Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.

A small family of novel basic leucine zipper proteins that includes abscisic acid (ABA)-INSENSITIVE 5 (ABI5) binds to the promoter region of the lea class gene Dc3. The factors, referred to as AtDPBFs (Arabidopsis Dc3 promoter-binding factors), were isolated from an immature seed cDNA library. AtDPBFs bind to the embryo specification and ABA-responsive elements in the Dc3 promoter and are unique in that they can interact with cis-elements that do not contain the ACGT core sequence required for the binding of most other plant basic leucine zipper proteins. Analysis of full-length cDNAs showed that at least five different Dc3 promoter-binding factors are present in Arabidopsis seeds; one of these, AtDPBF-1, is identical to ABI5. As expected, AtDPBF-1/ABI5 mRNA is inducible by exogenous ABA in seedlings. Despite the near identity in their basic domains, AtDPBFs are distinct in their DNA-binding, dimerization, and transcriptional activity.LEA (late embryogenesis abundant) genes as a group are highly expressed during late stages of embryo development (Hughes and Galau, 1991;Thomas, 1993;Parcy et al., 1994). lea gene products are ubiquitous among higher plants, and they are probably involved in the protection of cells from dehydration (Dure et al., 1989; Dure, 1993; Ingram and Bartels, 1996;Xu et al., 1996). Expression of many lea genes is not only seed specific but is also inducible by abscisic acid (ABA) or environmental stresses such as drought and high salinity (Skriver and Mundy, 1990; Chandler and Robertson, 1994; Ingram and Bartels, 1996).Dc3 is a carrot (Daucus carota) lea class gene that is abundantly expressed during somatic embryogenesis (Wilde et al., 1988). The Dc3 promoter drives ␤-glucuronidase (GUS) reporter gene expression in developing seeds of transgenic tobacco (Nicotiana tabacum) and in nonembryonic tissues exposed to exogenous ABA and conditions of water deficit (Seffens et al., 1990;Vivekananda et al., 1992;Siddiqui et al., 1998). Analysis of the Dc3 promoter revealed minimal sequences necessary for embryo-specific expression residing within a 117-bp region including the transcription start site (Thomas, 1993; Chung, 1996;Thomas et al., 1997). However, this proximal promoter region (PPR) is not sufficient for ABAinduced expression; the distal promoter region (DPR), located between Ϫ314 and Ϫ287, is also required for ABA response in addition to the PPR (Chung, 1996;Thomas et al., 1997). The PPR contains five related cis regulatory elements required for expression in embryogenesis; these elements (E motifs) share the consensus sequence ACACNNG. Elements with NNNCGTGT consensus are repeated within the minimal DPR. These latter elements are similar to the E motifs. The function of these elements has been demonstrated in planta (Chung, 1996).Protein-binding studies showed that similar, seedspecific, or ABA-inducible protein factors bind to the PPR and the DPR. Competition DNA-binding assays indicated that similar factors can bind to both the PPR and the DPR. Genes encoding these Dc3 promoterbindin...

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.

We used virtual subtraction, a new gene isolation strategy, to isolate several genes of interest that are expressed in Arabidopsis embryos. These genes have demonstrated biological properties or have the potential to be involved in important biological processes. One gene isolated by virtual subtraction is PEI. It encodes a protein containing a Cys3His zinc finger domain associated with a number of animal and fungal transcription factors. In situ hybridization results showed that PEI1 is expressed throughout the embryo from globular to late cotyledon stage. Transgenic Arabidopsis plants expressing a PEI1 antisense gene produced white seeds in which embryo development did not progress through heart stage. Aberrant embryos failed to form cotyledons, but the embryonic root appeared to be normal. Aberrant embryos did not turn green, and the expression of genes involved in photomorphogenesis was drastically attenuated. In culture, aberrant embryos did not form true leaves, but root formation was apparently normal. These results suggest that PEI1 is an embryo-specific transcription factor that plays an important role during Arabidopsis embryogenesis, functioning primarily in the apical domain of the embryo.