共查询到20条相似文献,搜索用时 671 毫秒
1.
W. Z. Wang B. P. Yang C. L. Feng J. G. Wang G. R. Xiong T. T. Zhao S. Z. Zhang 《Tropical plant biology》2017,10(2-3):77-85
Genetic engineering provides new opportunities for improving economically important traits in sugarcane cultivars. In this study, an efficient Agrobacterium-mediated transformation system that uses the bar gene (a herbicide resistance gene that is used in conjunction with the herbicide Basta) as a selection marker was developed. Using this transformation selection system, all of the resistant plants after selection were nearly 100% polymerase chain reaction (PCR) detection positive and showed herbicide resistance. Each gram of sugarcane calli used for transformation produced approximately 12 transgenic lines. It took approximately 4 months to generate transgenic plants that measured 10 cm in height for greenhouse transplantation. 相似文献
2.
C. R. van Beek J. J. Fernhout J. Kossmann J. R. Lloyd C. van der Vyver 《Tropical plant biology》2018,11(3-4):154-162
Genetic engineering can be used to introduce economically important traits in sugarcane cultivars. Part of any transformation process involves the selection of genetically transformed cells. In this study, an efficient sugarcane in vitro selection system was developed using mutated protophorhyrinogen oxidase (PPO) genes as selectable markers. Two PPO genes, that encode proteins targeted either to the mitochondria or plastid, were isolated from tobacco and maize. Site-directed mutagenesis was used to alter the nucleotide sequence of these genes so that the resulting proteins are less sensitive to diphenylether type herbicides. Sugarcane callus was genetically transformed through particle bombardment with constructs allowing expression of either transgene, and putative transgenic calli were selected on fomesafen. It took approximately 4 weeks to select herbicide resistant calli clones on 10 mg/l fomesafen in the presence of light, which increased the selection pressure, and a further 8 weeks to regenerate resistant plantlets. PCR analysis confirmed that all regenerated putative transgenic sugarcane plants contained the transgene. All transgenic plants showed levels of herbicide resistance when planted in soil. 相似文献
3.
Natacha Soto Celia Delgado Yuniet Hernández Yamilka Rosabal Aleines Ferreira Merardo Pujol Francisco J. L. Aragão Gil A. Enríquez 《Plant Cell, Tissue and Organ Culture》2017,128(1):187-196
Soybean is highly affected by weeds in tropical countries, causing significant losses in yields. Transgenic herbicide resistant soybeans have been produced in a limited number of varieties and parental lines. This study was conducted to obtain glyphosate herbicide resistant transgenic soybean plants through particle bombardment of embryonic axes in a Cuban variety. Shoot regeneration in 25 mg/L of glyphosate occurred within a short period and plantlets developed roots in a medium without selection pressure, which favored the in vitro growth of plants at a transformation frequency of 3.1–6.0?%. Expression and integration of the cp4epsps gene was confirmed in the progeny by an immune-detection assay, PCR and Southern blot. All greenhouse evaluated transgenic soybean lines (T1) displayed tolerance to 1.25 Kg/ha of glyphosate. Growth and seed development of transformed plants was similar to untransformed plants. The regeneration procedure using embryonic axes combined with the efficient selection of shoots in glyphosate enabled the production of transgenic plants of this Cuban genotype, showing high tolerance to the herbicide, good efficiency and reproducibility. 相似文献
4.
