Improved salt-tolerance of transgenic soybean by stable over-expression of AhBADH gene from Atriplex hortensis.

KEY MESSAGE: The salt-tolerance of transgenic soybean cleared for environmental release was improved by stable over-expression of AhBADH gene from Atriplex hortensis, which was demonstrated through molecular analysis and field experiments. An effective strategy for increasing the productivity of major crops under salt stress conditions is the development of transgenics that harbor genes responsible for salinity tolerance. Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the biosynthesis of the osmoprotectant, glycine betaine (GB), and osmotic balance in plants, and several plants transformed with BADH gene have shown significant improvements in salt tolerance. However, very few field-tested transgenic cultivars have been reported, as most of the transgenic studies are limited to laboratory or green house experiments. In this study, we demonstrated through field experiments that AhBADH from Atriplex hortensis confers salt tolerance when transformed into soybean (Glycine max L.). AhBADH was successfully introduced into soybean by Agrobacterium mediated transformation. A total of 256 transgenic plants were obtained, out of which 47 lines showed significant enhancement of salt tolerance compared to non-transgenic control plants. Molecular analyses of the transgenic line TL2 and TL7 with the highest salt tolerance exhibited stable inheritance and expression of AhBADH in progenies with a single copy insertion. TL1, TL2 and TL7 exhibited stable enhanced salt tolerance and improved agronomic traits when subjected to 300mM NaCl treatment. Currently, the transgenic line TL2 and TL7 with stable enhanced salt tolerance, which have been cleared for environmental release, are under biosafety assessment. TL 2 and TL7 stably expressing AhBADH could then be applied in commercial breeding experiments to genetically improve salt tolerance in soybean.

Comparative transcriptome analysis of Veratrum maackii and Veratrum nigrum reveals multiple candidate genes involved in steroidal alkaloid biosynthesis.

Veratrum (Melanthiaceae; Liliales) is a genus of perennial herbs known for the production of unique bioactive steroidal alkaloids. However, the biosynthesis of these compounds is incompletely understood because many of the downstream enzymatic steps have yet to be resolved. RNA-Seq is a powerful method that can be used to identify candidate genes involved in metabolic pathways by comparing the transcriptomes of metabolically active tissues to controls lacking the pathway of interest. The root and leaf transcriptomes of wild Veratrum maackii and Veratrum nigrum plants were sequenced and 437,820 clean reads were assembled into 203,912 unigenes, 47.67% of which were annotated. We identified 235 differentially expressed unigenes potentially involved in the synthesis of steroidal alkaloids. Twenty unigenes, including new candidate cytochrome P450 monooxygenases and transcription factors, were selected for validation by quantitative real-time PCR. Most candidate genes were expressed at higher levels in roots than leaves but showed a consistent profile across both species. Among the 20 unigenes putatively involved in the synthesis of steroidal alkaloids, 14 were already known. We identified three new CYP450 candidates (CYP76A2, CYP76B6 and CYP76AH1) and three new transcription factor candidates (ERF1A, bHLH13 and bHLH66). We propose that ERF1A, CYP90G1-1 and CYP76AH1 are specifically involved in the key steps of steroidal alkaloid biosynthesis in V. maackii roots. Our data represent the first cross-species analysis of steroidal alkaloid biosynthesis in the genus Veratrum and indicate that the metabolic properties of V. maackii and V. nigrum are broadly conserved despite their distinct alkaloid profiles.

Application of CRISPR/CasΦ2 System for Genome Editing in Plants.

CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg2+ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg2+ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg2+ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.

Agrobacterium-Mediated Genetic Transformation of the Medicinal Plant Veratrum dahuricum.

Veratrum dahuricum L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of Veratrum dahuricum mediated with Agrobacterium tumefaciens is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. A. tumefaciens LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. A. tumefaciens cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of Veratrum.

Transformation of alfalfa chloroplasts and expression of green fluorescent protein in a forage crop.

The ability to transform chloroplasts in multiple species is important for improving agricultural traits. Chloroplast transformation of alfalfa (Medicago sativa L.), a useful forage plant with high market value, was achieved using a vector carrying aadA and gfp genes being introduced into the chloroplasts of alfalfa via particle bombardment using leaves and calli as explants. Resistant somatic embryos were generated and developed into plantlets from explants. The transformation efficiency was 1.3% for callus explants and 2.7% for leaf explants. PCR and Southern blotting analyses revealed that the foreign genes were integrated into the transformed chloroplast genome. The occurrence of GFP was further confirmed by fluorescence microscopy. Expression of foreign genes in alfalfa chloroplasts is therefore possible, and provides a novel means for genetic improvement of agronomically important traits and production of value-added proteins.