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.

SummaryA member of the R2R3-MYB family of transcription factors was cloned from a cDNA library constructed from RNA isolated from differentiating pine xylem. This MYB, Pinus taeda MYB4 (PtMYB4), is expressed in cells undergoing ligni®cation, as revealed by in situ RT-PCR. Electrophoretic mobility shift assays (EMSAs) showed that recombinant PtMYB4 protein is able to bind to DNA motifs known as AC elements. AC elements are ubiquitous in the promoters of genes encoding lignin biosynthetic enzymes. Transcriptional activation assays using yeast showed that PtMYB4 could activate transcription in an AC-element-dependent fashion. Overexpression of PtMYB4 in transgenic tobacco plants altered the accumulation of transcripts corresponding to genes encoding lignin biosynthetic enzymes. Lignin deposition increased in transgenic tobacco plants that overexpressed PtMYB4, and extended to cell types that do not normally lignify. Taken together, these ®ndings are consistent with the hypothesis that PtMYB4 is suf®cient to induce ligni®cation, and that it may play this role during wood formation in pine.

SummaryOverexpression of a pine MYB, PtMYB4, in Arabidopsis caused ectopic lignin deposition and allowed the plants to undergo photomorphogenesis even when they were grown in the dark. The phenotype caused by PtMYB4 overexpression was reminiscent of the previously characterised dark-photomorphogenic mutant, de-etiolated 3 (det3); consequently, we tested the hypothesis that MYB misexpression may explain aspects of the det3 phenotype. We show here that AtMYB61, a member of the Arabidopsis R2R3-MYB family, is misexpressed in the det3 mutant. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that AtMYB61 was misexpressed in a det3 background relative to wild-type plants. Examination of AtMYB61 promoter activity in a det3 background showed that the spatial control of AtMYB61 expression was lost. In order to determine if such misexpression could explain the mutant phenotype, AtMYB61 was overexpressed in wild-type Arabidopsis plants. Transgenic plants that overexpressed AtMYB61 had the same ectopic ligni®cation and dark-photomorphogenic phenotype as that of the det3 mutant. In order to test if AtMYB61 was necessary for these aspects of the det3 phenotype, AtMYB61 expression was downregulated in det3 plants in both antisense and sense suppression experiments. Suppression of AtMYB61 in a det3 mutant background restored all mutant phenotypes of the det3 mutant associated with development in the dark. Taken together, these results suggest that AtMYB61 misexpression was both suf®cient and necessary to explain the ectopic ligni®cation and dark-photomorphogenic phenotypes of the det3 mutant.

A cDNA encoding a member of the R2R3-MYB family of transcription factors was cloned from a library constructed from differentiating Pinus taeda (loblolly pine) xylem RNA. This MYB family member, Pinus taeda MYB1 (PtMYB1), was most abundantly expressed in differentiating xylem, as assessed by both ribonuclease protection assays, and by northern blot analysis with poly(A)-enriched RNA. Similar to other plant R2R3-MYB family members, recombinant Pt MYB1 protein was able to bind to AC elements in electrophoretic mobility shift assays (EMSAs). AC elements are DNA motifs rich in adenosine and cytosine that have been implicated in the xylem-localised regulation of genes encoding lignin biosynthetic enzymes. Pt MYB1 not only bound to AC elements, but was also able to induce AC-element-dependent shifts in the electrophoretic mobility of a plant promoter that contains three AC elements, the minimal PHENYLALANINE AMMONIA-LYASE 2 (PAL2) promoter from Phaseolus vulgaris. Transcriptional activation assays conducted using yeast showed that Pt MYB1 also activated transcription, and that it did so in an AC-element-dependent fashion. Pt MYB1 also activated transcription from the minimal PAL2 promoter in plant cells in an AC-element-dependent fashion, as revealed by transient transcriptional activation assays with microprojectile-bombarded tobacco NT-1 cells. Taken together, these finding are consistent with the hypothesis that Pt MYB1 may regulate transcription from cis -acting AC elements in pine xylem.