Agrobacterium Inoculum Research Articles

High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD600≈2.0) in large cultures and resuspended in inoculation medium to OD600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees relatively high transformation rate in Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

Transient expression of bioactive recombinant human plasminogen activator in tobacco leaf

To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves (P < 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.

Optimization of transient GUS expression of Agrobacterium-mediated transformation in Dierama erectum Hilliard using sonication and Agrobacterium

Efficient Agrobacterium-mediated transformation was established via sonication of embryonic shoot apical meristems (ESAMs) of Dierama erectum Hilliard. Effects of explant types, co-cultivation time, acetosyringone concentration, Agrobacterium concentration and different gene delivery methods were evaluated for higher efficiency of genetic transformation in D. erectum. An explant type (ESAMs), concentration of Agrobacterium inoculum (OD600 of 1.6) and acetosyringone (50mgL−1), and co-cultivation duration (3days) were optimized for efficient genetic transformation of D. erectum. The transformation efficiency varied with explant types (from 0 to 60%), concentrations of bacteria (10 to 55%) and acetosyringone (50 to 90%) and period of co-cultivation (30 to 70%). The transformation efficiency was best with ESAMs explants compared with callus clusters. The gene delivery method via sonication-assisted Agrobacterium-mediated transformation (SAAT) provided higher transformation efficiency (40%) GUS expression compared with agrobacterial monolayer and agrobacterial suspension which gave less than 5% transformation efficiency. The putative transgenic plants which histochemically expressed GUS, were confirmed further with PCR and 35.3% of the plants were GUS positive. Stable integration of the transgene was not demonstrated hence the GUS expression observed was regarded as transient. This newly developed transformation system may facilitate improvement of D. erectum characteristics and other related geophytes.

Factors affecting Agrobacterium-mediated transformation efficiency of kumquat seedling internodal stem segments

Kumquat (Fortunella crassifolia Swingle.) has a short juvenile phase of 2–3 years and it can flower and bear fruit several times per year. It is, therefore, helpful for citrus to evaluate functional genes rapidly. In the present experimentation, a comprehensive and systematic study was performed to improve the transformation efficiency of kumquat, which was usually less than 4.1% in previous reports. Several factors, including explant status, Agrobacterium infection procedure, and co-cultivation conditions, were tested by using single factors or orthogonal experimental design. The higher regeneration and transformation rates were obtained from the seedlings cultured for 25days in darkness followed by 10days under light. The Agrobacterium cells suspended with co-cultivation medium to a final concentration 1×109 cfu/ml (OD600 value of 0.5) and immersing explants in Agrobacterium inoculum for 30min, resulted in better infection efficiency. The results indicated that the following conditions could improve the transformation efficiency. These treatments included adding 100μM AS into inoculum and co-cultivation media, placing internodal stem segments by end to end on co-cultivation medium for 3days at 25°C and placing co-cultivation medium in pH 5.4 with sterile filter paper support. With all these improvements, GUS-positive buds were recovered at the positive rate of 23.6% on selection medium. Polymerase chain reaction (PCR) analysis using gene-specific primers further confirmed the integration of the transgene for in vitro grafted plantlets at the transformation rate of 10.35%. Normal expression of nptⅡ and FLP genes was observed by RT-PCR and Real time q-PCR analysis.

Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum).

A new method of transgenic development called “In-planta” transformation method, where Agrobacterium is used to infect the plantlets but the steps of in vitro regeneration of plants is totally avoided. In this study, we have reported a simple In-planta method for efficient transformation of diploid cotton Gossypium hirsutum cv LRK-516 Anjali using Agrobacterium tumefaciens EHA-105 harbouring recombinant binary vector plasmid pBinAR with Arabidopsis At-NPR1 gene. Four day old plantlets were used for transformation. A vertical cut was made at the junction of cotyledonary leaves, moderately bisecting the shoot tip and exposing meristem cells at apical meristem. This site was infected with Agrobacterium inoculum. The transgenic events obtained were tested positive for the presence of At-NPR1 gene with promoter nptII gene. They are also tested negative for vector backbone integration and Agrobacterium contamination in T0 events. With this method a transformation frequency of 6.89% was reported for the cv LRK-516.

RETRACTEDARTICLE: Influence of Agrobacterium rhizogenes strains, acitosyringone, inoculum size and temperature on production of active ingredients from Picrorhiza kurrooa.

RETRACTEDARTICLE: Influence of Agrobacterium rhizogenes strains, acitosyringone, inoculum size and temperature on production of active ingredients from Picrorhiza kurrooa.

English

Sorghum is considered a recalcitrant crop based on genetic transformation. The main technical problem is callus browning in culture tissue and genetic transformation for sorghum. In this study, we developed a simple and effective method for sorghum transformation excluding tissue culture process. Sorghum mature embryos were pierced by a needle and then soaked in an Agrobacterium inoculum with acetosyringone. The best-performing concentration of acetosyringone was 100 μM in inoculation with pierced seeds. When these inoculated seeds grew to three leaves, a 1‰ hygromycin solution was used to brush the second and third leaf of inoculated seedlings to identify transformants. The identification results showed that the highest efficiency of transformation was 4% in the study. Molecular analysis for T0 and T1 plants identified that target gene was integrated into sorghum genomic DNA. And target gene can normally express in transformants’ leaves. Genetic analysis of T1 transformants showed that most of T1 transformants fitted to 3:1 Mendelian inheritance. All these results showed that the presented method is an easy and successful method for sorghum transformation. In the future, the method will be used for broad functional genomics studies and for biotechnological application of sorghum. © 2016 Friends Science Publishers

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.)

Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

Piercing and vacuum infiltration of the mature embryo: a simplified method for Agrobacterium-mediated transformation of indica rice

Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T (0)) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T (0) plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation was further confirmed by the genetic and molecular analyses of T (1) transformants.

Agrobacterium-mediated transformation of the winter jujube (Zizyphus jujuba Mill.)

Winter jujube, a species that originated in China, is the most prominent elite variety of jujube (Zizyphus jujuba Mill.). Due to its economic value and its recalcitrance to improvements through traditional plant breeding approaches, genetic transformation techniques may have a great potential in providing the means to transfer one or more selected desirable traits into the plant genome. We reported here an improved protocol for the Agrobacterium-mediated transformation of shoot tips of winter jujube. We have identified a set of optimum transformation conditions that take into account Agrobacterium inoculum density, Agrobacterium incubation period, co-cultivation conditions, and vacuum (use of a vacuum pump to create a negative-pressure environment). The highest transformation frequency (5.2%) was obtained when the shoot-tip explants were infected for 10 min and co-cultured for 4 days with Agrobacterium at OD600 0.8 under a negative pressure of 0.5 × 105 Pa. PCR and southern blot analyses confirmed the presence of transgenic plants and the stable integration of the target gene into the genome of regenerated plants. A histochemical staining analysis for GUS activity in the transgenic shoot tips also validated the efficiency of the transformation system.

The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and L-cysteine (600 mg l(-1)) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22 degrees C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l(-1) hygromycin for 10 days, 5 mg l(-1) hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l(-1) hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T(1) plants were analyzed and transmission of transgenes to the T(1 )generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.

Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD

Abstract Improved protocol for Agrobacterium mediated transformation of tomato ( Lycopersicon esculentum ) Micro-Tom was developed to use in corporation of the carotenoid biosynthetic genes CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase). From these experiment, a transformation methodology using explants from cotyledons cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/L kanamycin and 500 mg/L carabenillin for 6–8 weeks and resulted in a greater than 20% transformation efficiency in the concentration of Agrobacterium OD600 = 0.2 tested. In this transformation method, no feeder layers were used and the subculture media was minimal. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efficiency, 20.87%, was obtained by using OD600 = 0.2, which was significantly higher than that of OD600 = 1.0. The presence of the inserted target genes was checked using a rapid and efficient PCR test. The protocol was successfully employed in the production of transgenic Micro-Tom tomato containing the carotenoid biosynthesis CsZCD under constructive promoter. This procedure represents a simple, efficient and general means of transforming tomato.

Plate flooding as an alternative Agrobacterium-mediated transformation method for American chestnut somatic embryos

In an attempt to improve Agrobacterium-mediated transformation frequency of American chestnut somatic embryos, a novel method of inoculation/co-cultivation was developed. Plate flooding is a simple method where the Agrobacterium inoculum is poured onto the embryos while they remain on multiplication medium. This method tested the hypothesis that wounding tissues prior to co-cultivation was unnecessary or counterproductive. Two clones, WB296 and P1-1, were tested for differences in transformation efficiency as measured by the number of transformed embryogenic cell lines per Petri dish, the total number of transformed cell lines (embryos plus callus) and percentage of transformants that remained embryogenic. Plate flooding using clone WB296 produced significantly more transformed embryo cell lines and had a higher percentage of transformants remain embryogenic. The number of total transformed cell lines (embryos plus callus) was the same as obtained by other methods (desiccation, blot dry, sand abrasion, sonication and vacuum infiltration). With clone P1-1 there were no significant differences among the inoculation/co-cultivation treatments tested. Polymerase chain reaction and Southern hybridizations confirmed that the transgene of interest had been stably integrated into both American chestnut clones. Whole plants were regenerated from clone P1-1.

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying beta-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2 approximately 5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T(0) transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T(1) plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T(2) plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that beta-1, 3-glucanase gene was normally expressed in the T(2) plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene.

We have successfully transferred and expressed a reporter gene driven by an alpha-amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10-12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of beta-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice alpha-amylase gene (alpha Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice alpha-amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.

Agroinfection of wheat: Inoculation of in vitro grown seedlings and embryos

Various inoculation procedures of in vitro grown wheat seedlings with Agrobacterium tumefaciens carrying a wheat dwarf virus (WDV) dimer led to 13–46% agroinfection. Deliberate wounding of seedlings during or before inoculation gave higher agroinfection rates than in seedlings without wounding. Seedlings from 1–4 days old were equally receptive to inoculations in the meristematic regions. Vacuum infiltration of the Agrobacterium inoculum into excised cultured wheat embryos gave up to 35% agroinfection (with 4 ωg 1 −1 acetosyringone). WDV was detectable in seedlings by the ELISA assay 4 days after inoculation and there was good agreement between the ELISA and DNA dot blot assays of virus infection. Agrobacterium was found to deliver DNA (WDV) to the three wheat varieties tested and to Aegilops speltoides, Triticum monococcum and T. durum.

Der Einfluß verschiedener Osmotica auf die Tumorinduktion durch Agrobacterium tumefaciens

Summary Different osmotica [sucrose, KNO3, Ca(NO3)2] are reducing tumor induction in Kalanchoe leaves if they are added to an Agrobacterium inoculum in varying concentrations. The inhibitory effect begins at concentrations which are well below the isoosmotic value of the leaf tissues and are not seriously disturbing bacterial growth. It increases with increasing concentrations of the osmotica with no threshold concentration at all. The conclusion is drawn that for tumor induction in planta no intimate contact between neighbouring plant cells is necessary. This is in clear contrast to phenomena associated with virus infections of plant leaves.