Development of Agrobacterium tumefaciens Infiltration of Infectious Clones of Grapevine Geminivirus A Directly into Greenhouse-Grown Grapevine and Nicotiana benthamiana Plants.

Grapevine virus infectious clones are important tools for fundamental studies, but also because of their potential for translational applications for grapevine improvement. Although several grapevine virus infectious clones have been developed, there has been difficulty in directly infecting mature grapevine plants, and many of the viruses used still cause disease symptoms in grapevine plants, making them less likely candidates for biotechnological applications in grapes. Here, we developed an improved Agrobacterium tumefaciens infiltration method that can be used to deliver DNA plasmids and viral infectious clones directly into approximately 20- to 40-cm-high (above soil) greenhouse-grown grapevine plants. We also developed infectious clones for two isolates of grapevine geminivirus A (GGVA): Longyan (China; GenBank accession KX570611; GGVA-76) and Super Hamburg (Japan; GenBank accession KX570610; GGVA-93). Neither virus caused any obvious symptoms when inoculated to plants of grapevine varieties Colombard, Salt Creek, Cabernet Sauvignon, and Vaccarèse. However, the two GGVA isolates induced different symptom severity and viral titer in Nicotiana benthamiana plants. The two GGVA isolates used here were found to accumulate to different titers in different parts/branches of the infected grapevine plants. The GGVA infectious clones and the improved grapevine infiltration technique developed here provide new, valuable tools that can be applied to grapevine plants, possibly even for translational applications such as disease management and desired trait improvements.

Exogenous methyl jasmonate modulates antioxidant activities and delays pericarp browning in litchi.

Pericarp browning (PB) is a serious problem in harvested litchi and drastically affects consumer acceptability and marketability. Postharvest PB and subsequent decay in fruit are linked to reactive oxygen species (ROS) accumulation in tissues. Antioxidants neutralize or scavenge ROS and maintain the shelf-life of fruit, especially in non-climacteric ones such as litchi. This work was aimed to assess the effect of vacuum infiltrated methyl jasmonate (MeJA; 1 and 2 mM) on the quality of harvested litchi fruit (cv. Purbi) during ambient storage (28 °C, RH 70-75%). The exogenous MeJA infiltration (2 mM) significantly retained quality attributes of litchi fruit as evident by lowered PB, weight loss, disease occurrence, quinone, and ROS (H2O2 and O2 -) accumulation. Moreover, MeJA infiltrated fruit suppressed the activity of polyphenol oxidase and peroxidase resulting in higher anthocyanin, phenolics, antioxidant potential, phenylalanine ammonia lyase activity as well as membrane integrity throughout the storage. Control fruit showed an early quality deterioration marked by prominent PB and other biochemical degradative changes. Thus, exogenous MeJA infiltration (2 mM) could be suggested to increase the shelf life of litchi by four days under ambient conditions.

Production of active human FGF21 using tobacco mosaic virus-based transient expression system.

Fibroblast growth factor (FGF) family has a wide range of metabolic processes. FGF21 exerts critical physiological functions in clinical application. This study aimed to explore a convenient and highly efficient approach for rhFGF21 expression using TMV-TES. Firstly, the vector pTTEV-GFP was constructed, followed by optimisation of the expression parameters in Nicotiana benthamiana. Then, the rhFGF21 encoding gene harbouring vector pTTEV-rhFGF21 was constructed. Agrobacterium-mediated vacuum infiltration was performed with the optimised parameters and the expression of rhFGF21 was confirmed by the immunoblotting analysis. ELISA revealed that the protein accumulation of rhFGF21 accounts for 0.11% of total soluble proteins. The biological activity was evaluated and the results suggested that tobacco-expressed rhFGF21 could stimulate the glucose uptake in swiss 3T3-L1 adipocytes, which was similar to the activity of commercial products, suggesting its native biological activity. Therefore, using TMV-TES to express rhFGF21 will be a feasible approach for the mass production of rhFGF21.

Protein Extraction, Enrichment and MALDI MS and MS/MS Analysis from Bitter Orange Leaves (Citrus aurantium).

