The Experts below are selected from a list of 1905 Experts worldwide ranked by ideXlab platform
P. Winge - One of the best experts on this subject based on the ideXlab platform.
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transgene free genome editing in marine algae by bacterial conjugation comparison with Biolistic crispr cas9 Transformation
2018Co-Authors: Amit Sharma, M. Nymark, T. Sparstad, A. M. Bones, P. WingeAbstract:The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by Biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that Biolistic Transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative Transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both Transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the Biolistic Transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.
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Transgene-free genome editing in marine algae by bacterial conjugation – comparison with Biolistic CRISPR/Cas9 Transformation
2018Co-Authors: A. K. Sharma, M. Nymark, T. Sparstad, A. M. Bones, P. WingeAbstract:Abstract The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by Biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that Biolistic Transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative Transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both Transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the Biolistic Transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs
John C Sanford - One of the best experts on this subject based on the ideXlab platform.
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Biolistic Transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles
1994Co-Authors: Jeanette L Rasmussen, Julie R Kikkert, Mihir K Roy, John C SanfordAbstract:We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered Biolistic device. In addition, E. coli cells were used as microprojectiles for the Transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of Transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.
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major improvements in Biolistic Transformation of suspension cultured tobacco cells
1992Co-Authors: Julie A Russell, Mihir K Roy, John C SanfordAbstract:Suspension cultures of the NT1 line ofNicotiana tabacum L. were used as a model system to study plant Biolistic Transformation, because of their uniformity, rapid growth, and ease of handling. The β-glucuronidase gene and the neomycin phosphotransferase genes were used to assay transient and stable Transformation. Numerous factors were studied and optimized, such that the frequency of Transformation was increased roughly 60-fold for transient transformants and 20-fold for stable transformants. Both biological parameters (the promoter used to drive gene expression, osmotic preconditioning and posbombardment handling of the cells) and physical parameters of the bombardment process (particle acceleration device and accelerator parameters) were tested. The factors that increased Transformation rates the most were promoter strength, use of a helium-driven particle accelerator, and osmotic preconditioning of the cells.
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physical trauma and tungsten toxicity reduce the efficiency of Biolistic Transformation
1992Co-Authors: Julie A Russell, Mihir K Roy, John C SanfordAbstract:A cell suspension culture of tobacco (Nicotiana tabacum L.) was used as a model to study injury to cells during Biolistic Transformation. Lawns of cells were bombarded with tungsten particles that were coated with a plasmid containing the β-glucuronidase and the neomycin phosphotransferase II genes. When a gunpowder-driven Biolistic device was used, numerous transiently expressing cells were focused around the epicenter of the blast which was manifested by a hole blown in the filter paper supporting the cells. However, transformed cells nearest the blast epicenter were injured and could not be recovered as stable transformants. The injury was primarily caused by physical trauma to the cells from gas blast and acoustic shock generated by the device. Postlaunch baffles or meshes placed in the gunpowder device reduced cell injury and increased the recovery of kanamycin-resistant colonies 3.5- and 2.5-fold, respectively. A newly developed helium-driven device was more gentle to the cells and also increased the number of transformants. Cell injury could be further moderated by using a mesh and a prelaunch baffle in the helium device. Toxicity of the tungsten microprojectiles also contributed to cell injury. Gold microprojectiles were not toxic and resulted in fourfold more kanamycin-resistant colonies than when similar quantities of similarly sized tungsten particles were used.
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Biolistic Transformation of prokaryotes factors that affect Biolistic Transformation of very small cells
1992Co-Authors: Franzine Smith, Peter R Harpending, John C SanfordAbstract:Summary: Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for Biolistic Transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 104 per plate per 0·8 μg DNA. Although Transformation rates for the other species were low (<102 per plate per 0·8 μg DNA), successful Transformation without optimization for each species tested suggests wide utility of Biolistic Transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of Biolistic Transformation.
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Biolistic Transformation of a procaryote bacillus megaterium
1991Co-Authors: K B Shark, Franzine Smith, P R Harpending, J L Rasmussen, John C SanfordAbstract:We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed Biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a Biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although Transformation efficiency varied among strains. Physical variables of the Biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the Biolistic Transformation of a procaryote.
