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.

Oxalate oxidase (OXO) converts oxalic acid (OA) and O 2 to CO 2 and hydrogen peroxide (H 2 O 2 ), and acts as a source of H 2 O 2 in certain plant-pathogen interactions. To determine if the H 2 O 2 produced by OXO can function as a messenger for activation of defense genes and if OXO can confer resistance against an OA-producing pathogen, we analyzed transgenic sunflower (Helianthus annuus cv SMF3) plants constitutively expressing a wheat (Triticum aestivum) OXO gene. The transgenic leaf tissues could degrade exogenous OA and generate H 2 O 2 . Hypersensitive response-like lesion mimicry was observed in the transgenic leaves expressing a high level of OXO, and lesion development was closely associated with elevated levels of H 2 O 2 , salicylic acid, and defense gene expression. Activation of defense genes was also observed in the transgenic leaves that had a low level of OXO expression and had no visible lesions, indicating that defense gene activation may not be dependent on hypersensitive response-like cell death. To further understand the pathways that were associated with defense activation, we used GeneCalling, an RNA-profiling technology, to analyze the alteration of gene expression in the transgenic plants. Among the differentially expressed genes, full-length cDNAs encoding homologs of a PR5, a sunflower carbohydrate oxidase, and a defensin were isolated. RNA-blot analysis confirmed that expression of these three genes was significantly induced in the OXO transgenic sunflower leaves. Furthermore, treatment of untransformed sunflower leaves with jasmonic acid, salicylic acid, or H 2 O 2 increased the steady-state levels of these mRNAs. Notably, the transgenic sunflower plants exhibited enhanced resistance against the OA-generating fungus Sclerotinia sclerotiorum.Oxidative burst, including hydrogen peroxide (H 2 O 2 ) production, is one of the early events that are associated with a hypersensitive response (HR) in many plant-pathogen interactions (HammondKosack and Jones, 1996; Lamb and Dixon, 1997). Several defensive roles for H 2 O 2 have been proposed (Lamb and Dixon, 1997). For example, H 2 O 2 in plant tissues may reach levels that are directly toxic to microbes (Peng and Kú c, 1992). H 2 O 2 may contribute to the structural reinforcement of plant cell walls (Bolwell et al., 1995) and trigger lipid peroxide and salicylic acid (SA) synthesis (Leô n et al., 1995). Moreover, H 2 O 2 appears to have roles in signal transduction cascades that coordinate various defense responses, such as induction of HR and synthesis of pathogenesis-related (PR) proteins and phytoalexins (Greenberg et al., 1994; Hammond-Kosack and Jones, 1996). These important roles of H 2 O 2 have attracted molecular pathologists' interest in manipulating the H 2 O 2 level by overexpressing an H 2 O 2 -generating enzyme, such as Glc oxidase (Wu et al., 1995; Kazan et al., 1998), to combat diseases in plants.Oxalate oxidase (OXO; EC 1.2.3.4) is one of the enzymes that can produce H 2 O 2 in plants. It releases CO 2 and H 2 O 2 from O 2...

SUMMARYThe liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I-CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I-CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium-mediated transformation of immature embryos, and transgenic T 0 plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T 0 plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant.

Clonal populations regenerated from single-leaf cell protoplasts of the potato cultivar ;Russet Burbank' display a high frequency of variation for several horticultural and disease resistance characters. Observations over a period of three tuber generations suggest stable changes in tuber shape, yield, and maturity date, in photo-period requirements for flowering, and in plant morphology. Enhanced resistance to early blight (Alternaria solani) and late blight (Phytophthora infestans) diseases also regularly occurs within regenerated populations. These findings are discussed in the context of possible application to varietal improvement, particularly as they pertain to asexually propagated plants.

SUMMARYThe I-CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double-strand breaks and enhancing DNA recombination reactions in maize cells. The DNA-binding properties of this protein were re-designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single-chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium-mediated transformation, the cleavage resulted in mutations at a co-delivered extra-chromosomal ms26-site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26-site in 5.8% of transgenic T 0 plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of doublestrand break repair generated by non-homologous end joining. One of 21 mutagenized T 0 plants carried two mutated alleles of the MS26 gene. As expected, the bi-allelic mutant T 0 plant and the T 1 progeny homozygous for the ms26 mutant alleles were male-sterile. This paper described the second maize chromosomal locus (liguless-1 being the first one) mutagenized by a re-designed I-CreI-based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.