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Agrobacterium Mediated Transformation of Maize (Zea mays L.)

Agrobacterium Mediated Transformation of Maize (Zea mays L.)

system in these species are: i) high frequency of transformation; ii) proper integration of the foreign gene into the host genome; iii) low copy number of the gene inserted, resulting in most cases in a correct expression of the transgene itself. A limitation of the system is represented by the strict interaction between the genotype of the plant and the Agrobacterium strain, and the need to identify and to supplement specific signal molecules for the vir genes induction during the co- cultivation period (acetosiryngone or sinapinic acid) [8]. Although not being natural hosts for A. tumefaciens, monocotyledonous species seem to be in some instances susceptible to the infection. Studies on Agrobacterium infection of maize were first reported by Grimsley et al. [9] and Gould et al. [10], but the first evidences of the possibility of application of Agrobacterium mediated transformation of cereal species come from the works of Chan [11] and Hiei [12], who first obtained transgenic rice plants by means of transformation of immature embryos with A. tumefaciens. Most recently, the technique has been successfully applied to maize, and transgenic maize plants obtained at high frequency [5]. Ishida and co-workers [5] reported on the efficient transformation of maize inbred A188, and of some crosses between A188 and other inbreds. Agrobac- terium mediated transformation method has been used to transform tissue culture amenable genotypes including the Hi II hybrid [13, 6, 14] or inbred lines A188 and H99 [5, 15, 16]. A limited number of proprietary [17] or public inbred lines [15, 7, 18], and various recalcitrant inbred lines crossed to A188 [19, 20] have also been transformed using this method.
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High-Efficiency Agrobacterium-Mediated Transformation of Tobacco (Nicotiana tabacum)

High-Efficiency Agrobacterium-Mediated Transformation of Tobacco (Nicotiana tabacum)

Agrobacterium tumefaciens is a naturally occurring soil borne pathogenic bacterium that causes crown gall disease in dicotyledonous plants (3). The Agrobacterium-mediated transformation is an efficient and low-cost tool that exploits the natural ability of A. tumefaciens cells to transfer and integrate T-DNA into the host plant genome (27). The genetic transformation mediated by Agrobacterium has some important advantages than other transformation methods such as biolistic. The Agrobacterium-mediated plant transformation is a single-cell transformation system and not forming chimeras. As well as, the transfer of a single copy number of transgene results in fewer problems with transgene co-suppresion and instability (3). Efficiency of Agrobacterium-mediated transformation and delivery of T-DNA into plant cells is influenced by several factors, including genotype of the plant (10), type and age of explant (15, 27), strain of A. tumefaciens (24), the type of vector (29), bacterial cell density (19), pre-culture period (27), acetosyringone (AS) concentration (19), infection time (37), co-cultivation period (4), pH in co- cultivation medium (37), co-cultivation temperature
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Development of agrobacterium mediated transformation protocol for mature seed derived callus tissues of citrus cultivar ‘gailiangcheng orange x weizhang satsuma mandarin’

Development of agrobacterium mediated transformation protocol for mature seed derived callus tissues of citrus cultivar ‘gailiangcheng orange x weizhang satsuma mandarin’

Citrus breeding through conventional techniques is very difficult due to its complex reproductive biology such as low genetic diversity, apomixes, polyembryony, high heterozygosity, long juvenility, and auto-incompatibility (Boscariol et al., 2003). Biotechnological techniques including genetic transformation have made it possible to overcome some of these limitations (Hammerschlag and Litz, 1992). Among various transformation techniques, Agrobacterium- mediated transformation has been extensively used in the genetic improvement of Citrus species from the last two decades. This technique has made it possible to introduce a particular trait of interest from one cultivar to another elite Citrus cultivar without altering its original traits (Zanek et al., 2008). The currently available in vitro and Agrobacterium- mediated transformation protocols are species or even cultivar dependent (Pena et al., 2007; Zhao et al., 2010; Bachchu et al., 2011; Donmez et al., 2013; Orbovic et al., 2013). It is very necessary to develop an Agrobacterium - mediated transformation protocol for Citrus species particularly for the cultivar under study. Varying transformation efficiencies had been reported in previous Agrobacterium- mediated- transformation studies on Citrus. Li et al. (2002, 2003)
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REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION OF WATERMELON

REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION OF WATERMELON

supplimented with agar and phytagel individually or/and together were tested. Agrobacterium-mediated transformation system: The explants of watermelon cv Giza 1 and Giza 21 were co-cultivated with A. tumefaciens strain LBA4404 harboring the plasmid pSIV2678. This was performed through two replicates and the numbers of explants in each replicate were 60 with a total number of 120 explants. The treated explants were cultured onto medium 5MSABA for 3 days. Subsequently, they were transferred to the selection medium (5MSBABA medium supplemented with 250 µg/L bialaphos and 300 mg/L carbinicillin) and incubated for 2-3 weeks. Developed shoots were excised from their original explants and then transferred to the root formation medium. Plantlets were then acclimatized in the greenhouse.
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An improved protocol for rapid and efficient agrobacterium mediated transformation of tomato for salt tolerance (solanum lycopersicum l )

An improved protocol for rapid and efficient agrobacterium mediated transformation of tomato for salt tolerance (solanum lycopersicum l )

in identification and isolation of several genes that could potentially be involved in salt tolerance. According to literature published from 1993 till date, various authors have claimed enhancement of salt tolerance through either overexpression of endogenous genes or expression of genes that evidently act on mechanisms involved in the process of tolerance (Borsani et al., 2003, Flowers, 2004). Overall, the result obtained by previous researchers suggests that the expression of individual and Co-expression of genes in transgenic plants can increase salinity tolerance to some extent, which would be sufficient from breeding point of view. The advance development of transgenic plants is an effective approach for improving tolerance to stresses. Hence, establishment of an efficient transformation system is essential. The different agronomically useful traits have been incorporated into tomato using Agrobacterium mediated transformation (Raj et al., 2005; Roy et al., 2006). After first report on tomato leaf disc transformation by McCormick., 1986, there have been a number of publications on optimization of different factors involved in tomato transformation such as genotype (Sharma et al., 2009), type of explants (Bhatia et al., 2005), plant growth regulators (Gubis et al., 2003) and antibiotics used (Briza et al., 2008) in regeneration of tomato. However, transformation of tomato is still far from routine and it can show widely variable rates of success, depending on the cultivar and other factors (Park et al., 2003). In present study tomato plants of “Pusa ruby” over-expressing PgNHX1 gene a vacular Na + /H + antiporter gene from Pennisetum glaucum, AVP1 and Co-expression of
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Female reproductive system of Amaranthus as the target for Agrobacterium mediated transformation

Female reproductive system of Amaranthus as the target for Agrobacterium mediated transformation

Agrobacterium-mediated transformation through flo- ral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agro- bacterium tumefaciens strain (AGL1). The cell sus- pension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T 1 ). The T 1 seeds were germinated and grown to
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PEG mediated and Agrobacterium mediated transformation in the mycopathogen Verticillium fungicola

PEG mediated and Agrobacterium mediated transformation in the mycopathogen Verticillium fungicola

