Strain LBA4404 Research Articles

Setting of seeds during Agrobacterium-mediated winter wheat transformation by in planta method

Aim. To investigate the frequency of seeds setting and the formation of transgenic plants during Agrobacterium-mediated transformation of winter wheat by the in planta method using different methods of applying agrobacteria and the composition of inoculation media. Methods. Agrobacterium-mediated transformation by in planta method, molecular genetic analysis, methods of mathematical statistics. Results. A significant difference was found in the studied genotypes of winter wheat according to the indicator seed setting depending on the A. tumefaciens strain used and the inoculation medium. When comparing the effect of different strains of agrobacteria on this indicator, no significant differences between them were established, however, a tendency to increase the frequency of seed formation was observed when using the LBA4404 strain. When using the MS-22 inoculation medium, which additionally contained 12.5 mM MES, 4 mM NH4CI, 5.5 mM MgSO4, a certain increase in the number of formed seeds was also observed compared to the MS-21 medium supplemented with sodium thiosulfate, the frequency of seed setting increased in on average by 5 %. Conclusions. The genotypic dependence of the frequency of seed setting and the formation of transgenic plants with the use of different inoculation media and the method of the transformation procedure was established.

Enhancing Agrobacterium-mediated plant transformation efficiency through improved ternary vector systems and auxotrophic strains.

Agrobacterium-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence (vir) gene helper plasmid (ternary helper), demonstrated that including an additional vir gene helper plasmid into disarmed Agrobacterium strains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophic Agrobacterium strains to boost Agrobacterium-mediated plant transformation efficiency. Auxotrophic Agrobacterium strains are useful in reducing Agrobacterium overgrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from public Agrobacterium strains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transient GUS expression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries the virA gene from pTiBo542 in addition to other vir gene operons (virG, virB, virC, virD, virE, and virJ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improved Agrobacterium system, including auxotrophic disarmed Agrobacterium strains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.

Optimization of stable genetic transformation protocol in castor (Ricinus communis L. cv. TMV 5) using beta glucuronidase reporter gene for pioneer of desirable genes

The simple and stable protocol was standardised for castor (Ricinus communis L. cv. TMV 5) genetic transformation using Agrobacterium tumefaciens strain LBA4404 harbouring the binary plasmid pBAL2 (18.8 kb). Cotyledonary nodes from ten days old, in vivo seedlings were utilized as target cells for Agrobacterium mediated transformation. Explant pre-culture studies were carried out at 2, 4, 6, 8, 10 and 12 day intervals. The 4th day old explants cultivated on mMS medium (MS medium+B5 Vitamins) using plant growth regulators had the highest response percentage (50.6%). Kanamycin (0-175 mg/L) and Hygromycin (0-13 mg/L) sensitivity in well-developed shoots was investigated. Of the two antibiotics, Kanamycin 50 mg/L and Hygromycin 3 mg/L was found optimum. Different levels of acetosyringone (0-200 mg/L) were used in the co-cultivation medium to study the transformation efficiency of castor. Among the different concentrations, maximum number of explants showed GUS expression at 100 mg/L of acetosyringone in the co-cultivation medium at 2 days of co-cultivation period and the Cotyledonary node produced multiple shoots development and plantlet establishment in 0.3 mg/L TDZ, 0.6 mg/L PF-68, kanamycin 50 mg/L, 0.3 mg/L GA3, 1.5 mg/L IBA and 0.6 mg/L AgNO3. The rooted shoots were successfully acclimatized. Histochemical GUS assay was used to monitor T-DNA delivery into the target cells. PCR and Southern hybridization were used to confirm the transformants with the NPT II and GUS gene. A very high frequency (29.3%) of β-glucuronidase (GUS) gene expression was obtained through Agrobacterium-mediated gene transfer into cotyledonary node explants of Castor. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of Cator with desirable gene of agronomic importance.

Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation

Cotton is one of the world’s largest fiber-producing crops. It is also known as the important cash crop of Pakistan. Cotton crop products contribute to economy as cottonseed oil, animal feed and the fiber support the textile industries, thus, playing a significant role in uplifting the economy of a country. But over time, bollworm attacks had escalated, severely reducing the yield of cotton and damaging cotton production. Bt (Bacillus thuringiensis) cotton is being widely used nowadays due to its enhanced resistance against cotton bollworms which are responsible for reducing crop production leading to high economic loss. It greatly affects the economy by increasing the cost of production and protection. Using resistant strains of Bt cotton not only makes it easier to combat bollworm attacks but also reduces the substantial expense issues that farmers face. Bt has many toxic proteins among which the two crystal proteins, Cry1Ac and Cry2Ab, are used to develop resistance against bollworms in cotton. This study aimed to optimize the developing transgenic callus containing Cry1Ac and Cry2Ab genes in cotton. The dual gene construct (Cry1Ac + Cry2Ab) provides more resistance to the cotton crop as compared to the single gene construct against whom the most bollworms have developed resistance. The vector was inoculated into the hypocotyl segments and the transformation process was preceded by shifting the hypocotyls at different mediums Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation. The construct-containing vector was introduced into a standard cotton line. A molecular confirmation test was carried out utilizing PCR and gene-specific primers after the formation of the transgenic callus. As a result of the research an indigenous cotton line that expresses double Bt genes for insect resistance was developed.

Establishment of an <i>In Vitro</i> Propagation, Transformation, and Gene Editing System in Tomato (<i>Solanum lycopersicum</i>)

Tomato (<em>Solanum lycopersicum</em>), a family of Solanaceae, is an annual crop grown in fields and greenhouses. Tomato is the fourth-largest agricultural crop in the world, with a market value of more than $50 billion. Numerous attempts were conducted on the tomato for further improvement via <em>in vitro</em> regeneration and transformation. This study aims to establish <em>in vitro</em> multiplication, transformation, and gene editing systems in tomatoes by using cotyledon explants. Cotyledon explants were placed on the MS medium supported with 6-benzyl adenine (BA) and kinetin (Kin) at four different concentrations for shoot induction. The shoot induction from cotyledon explants significantly increased, and the regeneration rate was 45% after using BA at 8.8 µM compared to Kin. For root induction, indole-3-butyric acid (IBA) at 4.8 µM concentration revealed the highest number of explants producing root (55%). Three <em>Agrobacterium tumefaciens</em> strains (EHA105, GV3101, and LBA4404) were used to establish and develop a tomato transformation method. Each <em>Agrobacterium</em> strain harbors the plasmid pCAMBIA2301, including the <em>nptII </em>marker. <em>Agrobacterium</em> strain LBA4404 showed the lowest number of dead explants, besides increasing the number of explant-produced shoots (30%) and the number of shoots per transformed explants compared to GV3101 and EHA105 strains. Our results revealed that the success of tomato transformation depends on selecting a suitable <em>A.</em> <em>tumefaciens</em> strain. Finally, the tomato gene editing system was successfully established by transferring the pCAMBIA1300-pCas9-sgRNA-U3 binary vector into the cotyledon using the selective <em>Agrobacterium</em> strain LBA4404, and the transformation effectivity was confirmed by PCR analysis.

Tobacco Plant: A Novel and Promising Heterologous Bioreactor for the Production of Recombinant Bovine Chymosin.

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD600) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform.

Evaluation of antibacterial properties of nisin peptide expressed in carrots

Nisin, derived from Lactococcus lactis, is a well-known natural food preservative. In the present study, the gene of nisin was transformed to carrot by Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing neomycin phosphotransferase II (nptII) gene, peptide signal KDEL, and Kozak sequence. The integration of nisin and nptII transgenes into the plant genome was confirmed by polymerase chain reaction (PCR) and dot blot analysis. The gene expression was also performed by RT-PCR and Enzyme-Linked Immunosorbent Assay. The level of nisin expressed in one gram of transgenic plant ranged from 0.05 to 0.08 μg/ml. The stability of nisin varied in orange and peach juices depending on the temperature on the 70th day. The leaf protein extracted from the transgenic plant showed a significant preservative effect of nisin in peach and orange juice. A complete inhibition activity against Staphylococcus aureus and Escherichia coli in orange juice was observed within 24 h. After 24 h, log 1 and log 2 were obtained in a peach juice containing Staphylococcus aureus and Escherichia coli, respectively. Results of HPLC indicated that Chlorogenic and Chicoric acid compounds were increased in transgenic plants, but this increase was not significant. The study of determining the genetic stability of transgenic plants in comparison with non-transgenic plants showed high genetic stability between non-transgenic plants and transgenic plants. This study confirmed the significant inhibitory effect of nisin protein on gram-positive and gram-negative bacteria.

