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.

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.

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.

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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 have shown signi cant improvements in salt and drought tolerance. However, very few eld-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 eld experiments that BADHfrom Atriplex hortensis (AhBADH) 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 signi cant enhancement of salt tolerance compared to non-transgenic control plants. Molecular analyses of the transgenic line TL7 with the highest salt tolerance exhibited stable inheritance and expression of AhBADH in progenies with a single copy insertion. TL7 exhibited stable enhanced salt tolerance and improved agronomic traits when subjected to 300mM NaCl treatment. Currently, the transgenic line TL7 with stable enhanced salt tolerance, which has been cleared for environmental release, is under biosafety assessment. TL7 stably expressing AhBADH could then be applied in commercial breeding experiments in order to genetically improve salt tolerance in soybean. Key MessageThe salt-tolerance of transgenic soybean cleared for environmental release was improved by stable overexpression of AhBADH gene from Atriplex hortensis, which was demonstrated through molecular analysis and eld experiments.