Abstract
Constitutive expression of the Zea mays L. (maize) morphogenic transcription factors Baby Boom (Bbm) and Wuschel2 (Wus2) in maize can not only greatly increase transformation efficiency but can also induce phenotypic abnormalities and sterility. In an effort to alleviate the pleiotropic effects of constitutive expression, a genome wide search was undertaken to find suitable maize promoters to drive tissue and timing-specific expression of the transformation enhancing genes Bbm and Wus2. A promoter from a maize phospholipid transferase protein gene (Zm-PLTPpro) was identified based on its expression in leaves, embryos, and callus while being downregulated in roots, meristems, and reproductive tissues. When Zm-PLTPpro driving Bbm was transformed into immature maize embryos along with a Wus2 expression cassette driven by the nopaline synthase promoter (Nospro::Wus2) abundant somatic embryos rapidly formed on the scutella. These embryos were individual and uniformly transformed and could be directly germinated into plants without a callus phase. Transformed plants could be sent to the greenhouse in as little as 1 mo and regenerated plants matched the seed-derived phenotype for the inbred and were fertile. However, T1 seed from these plants had poor germination. Replacing Nospro with a maize auxin-inducible promoter (Zm-Axig1pro) in combination with Zm-PLTPpro::Bbm, allowed healthy, fertile plants to be regenerated. Single-copy T1 seed germinated normally and had a predominantly wild-type inbred phenotype. For maize, this callus-free transformation process has worked in all inbred lines tested.
Highlights
Since the discovery that immature maize embryos could be used to initiate regenerable embryogenic callus (Green and Phillips 1975), almost all high throughput transformation systems in cereals including maize have required callus formation and selection prior to plant regeneration
The RNA Sequencing by Expectation Maximization method (RSEM; Li and Dewey 2011) was used as the metric for the quantification of gene expression in transcripts per million reads observed normalized units (TPM) with each gene model’s TPM estimate summarizing the expression value for all its measurable isoforms in a way that is relative to the total number of sequences in a particular sample’s library
Lowe et al (2016) previously demonstrated that the maize morphogenic regulators Baby Boom (Bbm) and Wus2 can be used to stimulate growth of embryogenic callus from immature embryos and normally recalcitrant tissues such as leaves or explants derived from mature seeds
Summary
Since the discovery that immature maize embryos could be used to initiate regenerable embryogenic callus (Green and Phillips 1975), almost all high throughput transformation systems in cereals including maize have required callus formation and selection prior to plant regeneration (see Ji et al 2013 for review). Lowe et al (2016) have recently described a transformation method that uses the maize transcription factors Baby Boom (Bbm) and Wuschel (Wus2) to stimulate the proliferation of transformed cells In this case, cells were directly induced to form callus by the transgenes rather than transforming cells that had been induced to divide by conventional tissue culture methods. Cells were directly induced to form callus by the transgenes rather than transforming cells that had been induced to divide by conventional tissue culture methods Using this method, pleiotropic effects were observed in transgenic plants making excision of Bbm and Wus necessary to obtain normal transgenic plants. The Lowe et al (2016) method still required 3 mo of callus selection before transferring callus to dry filter papers for 3 d to induce excision
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