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Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

The moss Physcomitrella patens was engineered to produce the diterpenoid sclareol, an important precursor for the synthesis of ambergris substitutes for the perfume industry. The best total yield of sclareol was 2.84 mg/g dry weight (2.28 mg/l culture) obtained after 18 days of cultivation in liquid media (extracted from both media and cell pellet). The two active sclareol synthase genes were integrated in a random fashion, and linked with the ribosomal skip 2A under the control of the CaMV 35S promoter. We conclude that moss can produce sclareol and utilize the ribosomal skip 2A. In addition, we observed growth impairment in all our sclareol-producing lines and moss lines knocked out in the endogenous diterpene synthase (copalyl/kaurene synthase—PpCPS/KS). A RT-PCR study, with ubiquitin as the best reference gene, showed that there was a down-regulation of the transcription of the terpenoid biosynthetic genes in the PpCPS/KS knock out moss. This down-regulation was recovered by the introduction of the two sclareol synthases, suggesting that the regulation of the general terpenoid biosynthesis is very flexible and can be amended in future biotechnological engineering.

A highly sensitive and simple spectrofluorimetric method for the determination of reduced glutathione based on the fluorescence quenching effect of glutathione on the hemoglobin-catalyzed reaction of H2O2 with L-tyrosine was developed. The concentration of glutathione is linear with the fluorescence quenching (ΔF) of system under the optimal experimental conditions. The calibration graph is linear in the range 6.25 × 10−9 to 3.75 × 10−6 M with the detection limit of 2.23 × 10−9 M. This method can be used for the determination of glutathione in vegetables with satisfactory results.

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N6-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.

Plant metabolites vary at different stages of their life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-targeted squamosa promoter binding protein-like (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-β-caryophyllene in the flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of patchouli oil, largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by upregulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production but also suggests a new strategy to engineer plants for accelerated growth with enhanced production of terpenoids.

Improved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar beet germplasm. The effects of N 6-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production of transgenic sugar beet following Agrobacterium-mediated transformation.

We have developed a routine system, based on microprojectile bombardment of embryogenic cell clusters for recovery of fertile transgenic rice (Oryza sativa L.) plants. Embryogenic cell clusters were established within 3–4 months from calli derived from mature seeds of nine diverse cultivars. The cell clusters were bombarded with plasmids carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and β-glucuronidase (gus A), both driven by the cauliflower mosaic virus (CaMV)35S promoter. Resistant calli were readily recovered from hygromycin-containing medium. On average 4.6 resistant calli were recovered from each bombarded plate. Transgenic plants were routinely regenerated from such resistant calli. In this system, where the transformation procedure was based on bombardment of embryogenic cell clusters, there were no escapes. Data from Southern blot analysis of T0 and T1 plants confirmed that the transgene was stably integrated into the host genome and inherited in a Mendelian fashion.

The pulse-discharge electroporation system HPES-3 has been used to transfer the bialaphos acetyltransferase gene of plasmid pDM302 into three commercial spring wheat cultivars T2003, Huanong No. 400 and Qu an Mai No. 1. These three cultivars are widely farmed in South China. The immature embryos, collected at 12–15 days after anthesis, were electropo- rated and exposed to phosphinothricin-containing medium. Phosphinothricin-resistant embryogenic calli were induced and from these a plantlet of cultivar T2003 was recovered. PCR analysis and DNA dot blot hybridization showed that the bialaphos acetyltransferase gene was present in the cells of the transgenic plant.

Summary A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post-selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation-sensitive enzymes revealed that the DNA represented by the common bands was N6-adenine methylated (m6A DNA), and there was even m6A DNA in some 30 month samples. Although the source of m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma-like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.

The apical meristems in dry wheat seeds were exposed (dissected seeds) to provide a target for DNA uptake. Using wheat, dwarf virus as the marker DNA, various methods of delivery were compared. Dry dissected seeds imbided in wheat dwarf virus DNA solution gave infection in 16% of the seedlings growing from them. A wheat dwarf virus dimer placed between the T-DNA borders of a vector plasmid inAgrobacterium tumefaciens (disarmed C58) andA. rhizogenes (LBA 9402) gave high levels of infection (79%) when dissected seeds were soaked in theAgrobacterium inoculum (agroinfection). Bombardment of dry dissected seeds with tungsten particles coated in wheat dwarf virus DNA did not give infection, but when softened by presoaking in water for 14 h, infection was observed at a low level (3%). The exposure of the apical meristem in all three methods gave a higher frequency of infection compared with treating intact seeds and in some cases the difference was substantial. The significance of the approach for DNA uptake studies is discussed along with its relevance to achieving stable transformation with non-viral constructs.

Background The development of robust microbes with tolerance to the combined lignocellulose-derived inhibitors is critical for the efficient cellulosic ethanol production. However, the lack of understanding on the inhibition mechanism limited the rational engineering of tolerant strain. Here, through the metabolomic analysis of an adaptation process of Saccharomyces cerevisiae to representative inhibitors, i.e., furfural, acetic acid and phenol (FAP), we figured out the new candidates for improving inhibitor tolerance. Results After metabolomic analysis, proline and myo-inositol were identified as the potential metabolites responsible for strain tolerance to inhibitors. The deletion of genes involved in proline or myo-inositol synthesis weakened strain tolerance against FAP stress. On the contrary, the addition of proline or myo-inositol in medium exerted a protective effect on cell growth under FAP stress. Furthermore, the enhancement of proline or myo-inositol synthesis by overexpressing key gene PRO1 or INO1 conferred yeast strain significantly increased FAP tolerance. All the recombinant strains finished the fermentation within 60 h under FAP stress, while the control strain was still in the lag phase. Meanwhile, it was found that the intracellular level of reactive oxygen species (ROS) under FAP condition was decreased with the increase of proline content, suggesting the function of proline as a ROS scavenger to protect strains from inhibitor damage. Conclusion Increasing proline and myo-inositol were uncovered as the new determinants for improving strain tolerance to FAP under the guidance of metabolomics. Meanwhile, this study displayed the powerful application of metabolomics to develop rational strategies to increase stress tolerance and provided valuable insights into the design of recombinant microbes for the complex traits. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0329-5) contains supplementary material, which is available to authorized users.