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1.
Recovery of transgenic plants by pollen-mediated transformation in <Emphasis Type="Italic">Brassica juncea</Emphasis> 总被引:1,自引:0,他引:1
The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in
0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed
stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds
on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed
plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T1 progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated
that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication. 相似文献
2.
De Bolle MF Butaye KM Goderis IJ Wouters PF Jacobs A Delauré SL Depicker A Cammue BP 《Plant molecular biology》2007,63(4):533-543
Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression
of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene
expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why
chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs
on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs
do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results
show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have
an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene
expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression
in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.
Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work 相似文献
3.
Asad S Mukhtar Z Nazir F Hashmi JA Mansoor S Zafar Y Arshad M 《Molecular biotechnology》2008,40(2):161-169
A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for
cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were
treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and
selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic
callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene
copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1
AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is
first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate
fertile cotton transformants. 相似文献
4.
The most economically significant Chinese cotton cultivar (Gossypium hirsutum L. cv. Zhongmian 35) was transformed via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast-transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium containing glyphosate. Thirty-four independent transgenic lines were obtained after selection, giving a maximal 1.9% transformation frequency. The integration and expression of the aroA-M1 gene in T0 plants and T1 progeny were confirmed using DNA hybridization, Western blot and PCR techniques. An increased resistance of T0 and T1 transgenic plants towards glyphosate was also observed. 相似文献
5.
Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA 总被引:1,自引:0,他引:1
Yan Zhao Qian Qian Hui-Zhong Wang Da-Nian Huang 《In vitro cellular & developmental biology. Plant》2007,43(4):328-334
The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic
plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment
of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator)
without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present
paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R1 generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes
transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants,
which will promote the advancement in plant genetic engineering greatly. 相似文献
6.
A breakthrough in transgenic Arabidopsis thaliana research was the development of the floral dip transformation protocol, a simple and reliable method of obtaining transformants,
T1 transgenic lines, at high efficiency while avoiding the use of tissue culture. However, the traditional protocol (a “sterile”
method) of obtaining T2 transgenic lines has not evolved along with improvements in transformation technology as it continues to be laborious and
time-consuming. In this study, we report on the development of an improved protocol (a “nonsterile” method) for selecting
and growing A. thaliana transformants (T2 transgenic lines) resistant to kanamycin under nonsterile conditions. This protocol involves the use of a simple yet specialized
device that will aid in solium selection of T2 transgenic lines that can be rapidly grown in a hydroponic system. The “nonsterile” method reduces labor and time involved
as compared to the “sterile” method; moreover, it is easy to set up and maintain. This method may also be applicable to other
selecting agents, and perhaps to other plants. 相似文献
7.
The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacteriummediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants. 相似文献
8.
9.
A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually
with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the
number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared
to the whole plasmid. The Gfp expression was stable up to the T2 generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis
of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene
and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration
patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation
in bentgrass with possible applications to other plant species. 相似文献
10.
High molecular weight (HMW) glutenin polypeptides are critical contributors to the visco/elastic properties responsible for
the processing characteristics and utilizations of wheat flour. In order to improve bread making quality of flour and produce
transgenic plants free of selectable markers, a linear DNA construct consisting of a minimal expression cassette with the
HMW-GS 1Bx14 gene was transformed into wheat cultivar Mianyang19 by microprojectile bombardment. The transform ants were selected by PCR
instead of herbicidal markers. Seven transgenic plants were identified from a total of 1219 transformants, yielding a transformation
frequency of 0.28%. An SDS-PAGE analysis confirmed that the 1Bx14 gene was expressed in three T1 seeds of the transgenic plants. Our results demonstrated that it is feasible to obtain marker-free
trans-formants using the linear-expression-cassette-transformation approach coupled with PCR selection. 相似文献
11.
Michielse CB Salim K Ragas P Ram AF Kudla B Jarry B Punt PJ van den Hondel CA 《Molecular genetics and genomics : MGG》2004,271(4):499-510
Two transformation systems, based on the use of CaCl2/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS+ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl2/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl2/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at
Communicated by C. P. Hollenberg 相似文献
12.
Thomas Altmann Brigitte Damm Wolf B. Frommer Thomas Martin Peter C. Morris Dieter Schweizer Lothar Willmitzer Renate Schmidt 《Plant cell reports》1994,13(11):652-656
Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM
Arabidopsis medium
- ANOVA
analysis of variance
- DAPI
4,6-Diamidino-2-phenylindole
- PEG
polyethyleneglycol 相似文献
13.
We have developed a self-excision Cre-vector to remove marker genes from Brassica napus. In this vector cre recombinase gene and bar expression cassette were inserted between two lox sites in direct orientation. These lox-flanked sequences were placed between the seed-specific napin promoter and the gene of interest (vstI). Tissue-specific cre activation resulted in simultaneous excision of the recombinase and marker genes. The vector was introduced into B. napus by Agrobacterium-mediated transformation. F1 progeny of seven lines with single and multiple transgene insertions was subjected to segregation
and molecular analysis. Marker-free plants could be detected and confirmed by PCR and Southern blot in all transgenic lines
tested. The recombination efficiency expressed as a ratio of plants with complete gene excision to the total number of investigated
plants varied from 13 to 81% dependent on the transgene copy number. Potential application of this system would be the establishment
of marker-free transgenic plants in generatively propagated species. 相似文献
14.
Kumar V Satyanarayana KV Sarala Itty S Indu EP Giridhar P Chandrashekar A Ravishankar GA 《Plant cell reports》2006,25(3):214-222
A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct
regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained
with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having
Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA
but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for
the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single
and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders. 相似文献
15.
