A betaxanthin-based visible and fluorescent reporter for monitoring plant transformation

Commonly used reporters rely on a single property,such as the fluorescence of GFP and visible color of anthocyanins,therefore these reporters hardly handle the complicated condition in practice.Betaxanthins are a group of plant natural products derived from the amino acid tyrosine.Its visible yellow-orange color and green fluorescence under blue light make it a promising new reporter.Only two enzymatic reactions are required to convert tyrosine into betaxanthins.Here,we synthesized an open reading frame named Bx that contained all the betaxanthins biosynthetic genes and demonstrated its use as a powerful and efficient reporter in tobacco,carrot,and tomato.

Generating Marker-Free Transgenic Tobacco Plants by Agrobacteriummediated Transformation with Double T-DNA Binary Vector

We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.

Reliable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.

A simple and efficient in planta transformation method based on the active regeneration capacity of plants

Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants.However,the complicated operational protocols and low efficiency of cur-rent transformation strategies restrict the genetic modification of most plant species.This paper de-scribes the development of the regenerative activity–dependent in planta injection delivery(RAPID)method based on the active regeneration capacity of plants.In this method,Agrobacterium tumefaciens is delivered to plant meristems via injection to induce transfected nascent tissues.Stable transgenic plants can be obtained by subsequent vegetative propagation of the positive nascent tissues.The method was successfully used for transformation of plants with strong regeneration capacity,including different genotypes of sweet potato(Ipomoea batatas),potato(Solanum tuberosum),and bayhops(Ipo-moea pes-caprae).Compared with traditional transformation methods,RAPID has a much higher trans-formation efficiency and shorter duration,and it does not require tissue culture procedures.The RAPID method therefore overcomes the limitations of traditional methods to enable rapid in planta transformation and can be potentially applied to a wide range of plant species that are capable of active regeneration.

Agrobacterium-Mediated Transformation of Cry8db Gene in Vietnam Sweet Potato Cultivar

Sweet potato [Ipomoea batatas （L.） Lam.] is an important food crop in the world as well as in Vietnam. It is well known as a recalcitrant crop for gene transformation and tissue culture because of its genotype dependent in vitro responses. In present study, Agrobacterium-mediated transformation of cry8Db from Bacillus thuringiensis into KB 1 sweet potato variety has been studied. The C58cv strain carrying a pBl 121 backbone which contained cry8Db delta-endotoxin gene regulated under 35 S CaMV prom oter, and the selection marker gene, neomycin phosphotransferase （npt11） gene, was subjected for plant transformation. Callus induced from shoot tips and leaf explants were inoculated and cocultured with A. tumefaciens. The selection occurred during callus producing and plant regenerating steps. A total of 201 transgenic putative plant lines were produced, and 21 transgenic lines were positively confirmed by PCR and finalized by Southern blot. Four putative transgenic lines confirming a single copy of the crySEIb gene were transferred into soil pots in greenhouse. Biological activity evaluation for the insecticidal capacity of these transgenic lines under controlled conditions showed that the level of infestation by sweet potato weevils （Cylasformicarius） in untransformed plants was higher than that of transgenic lines.

PEG－mediated Gene Transfer into Orychophragmus Violaceus Cotyledon Protoplast and Regeneration of Transgenic Plants

Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.

Cultivating potential:Harnessing plant stem cells for agricultural crop improvement

Meristems are stem cell-containing structures that produce all plant organs and are therefore important targets for crop improvement.Developmental regulators control the balance and rate of cell divisions within the meristem.Altering these regulators impacts meristem architecture and,as a consequence,plant form.In this review,we discuss genes involved in regulating the shoot apical meristem,inflorescence meristem,axillary meristem,root apical meristem,and vascular cambium in plants.We highlight several examples showing how crop breeders have manipulated developmental regulators to modify meristem growth and alter crop traits such as inflorescence size and branching patterns.Plant transformation techniques are another innovation related to plant meristem research because they make crop genome engineering possible.We discuss recent advances on plant transformation made possible by studying genes controlling meristem development.Finally,we conclude with discussions about how meristem research can contribute to crop improvement in the coming decades.

