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Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency
Carvalho, Carlos Henrique S.;Zehr, Usha B.;Gunaratna, Nilupa;Anderson, Joseph;Kononowicz, Halina H.;Hodges, Thomas K.;Axtell, John D.;
Genetics and Molecular Biology , 2004, DOI: 10.1590/S1415-47572004000200022
Abstract: the results presented in this work support the hypothesis that agrobacterium-mediated transformation of sorghum is feasible, analogous to what has been demonstrated for other cereals such as rice, maize, barley and wheat. the four factors that we found most influenced transformation were: the sensitivity of immature sorghum embryos to agrobacterium infection, the growth conditions of the donor plant, type of explant and co-cultivation medium. a major problem during the development of our protocol was a necrotic response which developed in explants after co-cultivation. immature sorghum embryos proved to be very sensitive to agrobacterium infection and we found that the level of embryo death after co-cultivation was the limiting step in improving transformation efficiency. the addition of coconut water to the co-cultivation medium, the use of vigorous and actively growing immature embryos and the removal of excess bacteria significantly improved the survival rate of sorghum embryos and was critical for successful transformation. hygromycin phosphotransferase (hpt) proved to be a good selectable marker for sorghum. we also found that b-glucuronidase (gus) activity was low in most of the transgenic plant tissues tested, although it was very high in immature inflorescences. although promising, the overall transformation efficiency of the protocol is still low and further optimization will require particular attention to be given to the number of agrobacterium in the inoculum and the selection of sorghum genotypes and explants less sensitive to agrobacterium infection.
Transient Expression of gusA and gfp Gene in Agrobacterium-mediated Banana Transformation Using Single Tiny Meristematic Bud  [PDF]
S. Sreeramanan,M. Maziah,M.P. Abdullah,M. Sariah
Asian Journal of Plant Sciences , 2006,
Abstract: Several factors were investigated for the transfer of an intron-containing β-glucuronidase (gusA) and green fluorescent protein (gfp) gene into banana cultivar, Pisang Rastali (AAB) single bud using the Agrobacterium-mediated transformation system. Two disarmed Agrobacterium tumefaciens strains, EHA 101 (pIG121-Hm) and LBA 4404 (pCambia 1304) were evaluated as vector systems. A number of parameters such as pre-culture period of buds prior to inoculation, co-cultivation period, acetosyringone concentration used during co-cultivation in MS medium, methods of wounding, Agrobacterium strains, influence of different bud sizes and post cultivation period were evaluated to maximise transformation efficiency. Agrobacterium tumefacien, EHA101 (pIG121-Hm), a supervirulent strain proved to be significantly better than LBA 4404 (pCambia 1304) based on higher expression intensity and even spread of GUS staining (pIG121-Hm harbours gusA gene only and pCambia 1301 harbour both gusA and gfp gene). The GFP expression was higher than the GUS using Agrobacterium tumefacien, LBA 4404 (pCambia 1304). The results from transient transformation of single tiny meristematic bud suggested that Agrobacterium-mediated transfer of T-DNA to banana cells is highly efficient. By combining the best treatments, an efficient and reproducible transformation procedure has been established for the production of transgenic banana using this system.
Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene
Shawkat Ali, Abdul Mannan, Mohamed El Oirdi, Abdul Waheed, Bushra Mirza
BMC Research Notes , 2012, DOI: 10.1186/1756-0500-5-285
Abstract: Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100?mg?L-1 along with 250?mg?L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200?μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3?mg?L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis.Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated.Genetic transformation of Citrus is a valuable technique for Citrus improvement due to difficulties of conventional Citrus breeding. In Citrus, gene transformation is carried out by three different techniques i.e., particle bombardment [1] protoplast transformation [2] and Agrobacterium[3-7]. Agrobacterium mediated transformation is the most commonly used method for gene transfer to Citrus as it does not need embryogenic calli and is protoplast independent [2]. Agrobacterium mediated gene transformation have been reported for a number of Citrus species by different groups [8-10]. However the transformation efficiency is still relatively low for some Citrus species and is cultivar dependent [11-13]. Rough lemon (C. jambhiri Lush.) originated from Himalayan foothills in India and Pakistan, is the commonly used Citrus rootstock in riverland of South Australia, lemon growing area of Arizona [14], south Asia, and in Pakistan it is used as rootstock for more than 90% of citrus fruit plants. Rough lemon is very vigorous rootstock and produce high yield performance in early years [14]. Besides this it propa
Effect of Polarity on in vitro Tumor Formation by Agrobacterium tumefaciens and Necrotic Response of Grape Cultivars
M.I. Haque,R.N. Goodman,N.U. Raja
Pakistan Journal of Biological Sciences , 1998,
Abstract: Tumor formation by Agrobacterium tumefaciens (AT) was significantly increased when the grape stem explants were planted and inoculated basal end upwards. When stem pieces of Catawba and Chancellor cultivars of Vitis vinifera were inoculated basal end upwards, these exhibited a 40% increase in tumor occurrence and a 3-4 times increase in tumor size. The enhanced tumor production reflects basipetal auxin accumulation in the grape stem explants. This in vitro inoculation procedure provided an efficient method to study pathogenicity of AT strains on a large scale in grape cultivars. Some necrosis accompanied tumor formation following in vitro inoculation in normal upwards direction. In some cases, tumors were produced in early stages but they became necrotic later. It might be possible that the higher auxin production in transformed cells lead to higher auxin oxidase activity which might be responsible for this necrosis.