Yidong Ran Nicola Patron Qin Yu Suzan Georges John Mason German Spangenberg 《Plant Cell, Tissue and Organ Culture》2014,118(1):67-75
Lolium rigidum Gaud. is an annual grass grown for forage but also an economically damaging crop weed. A single genotype somatic embryogenic callus line, VLR1-60, was identified from a herbicide susceptible L. rigidum population, VLR1, and proved to be amenable to Agrobacterium tumefaciens-mediated transformation. Somatic embryogenic calli were continuously induced from the meristematic region of VLR1-60 plants multiplied in vitro and the basic tolerance level of VLR1-60 to hygromycin B was determined. A hygromycin phosphotransferase gene was used as a selectable marker for hygromycin B selection. Somatic embryogenic calli derived from in vitro grown vegetative tillers were co-cultivated with the A. tumefaciens strain EHA105 harbouring binary vector carrying reporter genes and selectable marker in the presence of acetosyringone for 3 days. Inoculated calli were recovered on callus proliferation medium containing Timentin? but lacking hygromycin and were then subcultured onto media with hygromycin concentrations increased progressively through time for selection of transformed plant cells. Putative transgenic plants were recovered and integration of transgenes was confirmed by Southern hybridization analysis and by detection of DsRed or GUS activity in transgenic plants. The frequency of plant transformation was 1.3 %. The ability to transform L. rigidum will provide opportunities for functional characterization of genes to improve forage quality and increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make L. rigidum a damaging crop weed. 相似文献
5.
Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR)
tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay
showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving
the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation. 相似文献
6.
Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems.
Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol
Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant
to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato. 相似文献
7.
Shen-Jie Wu Hai-Hai Wang Fei-Fei Li Tian-Zi Chen Jie Zhang Yan-Jie Jiang Yezhang Ding Wang-Zhen Guo Tian-Zhen Zhang 《Plant Molecular Biology Reporter》2008,26(3):174-185
Agrobacterium-tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency
of coculture, selection cultivation, and plant regeneration. After 48-h cocultivation, the number of β-glucuronidase (GUS)-positive
calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse-granule
EC. It indicated that efficiency of transient transformation was affected by EC morphology. And transient transformation efficiency
was also improved by cocultivation on the medium adding 50 mg l−1 acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density was beneficial to production
of more kanamycin-resistant (Km-R) calli lines. From an original 0.3-g EC, an average of 20 Km-R calli lines were obtained
from a selection dish and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated
on the differentiation medium with dehydration treatments and the GUS-positive rate of regeneration plants was about 72.60%.
Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase
II gene and uidA. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly
with the low copy number, has been integrated into the cotton genome.
Shen-Jie Wu and Hai-Hai Wang should be considered as joint first authors 相似文献
8.
Hyun A. Kim Setyo Dwi Utomo Suk Yoon Kwon Sung Ran Min Jin Seog Kim Han Sang Yoo Pil Son Choi 《Plant biotechnology reports》2009,3(4):277-283
One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 × 14 days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l?1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R1 generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide. 相似文献
9.
Y. Y. Wu Q. J. Chen X. H. Cui H. Chen J. Chen X. C. Wang 《Russian Journal of Plant Physiology》2007,54(4):524-529
We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were
tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest
callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine.
Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized
regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin,
a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques,
we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results
indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and
transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596.
The text was submitted by the authors in English. 相似文献
10.
11.
Y A Badr M A Kereim M A Yehia O O Fouad A Bahieldin 《Photochemical & photobiological sciences》2005,4(10):803-807
A modified, non-damaging, protocol for the production of fertile transgenic wheat (Triticum aestivum L. cultivar Giza 164) plants by laser micropuncture was developed. The new homemade setup secures the transformation of as many as 60 immature embryo-derived calli (10000 cells each) in less than one hour using a UV excimer laser with two dimensional translation stages, a suitable computer program and a proper optical system. Five-day-old calli were irradiated by a focused laser microbeam to puncture momentarily made self-healing holes ( approximately 0.5 microm) in the cell wall and membrane to allow uptake of the exogenous DNA. The plant expression vector pAB6 containing bar gene as a selectable marker for the herbicide bialaphos resistance and GUS (uidA) gene as a reporter gene was used for transformation. No selection pressure was conducted during the four-week callus induction period. Induced calli were transferred to a modified MS medium with 1 mg l(-1) bialaphos for regeneration, followed by selection on 2 mg l(-1) bialaphos for rooting. Three regenerated putative transgenic events were evaluated for the integration and stable expression of both genes and results indicated that this modified procedure of laser-mediated transformation can be successfully used in transforming wheat. 相似文献
12.