Citrus aurantium is a widespread tree in the Mediterranean area, and it is mainly used as rootstock for other citrus. In the present study, a vacuum infiltration centrifugation procedure, followed by solid phase extraction matrix-assisted laser desorption ionization tandem mass spectrometry (SPE MALDI MS/MS) analysis, was adopted to isolate proteins from leaves. The results of mass spectrometry (MS) profiling, combined with the top-down proteomics approach, allowed the identification of 78 proteins. The bioinformatic databases TargetP, SignalP, ChloroP, WallProtDB, and mGOASVM-Loc were used to predict the subcellular localization of the identified proteins. Among 78 identified proteins, 20 were targeted as secretory pathway proteins and 36 were predicted to be in cellular compartments including cytoplasm, nucleus, and cell membrane. The largest subcellular fraction was the secretory pathway, accounting for 25% of total proteins. Gene Ontology (GO) of Citrus sinensis was used to simplify the functional annotation of the proteins that were identified in the leaves. The Kyoto Encyclopedia of Genes and Genomes (KEGG) showed the enrichment of metabolic pathways including glutathione metabolism and biosynthesis of secondary metabolites, suggesting that the response to a range of environmental factors is the key processes in citrus leaves. Finally, the Lipase GDSL domain-containing protein GDSL esterase/lipase, which is involved in plant development and defense response, was for the first time identified and characterized in Citrus aurantium.

Agrobacterium-mediated vacuum infiltration and floral dip transformation of rapid-cycling Brassica rapa.

BACKGROUND: Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier. RESULTS: Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T1 seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot. CONCLUSION: A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.

Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

BACKGROUND: Strawberry vein banding virus (SVBV) is a double-stranded DNA plant virus, which has been found in North America, Australia, Brazil, Japan, Europe and several provinces of China. Infected strawberry plants exhibit mild vein-banding symptoms and chlorosis along the veins. It is one of the most economically important diseases in Asiatic, European and North American strawberry-growing areas. FINDINGS: The complete genome of an SVBV Chinese isolate (SVBV-CN) was isolated and cloned from a naturally infected strawberry (Fragaria × ananassa cv. Sachinoka) sample found in Shenyang city of Liaoning province. Sequence analysis revealed a complete genome of 7864 nucleotides (nts) that indicated SVBV-CN was most closely related to SVBV from the United States (SVBV-US) with a sequence similarity of 85.8 %. Two major clades were identified based on phylogenetic analysis of the complete genome sequences of caulimoviruses. SVBV-CN clustered together with SVBV-US, whereas other caulimoviruses formed a separate branch. Agrobacterium-mediated inoculation of Fragaria vesca with an infectious clone of SVBV-CN results in systemic infection with distinct symptoms of yellowing bands along the main leaf veins. This suggests that the SVBV-CN infectious clone can recapitulate the symptoms observed in naturally infected strawberries, and therefore is likely the causal agent of the original disease observed in strawberries. Furthermore, strawberry plants inoculated with the infectious clone using vacuum infiltration developed symptoms with a very high infection rate of 86-100 % in 4-5 weeks post-inoculation. This compares to an infection rate of 20-40 % in 8-9 weeks post-inoculation using syringe-inoculation. CONCLUSIONS: The complete nucleotide sequence of SVBV from a naturally infected strawberry was determined. Agroinfiltration of strawberry plants using an infectious clone of SVBV-CN resulted in symptoms typically found in infected strawberries from Shenyang city of Liaoning province in China. This is the first report describing an infectious clone of SVBV-CN, and that vacuum infiltration can be potentially used as a new and highly efficient means for inoculation of strawberry plants.

Agrobacterium-mediated in planta genetic transformation of sugarcane setts.