Infectivity analysis of chilli leaf curl Ahmedabad virus and its associated tomato leaf curl Bangladesh betasatellite complex in chili

Chilli leaf curl virus is responsible for inducing leaf curl disease in Chili (Capsicum annum L.), with whitefly (Bemisia tabaci) serving as the vector for transmission. Chili leaf samples with leaf curl symptoms were collected from Guntur, Andhra Pradesh, India. PCR-based detection screened for begomoviral infection and betasatellite in the collected samples. In this study, we have characterized tomato leaf curl Bangladesh betasatellite (ToLCBB) (OQ184745) using universal betasatellite primers. Partial dimer constructs were developed using the betasatellite and the DNA A genome of the chilli leaf curl Ahmedabad virus (ChiLCAV) (MW316419) in pCAMBIA 1302 vector. Both the constructs were mobilized separately into the Agrobacterium strain LBA4404, followed by agroinfiltration into healthy chili plants. Plants co-infiltrated with DNA A and betasatellite dimers exhibited symptoms after 21 days of infiltration, whereas plants infiltrated separately with the partial dimer constructs did not display any symptoms. Prominent symptoms include upward curling, thickening of stem ribs and stunted plant growth. Chili plants infiltrated with the infectious clones of ChiLCAV + ToLCBB and ChiLCAV alone showed positive amplification with ChiLCAV-specific and betasatellite primers. In contrast, plants infiltrated with ToLCBB partial dimer construct did not show any amplification with betasatellite primers. ChiLCAV DNA A titer accumulation was further quantified with qPCR and we observed that there is a significant increase (p < 0.0001) in the accumulation level with ChiLCAV + ToLCBB construct when compared to the ChiLCAV construct alone. Thus, this study highlights the pathogenesis of leaf curl disease in C. annuum caused by the begomovirus complex of ChiLCAV and its associated betasatellite ToLCBB.

Efficient genetic transformation of papaya using RNAi CP gene against papaya ringspot virus

Papaya Ring Spot Virus disease is wide spread across papaya growing countries of the world and is one of the major impediments in successful papaya cultivation. Genetically engineered papaya varieties viz., SunUp and Rainbow have already been developed and commercialized in USA using coat protein mediated resistance. However, transgenic papaya conferring resistance to papaya ringspot virus has not been developed in India till date due to lack of suitable genetic transformation protocol for Indian papaya varieties and unavailability of coat protein gene construct for harbouring broad-spectrum resistance. The current study reports an efficient gene delivery mechanism in papaya. Young globular embryos infected for 30 minutes with Agrobacterium tumefaciens strain LBA 4404 harbouring hairpin loop of truncated coat protein gene and subsequently co-cultivated in presence of 100 pM acetosyringone and 1mM spermidine in dark for 72 hours gave rise to independent transgenic events characterized by PCR, dot blot hybridisation and RT-PCR.

Agrobacterium-mediated Genetic Transformation of two Peanut (Arachis hypogaea L.) Varieties using Salinity Tolerant PDH45 Gene

An efficient Agrobacterium-mediated genetic transformation protocol has been developed in the present investigation to integrate salinity tolerant gene pea DNA helicase (PDH45) in two peanut varieties, Dhaka-1 and BARI Chinabadam-8. Decapitated half-embryo explants of the above-mentioned varieties were also used to develop a transformation compatible in vitro regeneration system. MS medium supplemented with 5.0 mg/l BAP and 0.5 mg/l Kn showed maximum response for shoot induction in both varieties. Half strength of MS medium supplemented with 0.2 mg/l IBA showed better response towards root induction for both varieties. Genetic transformation experiments were performed using Agrobacterium strain LBA4404 containing pCAMBIA1301-PDH45 conferring reporter gus gene, hygromycin resistance hpt gene, and salinity tolerant gene pdh45. The transformation variables were optimized, OD600 1.4 with 45 minutes of incubation period, and three days of co-culture showed the best performance. The transformed shoots were selected using 30 mg/l hygromycin. The overall transformation frequency was very low, where 0.10% shoots of Dhaka-1 and 0.07% shoots of BARI Chinabadam-8 survived the selection pressure. The stable integration of the transgene in the transformed shoots was confirmed through PCR analysis. Plant Tissue Cult. &amp; Biotech. 33(1): 37-46, 2023 (June)

