Biology subjects

Simon, M. K.

Publications and source records attributed to Simon, M. K..

4 recordsLinked to original sources

bioRxiv2025

Induction of synthetic apomixis in two sorghum hybrids enables seed yield and genotype preservation over multiple generations

Apomixis, a process of clonal reproduction through seed, has the potential to significantly change agriculture by enabling a clonal seed propagation system for hybrid crops. Here, we demonstrate that hybrid seed from synthetically induced apomictic sorghum hybrids can be generated and maintained across multiple seed generations. This was achieved through the combination of avoidance of meiosis and induced parthenogenesis. Avoidance of meiosis was generated by the CRISPR/Cas9 knockout of the sorghum meiosis genes Spo11, Rec8, and OsdL1 and OsdL3. Parthenogenesis was induced in the resultant diploid egg cell using a maize egg cell promoter to express the Cenchrus ASGR-BBML2 gene coding sequence. Two strategies incorporating these components were used to induce synthetic apomixis in two different sorghum hybrids. Each hybrid used Tx623 as a female parent and either Tx430 or the African landrace Macia as a male parent. Seed yields in the induced apomictic hybrids were consistent and stable for multiple generations following self-pollination but reduced relative to the sexual hybrids. Sorghum contains two copies of the Osd1 gene that function in meiotic non-reduction. CRISPR/Cas9 knockout of both OsdL1 and OsdL3 loci was sufficient to produce clonal hybrid progeny in conjunction with the other apomixis induction components, but this led to a significant reduction in seed set. By contrast, a single in-frame edit of either OsdL1 or OsdL3 significantly improved seed set of clonal hybrid progeny. Fine-tuning OsdL activity appears to be essential to optimizing fertility. As the efficiency of seed set in the induced synthetic sorghum apomicts was lower than that of the sexual hybrid control, additional improvements are required to unlock the agronomic potential of synthetically induced apomictic sorghum in the field.

plant biology↗

bioRxiv2022

Trait Stacking Simultaneously Enhances Provitamin A Carotenoid and Mineral Bioaccessibility in Biofortified Sorghum bicolor

Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including sorghum (Sorghum bicolor). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Kernels from transformed sorghum events were processed into model porridges and evaluated for carotenoid and mineral content as well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 {micro}g/g) compared to null segregants (4.2 {micro}g/g) and wild-type control (Tx430; 3.7 {micro}g/g). A 200 g serving of porridge made with these transgenic events represents a projected 53.7% of a 4- 8-year-old childs vitamin A estimated average requirement. Phytase activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg/g), altered the profile of inositol phosphate metabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1); and zinc (19.0 to 4.9) in engineered material; suggesting improved mineral bioaccessibility. Improved phytate:mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were over 2300% greater in transgenic events compared to corresponding null segregants and wild-type controls. These data suggest that combinatorial approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.

plant biology↗

bioRxiv2021

Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum

For many important crops, including sorghum, the use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but more critically, by the ability to regenerate a fertile plant from the engineered cell through conventional tissue culture methods. Here, we report that Wuschel2 (Wus2)-enabled sorghum (Sorghum bicolor L.) transformation increases not only the efficiency of transgenic plant recovery, but also increases the observed efficiency of CRISPR/Cas-targeted genome editing within the population of T0 plants. Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased regeneration capacity, resulting in higher transformation efficiency across different sorghum genotypes. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrated up to an additional 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled, Agrobacterium-mediated transformation (compared to the control treatment with no Wus2), across various targeted loci in different sorghum genotypes. Wus2-enabled genome editing may be applicable to other crops and increasingly popular in planta transformation strategies.

developmental biology↗

bioRxiv2019

Developing a rapid and highly efficient cowpea regeneration and transformation system using embryonic axis explants

Cowpea is one of the most important legume crops planted worldwide, especially in Sub-Saharan Africa and Asia. Despite decades of effort, genetic engineering of cowpea is still challenging due to inefficient in vitro shoot regeneration, Agrobacterium-mediated T-DNA delivery and transgenic selection. Here, we report a rapid and highly efficient cowpea transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem by cutting through the middle of the epicotyl stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. Furthermore, the application of a ternary transformation vector system using an optimized pVIR accessory plasmid provided high levels of Agrobacterium-mediated gene delivery. The utilization of spectinomycin as the selection agent enabled more efficient transgenic selection and plant recovery. Transgenic cowpea shoots developed exclusively from the cotyledonary nodes at high frequencies of 4.5 to 37% across a wide range of cowpea genotypes. We believe that the transformation principles established in this study could also be applied to other legumes to increase transformation efficiencies.

plant biology↗