Bruce I Reisch - One of the best experts on this subject based on the ideXlab platform.
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Biolistic Transformation of grapevine using minimal gene cassette technology
2006Co-Authors: Jose R Vidal, Julie R Kikkert, Bruno D Donzelli, Patricia G Wallace, Bruce I ReischAbstract:The use of minimal gene cassettes (MCs), which are linear DNA fragments (promoter+open reading frame+terminator) lacking the vector backbone sequence, was compared to the traditional use of whole circular plasmids (CPs) for Transformation of grapevine. Embryogenic cell suspensions of ‘Chardonnay’ (Vitis vinifera L.) were transformed via particle co-bombardment using two nonlinked genes in either MCs or CPs. One construct contained the npt-II selectable marker and the second construct contained the MSI99 antimicrobial peptide gene. A total of five lines each from MC and CP treatments that showed positive signals by PCR for both the npt-II and MSI99 genes were selected. Southern blot analyses revealed up to five integration events in the DNA treatments. Transcription levels determined by semi-quantitative RT-PCR varied among transgenic lines. No significant differences were found in transgene transcription between lines from MC and CP Transformation. The correlation between npt-II and MSI99 transcription levels was positive (P<0.05), however, no correlation between the transcription level and the number of integration events was observed. Transgenic lines presented a similar phenotype in leaf morphology and plant vigor compared to non-transgenic lines. Moreover, transgenic lines from both MC and CP DNA treatments produced fruit as did the non-transgenic lines in the third year of growth in the greenhouse. Our data confirm the effectiveness of the minimal cassette technology for genetic Transformation of grapevine cultivars.
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stable Transformation of plant cells by particle bombardment Biolistics
2005Co-Authors: Julie R Kikkert, Jose R Vidal, Bruce I ReischAbstract:Particle bombardment, or Biolistics, is a commonly used method for genetic Transformation of plants and other organisms. Millions of DNA-coated metal particles are shot at target cells or tissues using a Biolistic device or gene gun. The DNA elutes off the particles that lodge inside the cells, and a portion may be stably incorporated in the host chromosomes. A protocol for the generation of transgenic grapevines via Biolistic Transformation of embryogenic cell suspension cultures is detailed in this chapter. In a typical experiment, transient gene expression averaged nearly 8000 "hits" per bombarded plate. Five months after bombardment, there were nearly five putative transgenic embryos per bombarded plate. About half of the embryos were regenerated into confirmed transgenic plants. The basic bombardment procedures described are applicable to a wide range of plant genotypes, especially those for which embryogenic cell cultures are available. All users of particle bombardment technology will find numerous useful tips to maximize the success of Transformation.
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transgenic plantlets of chancellor grapevine vitis sp from Biolistic Transformation of embryogenic cell suspensions
1996Co-Authors: Julie R Kikkert, Patricia G Wallace, Dominique Hebertsoule, Michael J Striem, Bruce I ReischAbstract:Transgenic plantlets of ‘Chancellor’ grapevine (Vitis L. complex interspecific hybrid) were produced via Biolistic Transformation. Embryogenic cell suspensions were bombarded with 1 μm tungsten particles coated with pBI426 which encodes a fusion peptide between β-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for Transformation of ‘Chancellor’ and should be applicable to other important grape cultivars.
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optimization of Biolistic Transformation of embryogenic grape cell suspensions
1993Co-Authors: Dominique Hebert, Julie R Kikkert, Franzine Smith, Bruce I ReischAbstract:Embryogenic suspensions of ‘Chancellor’ (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding s-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 μM kanamycin. Factors that affect Biolistic Transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 μm (M10) resulted in more transient gene expression than 0.771 μm diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the Biolistic device holds much promise as a method to achieve stable Transformation of grapevines.
Julie R Kikkert - One of the best experts on this subject based on the ideXlab platform.
