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Abstract. Immature embryos and embryogenic calli of rice, both japonica and indica subspecies, were bombarded with tungsten particles coated with plasmid DNA that contained a gene encoding hygromycin phosphotransferase (HPH, conferring hygromycin resistance) driven by the CaMV 35s promoter or Agrobacterium tumefacieiis NOS promoter. Putatively transformed cell clusters were identified from the bombarded tissues 2 weeks after selection on hygromycin B. By separating these cell clusms from each other, and by stringent selection not only at the callus growth stage but also during regeneration and plantlet growth, the overall transformation and selection efficiencies were substantially improved over those previously reported. From the most responsive cultivar used in these studies, an average of one transgenic plant was produced from 1.3 immature embryos or from 5 pieces of embryogenic calli bombarded. Integration of the introduced gene into the plant genome, and inheritance to the offspring were demonstrated. By using this procedure, we have produced several hundred transgenic plants. The procedure described here provides a simple method for improving transformation and selection efficiencies in rice and may be applicable to other monocots.

BackgroundCamelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits.ResultsExpressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50–57 %, while expression of full bypass increased seed yield by 57–73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions.ConclusionsThe photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0357-1) contains supplementary material, which is available to authorized users.

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1,140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1,140 bp 5' UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5' UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5' regulatory sequence, consisting of the rubi3 promoter, 5' UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5' UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5' UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5' regulatory sequences of other plant polyubiquitin genes.

Introns play a very important role in regulating gene expression in eukaryotes. In plants, many introns enhance gene expression, and the effect of intron-mediated enhancement (IME) of gene expression is reportedly often more profound in monocots than in dicots. To further gain insight of IME in monocot plants, we quantitatively dissected the effect of the 5' UTR intron of the rice rubi3 gene at various gene expression levels in stably transformed suspension cell lines. The intron enhanced the GUS reporter gene activity in these lines by about 29-fold. Nuclear run-on experiments demonstrated a nearly twofold enhancement by the 5' UTR intron at the transcriptional level. RNA analysis by RealTime quantitative RT-PCR assays indicated the intron enhanced the steady state RNA level of the GUS reporter gene by nearly 20-fold, implying a strong role of the intron in RNA processing and/or export. The results also implicated a moderate role of the intron in enhancement at the translational level ( approximately 45%). Moreover, results from a transient assay experiment using a shortened exon 1 sequence revealed an important role of exon 1 of rubi3 in gene expression. It may also hint a divergence in IME mechanisms between plant and animal cells. These results demonstrated transcriptional enhancement by a plant intron, but suggested that post-transcriptional event(s) be the major source of IME.

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The development of wheat (Triticum aestivum L.) cultivars that are resistant to Wheat streak mosaic virus (WSMV), yet competitive in yield under nondiseased conditions, is an objective for breeding programs in the Great Plains. This field study was conducted to compare classical and transgenic sources of resistance to WSMV. Three sets of germplasm were evaluated. These included adapted cultivars with various levels of tolerance, transgenic wheat lines containing viral coat protein or replicase sequences from WSMV that showed resistance in greenhouse trials, and germplasm with resistance to WSMV due to a translocated segment of chromosome 4Ai-2 from Thinopyrum intermedium (Host) Barkworth and Dewey containing Wsm1. A replicated field trial was conducted at Bozeman, MT, over a two-year period to evaluate the effectiveness of these different sources of resistance to mechanical inoculation of WSMV. Adapted cultivars differed in their ability to tolerate WSMV with mean reductions in yield over the two years ranging from 41 to 74%. Incorporation of the replicase or coat protein gene from WSMV did not provide field resistance to viral infection and in general, transgenic lines yielded less than their parent cultivar, 'Hi-Line'. Wheat-Thinopyrum lines positive for a DNA marker linked to the Wsm1 gene had significantly reduced yield losses ranging from 5 to 39% compared with yield losses of 57 to 88% in near isogenic lines not having the Wsm1 gene. Yield of lines with Wsm1 in the absence of disease ranged from 11 to 28% less than yield of lines without Wsm1. Our results suggest Wsm1 provides the best source of WSMV resistance but a yield penalty may exist because of the presence of the translocation.