SummaryThe ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation‐based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non‐responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.

Seed increase through manual pollination is a critical part of maize breeding and genetics research to advance generations in breeding programs, to create desired research crosses, and produce hybrid seed for trials. Pollination in the field and in controlled environments relies on the availability of high‐quality pollen at the time that recipient silks are receptive. Generally, pollinations are made by capturing pollen from the tassel in a paper pollinating bag placed on the tassels one day prior to pollination and newly released pollen is then transferred to silks on the target plant. In the field, maize pollen is only viable for one to four hours following dehiscence and the rate of desiccation is influenced by environmental conditions. We have developed a method which increases the lifespan of pollen and allows pollen from a single tassel to be used to pollinate many ears by mixing fresh pollen with a dilutant that can be stored for multiple days. We identified characteristics of the size of suitable substrates and selected a PEEK‐based substrate for regular utilization. We evaluated pollen viability and empirically demonstrated the capability to store pollen up to 9 days when pollen is mixed with a PEEK substrate and stored at 6°C. The pollen storage method was used to make successful pollinations across 24 maize inbred lines tested and was generally equivalent to the standard manual pollination process. This method has the potential to increase the efficiency of breeding operations and may be useful in an array of genetic studies.

Seed increase through manual pollination is a critical part of maize breeding and genetics research to advance generations in breeding programs, to create desired research crosses, and produce hybrid seed for trials. Pollination in the field and in controlled environments relies on the availability of high-quality pollen at the time that recipient silks are receptive. Generally, pollinations are made by capturing pollen from the tassel in a paper pollinating bag placed on the tassels one day prior to pollination and newly released pollen is then transferred to silks on the target plant. In the field, maize pollen is only viable for one to four hours following dehiscence and the rate of desiccation is influenced by environmental conditions. We have developed a method which increases the lifespan of pollen and allows pollen from a single tassel to be used to pollinate many ears by mixing fresh pollen with a dilutant that can be stored for multiple days. We identified characteristics of the size of suitable substrates and selected a PEEK based substrate for regular utilization. We evaluated pollen viability and empirically demonstrated the capability to store pollen up to nine days when pollen is mixed with a PEEK substrate and stored at 6 degrees Celsius. The pollen storage method was used to make successful pollinations across 24 maize inbred lines tested and was generally equivalent to the standard manual pollination process. This method has the potential to increase the efficiency of breeding operations and may be useful in an array of genetic studies.