Melatonin strongly enhances the Agrobacterium- mediated transformation of carnation in nitrogen-depleted media

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

Construction of an Agroinfectious Clone of a Korean Isolate of Sweet Potato Symptomless Virus 1 and Comparison of Its Infectivity According to Agrobacterium tumefaciens Strains in Nicotiana benthamiana

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus &lt;i&gt;Mastrevirus&lt;/i&gt; (family &lt;i&gt;Geminiviridae&lt;/i&gt;) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into &lt;i&gt;Nicotiana benthamiana&lt;/i&gt; using three &lt;i&gt;Agrobacterium tumefaciens&lt;/i&gt; strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The &lt;i&gt;A. tumefaciens&lt;/i&gt; strain LBA4404 was the most effective at transferring the SPSMV-1 genome to &lt;i&gt;N. benthamiana&lt;/i&gt;. We confirmed the viral replication in &lt;i&gt;N. benthamiana&lt;/i&gt; samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

Advances in the Transformation of Cyclamen persicum Mill. Through Direct Regeneration Based on an Optimized Kanamycin Selection Scheme.

Gene transfer technology has great value in ornamental plants toward the generation of varieties with new ornate characteristics. In the previous studies through the transformation of cyclamen, hygromycin was mainly used as a selective marker. However, there have been some drawbacks associated with hygromycin usage as a selecting agent. Therefore, in the current study, the optimization of kanamycin concentration in the regeneration media has been considered. Subsequently, the plant transformation using three different in vitro explants from three Cyclamen persicum cultivars using three Agrobacterium tumefaciens strains has been examined. Accordingly, the optimal kanamycin concentrations for regeneration from root and leaf explants were determined as 10 mg/L and for microtuber explants as 30 mg/L. The successful gene transformation in the antibiotic-resistant shoots were examined by PCR and UV-equipped microscopes. The gfp reporter gene transfer resulted in the highest efficiency of transformation (60%) to date, from the leaf explants of cv. Pure White inoculated with Agrobacterium tumefaciens strain LBA4404. In contrast, the lowest gene transfer efficiency (25%) was observed in root explants of cv. Dark Violet and cv. Neon Pink inoculated with strains GV3101 and AGL-1, respectively. The results of the current project are expandable to the subsequent investigations of Cyclamen persicum transformation.

Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus

Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

Protocol for efficient ginseng transformation

Panax ginseng is a major medicinal crop with pharmaceutical efficacy derived from ginsenoside metabolites. Despite its genome information, the inefficiency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and efficient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher rate compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the selection media until identification of newly growing cells. In β-glucuronidase (GUS) assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO4 before infection. After MgSO4 pre-treatment, the transformation efficiency of growing cells around infected callus was increased than non-treated control. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies. We re-designed the ginseng transformation protocol using ginseng calli for explant material, and MgSO4 pretreatment was conducted before Agrobacterium infection. RUBY reporter was successfully introduced in ginseng.

A new simple and effective method for PLRV infection to screen for virus resistance in potato

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

In-planta transient transformation of avocado (Persea americana) by vacuum agroinfiltration of aerial plant parts

