fully restored by coexpressing Escherichia coli glgC. The transcript level of miRNA156 target SQUAMOSA promoter binding-like (SPL) transcription factors was suppressed severely in Cg1-expressing lines as compared to the wild type. Expression of glgC alone or in combination with Cg1 enhanced biomass yield and total sugar content per plant, suggesting the potential of these genes in improving economically important biofuel feedstocks. A possible mechanism of the Cg1 phenotype is discussed. However, a more detailed study including genome-wide transcriptome and metabolic analysis is needed to determine the underlying genetic elements and pathways regulating the observed developmental and metabolic changes

Regeneration of a wide range of African cassava genotypes via shoot organogenesis from cotyledons of maturing somatic embryos and conformity of the fieldestablished regenerants

Genotypic differences in the ability of immature leaf lobes and apical shoot meristems of cassava to form primary somatic embryos in P-CIM were observed (p ≤ 0.05). The mean number of apical meristems forming primary organized embryogenic structures when cultured in embryo induction medium supplemented with picloram (P-CIM) had greatest variability between genotypes (C.V.=22.70%). Maturation frequencies of primary embryos were genotype-dependent and ranged from 17 to 100%. Secondary embryo formation was also genotype-dependent and their maturation frequencies varied from 48 to 100%. Cyclic somatic embryogenesis was successfully established and maintained in 11 genotypes in P-CIM. All genotypes underwent organogenesis with significant genotypic variation (p ≤ 0.05), and organogenic potential ranging from 5.4 to 76.8%. The number of somatic cotyledons forming multiple shoot buds or more than 10 shoot buds per cluster had the greatest variability between genotypes (C.V.=36.96%) as compared with the overall embryogenic potential. Shoot regeneration ability was neither related to primary embryogenic potential nor to explant type for primary embryo induction. Plantlet regeneration per responding explant ranged from 0.1 to 12. Regenerants established in the field at the frequency ranging from 60 to 100%. DNA content of regenerants was homogeneous and similar to that of mother plants and ploidy level was unchanged (2n = 36). The potential benefits of a systematic tissue culture approach for screening agronomically superior genotypes for regeneration capability and its usefulness in selecting those suited for transgenic programs are discussed

Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos

The impact of cassava transformation technologies for agricultural development in Africa will depend largely on how successfully these capabilities are transferred and adapted to the African environment and local needs. Here we report on the first successful establishment of cassava regeneration and transformation capacity in Africa via organogenesis, somatic embryogenesis and friable embryogenic callus (FEC). As a prerequisite for genetic engineering, we evaluated six African cassava genotypes for the ability of a) induction of FEC b) hygromycin sensitivity and c) T-DNA integration potential by different Agrobacterium strains. FEC was induced in genotypes TMS 60444, TME 1 and TMS 91/02327. Potential tissues for FEC formation were induced in TMS 91/02324, TME 12 and TME 13. Pure and proliferating FEC was obtained and maintained only in TMS 60444. FEC growth and shoot organogenesis were completely suppressed when hygromycin was used at a concentration of 20 mg/l in all tissue types and genotypes. With somatic cotyledons, statistically significant differences (p 0.05) were observed between Agrobacterium strains and genotypes with respect to T-DNA transfer efficiency. Using somatic cotyledons, TME 8 was found to be the most amenable to transformation with maximum blue spots per GUS-positive explants, and Agrobacterium GV3101 proved to be superior to EHA105, LBA4404, and AGl-1 for T-DNA transfer based on transient assays with a reporter gene (GUS). With FEC, Agrobacterium LBA4404 was superior to other strains. This study also identified EHA105 as a new virus helper strain to recover transgenic cassava plants. PCR and Southern hybridization of genomic DNA of the hygromycin-resistant cassava plants to a hpt probe confirmed the integration of hpt with integration events varying between 1 and 2 insertions. The benefit of combining the FEC and shoot organogenesis systems for recovering transgenic cassava plants is described. The contributions of this report to enhancing the development and deployment of genetic engineering of cassava for agricultural biotechnology development in Africa are discussed

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability