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D. N. Miroshnichenko G. N. Poroshin S. V. Dolgov 《Applied Biochemistry and Microbiology》2011,47(8):767-775
A protocol for biolistic transformation of bread wheat based on using mature seed tissues as explants has been developed.
Embryogenic callus obtained from mature seed tissues was transformed with a psGFP-BAR plasmid containing gfp reporter gene and bar selectable marker gene. The influence of hormone composition of the medium on the efficiency of transformation of mature
wheat seed tissues has been demonstrated. The use of auxin 2,4-D resulted in the formation of transgenic plants with a frequency
of 0.75%, while the use of Dicamba auxin for the regeneration of plants did not result in transformant development. The transgenic
status of the plants obtained in the experiments has been confirmed by PCR and RT-PCR. Stable inheritance of transgenic features
in the following generations of wheat (T1, T2) has been demonstrated and transgenic plants exhibiting high resistance to herbicides have been obtained. The protocol developed
allows for a simplified transformation of wheat in order to obtain transgenic plants with novel features. 相似文献
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Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling 总被引:1,自引:0,他引:1
To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying β-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2∼5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T0 transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T1 plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T2 plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that β-1, 3-glucanase gene was normally expressed in the T2 plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.Tong-Jin Zhao and Shuang-Yi Zhao contributed equally to this paper. 相似文献
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Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties
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Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants. 相似文献
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Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment 总被引:5,自引:0,他引:5
Dai Shunhong Zheng Ping Marmey Philippe Zhang Shiping Tian Wenzhong Chen Shouyi Beachy Roger N. Fauquet Claude 《Molecular breeding : new strategies in plant improvement》2001,7(1):25-33
We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T0, T1 and T2 transgenic plants. Oryza
sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hygr), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T1 and T2 generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T1 lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression. 相似文献
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To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium
tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD600 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion
of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding
explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea,
binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T0 transgenic plants were generated, representing 3.6% transformation frequency. T0 transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T1 progeny. PCR, RT-PCR, and Southern hybridization analysis of T0 and T1 transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein
in T0 and T1 transgenic chickpea plants was achieved maximum up to 116 ng mg−1 of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea
for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development
of chickpea plants with novel traits. 相似文献
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Anther culture–derived haploid embryos were used as explants for Agrobacterium‐mediated genetic transformation of bread wheat (Triticum aestivum L. cv CPAN1676) using barley HVA1 gene for drought tolerance. Regenerated plantlets were checked for transgene integration in T0 generation, and positive transgenic haploid plants were doubled by colchicine treatment. Stable transgenic doubled haploid plants were obtained, and transgene expression was monitored till T4 generation, and no transgene silencing was observed over the generations. Doubled haploid transgenic plants have faster seed germination and seedling establishment and show better drought tolerance in comparison with nontransgenic, doubled haploid plants, as measured by per cent germination, seedling growth and biomass accumulation. Physiological evaluation for abiotic stress by assessing nitrate reductase enzyme activity and plant yield under post‐anthesis water limitation revealed a better tolerance of the transgenics over the wild type. This is the first report on the production of double haploid transgenic wheat through anther culture technique in a commercial cultivar for a desirable trait. This method would also be useful in functional genomics of wheat and other allopolyploids of agronomic importance. 相似文献
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Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker 总被引:13,自引:0,他引:13
W. P. Chen X. Gu G. H. Liang S. Muthukrishnan P. D. Chen D. J. Liu B. S. Gill 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(8):1296-1306
Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression
of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was
to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter
and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded
with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration
medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T0) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected
35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal
and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T0 and T1 transgenic plants. Mendelian segregation of herbicide resistance was observed in some T1 progenies. Interestingly, a majority of the T1 progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression
under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated
in T1 transgenic wheat plants.
Received: 12 May 1998 / Accepted: 15 May 1998 相似文献
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Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance 总被引:9,自引:0,他引:9
G. Leckband H. Lörz 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,96(8):1004-1012
Transformation of barley and wheat via particle bombardment with a gene derived from Vitis vinifera L. (Vst1 gene) resulted in the expression of the foreign phytoalexin, resveratrol, in the transformed plants. Transgenic barley plants
were regenerated from microspores and transgenic wheat plants from immature embryos were both selected on Basta. Stable integration
of the gene in the genomes of transgenic barley and wheat plants, as well as their progeny, was analysed by Southern-blot
analysis. The induction of the stilbene synthase promoter and the transient expression of stilbene synthase-specific mRNA
after induction by wounding and infection were proofed in T1 and T2 progeny plants. An enhanced expression of the Vst1 gene under control of the stilbene synthase promoter was observed with enhancer sequences from the cauliflower mosaic virus
35s (CaMV 35s) promoter. The enzyme activity of the stilbene synthase was analysed in T1 progeny plants. The first pathological results indicated an increased resistance of transgenic barley plants to Botrytis cinerea used as a model experimental system.
