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.

SummaryWe investigated whether termination of transcripts with a self-cleaving ribozyme can enhance nuclear retention and serve as a tool to decrease speci®c plant gene expression. Nuclear retention was ®rst monitored in tobacco using the b-glucuronidase gene terminated with either the 35S CaMV 3¢ untranslated sequence (UTR) or a cis-acting ribozyme. Northern blot analysis of nuclear RNA and total RNA, and in situ hybridizations showed that the ribozyme-terminated transcripts were preferentially retained in the nucleus of transgenic tobacco. Ribozyme-terminated transcripts were subsequently tested as a gene down-regulation strategy in soybean. The embryo-speci®c D-12 fatty acid desaturase FAD2-1 gene was targeted because its down-regulation elevates oleic acid content of seed storage lipids. Both ribozyme-terminated antisense and standard antisense constructs were capable of gene downregulation, producing over 57% oleic acid compared with less than 18% in wild-type seed. Ribozyme termination cassettes were also constructed to evaluate sense transcripts for single gene downregulation and the simultaneous down-regulation of two embryo-speci®c genes in soybean using a single promoter. Eight independent soybean transformants were screened that harboured standard plus sense or ribozyme terminated FAD2-1 cassette. Two of the eight ribozyme terminated transformants displayed oleic acids levels in the seed storage lipids of over 75%, while none of the standard plus sense FAD2-1 lines showed elevated oleic acid phenotypes. The dual constructs targeted FAD2-1 and the FatB gene encoding a palmitoyl-thioesterase. Five transgenic soybean lines harbouring the dual constructs had oleic acid levels, greater than 85%, and saturated fatty acids levels, less than 6%. Thus, ribozyme termination of transcripts can be utilized to speci®cally down-regulate endogenous gene expression in soybean.

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.

A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T 0 ) representing 10 independent events were characterized. Seven of the 10 independent T 0 events co-expressed GUS. Progeny analysis was conducted by sowing the T 1 seeds and monitoring the expression of the GUS gene after 21 d. Individual T 1 plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mg l 21 solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either beta-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

and black currents (Ribes nigrum L.) (Goffman and Galletti, 2001). The yield potential of these herbs is Through a single desaturation step, the Borago officinalis L. ⌬ 6 rather limited and levels of GLA and STA in the seed desaturase can convert linoleic acid and ␣-linolenic acid to ␥-linolenic acid (GLA) and stearidonic acid (STA), respectively. Both GLA and storage lipids constitute Ͻ16 and 5%, respectively. STA are of interest to the pharmaceutical and nutraceutical industries.In plants, GLA is produced by the desaturation of Production of these fatty acids is costly. One potential strategy to linoleic acid via a ⌬ 6 desaturase (Sayanova et al., 1997). reduce production cost would be to generate them in a major oilseed A cDNA clone of a ⌬ 6 desaturase from borage excrop. To this end, a cDNA of the B. officinalis ⌬ 6 -desaturase gene pressed in tobacco was capable of generating both GLA was cloned downstream of the embryo-specific promoter ␤-conglyciand STA in the transgenic plants at 12.9 and 8.5%, nin. The resultant cassette was assembled into a two T-DNA binary respectively, in young leaves (Sayanova et al., 1997). vector, in which the second T-DNA element harbored a selectable Accumulation of the novel fatty acids in the transgenic marker cassette. The final plasmid was subsequently used to transform tobacco lines varied across tissue, with the highest levels soybean [Glycine max (L.) Merr.]. The simultaneous delivery of two of 27.2% GLA and 8.7% STA being observed in stem T-DNA elements was used as a strategy to derive soybean progeny transgenic for the ⌬ 6 desaturase T-DNA and free of the marker gene tissue (Sayanova et al., 1999). Introduction of a fungal T-DNA. Twenty-nine transgenic soybean lines were recovered that (Mortierella alpina Peyronel) ⌬ 6 desaturase into canola harbored both T-DNA elements, of which 17 produced GLA and (Brassica napus L.) resulted in accumulation of GLA STA in the seed storage lipids. Average GLA levels ranged from 3.4 in the seed to approximately 13%, while dual expression up to 28.7%, while STA levels varied from just under 0.6 to 4.2% in of the fungal ⌬ 12 -and ⌬ 6 -desaturase genes resulted in the T 1 generation. Among the 17 lines that produced GLA and STA, elevated levels of GLA in seeds, 43%, and production four lines were identified that were free of the selectable marker of STA levels of ≈2% (Liu et al., 2001). The combination T-DNA element.of ⌬ 12 -and ⌬ 6 -desaturase genes was required to enhance GLA levels in canola since the predominate fatty acid in B. napus seed is oleic. Expression of the ⌬ 12 desaturase Univ. of Nebraska, Lincoln, NE 68583; B. Schweiger and A. Kinney, DuPont Exp. Stn., Wilmington, DE 19880. This publication is a contri-nology by implementing the identical marker gene for

Recombinase-mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of transgenes can be stacked to the same site through multiple rounds of RMCE. A gene-silencing cassette, designed to simultaneously silence soybean (Glycine max) genes fatty acid ω-6 desaturase 2 (FAD2) and acyl-acyl carrier protein thioesterase 2 (FATB) to improve oleic acid content, was first inserted by RMCE at a precharacterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley (Hordeum vulgare) high-lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS), to improve the contents of the essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four overexpression cassettes to the previously integrated FAD2-FATB gene-silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes.

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