KEY MESSAGE: An efficient, reproducible, and genotype-independent in planta transformation has been developed for sugarcane using setts as explant. Traditional Agrobacterium-mediated genetic transformation and in vitro regeneration of sugarcane is a complex and time-consuming process. Development of an efficient Agrobacterium-mediated transformation protocol, which can produce a large number of transgenic plants in short duration is advantageous. Hence, in the present investigation, we developed a tissue culture-independent in planta genetic transformation system for sugarcane using setts collected from 6-month-old sugarcane plants. The sugarcane setts (nodal cuttings) were infected with three Agrobacterium tumefaciens strains harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA(®). Several parameters influencing the in planta transformation such as A. tumefaciens strains, acetosyringone, sonication and exposure to vacuum pressure, have been evaluated. The putatively transformed sugarcane plants were screened by GUS histochemical assay. Sugarcane setts were pricked and sonicated for 6 min and vacuum infiltered for 2 min at 500 mmHg in A. tumefaciens C58C1 suspension containing 100 µM acetosyringone, 0.1 % Silwett L-77 showed the highest transformation efficiency of 29.6 % (with var. Co 62175). The three-stage selection process completely eliminated the chimeric transgenic sugarcane plants. Among the five sugarcane varieties evaluated using the standardized protocol, var. Co 6907 showed the maximum transformation efficiency (32.6 %). The in planta transformation protocol described here is applicable to transfer the economically important genes into different varieties of sugarcane in relatively short time.

Quantitative Estimation of Promoter Activity in Cannabis sativa Using Agroinfiltration-Based Transient Gene Expression.

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

Critical parameters for robust Agrobacterium-mediated transient transformation and quantitative promoter assays in Catharanthus roseus seedlings.

Agrobacterium-mediated transient expression methods are widely used to study gene function in both model and non-model plants. Using a dual-luciferase assay, we quantified the effect of Agrobacterium-infiltration parameters on the transient transformation efficiency of Catharanthus roseus seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre- and post-infiltration dark incubation and is less sensitive to the Agrobacterium growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven- to eight-fold while a dark incubation pre- and post-infiltration increased transformation efficiency by five- to 13-fold. Agrobacterium in exponential compared with stationary phase increased transformation efficiency by two-fold. Finally, we quantified the variation in our Agrobacterium-infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6-fold in raw firefly luciferase (FLUC) and raw Renilla luciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of Agrobacterium infiltration in C. roseus seedlings will facilitate the study of this important medicinal plant and will expand the application of Agrobacterium-mediated transformation methods in other plant species.

A simple and efficient genetic transformation system for soybean (Glycine max (L.) Merrill) targeting apical meristem of modified half-seed explant.

The present study is an attempt to establish a fast, highly reproducible transformation with a simplified regeneration system in soybean targeting the apical meristem. The modified half-seed explants from soybean cultivar (cv.) JS335 were subjected to different time intervals of sonication (0, 1, 10, 20, and 30 min) and vacuum infiltration (0, 1, 10, 20, and 30 min) in the presence of Agrobacterium tumefaciens strain EHA105 harbouring pCAMBIA1301. The explants were then co-cultivated and subjected to a modified plant regeneration process that involves only two steps (1) primary shoot regeneration, and (2) in vitro rooting of primary shoot. The rooted plantlets were hardened and maintained in the greenhouse until maturity. Sonication treatment of 10 min, followed by plant regeneration using a modified method, recorded the highest transformation efficiency of 26.3% compared to other time duration tested. Furthermore, 10 min of vacuum infiltration alone resulted in even higher transformation efficiency after regeneration, reaching 28.0%. Interestingly, coupling sonication and vacuum infiltration for 10 min respectively produced the highest transformation efficiency after regeneration of 38.0%. The putative transformants showed gus expression in mature leaves, trifoliate leaves, flowers, and pods. The presence of hpt II was also confirmed in putative transformants, with an amplicon size of 500 bp. Quantitative real-time PCR confirmed the existence of hpt II as one to two copies in the soybean genome of T0 plants. Furthermore, the segregation pattern was observed in the T1 generation soybean plants which were confirmed using PCR for hpt II. The optimized protocol when tested with other Indian soybean cultivars showed an enhanced transformation efficiency ranging from 19.3% (cv. MAUS47) to 36.5% (cv. CO1). This optimized protocol could provide a reliable platform to overcome the challenges that are associated with the genetic engineering of soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03715-8.