We report here the transformation of two species of orchid, Dendrobium phalaenopsis and D. nobile,by biolistic bombardment. Calli or protocorm-like bodies (PLBs) were used as target explants. Gold particles (1.0 microm) coated with plasmid DNA (pCAMBIA1301) encoding an intron-containing beta-glucuronidase gene (gus-int) and a hygromycin phosphotransferase (hpt) gene were introduced into the PLBs or calli using the Bio-Rad PDS-1000/He Biolistic Particle Delivery System. Calli and PLBs were then chopped up and pre-cultured in 1/2-strength MS medium supplemented with 0.4 M mannitol for a 1-h osmoticum treatment before bombardment. Immediately after bombardment, the calli and PLBs were transferred to 1/2-strength MS medium without mannitol for recovery. Putatively transformed plantlets were obtained by selection and regeneration on medium supplemented with 30 mg/l hygromycin. The highest efficiency of transformation was obtained when selection was conducted at 2 days post-bombardment. For D. phalaenopsis and D. nobile, respectively, about 12% and 2% of the bombarded calli or PLBs produced independent transgenic plants. Integration and expression of the transgenes were confirmed by Southern hybridization and Northern hybridization. No nontransformed plants were regenerated, indicating a tight selection scheme. However, separate incorporation of the gus gene and the hpt gene was observed, and in one transgenic line the gus gene was integrated into the genome of the transgenic plant, but not expressed. 相似文献
13.
The parameters for delivery of expression cassettes to cells of wheat morphogenic callus induced from immature embryos were optimized. Three systems (gradation, delayed, and regeneration) for in vitro selection of transgenic wheat tissue using the bar gene, providing resistance to the herbicide phosphinothricin (PPT), were compared. The efficiency of gene delivery to the cells competent for plant regeneration was assessed by comparing the number of spots transiently expressing uidA gene (encoding β-glucuronidase) per unit surface of the morphogenic calluses treated under various conditions. The selection systems in question were evaluated by comparing the transformation efficiency frequencies. The optimal parameters for wheat biolistic transformation using a particle inflow gun were determined, namely, the distance between the particle source and the target tissue (12 cm) and helium pressure during the shot (6 atm). The optimal time of callus tissue development on the medium inducing callus formation was determined (10–14 days). Comparison of the three selection variants demonstrated that the regeneration system was the most efficient for producing true transgenic plants of common wheat. 相似文献
14.
A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis. 相似文献
15.
Park Mee Yeon Yi Na Ri Lee Han Yong Kim Sung Tae Kim Minkyun Park Ju-Hyun Kim Ju-Kon Lee Jong Seob Cheong Jong-Joo Choi Yang Do 《Molecular breeding : new strategies in plant improvement》2002,9(3):171-181
We established an effective biolistic transformation procedure fortransferring foreign genes into garlic (Allium sativumL.),which we demonstrated by generating transgenic plants resistant tochlorsulfuron, a sulfonylurea herbicide. We subcultured callus tissue from theapical meristem of garlic cloves and repeatedly selected calli with brittle,non-mucilaginous surfaces for over six months, to increase transformationefficiency. We then constructed recombinant DNA that contained the acetolactatesynthase (ALS) gene from a chlorsulfuron-resistantArabidopsis mutant, the cauliflower mosaic virus 35Spromoter, the -glucuronidase (GUS) reporter gene, and the hygromycinphosphotransferase (HPT) selectable marker gene. The garlic calli werebombarded twice with tungsten particles coated with the DNA constructs. Transformed calliwere efficiently selected by embedding them in solid agar medium containing 50mg l–1 hygromycin B. Selected propagules wereregenerated into 12 independent plants. We confirmed that the transgenes wereintegrated and expressed in the plants using PCR-Southern and Northern blotanalyses and by -glucuronidase expression assay forGUS. The regenerated plants survived in the presence of 3mg l–1 chlorsulfuron, demonstrating that theirALS was insensitive to this herbicide. These results illustrate the successfultransformation of foreign genes into garlic plants. The set of proceduresdeveloped in this study is applicable to the generation of transgenic garlicplants with other agronomically beneficial traits.
These authors contributed equally to this work 相似文献
16.