Fig. 4. PEG-mediated transformation, with transforming DNA integrating randomly and in varying copy number into the target DNA. (a)–(b) Genomic DNA of 440GUS (a) and 440GFP (b) was digested with EcoRI (lanes 1 and 4), KpnI (lanes 2 and 5) and SalI (lanes 3 and 6) and was probed with a 600 bp hph fragment obtained by PCR using pAN7-1 as a template. Single bands of varying size in lanes 1 and 3 indicate single copy random integration of the pAN7-1 DNA into the genome of the GUS co-transformant. Lane 2 did not resolve successfully. Multiple bands of varying sizes in lanes 4–6 indicate multi-copy, multi-locus random integration of the pAN7-1 DNA into the genome of the GFP co-transformant. (c) 440GUS genomic DNA digested with EcoRI, KpnI and BamHI in lanes 7–9 respectively, was probed with a 2000 bp NcoI pNOM102 fragment. Single bands of varying sizes in lanes 7–9 indicate single copy random integration of the pNOM102 transforming DNA into the target genome. (d) 440GFP genomic DNA digested with EcoRI, PstI and KpnI in lanes 10–12 respectively was probed with a 1 kb EcoRI pUCGFP fragment. Single bands in lanes 10–12 of varying sizes indicate single copy random integration of the pGPDGFP transforming DNA into the target genome. Selected molecular weight size markers are indicated.
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EFFICIENT REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION PROTOCOL FOR RECALCITRANT INDICA RICE (ORYZA SATIVA L.).

EFFICIENT REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION PROTOCOL FOR RECALCITRANT INDICA RICE (ORYZA SATIVA L.).

Most rice transformation protocol involved the use of embryogenic calli as the starting explant. Rice calli induction and regeneration were often cultivar-dependent and time-consuming thereby limiting the transformation efficiency of indica rice varieties (Danilova, 2007). Broadening the choice of rice target tissues that are competent for genetic engineering is a crucial step in the improvement of transformation efficiency. The use of rice shoot apex in Agrobacterium-mediated transformation has been performed for some indica rice cultivars. Utilizing shoot apex as explant for genetic transformation presents several advantages over callus tissues. Shoot apex culture is a reproducible and economically feasible method for producing plants that are free from pathogens (Badoni and Chauhan, 2009). Meristems are often devoid of such systemic pathogen since there are yet any differentiated conducting tissues (Alam et al., 2013). Also, Bairu et al. (2010) revealed that because shoot apex did not undergo dedifferentiation stage as in callus cultures, the chances of obtaining somaclonal variant or genetic mutations were low. Another major advantage is that meristematic tissue in the shoot apex region can develop and regenerate directly into shoots indicating the sustainability and plasticity of the meristematic region (Gamborg et al., 2002; Atak and Celik, 2009). So far, the regeneration of MR219 shoot apex have been studied but the regeneration process was rather lengthy and the survival rate of the shoot apex was very low (Silvarajan et al., 2011). Moreover, no systematic optimization of Agrobacterium-mediated transformation protocol for MR219 shoot apices has been conducted at the moment. Since transformation efficiency depends largely on basic transformation parameters, an optimized transformation protocol is thus important fundamentally prior to gene insertion. Therefore, this study sought to address these problems by developing an efficient and rapid method for the regeneration and Agrobacterium transformation system for MR219 using rice shoot apices as the preferred target tissue.
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Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis

Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis

Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 10 4 conidia or 217 transformants per 10 7 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T- DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus.
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Unique Signaling Properties of CTAR1 in LMP1-Mediated Transformation

