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.

A late embryogenesis abundant (LEA) protein gene, HVA7, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA 7 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVAl protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerante correlated with the leve1 of the HVAl protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under wateror salt-stress conditions. Thus, LEA genes hold considerable potentia1 for use as molecular tools for genetic crop improvement toward stress tolerance.

We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

DELLA proteins are nuclear-localized negative regulators of gibberellin signaling found ubiquitously throughout higher plants. Dominant dwarfing mutations of DELLA proteins have been primarily responsible for the dramatic increases in harvest index of the 'green revolution'. Maize contains two genetic loci encoding DELLA proteins, dwarf plant8 (d8) and dwarf plant 9 (d9). The d8 gene and three of its dominant dwarfing alleles have been previously characterized at the molecular level. Almost 20 years after the initial description of the mutant, this investigation represents the first molecular characterization of d9 and its gibberellin-insensitive mutant, D9-1. We have molecularly, subcellularly and phenotypically characterized the gene products of five maize DELLA alleles in transgenic Arabidopsis. In dissecting the molecular differences in D9-1, a critical residue for normal DELLA function has been uncovered, corresponding to E600 of the D9 protein. The gibberellin-insensitive D9-1 was found to produce dwarfing and, notably, earlier flowering in Arabidopsis. Conversely, overexpression of the D9-1 allele delayed flowering in transgenic maize, while overexpression of the d9 allele led to earlier flowering. These results corroborate findings that DELLA proteins are at the crux of many plant developmental pathways and suggest differing mechanisms of flowering time control by DELLAs in maize and Arabidopsis.