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Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion 总被引:1,自引:0,他引:1
Asako Sigeno Sugane Hayashi Toru Terachi Hiroshi Yamagishi 《Plant cell reports》2009,28(11):1633-1640
Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed
plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant
of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, ‘Telstar’, were used as cell fusion partners. An
efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus
development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell
fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing
the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants
demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco
transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids
of tobacco into petunia. 相似文献
3.
Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding. 相似文献
4.
Chen Yuh-Kun Lohuis Dick Goldbach Rob Prins Marcel 《Molecular breeding : new strategies in plant improvement》2004,14(3):215-226
The application of RNA-mediated resistance against Cucumber mosaic virus (CMV) by using single transgene constructs generally results in only a small portion of resistant individuals. Inverted repeat constructs encoding self-complementary double-stranded RNA have been demonstrated a potential way to obtain RNA-mediated resistance at high efficiency. To test this observation as a possible method for high frequency induction of CMV resistance, Nicotiana benthamiana plants were transformed with transgenes designed to produce double strand RNA molecules of CMV RNA 2 or coat protein (CP) gene sequences. Seventy-five percent of the tested R0 plants transformed with an RNA 2-derived inverted repeat construct (1534 nt CMV sequence) showed extreme resistance to CMV, while a lower percentage of resistance (30%) was observed in R0 lines transformed with a similar construct of a shorter viral RNA 2 sequence (490 nt). The resistance level conferred by CP sequences was also efficient by using a dsRNA construct, reaching a level of 50%. Self-pollinated (S1) progenies obtained from most resistant R0 plants all showed resistance levels of 100%, perfectly correlating with the expression of transgenic siRNAs. The results indicate that the use of inverted repeat viral transgenes is a highly efficient approach to obtain CMV resistant transgenic plants. Consequently, only a handful of transgenic plants will have to be generated using such constructs for successful resistance, which enables the implementation of this protocol for crops that are difficult to transform, such as ornamental plants in which CMV is an important pathogen. 相似文献
5.
Gunvant Patil Amit Deokar P. K. Jain R. J. Thengane R. Srinivasan 《Plant cell reports》2009,28(11):1669-1676
To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose
isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented
medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed
using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of
20 g l−1 mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection
pressure of 15 g l−1 mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been
transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into
the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker
genes. 相似文献
6.
Vidadi M. Yusibov Pak Chun II Viacheslav M. Andrianov Eleonora S. Piruzian 《Plant molecular biology》1991,17(4):825-836
The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress. 相似文献
7.
Over Expression of Rice chitinase Gene in Transgenic Peanut (Arachis hypogaea L.) Improves Resistance Against Leaf Spot 总被引:1,自引:0,他引:1
A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP,
1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative
transformed plants (T0) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets
was over 60% while healthy putatively transgenic (T0) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants).
chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation
was observed between chitinase activity and fungal pathogen resistance. 相似文献
8.
Kamo KK 《Plant cell reports》2003,21(8):797-803
UidA silencing did not occur following three seasons of dormancy for 23 independently transformed lines of Gladiolus plants carrying the bar- uidA fusion gene under control of either the cauliflower mosaic virus 35S (CaMV 35S), ubiquitin ( UBQ3), mannopine synthase ( mas2), or rolD promoters. The highest levels of GUS (beta-glucuronidase) expression were observed in callus, shoots, and roots of plants carrying the bar- uidA fusion gene under control of the CaMV 35S promoter and in shoots and roots of greenhouse-grown plants that contained the rolD promoter. There was no major difference in GUS expression when plants carrying the fusion gene driven by either the CaMV 35S, mas2, or UBQ3 promoters were grown in vitro as compared to growth in the greenhouse, although plants containing the rolD promoter expressed at 4- to 11-fold higher levels in shoots and roots, respectively, when grown in the greenhouse. The levels of GUS expression in greenhouse-grown plants were higher in roots than shoots for all four promoters. Of the 21 plants analyzed, 20 contained one to three copies of the bar- uidA fusion gene. Of the 23 plants analyzed, 11 had rearrangements of the transgene, but without apparent effects on levels of GUS expression. 相似文献
9.
Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein
accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated
for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and
in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf
explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the
PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed
shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation
would be limited to function as a secondary selective marker. 相似文献
10.
Frank T. Röder Thomas Schmülling Christiane Gatz 《Molecular & general genetics : MGG》1994,243(1):32-38
We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product — when highly expressed — interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype. 相似文献
11.
Regeneration of transformed flax shoots after inoculation withAgrobacterium tumefaciens carrying a binary vector with either a neomycin phosphotransferase (nptII) gene and a -glucuronidase (GUS) reporter gene or a spectinomycin resistance gene was examined. Hypocotyls from 4-day-old seedlings were inoculated with either of the twoA. tumefaciens strains. Selection and regeneration were achieved on a medium containing 0.1 M thidiazuron, 0.01 M napthalene acetic acid, 100 mgl–1 kanamycin sulphate or spectinomycin sulphate and 300 mgl–1 cefotaxime. Use of different neomycins for the selection of transformed tissues did select transformed calli but not transformed shoots either directly or via a callus phase. Selection based on spectinomycin resistance allowed the growth of transformed shoots. Transgenic shoots were rooted on a medium containing 100 mgl–1 spectinomycin sulphate. Integration of the spectinomycin resistance gene into the flax genome was confirmed by Southern blot hybridizations and spectinomycin resistance was shown to be inherited as a dominant Mendeliant trait. Therefore, spectinomycin resistance is more suitable for genetic engineering of flax than aminoglycoside resistance. 相似文献
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13.
E. Firoozabady Y. Moy W. Tucker K. Robinson N. Gutterson 《Molecular breeding : new strategies in plant improvement》1995,1(3):283-293
We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis. 相似文献
14.
Zhiqiang Pan Jingkun Ho Qin Feng Dao-Shun Huang Ralph A. Backhaus 《Plant Cell, Tissue and Organ Culture》1996,46(2):143-150
In vitro grown shoot tissue of facultative apomictic lines of guayule (Parthenium argentatum Gray), a rubber producing desert shrub, were transformed by Agrobacterium-mediated DNA transfer and regenerated into complete plants. Guayule shoots of lines 11591, UC101 and UC104 were inoculated with A. tumefaciens strains LBA4404 or PC2760 harboring the binary vector pCGN1557. Axillary shoots were regenerated from transformed cells and rooted in vitro in the presence of kanamycin. Genetic transformation in all cases was verified by Southern blot analysis. Transgenic plants were grown to maturity in the greenhouse and, as predicted for apomictic species, all seed produced possessed kanamycin resistance. Because apomicts have limitations for gene transfer by normal sexual crosses, this method offers a new means of transferring genes into this species.Abbreviations BA
benzyladenine
- EDTA
ethylene diamine tetraacetate
- kanR
kanamycin resistance
- MS salts
salts of Murashige and Skoog medium (1962)
- NAA
naphthalene acetic acid
- NPT-II
neomycin phosphotransferase
- SDS
sodium dodecyl sulfate 相似文献
15.
An efficient protocol for shoot regeneration and genetic transformation was applied to root segments of a new Lotus corniculatus L. cultivar Bokor. The shoots, that regenerated on root segments, were inoculated with Agrobacterium rhizogenes A4M70GUS, and produced hairy roots, which on media with 0.2 mg dm−3 benzylaminopurine, regenerated shoots. After rooting and acclimation, the transformed plants were planted in the experimental
field. Their morphological traits were compared to controls. No signs of the rol genes phenotype were present. The transformants were significantly taller than controls, while there were no significant
differences in the leaf area. The glucuronidase activity and the presence of uidA gene was demonstrated in transformed plants of T0 and in seedlings of T1 generations. It is concluded that A. rhizogenes could be a vector of choice for the transfer of desirable genes into the bird's foot trefoil genome.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
16.
Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability 总被引:11,自引:0,他引:11
van der Salm Theo P.M. van der Toorn Caroline J.G. Bouwer Reinoud Hänisch ten Cate Charlotte H. Dons Hans J.M. 《Molecular breeding : new strategies in plant improvement》1997,3(1):39-47
Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable.The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics. 相似文献
17.