Unique Signaling Properties of CTAR1 in LMP1-Mediated Transformation

LMP1-mediated transformation is impaired by dnTRAF2 and dnTRAF3. The interaction of CTAR1 with several TRAFs, including TRAF1, -2, -3, and -5, mediates the activa- tion of signal transduction pathways, such as NF- ␬ B and in- duction of key progrowth cellular molecules like the EGFR (5, 12, 13, 35, 37, 53). To determine whether TRAF2 and/or TRAF3 mediates the ability of LMP1 to block contact-inhib- ited growth, Rat-1 cell lines stably expressing pBabe-puromy- cin-M3, dnTRAF2, or dnTRAF3 were established and then transduced with serial dilutions of either pBabe-puromycin or LMP1. Cells on duplicate plates were harvested, and expres- sion of the dnTRAFs and LMP1 was confirmed by Western blot analysis indicating reduced levels of nuclear NF- ␬ B iso- forms p50 and p65 (data not shown). dnTRAFs contain the TRAF-binding domain but have been deleted of their RING and zinc finger domains, thus allowing interaction with their target molecule while impairing their ability to induce signaling upon their recruitment. Fourteen days posttransduction, cells were observed for focus formation (Fig. 8A). Transduction of the pBabe-puromycin-M3 (vector) stable cells with pBabe-pu- romycin (pBabe-puromycin-M3–pBabe-puromycin) or the sta- ble cells expressing only dnTRAF2 or dnTRAF3 did not show signs of focus formation. In contrast, transduction of the pBabe-puromycin-M3, dnTRAF2, or dnTRAF3 stable cells with LMP1 resulted in focus formation. Although dnTRAF2 or dnTRAF3 did not completely ablate focus formation, they markedly reduced the number of foci.
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Agrobacterium mediated Transformation of rice, var. Pusa Basmati-1

Agrobacterium mediated Transformation of rice, var. Pusa Basmati-1

high fertility of transgenics and transmission of transgenes in a Mendelian fashion. Hence, this technique has become the most preferred option to transform desired genes in rice varieties when compare to direct gene transfer techniques, in which, multi-copy integration, transgene silencing, reduced fertility and not genotype-independent in terms of efficiency are found [10-13]. As the former has more advantages than the latter technique, in this study, an attempt has been made on the Agrobacterium mediated gene transformation of pusa basumathi-1 rice variety. Hiei et al., [14] reported the unequivocal evidence of Agrobacterium based transformation in japonica rice. Later, the host range was extended to a few javanica and indica rice varieties [15–17]. However, the efficiency of Agrobacterium based transformation in rice is modulated by genotype, choice of tissue and choice of vector besides culture conditions [18]. In rice, different binary vectors have been used for achieving genetic transformation. Hiei et al., [14], Rashid et. al., [17], and Cheng et. al., [19] used pIG121Hm vectors, which is a derivative of the most commonly used vectors, viz. pBI121 [20]. Through this study, Agrobacterium mediated transformation of an elite indica variety Pusa Basmati-1 has been reported and this is confirmed by molecular analysis.
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Review of methodologies and a protocol for the Agrobacterium -mediated transformation of wheat

Review of methodologies and a protocol for the Agrobacterium -mediated transformation of wheat

Agrobacterium tumefaciens strains and binary vectors The ability of particular Agrobacterium strains to transform plant cells is defined by their chromosomal and plasmid genomes which between them must encode all the machinery necessary for attachment and DNA-transfer. The Agrobacterium strains that have been successfully used for wheat transformation are based on only two chromo- somal backgrounds, LBA4404 (Ach5) and C58 but these have been used with a wide range of Ti and binary plas- mids. Some strains, notably AGL0 and AGL1 have been engineered to contain the so-called hypervirulent Ti plas- mid, pTiBo542 harbouring additional vir genes originat- ing from the Agrobacterium strain A281 which in its oncogenic form possesses a broad host range and a induces large, rapidly appearing tumours [31]. The strains used in the papers reviewed (see Table 2), also contain a binary and sometimes helper plasmids, often conferring yet more copies of virulence genes. A comparison of dif- ferent Agrobacterium strains demonstrated that AGL0, a hypervirulent strain containing a disarmed pTiBo542 plasmid [32], was better at generating wheat transform- ants than other strains tested [19]. The ability of the Ti plasmid pTiBo542 to confer higher transformation effi- ciencies was first observed in dicots [33-35] and the vir genes from this plasmid have been widely adopted for monocot transformation vectors (reviewed by [11]). The weakly virulent Agrobacterium strain LBA4404, was suc- cessful in transforming wheat only when augmented by the superbinary plasmid pHK21 which possessed extra copies of vir B, C and G genes from pTiBo542 but not when carrying a standard binary plasmid [20]. Further evi- dence of the positive effect of additional vir genes was pro- vided by the demonstration that a 15 Kb fragment of pTiBo542 on a pSOUP helper plasmid [36] enhanced T- DNA delivery and the production of transgenic wheat plants, even when in a hypervirulent AGL1 background already containing pTiBo542 as a resident Ti plasmid [21,37]. Although there has been a tendency to incorpo- rate additional vir genes, particularly virG, into binary vec- tors this is not always necessary, at least for cv Bobwhite, in which a large number of transgenic lines have been reported using apparently standard Agrobacterium strains and binary vectors [16-18]. There is also one report [15] of transformation with a normal binary in the Agrobacterium strain C58C1 which the authors describe as disarmed, however it is our understanding that the C58C1 strain is actually cured of its pTiC58 plasmid [38,39]. There is cur- rently insufficient data to define precisely which vir genes are necessary and where they should reside for optimal wheat transformation in different genotypes. There is also scope for further research into the effect on wheat trans-
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Tissue culture and biolistic–mediated transformation of Impatiens balsamina