Soo Jung Kim Jee-Yeon Lee Yong-Min Kim Song-Sook Yang Ok-Jin Hwang Nam-Ju Hong Kyung-Moon Kim Hyo-Yeon Lee Pill-Soon Song Jeong-II Kim 《Journal of Plant Biology》2007,50(5):577-585
We performedAgrobacterium-mediated genetic transformation of creeping bentgrass(Agrostis stolonifera L.) and produced herbicide-resistant transformants from commercial cultivars Crenshaw and Penncross. Seed-derived embryogenie calli were infected withA. tumefaciens EHA105 harboring pCAMBIA 3301, which includes an intron-containinggus reporter and abar selection marker. To establish a stable system, we examined various factors that could potentially influence transformation efficiency during the pre-culture, infection, and co-cultivation steps. The addition of kinetin to the callus pre-culture media increased efficiency about three-fold. Once the optimum infection and co-cultivation conditions were identified, this protocol was used successfully to bulk-produce herbicide-resistant transgenic plants whose herbicide resistance was confirmed using the BASTA® resistance test. Southern blot analysis demonstrated integration and low copy numbers of the integrated transgenes, and northern blot analysis verified their expression. Thus, we have established an efficient genetic transformation system for creeping bentgrass and confirmed a high frequency of single-copy transgene integration and functional gene expression. 相似文献
17.
The selectable marker gene phospho-mannose isomerase (pmi), which encodes the enzyme phospho-mannose isomerase (PMI) to enable selection of transformed cell lines on media containing mannose (Man), was evaluated for genetic transformation of papaya (Carica papaya L.). We found that papaya embryogenic calli have little or no PMI activity and cannot utilize Man as a carbon source; however, when calli were transformed with a pmi gene, the PMI activity was greatly increased and they could utilize Man as efficiently as sucrose. Plants regenerated from selected callus lines also exhibited PMI activity but at a lower specific activity level. Our transformation efficiency with Man selection was higher than that reported using antibiotic selection or with a visual marker. For papaya, the PMI/Man selection system for producing transgenic plants is a highly efficient addition to previously published methods for selection and may facilitate the stacking of multiple transgenes of interest. Additionally, since the PMI/Man selection system does not involve antibiotic or herbicide resistance genes, its use might reduce environmental concerns about the potential flow of those genes into related plant populations. 相似文献
18.
Andrew Montague Asma Ziauddin Raymond Lee W. Michael Ainley Judith Strommer 《Plant Cell, Tissue and Organ Culture》2007,91(1):29-36
Despite the significant advantages of using herbicide resistance for selection of genetically engineered plants, alfalfa transformation
has relied primarily on selection for antibiotic resistance. In the few studies reporting the use of resistance to the herbicide
phosphinothricin (PPT), transformation efficiencies were low. The present investigation describes a PPT-based selection system
for alfalfa transformation that uses the phosphinothricin acetyl-transferase (pat) gene as a selectable marker and 5.0 mg l−1 of bialaphos as the selective agent. The method achieves transformation efficiencies, measured as the percentage of explants
giving rise to one or more transformed plantlets, greater than 50%. These plantlets accumulated detectable amounts of PAT
at levels varying from 2 to 1367 pg μg−1 total protein. Transformed plants transferred to soil in the greenhouse were phenotypically normal and exhibited resistance
to bialaphos leaf painting at 5 g l−1 and applications of PPT equivalent to field-level use (0.5 kg ha−1). 相似文献
19.
Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.) 总被引:4,自引:0,他引:4
Zhang CL Chen DF McCormac AC Scott NW Elliott MC Slater A 《Molecular biotechnology》2001,17(2):109-117
Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely
upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic
plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that
was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged
that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes”
and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance
genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for
enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet
via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several
days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained
at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic
calli were regularly monitored under the microscope with blue light when they were transferred to media without selective
agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has
potential for the development of selectable marker-free transgenic sugar beet. 相似文献
20.
Thi Thu Huong Khuong Patrice Crété Christophe Robaglia Stefano Caffarri 《Plant cell reports》2013,32(9):1441-1454