N. Bohorova R. Frutos M. Royer P. Estañol M. Pacheco Q. Rascón S. McLean D. Hoisington 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(6-7):817-826
In order to develop a resistance management strategy to control tropical pests based on the co-expression of different toxins,
a fully modified Bacillus thuringiensis
cry1B gene and the translational fusion cry1B-cry1Ab gene have been developed. Both constructs were cloned under the control of a maize ubiquitin-1 or a rice actin-1 promoter
and linked to the bar gene driven by the CaMV 35S promoter. Immature embryos from the tropical lines CML72, CML216, and their hybrids, were used
as the target for transformation by microprojectile bombardment. Twenty five percent of the transformed maize plants with
cry1B expressed a protein that is active against southwestern corn borer and sugarcane borer. Ten percent of the transgenic maize
expressed single fusion proteins from the translational fusion gene cry1B-1Ab and showed resistance to these two pests as well as to the fall armyworm. Transgenic maize plants that carried the cry1B gene in T1 to T3 progenies transmitted trangenes with expected Mendelian segregation and conferred resistance to the two
target insects. Molecular analyses confirmed the cry genes integration, the copy number, the size of protein(s) expressed in maize plants, the transmission, and the inheritance
of the introduced cry gene. These new transgenic products will provide another recourse for reducing the build-up of resistance in pest populations.
Received: 25 September 2000 / Accepted: 15 December 2000 相似文献
18.
Anju Gulati Pat Schryer Alan McHughen 《In vitro cellular & developmental biology. Plant》2002,38(4):316-324
Summary A reproducible system for gene transfer in lentil through particle bombardment is presented. Lentil cotyledonary nodes excised
from germinated seedlings were bombarded with a plasmid containing a mutant acetolactate synthase gene (ALS) from tobacco
conferring resistance to sulfonylurea herbicides. Putative transgenic shoots regenerated on Murashige and Skoog medium supplemented
with 6-benzylaminopurine (BA) and chlorsulfuron (5 nM for first 4 wk followed by 2.5 nM for the remainder of the culture period) were micrografted and successfully transferred to soil. T0 and selfed progeny plants were screened using metsulfuron herbicide leaflet painting. The non-transformed escapes died and
transformed plants survived the test. The surviving plants were phenotypically normal and produced viable seeds. The presence
and stable transmission of the transgene into genomic DNA of screened T1 transformants was confirmed by PCR and Southern hybridization. This method for producing transformed plants will allow new
opportunities for lentil breeding to produce improved cultivars. 相似文献
19.
Manjul Dutt Dong H. Lee Jude W. Grosser 《In vitro cellular & developmental biology. Plant》2010,46(6):467-476
Agrobacterium-mediated transformation of Carrizo citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] with a binary vector containing a novel bifunctional reporter–selection fusion gene comprising an in-frame fusion
between the manA gene and egfp gene is detailed. This system combined the phosphomannose isomerase positive selection system with the ability to monitor
gene expression in a non-destructive manner using EGFP. Transgenic plants stably expressing the EGFP protein were regenerated
following Agrobacterium-mediated transformation using a vector containing this fusion gene. We also obtained comparable transformation efficiencies
when Carrizo explants were transformed using another Agrobacterium strain containing a binary vector with a bifunctional egfp–nptII fusion gene. Regenerating shoots were selected on medium containing 15 g L−1 mannose supplemented with 5 g L−1 sucrose for the manA-based selection or on medium containing 100 mg L−1 kanamycin for the nptII-based selection. Our results indicated that the mannose-based antibiotic-free selection combined with visual identification
of transgenic shoots using EGFP allows for early elimination of escape non-transgenic shoots and can provide a viable alternative
to antibiotic-based selection systems in the genetic transformation of citrus and other crops. 相似文献
20.
Donaldo Meynard Emmanuel Guiderdoni Blanca San Segundo 《Plant biotechnology journal》2014,12(3):367-377
Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants. 相似文献