Expression of Human Interferon α 2b Gene in Ginseng Cells

The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. Then the hIFN-α2b gene was introduced into ginseng callus cells via Agrobacterium-mediated transformation and the ginseng cell line carrying hIFN-α2b gene was selected on G418-containing medium. The presence of target gene in transformed cells was confirmed by PCR and RT-PCR. The results indicate that hIFN-α2b gene has been integrated into the ginseng cells＇ genome, with transcription products, hIFN-α2b expressed by the transgenic ginseng cells was detected by Western blot. It was shown that a specific protein band at 19000 could be observed. Cytopathic effect（CPE） inhibition assay using the W1SH-VSV system shows that the mean antiviral activity of expressed hlFN-a2α was 6.0× 10^4 IU/mL.

An in vivo Transient Expression System Can Be Applied for Rapid and Effective Selection of Artificial MicroRNA Constructs for Plant Stable Genetic Transformation

The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable,In this study,expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation.A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments.Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs.This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells

Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills 〉500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized （~0.8 mg/g dry weight） at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments （chloroplast, nucleus, and mitochondria）. The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of di- hydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants.

Plant genetic engineering and genetically modified crop breeding: history and current status

This review charts the major developments in the genetic manipulation of plant cells that have taken place since the first gene transfer experiments using Ti plasmids in 1983. Tremendous progress has been made in both our scientific understanding and technological capabilities since the first genetically modified(GM)crops were developed with single gene resistances to herbicides, insects, viruses, and the silencing of undesirable genes. Despite opposition in some parts of the world, the area planted with first generation GM crops has grown from 1.7 Mhm^2 in 1996 to 179.7 Mhm^2 in 2015.The toolkit available for genetic modification has expanded greatly since 1996 and recently Nobel Laureates have called on Greenpeace to end their blanket opposition,and plant scientists have urged that consideration be given to the benefits of GM crops based on actual evidence. It is now possible to use GM to breed new crop cultivars resistant to a much wider range of pests and diseases, and to produce crops better able to adapt to climate change.The advent of new CRISPR-based technologies makes it possible to contemplate a much wider range of improvements based on transfer of new metabolic pathways and traits to improve nutritional quality, with a much greater degree of precision. Use of GM, sometimes in conjunction with other approaches, offers great opportunities for improving food quality, safety, and security in a changing world.

pHairyRed： A Novel Binary Vector Containing the DsRed2 Reporter Gene for Visual Selection of Transgenic Hairy Roots

We developed a new plant transformation vector, pHairyRed, for enabling high throughput, non-destructive selection of Agrobacterium rhizogenes-mediated ＇hairy-root＇ transformation, pHairyRed allows easy in planta visualiza- tion of transgenic tissue with minimal disturbance to the plant. The DsRed2 reporter gene, encoding a red fluorescent protein, was cloned to yield pHairyRed （harbouring a multiple cloning site）, which was used with the highly efficient K599 A. rhizogenes strain to infect soybean （Glycine max L. Merrill） plants. DsRed2 fluorescence was easily detected in planta for the duration of a 5-week study with negligible levels of background autofluorescence. This enabled visual selection of transformed roots and subsequent excission of non-transformed roots, pHairyRed-transformed roots nodu- lated normally when inoculated with Bradyrhizobiumjaponicum. Within the nodule, DsRed2 fluorescence was plant-spe- cific, being absent in the bacteroid-dominated nodule infected zone. To test the reliability of pHairyRed as a high-fidelity binary vector reporter system, the gene encoding the soybean Nod factor receptor, GmNFRloL, was cloned into the vector for use in a complementation study with a non-nodulating nfrloL mutant of soybean. Complementation was achieved and, without exception, DsRed2 fluorescence was detected in all hairy roots that successfully formed nodules （100%, n = 34）. We anticipate broad application of this reporter system for the further analysis of root-related events in soybean and related legumes.