Tissue culture and biolistic–mediated transformation of Impatiens balsamina

This method involves the use of a high velocity of particles to penetrate the cells and introduce the DNA into the cells. The introduced DNA will be expressed in the plant cell if it is into plant chromosomal DNA (Wong, 1994). The particles that are commonly used are gold and tungsten. For biolistic transformation, DNA is coated onto the surface of gold or tungsten by precipitation with calcium chloride and spermidine. Generally with the high velocity, the DNA was delivered into the cells and once inside the cells, the DNA will elute off. If the foreign DNA reaches the nucleus, then transient expression will likely result and the transgene may be stably incorporated into host chromosomes (Kikkert et al., 2003; Birch and Bower, 1997).
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Agrobacterium-mediated transformation systems of Primula vulgaris

Agrobacterium-mediated transformation systems of Primula vulgaris

Pre-culture of explants was found crucial in this study, improving explant viability, reducing explant overgrowth from A. tumefaciens and assisting in the identification and removal of bacterial contaminated material prior to A. tumefaciens inoculation. Pre-induction of the regen- eration capacity via a pre-culture treatment has been reported to improve the adhesion of A. tumefaciens dur- ing co-cultivation [29]. Pre-culture activates cell division and the phase of the cell-cycle has been shown to influ- ence stable transformation [30] The presence of actively dividing cells improved cell competence for A. tume- faciens infection in regenerable tissue [29]. In Brassica rapa, seedling hypocotyls pre-cultured prior to expo- sure to A. tumefaciens showed greater shoot regenera- tion and improved transformation efficiency [31]. Similar improvements in regeneration and transformation effi- ciencies were reported for pre-cultured explants in tomato Solanum lycopersicum [32] and potato S. tubero- sum [33].
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Assessment of plant transformation technology of maize

Assessment of plant transformation technology of maize

Crop improvement is important criteria in the world. For this, conventional breeding is used to increase food production and create thousands of crop varieties to fulfil the demands of agricultural products. But conventional eeding is failure due to time consuming, unpredictability and lack of relative varieties. So, to overcome such problem, genetic engineering was introduced. It allows gene transfer without conventional breeding, for unrelated genera or species. It helps in increasing genetic resource to improve crop and also introduce known function of genes to achieve the goals of crop improvement by genetic engineering programme which is more predictable. There are no robust transformation technologies available in maize for higher transformation efficiency and quality of products. For this, two methods are developed. They are tissue culture dependent and independent for gene transfer technology. The methods are Agrobacterium mediated transformation and particle bombardment these methods, results are evaluated and assessed for both Agrobacterium
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Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H- ras

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H- ras

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non- transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras- transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.
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Tissue Culture-based Agrobacterium -mediated and in planta Transformation Methods