Emerging factors that influence efficiency of T-DNA gene transfer into Phalaenopsis violacea orchid via Agrobacterium tumefaciens–mediated transformation system  [cached]
Sreeramanan Subramaniam,Xavier Rathinam
International Journal of Biology , 2010, DOI: 10.5539/ijb.v2n2p64
Abstract: An early step in the Agrobacterium-mediated transformation of Phalaenopsis violacea orchid was investigated in the plant-bacterium interaction. Directed movement in response to chemical attractants is the crucial importance to Agrobacterium tumefaciens strains. Chemotaxis of Agrobacterium tumefaciens strains (EHA 101 and 105) towards wounded orchid tissues has been studied by using swarm agar plates. The results obtained indicate a minor role of chemotaxis in determining host specificity and suggest that it could not be responsible for the absence of tumorigenesis in Phalaenopsis violacea orchid under natural conditions. Various aspects of transformation were examined using transient gusA gene expression in efforts to improve the efficiency of producing transformants. Hypervirulent Agrobacterium tumefaciens strains, EHA 101 and 105, harboring the pCAMBIA 1304 plasmid containing gusA gene as a reporter marker, were used for transformation study. The effects of different concentrations of L-cysteine, silver nitrate, and various temperatures (0C) during co-cultivation in half-strength MS medium supplemented with 5% of banana Mas extract on transformation efficiency were evaluated. Strain EHA 105 proved significantly better than EHA 101 based on higher percentage of gusA gene expression and even spread of GUS staining on PLBs. In addition, 200mg.L-1 L-cysteine, 60μM silver nitrate, and under the temperature at 240C gave the highest percentage of transient GUS expression. The results from transient transformation of PLBs suggested that Agrobacterium-mediated transfer of T-DNA is highly efficient. Therefore, by combining the best treatments, an efficient and reproducible of Agrobacterium-mediated transformation procedure could be continued for the production of transgenic Phalaenopsis violacea orchid.
Investigation of Factors in Optimizing Agrobacterium-Mediated Gene Transfer in Citrullus lanatus cv. Round Dragon
G. Kavitah,F. Taghipour,F. Huyop
Journal of Biological Sciences , 2010,
Abstract: Agrobacterium tumefaciens strain LBA 4404 harboring a binary plasmid pCambar (containing Basta resistance (bar) gene, neomycin phosphotransferase (npt II) gene and β-Glucuronidase (GUS) gene) was used to optimize the transformation efficiency in Citrullus lanatus cv. Round Dragon. In this study, the ability of Agrobacterium tumefaciens to mediate a gene transfer in Citrullus lanatus was highly dependent on various transformation factors. In current investigation, we have established factors influencing gene expression including explant ages of cotyledon- (5 days old cotyledon), pre-culture condition (2 days), wounding technique (multi wired with massive wounding technique), Agrobacterium concentration (A600 nm 0.8), co-incubation period of explants with Agrobacterium (30 min) and acetosyringone concentration (200 μM) in co-cultivation medium. These factors gave maximized transformation efficiency. The expression of the foreign functional gene in the plant genome was confirmed by histochemical GUS assay activity after 3 days of co-cultivation period. By combining the best conditions from each evaluated factors, we successfully established an efficient and reproducible Agrobacterium-mediated transformation protocol for Citrullus lanatus cv. Round Dragon which yielded 100% of transgene expression with 181.18±0.57 blue spots per responding explant. In conclusion, the transformation system for Citrullus lanatus cv. Round Dragon was optimized. The transformation procedure was proved to influence the transformation efficiency in Citrullus lanatus at 100% transient expression. Present study also suggested that different plant varieties may affect the transformation rate. The optimized conditions will then be used in future research to make a transgenic plant.
