Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

Background Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi. Results In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots. Conclusion We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

Towards A Genetic Model Organism: An Efficient Method For Stable Genetic Transformation of Eschscholzia Californica (Ranunculales)

California poppy (Eschscholzia californica) is a member of the Ranunculales, the sister order to all other eudicots and as such in a phylogenetically highly informative position. Ranunculales are known for their diverse floral morphologies and biosynthesis of many pharmaceutically relevant alkaloids. E. californica it is widely used as model system to study the conservation of flower developmental control genes. However, within the Ranunculales, options for stable genetic manipulations are rare and genetic model systems are thus difficult to establish. Here, we present a method for the efficient and stable genetic transformation via Agrobacterium tumefaciens-mediated transformation, somatic embryo induction, and regeneration of E. californica. Further, we provide a rapid method for protoplast isolation and transformation. This allows the study of gene functions in a single-cell and full plant context to enable gene function analysis and modification of alkaloid biosynthesis pathways by e.g. genome editing techniques providing important genetic resources for the genetic model organism E. californica.

A cassava common mosaic virus vector for virus-induced gene silencing in cassava

Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut (Juglans regia L.) Fruits

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

Development of a robust transient expression screening system in protoplasts of Cannabis

Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed in Cannabis due to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from aseptic in vitro Cannabis explants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106 were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106 protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.

A Cassava Common Mosaic Virus Vector for Virus-induced Gene Silencing in Cassava

Background: Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing.Results:In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus strain CM (CsCMV-CM) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions: This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.