Production and Purification of Virus-Like Particles by Transient Expression in Plants.

Transient expression in plants has become a useful production system for virus-like particle (VLP) expression. High yields and flexible approaches to assembling complex VLPs, combine with ease of scale-up and inexpensive reagents to provide an attractive method for recombinant protein expression in general. Plants have demonstrated excellent capacity for the assembly and production of protein cages for use in vaccine design and nanotechnology. Furthermore, numerous virus structures have now been determined using plant-expressed VLPs, showing the utility of this approach in structural virology. Transient protein expression in plants uses common microbiology techniques, leading to a straightforward transformation procedure that does not result in stable transgenesis. In this chapter, we aim to provide a generic protocol for transient expression of VLPs in Nicotiana benthamiana using soil-free plant cultivation and a simple vacuum infiltration procedure, along with methodology for purifying VLPs from plant leaves.

Delivery of progeny virus from the infectious clone of cucumber green mottle mosaic virus and quantification of the viral load in different host plants.

Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is a widely occurring tobamovirus in cucurbits. The genome of CGMMV has been used previously for the expression of foreign genes in the plant. High throughput delivery and high viral titer are important requirements of foreign protein expression in plant through virus genome-based vector, in this study, Agrobacterium containing infectious construct of CGMMV was infiltrated through syringe, vacuum and high-speed spray to N. benthamiana, cucumber and bottle gourd leaves. The success rate of systemic infection of CGMMV agro-construct through all three methods was higher (80-100%) in N. benthamiana compared to the cucurbits (40-73.3%). To determine the high-throughput delivery of CGMMV in the plant system, four delivery methods viz. rubbing, syringe infiltration, vacuum infiltration and high-speed spray using the progeny virus derived through CGMMV agro-construct were compared in the three different plant species. Based on the rate of systemic infection and time required to perform delivery by different methods, vacuum infiltration was found most efficient for the high-throughput delivery of CGMMV. The quantification of CGMMV through qPCR revealed that CGMMV load varied considerably in leaf and fruit tissues depending with the time of infection. Immediately after expression of symptoms, a high load of CGMMV (~ 1 µg/100 mg of tissues) was noticed in young leaves of N. benthamiana and cucumber. In bottle gourd leaves, the CGMMV load was far low compared to N. benthamiana and cucumber plants. In the fruit tissues of cucumber and bottle gourd higher virus load was observed in mature fruit but not in immature fruit. The findings of the present study will serve as an important base line information to produce foreign protein through CGMMV genome-vector. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03630-y.

EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings.

Catharanthus roseus produces medicinal terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid biosynthesis in C. roseus . We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment.

Hydrogen sulphide infiltration downregulates oxidative metabolism and extends postharvest life of banana.

Hydrogen sulphide (H2 S) has emerged as a potential regulator of plant defence against different abiotic stresses. As a climacteric fruit, banana undergoes oxidative stresses shortly after harvest, resulting in faster ripening and senescence. This work examines the effects of vacuum infiltrated H2 S on ripening inhibition of banana. Banana fruits were vacuum infiltrated with 1 mm H2 S. Effects on oxidative stress markers, physiological changes, bioactive compounds and antioxidant potentials were examined during storage at 25 °C and 75-80% RH. Results indicate that treated fruits were less affected by oxidative stress, as evident by lower accumulation of ROS (superoxide and peroxide ions), elevated phenols content and antioxidant capacity. The ripening inhibitory effects of H2 S delayed chlorophyll loss and reduced ethylene and CO2 production. H2 S infiltration also reduced MDA accumulation and electrolytic leakage, resulting in longer shelf life. Vacuum infiltration with H2 S had a protective effect on postharvest banana through overcoming the deleterious effect of ROS and strengthening antioxidant potential. Thus, this method could be promising for enhanced preservation of banana during storage.

Production of Recombinant Proteins by Agrobacterium-Mediated Transient Expression.