An Easy Method for Agrobacterium tumefaciens-Mediated Gene Transfer to Nicotiana tabacum cv. TAPM26  [PDF]
P.C. Kutty,G.K.A. Parveez,F. Huyop
Journal of Biological Sciences , 2010,
Abstract: Research in Agrobacterium tumefaciens mediated gene transfer has advanced and opened new avenue for plant improvement via introduction of various beneficial genes. Current study investigates transformation parameters during co-cultivation to improve T-DNA delivery into Nicotiana tabacum cv. TAPM 26 by monitoring GUS expression level. The techniques involved basic plant tissue culture and establishment of plant transformation systems. Conditions assessed were bacterial inoculum concentration, infection period, wounding and pre-culture of explants, acetosyringone (AS) concentration and co-cultivation temperature. Optimized conditions resulted in high transformation efficiency at transient level were as follows; Agrobacterium tumefaciens growth phase of A600nm 0.8, infection period of 30 min, pre-culture of wounded explants prior to infection, addition of acetosyringone (AS) in bacterial growth culture (100 μM) and in co-cultivation medium (200 μM) and co-cultivation temperature of 22°C. Although higher density bacterial culture used for the infection process gave higher transformation rate, however, it compromised the viability of the explants. On the other hand, dilution of bacterial suspension reduced necrosis in explants and improved transformation events greatly. The transformation efficiency was increased 9 fold when the infection process was carried out at low temperature of 22°C. Current study has proven among the parameters assessed, temperature was the critical factor during co-cultivation process in Agrobacterium tumefaciens mediated gene transfer.
Agrobacterium-Mediated Gene Transfer to Cereal Crop Plants: Current Protocols for Barley, Wheat, Triticale, and Maize  [PDF]
Goetz Hensel,Christine Kastner,Sylwia Oleszczuk,Jan Riechen,Jochen Kumlehn
International Journal of Plant Genomics , 2009, DOI: 10.1155/2009/835608
Abstract: The development of powerful “omics” technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize.
Transformation system of Aspergillus japonicus mediated by Agrobacterium tumefaciens
根癌农杆菌介导的日本曲霉转化体系的建立

Hui Guo,Zhe Yang,Laijun Xing,Mingchun Li,
郭慧
,杨哲,邢来君,李明春

微生物学报 , 2011,
Abstract: Abstract: Objective]n order to select Aspergillus japonicus mutant strains that express high-yield of glycerol oxidase, we constructed a mutant library of Aspergillus japonicus that mediated by Agrobacterium tumefaciens. Methods]Tri-relative hybridization experiments were performed to transfer binary vector pBI-hphII into Agrobacterium tumefaciens EHA105, which was used as the infective strain and Aspergillus japonicus was used as the host strain. We established the transformation system of Aspergillus japonicus mediated by Agrobacterium tumefaciens and constructed a mutant library. At the same time, we analyzed the factors affecting the transformation efficiency such as the concentration of Agrobacterium tumefaciens, adding and not adding Acetosyringone, co-culture time, co-culture temperature and so on. Results]The results of PCR detection and Southern analysis showed that the T-DNA was integrated into genome of the Aspergillus japonicus. All of the nine randomly selected transformants were quite stable after ten rounds of successive cultures. Conclusion]The transformation system is a good basis for selection of Aspergillus japonicus mutant strains which give a high-yield of glycerol oxidase.
Agrobacterium-mediated transformation of plants: Basic principles and influencing factors
M Alimohammadi, MB Bagherieh-Najjar
African Journal of Biotechnology , 2009,
Abstract: Transformation is an important topic in plant biology and transgenic plants have become a major focus in plant research and breeding programs. Agrobacterium-mediated transformation as a practical and common method for introducing specific DNA fragments into plant genomes is well established and the number of transgenic plants produced using this method is increasing. Despite the popularity of the method, low efficiency of transformation is a major challenge for scientists. Modification of different genetic and environmental aspects of transformation method may lead to better understanding of the system and result in high efficiency transformation. In this review, we deal with recent genetic findings as well as different environmental factors which potentially influence Agrobacterium-mediated transformation.
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