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Biolistic Transformation of grapevine using minimal gene cassette technology
2006Co-Authors: Jose R Vidal, Julie R Kikkert, Bruno D Donzelli, Patricia G Wallace, Bruce I ReischAbstract:The use of minimal gene cassettes (MCs), which are linear DNA fragments (promoter+open reading frame+terminator) lacking the vector backbone sequence, was compared to the traditional use of whole circular plasmids (CPs) for Transformation of grapevine. Embryogenic cell suspensions of ‘Chardonnay’ (Vitis vinifera L.) were transformed via particle co-bombardment using two nonlinked genes in either MCs or CPs. One construct contained the npt-II selectable marker and the second construct contained the MSI99 antimicrobial peptide gene. A total of five lines each from MC and CP treatments that showed positive signals by PCR for both the npt-II and MSI99 genes were selected. Southern blot analyses revealed up to five integration events in the DNA treatments. Transcription levels determined by semi-quantitative RT-PCR varied among transgenic lines. No significant differences were found in transgene transcription between lines from MC and CP Transformation. The correlation between npt-II and MSI99 transcription levels was positive (P<0.05), however, no correlation between the transcription level and the number of integration events was observed. Transgenic lines presented a similar phenotype in leaf morphology and plant vigor compared to non-transgenic lines. Moreover, transgenic lines from both MC and CP DNA treatments produced fruit as did the non-transgenic lines in the third year of growth in the greenhouse. Our data confirm the effectiveness of the minimal cassette technology for genetic Transformation of grapevine cultivars.
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stable Transformation of plant cells by particle bombardment Biolistics
2005Co-Authors: Julie R Kikkert, Jose R Vidal, Bruce I ReischAbstract:Particle bombardment, or Biolistics, is a commonly used method for genetic Transformation of plants and other organisms. Millions of DNA-coated metal particles are shot at target cells or tissues using a Biolistic device or gene gun. The DNA elutes off the particles that lodge inside the cells, and a portion may be stably incorporated in the host chromosomes. A protocol for the generation of transgenic grapevines via Biolistic Transformation of embryogenic cell suspension cultures is detailed in this chapter. In a typical experiment, transient gene expression averaged nearly 8000 "hits" per bombarded plate. Five months after bombardment, there were nearly five putative transgenic embryos per bombarded plate. About half of the embryos were regenerated into confirmed transgenic plants. The basic bombardment procedures described are applicable to a wide range of plant genotypes, especially those for which embryogenic cell cultures are available. All users of particle bombardment technology will find numerous useful tips to maximize the success of Transformation.
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transgenic plantlets of chancellor grapevine vitis sp from Biolistic Transformation of embryogenic cell suspensions
1996Co-Authors: Julie R Kikkert, Patricia G Wallace, Dominique Hebertsoule, Michael J Striem, Bruce I ReischAbstract:Transgenic plantlets of ‘Chancellor’ grapevine (Vitis L. complex interspecific hybrid) were produced via Biolistic Transformation. Embryogenic cell suspensions were bombarded with 1 μm tungsten particles coated with pBI426 which encodes a fusion peptide between β-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for Transformation of ‘Chancellor’ and should be applicable to other important grape cultivars.
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Biolistic Transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles
1994Co-Authors: Jeanette L Rasmussen, Julie R Kikkert, Mihir K Roy, John C SanfordAbstract:We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered Biolistic device. In addition, E. coli cells were used as microprojectiles for the Transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of Transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.
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optimization of Biolistic Transformation of embryogenic grape cell suspensions
1993Co-Authors: Dominique Hebert, Julie R Kikkert, Franzine Smith, Bruce I ReischAbstract:Embryogenic suspensions of ‘Chancellor’ (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding s-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 μM kanamycin. Factors that affect Biolistic Transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 μm (M10) resulted in more transient gene expression than 0.771 μm diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the Biolistic device holds much promise as a method to achieve stable Transformation of grapevines.