Avocado, Persea americana Mill, is one of the most traded tropical fruits in the international market. To date, stable and transient transformation has only been achieved for of zygotic embryos and not of adult plant tissue, which limits functional genomics research. We provide the first transient Agrobacterium-mediated transformation methodology in avocado leaves that overcomes the recalcitrance to transformation of this species. We investigated the effect of Agrobacterium strain, leaf stage, wounding pre-treatment, the phytohormone jasmonic acid, and vacuum infiltration on transient transformation of avocado leaves. Using the Agrobacterium strain LBA4404 and the RUBY reporter a transformation frequency of up to 27% was obtained for avocado detached leaves. The transformation efficiency depended on the age of the leaf, with an intermediate stage of leaf development showing the highest efficiency of transient reporter gene expression. Microwounding pre-treatment facilitates agroinfiltration and coupled with leaf age are the primary factors influencing competence for transient transformation. Jasmonic acid did not significantly affect transient transformation in the absence of microwounding. However, microwounding and 250 µM of jasmonic acid acted synergistically to significantly enhance transient expression. Using this methodology with localized vacuum agroinfiltration, transient transformation of attached avocado leaves was achieved. This method unlocks the use of Agrobacterium-mediated transient transformation as a tool for explore gene function and metabolic pathways in both, detached and attached avocado leaves.

Overexpression of rice acyl-CoA-binding protein OsACBP5 protects Brassica napus against seedling infection by fungal phytopathogens

Context Class III acyl-CoA-binding proteins such as those from dicots (Arabidopsis and grapevine) play a role in defence against biotrophic pathogens. The overexpression of the monocot Oryza sativa (rice) OsACBP5 in Arabidopsis and rice has been demonstrated to enhance broad-spectrum disease resistance against selected phytopathogens in OsACBP5-overexpressing (OsACBP5-OE) lines. Aims We aimed to develop transgenic rapid-cycling Brassica napus (B. napus-RC) and canola cv. Westar OsACBP5-OEs using tissue culture-based Agrobacterium-mediated transformation and to evaluate transgenic plants for resistance against Alternaria blight, blackleg and Sclerotinia rot diseases. Methods Transgenic B. napus-RC and cv. Westar OsACBP5-OEs were generated through Agrobacterium-mediated transformation using Agrobacterium strain LBA4404 harbouring a plasmid with the rice complementary DNA encoding OsACBP5 driven by the cauliflower mosaic virus 35S promoter. Alternaria blight and blackleg pathogen assays were based on infecting young cotyledons, while detached leaf assay was used to test the tolerance of B. napus plants toward Sclerotinia sclerotiorum. Key results Average transformation efficiencies of 24.2% and 29.1% were obtained for B. napus-RC and B. napus cv. Westar cotyledons respectively. OsACBP5-OE plants exhibited resistance 5 days after inoculation with Alternaria brassicae, 12 days after inoculation with Leptosphaeria maculans, and 24 h after inoculation with S. sclerotiorum. Conclusions Transformation of B. napus-RC was shown herein to be an effective trait testing platform for canola. This study also provides an insight into the usefulness of OsACBP5 in enhancing resistance to necrotrophic phytopathogens. Implications OsACBP5 can be overexpressed in other crops to generate pathogen-resistant varieties.

Cloning and Analysis of T-DNA Targeted Gene of Colletotrichum gloeosporioides Pathogenicity-Defective Strain Cs16

In preliminary stage, the nucleotide sequence of Cs16 Colletotrichum gloeosporioides associated gene obtained from database of (NCBI), analyzed with Clustalx 1.83 in order to assess the deduced amino acid sequences. The full-length sequences of Cs16 gene is including with specific Open Reading Frame (ORF) having 3’ and 5’ UTR was obtained. In order to assess the relationship of C. gloeosporioides associated CS16 gene with other members, a combined rooted neighbor-joining (NJ) tree was generated through MEGA 4.5 by following 1000 bootstrap replicates. The resulted alignment predicts that Cs16 gene showed entirely diverse sequences from other species and has no any conserved domain sequences between them. In order to scrutinize the full length nucleotide sequences, the gene functional cloning of Cs16 gene has been carried out by PCR amplification. To examine the functional analysis of Cs16 gene in C. gloeosporioides and its regulatory mechanisms, the over expressing vector (pGapneoR26) was transformed into Agrobacterium strain LBA4404 that resulted successful detection of GFP-Cs16 expression into C. gloeosporioides transformants and it was also substantially detected by fluorescence microscopy. The T-DNA intentional mutants of C. gloeosporioides mycelia illustrated phonotypic different intensification, colour, colony growth rate than wild type isolated C. gloeosporioides STIJ6 strain.

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.