Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

Abstract

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

“…Embryos bombarded with Cas9 protein alone were used as negative controls, while DNA vectors encoding Cas9 and the four gRNAs served as positive controls. In these experiments, DNA vectors encoding ‘helper genes'—cell division promoting transcription factors (maize ovule developmental protein 2 ( ODP2 ) and maize Wuschel ( WUS )21), and selectable and visible marker genes ( MOPAT-DSRED fusion) were co-bombarded with the RNPs.…”

Tissue culture-based Agrobacterium-mediated and in planta transformation methods

“…Sonication and vacuum infiltration are the treatments that can be used in both TCBAT and in planta transformation. Although all the above-mentioned in planta transformation methods can help to enhance transformation efficiency in plants, other alternatives such as transcription factors (Lowe et al 2016) can help to achieve the genotype-independent high efficiency protocol for plant transformation and enhance the regeneration and transformation efficiency in tissue culture and Agrobacterium-recalcitrant plants.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…This system allows drastic increases in regeneration rates from various types of tissues and organs (such as leaves, calli and protoplasts), which makes it adapted to different transformation protocols (personal comm.). High transformation frequencies could be obtained using the same strategy in other monocot species including sorghum, sugarcane and rice [21]. Overexpression of Phosphomannose isomerase (PMI) and Ovule development protein2 (ODP2) also stimulated transformation and regeneration in maize [6].…”

“…Recently, maize inbred lines that were recalcitrant to transformation could be transformed with 40% efficiency by overexpressing the maize BABY BOOM (BBM) and WUSCHEL2 (WUS2) genes [21]. This system allows drastic increases in regeneration rates from various types of tissues and organs (such as leaves, calli and protoplasts), which makes it adapted to different transformation protocols (personal comm.).…”

“…For example, the labour and space-consuming production of immature embryos necessary for maize classical transformation could be replaced by alternative target tissues, like embryo slices from mature seed or leaf segments from seedlings whose preparation can be automated [21]. To overcome the constraints of plant regeneration (complexity and duration of the protocols, exposure to somaclonal variation), in planta transformation methods, independent from in vitro culture, are also being developed using whole plants or seeds [23][24][25].…”