Tissue Culture-based Agrobacterium -mediated and in planta Transformation Methods

takes months; but the lack of efficient plant tissue culture method in target plant is the factor that can prolong this process significantly. The lack of highly efficient tissue culture regeneration systems is among the main obstacles for generating transgenic plants with modified nuclear or plastid genome in many important crops, such as corn, rice, or tea (Mey- ers et al. 2010). The efficiency of transformation also depends on the ability of selection procedure and the frequency of shoot regeneration and (pol- len or somatic) embryogenesis (Sobhanian et al. 2012). The difficulties in DNA delivery as well as regeneration of the target plant species are the main challenges for genetic transformation of cereals and monocotyledonous plants using routine methods of gene transformation (Mrízová et al. 2014). There are some plant gene transformation methods that are independent of tissue culture procedure and can facilitate gene transformation in plants that do not have developed plant tissue culture protocol. These tissue culture-independent gene transformation methods have their own advantages and disadvan- tages. Here, we divide Agrobacterium-mediated gene transformation into regular tissue culture-based Agrobacterium-mediated gene transformation and in planta transformation (Figure 1). We then de- scribe the generally used method and recent suc- cessful achievements in both tissue culture-based Agrobacterium-mediated transformation (TCBAT) and in planta transformation.
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COMPARISON OF TRANSFORMATION EFFICIENCY OF FIVE AGROBACTERIUM TUMEFACIENS STRAINS IN NICOTIANA TABACUM L.

COMPARISON OF TRANSFORMATION EFFICIENCY OF FIVE AGROBACTERIUM TUMEFACIENS STRAINS IN NICOTIANA TABACUM L.

The transgenic tobacco plant expressing foreign genes was reported at the beginning of the last decade for the first time, although many of the molecular characteristics of this process were not discovered at that moment (Herrera-Estrella, 1983). With the passage of the time, as plant sciences developed, a great progress in understanding the Agrobacterium-mediated gene transfer to plant cells has been explored. The significant advantages of Agrobacterium-mediated transformation have been reported over direct transformation methods. The reduced copy number of the transgene has been notably observed resulting in fewer problems with transgene cosuppresion and instability (Koncz et al., 1994, Hansen et al., 1997). Agrobacterium- mediated transformation additionally is a single-cell transformation system and not forming mosaic
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A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana

Genetic transformation of banana can be achieved using different methods, such as Agrobacterium-mediated transformation, electroporation, and micro-projectile bombardment. However, Agrobacterium-mediated transformation is the most preferred method due to its advantages, such as integration of low copy number of transgenes into the host plant genome and transfer of relatively large segments of DNA with minimal rearrangements (Lindsey, 1992; Gelvin, 2003). Several protocols for Agrobacterium- mediated transformation are available using different explants such as embryogenic cell suspension (ECS) cultures (Ganapathi et al., 2001; Khanna et al., 2004; Kosky et al., 2010; Tripathi et al., 2012) and apical meristematic tissues for various varieties of banana and plantain (May et al., 1995; Tripathi et al., 2005, 2008). The most commonly used target tissue for transformation is ECS; however, production of such cell suspensions is laborious, time consuming, and extremely variety dependent. Production of ECS and the optimization of a transformation system for each specific variety therefore becomes a prerequisite for genetic improvement. Cell suspensions of various varieties have been developed using basal leaf sheaths and corm section (Novak et al., 1989), highly proliferating multiple meristems (Dheda et al., 1991; Strosse et al., 2006), zygotic embryos (Marroquin et al., 1993), and immature male flowers (Escalant et al., 1994; Cote et al., 1996; Navarro et al., 1997; Becker et al., 2000; Grapin et al., 2000).
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Transformation Studies in Solanaceous Plants

Transformation Studies in Solanaceous Plants

Agrobacterium-mediated transient gene expression system in intact plant leaves is a rapid and useful method for analysis of gene expression. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tomato and tobacco leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for the subcellular localization of rice proteins in the heterologous host systems of tomato and tobacco leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18 – C 2 H 2 zinc-finger protein (OsZF1,
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