The agroinfiltration of plant tissue is a robust method that allows the rapid and transient expression of recombinant proteins. Using wild-type plants as biomass, agroinfiltration exploits the ability of plants to synthesize even complex multimeric proteins that require oxidative folding and/or post-translational modifications, while avoiding the expensive and time-consuming creation of stably transformed plant lines. Here we describe a generic method for the transient expression of recombinant proteins in Nicotiana benthamiana at the small to medium laboratory scale, including appropriate binary vectors, the design and cloning of expression constructs, the transformation, selection, and cultivation of recombinant Agrobacterium tumefaciens, the infiltration of plants using a syringe or vacuum device, and finally the extraction of recombinant proteins from plant tissues.

3D-printed autoclavable plant holders to facilitate large-scale protein production in plants.

The Australian tobacco plant Nicotiana benthamiana is becoming increasingly popular as a platform for protein production and metabolic engineering. In this system, gene expression is achieved transiently by infiltrating N. benthamiana plants with suspensions of Agrobacterium tumefaciens carrying vectors with the target genes. To infiltrate larger numbers of plants, vacuum infiltration is the most efficient approach known, which is already used on industrial scale. Current laboratory-scale solutions for vacuum infiltration, however, either require expensive custom-tailored equipment or produce large amounts of biologically contaminated waste. To overcome these problems and lower the burden to establish vacuum infiltration in new laboratories, we present here 3D-printed plant holders for vacuum infiltration. We demonstrate that our plant holders are simple to use and enable a throughput of around 40 plants per hour. In addition, our 3D-printed plant holders are made from autoclavable material, which tolerate at least 12 autoclave cycles, helping to limit the production of contaminated waste and thus contributing to increased sustainability in research. In conclusion, our plant holders provide a simple, robust, safe and transparent platform for laboratory-scale vacuum infiltration that can be readily adopted by new laboratories interested in protein and metabolite production in Nicotiana benthamiana. Practical application Transient expression in Nicotiana benthamiana provides a popular and rapid system for producing proteins in a plant host. To infiltrate larger numbers of plants (typically >20), vacuum infiltration is the method of choice. However, no system has been described so far which is robust to use and can be used without expensive and complex equipment. Our autoclavable 3D-printed plant holders presented here will greatly reduce the efforts required to adopt the vacuum infiltration technique in new laboratories. They are easy to use and can be autoclaved at least 12 times, which contributes to waste reduction and sustainability in research laboratories. We anticipate that the 3D printing design provided here will drastically lower the bar for new groups to employ vacuum infiltration for producing proteins and metabolites in Nicotiana benthamiana.

A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Catharanthus roseus.

Functional genomics analyses in planta can be hampered in non-model plants that are recalcitrant to the genetic transformation such as the medicinal plant Catharanthus roseus (Apocynaceae). No stable transformation and regeneration of plantlets have been achieved with a high efficiency in this plant to date. In addition, while virus-mediated transient gene silencing has been reported a decade ago in C. roseus, tools for transient overexpression remain scarce. Here, we describe an efficient and reliable methodology for transiently overexpressing any gene of interest in C. roseus leaves. This protocol combines a vacuum-based Agroinfiltration approach and the high translational efficiency of a deconstructed virus-based binary vector (pEAQ-HT). The described methodology is robust, easy to perform, and results in high amount of transient expression in C. roseus. This protocol is expected to serve as valuable tool to enhance the in planta characterization of gene functions or even transiently knock-in novel enzymatic activities.

An efficient overexpression method for studying genes in Ricinus that transport vectorized agrochemicals.

