Search for: All records

Abstract This report provides an overview of the content and data collected from the “Successes, Challenges, and Opportunities Plant Transformation Research in Africa” panel discussion. Organized by PlantGENE, this event brought together scientists and stakeholders across the globe to examine the complex challenges and emerging opportunities in plant transformation research in laboratories across Africa. The discussion, rooted in insights from a panel of six leading scientists, highlights critical issues including restrictive regulatory environments, prohibitive costs, and the inconsistent availability of essential research materials. Additionally, the pervasive “brain drain” phenomenon, where skilled researchers leave the continent for better opportunities, exacerbates the difficulties faced by African scientists. Despite these challenges, the report also identifies significant advancements, particularly in the growing recognition of African leadership within universities and national agricultural research systems (NARS). These institutions, supported by highly skilled faculty and motivated graduate students, are producing high-quality research that contributes to global scientific knowledge. The panelists emphasized the necessity of creating an environment that encourages African scientists to remain on the continent and address local challenges through innovative research. Strengthening intra-African networks and fostering collaborations with the global scientific community are proposed as essential strategies to achieve this. This report underscores the critical need for substantial investments from both global and African organizations, working with African governments, to support these efforts. Furthermore, it calls for science-based decision-making and fair regulatory frameworks to align with unique opportunities and risks associated with technological advancements in Africa. This paper details the observations of six panelists and analyzes the results of attendee surveys in order to document these challenges and opportunities while advocating for sustained investment and strategic partnerships to build a thriving bioeconomy across Africa. 

Agrobacterium-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence (vir) gene helper plasmid (ternary helper), demonstrated that including an additionalvirgene helper plasmid into disarmedAgrobacteriumstrains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophicAgrobacteriumstrains to boostAgrobacterium-mediated plant transformation efficiency. AuxotrophicAgrobacteriumstrains are useful in reducingAgrobacteriumovergrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from publicAgrobacteriumstrains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transientGUSexpression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries thevirAgene from pTiBo542 in addition to othervirgene operons (virG,virB,virC,virD,virE, andvirJ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improvedAgrobacteriumsystem, including auxotrophic disarmedAgrobacteriumstrains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications. 

Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing. 

SUMMARY Plant transformation is an important part of plant research and crop improvement. Transformation methods remain complex, labor intensive, and inefficient. PlantGENE is a community of scientists from academia, industry, non‐profit research institutes, and government organizations working to improve plant transformation. PlantGENE hosts virtual training, interactive webinars, and a website with career opportunities, directories, and more. The plant science community has shown great interest and support for PlantGENE. 

For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers. 

Maize ( Zea mays ssp. mays ) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an Agrobacterium -mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.