The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their
public acceptance and future commercialization. In addition, their elimination may allow gene stacking by the same selection
strategy. In apricot, selection using the selectable marker gene nptII, that confers resistance to aminoglycoside antibiotics, is relatively effective. An attractive alternative is offered by
the MAT system (multi-auto-transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with an MAT vector has been attempted
in the apricot cultivar ‘Helena’. Regeneration from infected leaves with Agrobacterium harboring a plasmid containing the ipt gene was significantly higher than that from non-transformed controls in a non-selective medium. In addition, transformation
efficiencies were much higher than those previously reported using antibiotic selection, probably due to the integration of
the regeneration-promoting ipt gene. However, the lack of an ipt expression-induced differential phenotype in apricot made difficult in detecting the marker genes excision and plants had
to be evaluated at different times. PCR analysis showed that cassette excision start occurring after 6 months approximately
and 1 year in culture was necessary for complete elimination of the cassette in all the transgenic lines. Excision was confirmed
by Southern blot analysis. We report here for the first time in a temperate fruit tree that the MAT vector system improves
regeneration and transformation efficiency and would allow complete elimination of marker genes from transgenic apricot plants
by site-specific recombination. 相似文献
16.
A tissue culture-independent plant transformation method, called ovary-drip transformation, was established in which a minimal
linear gene cassette [35S CaMV promoter, open reading frame of soluble modified green fluorescent protein (smGFP), and NOS
terminator] was transformed into soybean. The method is characterized by directly dripping a DNA solution, which is supplemented
with a surfactant, onto the ovary wound 6–8 h after self-pollination. The growth of the pollen tube was measured after self-pollination.
The movement of smGFP across the passageway toward the embryo sac was monitored using fluorescein isothiocyanate-labeled DNA. The transformation
frequency reached 3.2% by PCR analysis. Southern analysis of the primary transformants denoted the integration of a single
site smGFP. The transgenic plants exhibited a high level of smGFP expression which was visible in the immature embryos of the transgenic soybean. 相似文献
17.
We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation
frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%,
in industrial production scale transformation experiments. For CP4, with glyphosate selection, the average transformation
frequency was 57%. Stable transformation frequency was positively correlated with transient expression (R=0.85), and variable with the genes of interest. DNA integration and germline transformation were confirmed by biological
assay, Southern Blot analysis, and R1 phenotype segregation. Transgene expression was observed in leaf, root, stem, flower, and fruit tissues of the transgenic
plants. Ninety-five percent of transgenic events coexpressed two introduced genes based on β-glucuronidase (GUS) and neonmycin phosphotransferase II (NPTII) expression. Seventy-five percent of transgenic events contained one to two copies of the introduced uidA (GUS) gene based on Southern analysis. Transgenic plants from the cotyledon explants to the transgenic plants transferred to soil
were produced within about 2–3 months depending on the genes of interest. The utility of this MicroTom model transformation
system for functional genomic studies, such as identification of genes related to important agricultural traits and gene function,
is discussed. 相似文献
18.
Suchandra Deb Roy Mukesh Saxena Prasanna S. Bhomkar Mikhail Pooggin Thomas Hohn Neera Bhalla-Sarin 《Plant Cell, Tissue and Organ Culture》2008,95(1):1-11
Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene
to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker
genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study
employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive
the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to
a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of
the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress. 相似文献
19.
Altpeter Fredy Xu Jianping Ahmed Salahuddin 《Molecular breeding : new strategies in plant improvement》2000,6(5):519-528
Perennial ryegrass (Lolium perenne L.) is the most important grass species in areas with a temperate climate. Biolistic transfer of a ubiquitin promoter driven nptII expression cassette into mature or immature tissue derived calli of perennial ryegrass followed by paromomycin selection, resulted in the rapid and efficient production of fertile transgenic ryegrass plants. Transformation efficiencies after paromomycin selection in combination with the nptII selectable marker compared favourably with hygromycin selection in combination with the hph selectable marker. In total 83 independent nptII expressing plants were produced. Transformation frequency was highly affected by genotype, explant, selection regime and the duration of the callus induction period. The optimised transformation protocol for mature embryo derived calli of turf-type or forage-type cultivars resulted in an average transformation efficiency of 5.2% or 6.6% respectively. This converts into 1.7 or 2.2 independent transgenic plants per bombardment. Immature inflorescence- and immature embryo-derived calli were also successfully used as target for the gene transfer, resulting in transformation efficiencies of up to 3.7% or 11.42% respectively. Transgenic plants were transferred to soil 12 or 9 weeks after excision of mature and immature embryos or inflorescences respectively. Transgene integration and expression were confirmed by PCR and ELISA or western blot analysis. Southern blot analysis confirmed the independent nature of the transgenic lines. The majority of lines showed the integration of two to six transgene copies, while 21% of the analysed lines had a single copy insert. A short tissue culture period in comparison to recently published reports seems to be beneficial for the production of normal and fertile transgenic ryegrass plants. Consequently we report for the first time molecular evidence for sexual transgene transmission in fertile transgenic perennial ryegrass. 相似文献
20.
Lamblin F Aimé A Hano C Roussy I Domon JM Van Droogenbroeck B Lainé E 《Plant cell reports》2007,26(6):765-772
In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants,
we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into
fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective
medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root
on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L−1 sucrose and 10 g L−1 mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in
leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached
6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the
Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully
used for the recovery of flax transgenic plants under safe conditions for human health and the environment. 相似文献