Received: 5 November 1997 / Accepted: 11 November 1997 相似文献
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Milligan A.S. Daly A. Parry M.A.J. Lazzeri P.A. Jepson I. 《Molecular breeding : new strategies in plant improvement》2001,7(4):301-315
Maize (Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat (Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase (pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T1 progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T2 plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T2 plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T2 wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation. 相似文献
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Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene 总被引:21,自引:0,他引:21
W. P. Chen P. D. Chen D. J. Liu R. Kynast B. Friebe R. Velazhahan S. Muthukrishnan B. S. Gill 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(5):755-760
The possibility of controlling wheat scab (caused by Fusarium graminearum Schw.) was explored by engineering wheat plants for constitutive expression of pathogenesis-related (PR) protein genes. A
rice thaumatin-like protein (TLP) gene (tlp) and a rice chitinase gene (chi11) were introduced into the spring wheat cultivar ’Bobwhite’ by co-transformation of the plasmids pGL2ubi-tlp (ubiquitin/tlp//CaMV 35S/hpt) and pAHG11 (CaMV 35S/chi11//ubiquitin/bar). The transformation was by biolistic bombardment. Bialaphos was used as the selection reagent. The integration and expression
of the tlp, bar, chi11 and hpt genes were analyzed by Southern, Northern and Western blot analyses. The four transgenes co-segregated in the T1 progeny of the transgenic plant and were localized at the telomeric region of the chromosome 6A long arm by sequential N-banding
and fluorescent in situ hybridization (FISH) using pAHG11 or pGL2ubi-tlp as the probes. Only the transgenes tlp and bar, under the control of the ubiquitin promoter-intron, were expressed. No expression of the chi11 and hpt genes, controlled by the CaMV 35S promoter, was detected in T1 plants. After inoculation with conidia of F. graminearum, the symptoms of scab developed significantly slower in transgenic plants of the T1, T2 and T3 generations expressing the tlp gene than in non-transformed control plants. This is the first report of enhanced resistance to F. graminearum in transgenic wheat plants with constitutive expression of TLP.
Received: 15 December 1998 / Accepted: 30 January 1999 相似文献
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K. M. Doshi F. Eudes A. Laroche D. Gaudet 《In vitro cellular & developmental biology. Plant》2007,43(5):429-435
Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the
two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene
at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T0 lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time
PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene
copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with
a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale.
Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory
genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented
embryos were only observed visually in mature T1 seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was
likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic
lines. 相似文献
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Host‐induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust
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Tuo Qi Xiaoguo Zhu Chenlong Tan Peng Liu Jia Guo Zhensheng Kang Jun Guo 《Plant biotechnology journal》2018,16(3):797-807
Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T3 to T4 generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases. 相似文献
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Chidambaram Parameswari Rajasekaran Sripriya Karuppannan Veluthambi 《In vitro cellular & developmental biology. Plant》2010,46(5):395-402
In an analysis of 339 independent T
0 transgenic rice lines generated by Agrobacterium-mediated transformation, albino plants appeared in the T
1 generation in two single-copy transgenic lines, O54 and O36 and in one double-copy transgenic line, C18. While the T
0 plants of these three lines were green, albino and green plants emerged in a 1:3 ratio in the T
1 generation. The albino phenotype segregated as a monogenic recessive trait. Southern blot analysis of the green and albino
plants in the T
1 generation confirmed that the albino trait and the T-DNA insertion events were unlinked. Segregation of the albino trait
from the transgenic trait in the lines O54 and O36 was confirmed in T
2 and T
3 generations, respectively. Homozygous transgenic plants free from the albino trait were also identified. In the double-copy
transgenic line C18, we genetically separated the two transgenic loci, out-segregated the albino locus from both transgene
loci, and identified homozygous plants for each of the transgenic events by Southern blot analysis in the T
1 generation itself. Thus, we demonstrate that when an albino trait appears in the T
1 generation and is unlinked to a transgene locus, the albino locus can be segregated from the transgene locus and homozygous
transgenic lines free from albinos can be established. 相似文献