BACKGROUND: Plant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals. Therefore, transporters associated with phloem loading of vectorized agrochemicals have drawn increasing attention. As a model system, castor bean (Ricinus communis L.) has been widely used to detect the phloem mobility of agrochemicals. However, there is still a lack of an efficient protocol for the Ricinus seedling model system that can be directly used to investigate the recognition and phloem loading functions of plasmalemma transporters toward vectorized agrochemicals. RESULTS: Here, using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Strong fluorescence signals were observed in leaves, demonstrating that exogenous genes can be successfully overexpressed in seedlings. Subsequently, gene expression time and vacuum infiltration parameters were optimized. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Parameter optimization showed Agrobacterium concentration at OD600 = 1.2, and infiltration for 20 min (0.09 MPa), return to atmospheric pressure, and then infiltration for another 20 min, were the suitable transformation conditions. To test the application of vacuum agroinfiltration in directly examining the loading functions of plasma membrane transporters to vectorized agrochemicals in seedlings, two LHT (lysine/histidine transporter) genes, RcLHT1 and RcLHT7, were overexpressed. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. Furthermore, the concentrations of L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of L-Val-PCA. CONCLUSIONS: We successfully constructed a transient expression system in Ricinus seedlings and laid the foundation for researchers to directly investigate the loading functions of plasma membrane transporters to vectorized agrochemicals in the Ricinus system.

Interaction between Dickeya dianthicola and Pectobacterium parmentieri in Potato Infection under Field Conditions.

Dickeya and Pectobacterium spp. both cause blackleg and soft rot of potato, which can be a yield-reducing factor to potato production. The purpose of this study was to examine the interaction between these two bacterial genera causing potato infection, and subsequent disease development and yield responses under field conditions. Analysis of 883 potato samples collected in Northeastern USA using polymerase chain reaction determined that Dickeya dianthicola and P. parmentieri were found in 38.1% and 53.3% of all samples, respectively, and that 20.6% of samples contained both D. dianthicola and P. parmentieri. To further investigate the relationship between the two bacterial species and their interaction, field trials were established. Potato seed pieces of "Russet Burbank", "Lamoka", and "Atlantic" were inoculated with bacterial suspension of D. dianthicola at 107 colony-forming unite (CFU)/mL using a vacuum infiltration method, air dried, and then planted in the field. Two-year results showed that there was a high correlation (p < 0.01) between yield loss and percent of inoculated seed pieces. In a secondary field trial conducted in 2018 and 2019, seed pieces of potato "Shepody", "Lamoka" and "Atlantic" were inoculated with D. dianthicola, P. parmentieri, or mixture of both species, and then planted. In 2019, disease severity index, as measured by the most sensitive variety "Lamoka", was 16.2 with D. dianthicola inoculation, 10.4 with P. parmentieri, 25.4 with inoculation with both bacteria. Two-year data had a similar trend. Thus, D. dianthicola was more virulent than P. parmentieri, but the co-inoculation of the two species resulted in increased disease severity compared to single-species inoculation with either pathogen.

Tissue Culture- and Selection-Independent Agrobacterium tumefaciens-Mediated Transformation of a Recalcitrant Grain Legume, Cowpea (Vigna unguiculata L. Walp).

A simple and generally fast Agrobacterium-mediated transformation system with no tissue culture and selection steps has been developed for the first time in a recalcitrant food legume, cowpea. The approach involves wounding of 1-day-old germinated seeds with a needle or sonication either alone or in combination of vacuum infiltration with A. tumefaciens EH105 (pCAMBIA2301) carrying a ß-glucuronidase (GUS) gene (uidA) and a neomycin phosphotransferase (nptII) gene for stable transformation. Sonicated and vacuum infiltrated seedlings showed the highest transient GUS activity in 90% of the explants. The sprouted co-cultured seeds directly established in soil and without selection were allowed to develop into plants which on maturity produced T0 seeds. The presence of the alien genes, nptII and uidA in T0 plants and their integration into the genome of T1 plants were confirmed by polymerase chain reaction (PCR) and Southern blot analyses, respectively. The transgenes were inherited in the subsequent T2 generation in a Mendelian fashion and their expression was confirmed by semi-quantitative PCR. The transformation frequency of 1.90% was obtained with sonication followed by vacuum infiltration with Agrobacterium. This approach provides favorable circumstances for the rapid meristem transformation and likely makes translational research ease in an important recalcitrant food legume, cowpea.