G. K. A. Parveez - One of the best experts on this subject based on the ideXlab platform.
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ISSN 1682-296X © 2010 Asian Network for Scientific Information Biolistic Transformation of Citrullus vulgaris Schrad (Watermelon)
2014Co-Authors: F. Suratman, F. Huyop, A. Wagiran, Z. Rahmat, H. Ghazali, G. K. A. ParveezAbstract:Abstract: In this study, DNA-coated gold particles were used to transform cotyledon sections including axillary meristem of Citrullus vulgaris using the Biolistic method for optimizing the Transformation parameters and eventually regeneration of putative transgenic watermelon plants. Initially, five Biolistic parameters namely helium pressure, macrocarrier to target tissue distance, explant ages, DNA concentrations and osmotic treatment were optimized using plasmid pRQ6 (carrying both gusA and hpt genes). The optimized Biolistic parameters for cotyledon sections of Citrullus vulgaris explants were determined as follow: 1100 psi helium pressure, 6 cm target distance, 4-day-old explants, concentration of DNA at 1.2 µg/bombardment and osmotic treatment for 24 h on medium supplemented with 0.6 M mannitol prior to bombardment. Using the optimized parameters, Transformation of watermelon cotyledons was carried out using pAHG11, pCAMBIA 1301, co-Transformation (pAHG11+pCAMBIA 1301) and pCambar plasmids. Bombarded tissue’s were selected on either 5 mg LG 1 1 hygromycin or 2 mg LG PPT. Finally, the transformants produced were subjected to GUS histochemical assay. Integration of the transgenes into transgenic watermelon genome was confirmed by PCR analysis. Key words: Biolistics, transgenic watermelon, Citrullus vulgaris, optimizatio
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Biolistic Transformation of oil palm using the phosphomannose isomerase pmi gene as a positive selectable marker
2013Co-Authors: Bohari Bahariah, G. K. A. Parveez, Mat Yunus Abdul Masani, Subhi Siti Masura, Norzulaani Khalid, Rofina Yasmin OthmanAbstract:The selectable marker system based on the Escherichia coli phosphomannose isomerase (pmi) gene was evaluated for the genetic Transformation of oil palm. Four Transformation vectors were constructed for transforming oil palm. The Transformation vectors both carried the pmi gene alone or in combination with the β-glucuronidase (gusA) gene, and were driven by either the maize ubiquitin promoter (pMI11 and pMI11G) or the CaMV35S promoter (pMI3 and pMI3G). The four Transformation vectors were transformed into oil palm embryogenic calli via Biolistic-mediated Transformation. For stable Transformation experiments, bombarded oil palm embryogenic calli were selected on a medium supplemented with mannose as the only carbon source (without sucrose) one month after bombardment. Transformed embryogenic calli that survived selection on mannose were later isolated, proliferated and regenerated into whole plantlets on a regeneration medium containing mannose. The status of the regenerated transgenic plantlets was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Transgene expression was detected by reverse transcription (RT)-PCR analysis. The results of this study indicate that a mannose-based selection system can be successfully used in oil palm Transformation.
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Biolistic Transformation of citrullus vulgaris schrad watermelon
2010Co-Authors: F. Suratman, F. Huyop, A. Wagiran, Z. Rahmat, H. Ghazali, G. K. A. ParveezAbstract:In this study, DNA-coated gold particles were used to transform cotyledon sections including axillary meristem of Citrullus vulgaris using the Biolistic method for optimizing the Transformation parameters and eventually regeneration of putative transgenic watermelon plants. Initially, five Biolistic parameters namely helium pressure, macrocarrier to target tissue distance, explant ages, DNA concentrations and osmotic treatment were optimized using plasmid pRQ6 (carrying both gusA and hpt genes). The optimized Biolistic parameters for cotyledon sections of Citrullus vulgaris explants were determined as follow: 1100 psi helium pressure, 6 cm target distance, 4-day-old explants, concentration of DNA at 1.2 µg/bombardment and osmotic treatment for 24 h on medium supplemented with 0.6 M mannitol prior to bombardment. Using the optimized parameters, Transformation of watermelon cotyledons was carried out using pAHG11, pCAMBIA 1301, co-Transformation (pAHG11+pCAMBIA 1301) and pCambar plasmids. Bombarded tissue’s were selected on either 5 mg L-1 hygromycin or 2 mg L-1 PPT. Finally, the transformants produced were subjected to GUS histochemical assay. Integration of the transgenes into transgenic watermelon genome was confirmed by PCR analysis.