共查询到20条相似文献,搜索用时 31 毫秒
1.
Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica) 总被引:2,自引:0,他引:2
An improved broccoli transformation system was developed by optimising several factors that affect the rate of effective
Agrobacterium-mediated transformation. Leaf explants of cultivar Shogun were co-cultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pART278. The T-DNA of this binary vector contains a neomycin phosphotransferase II
(NOS-NPTII-NOS) gene for kanamycin resistance and a β-glucuronidase (35S-GUS-OCS) gene. Several media and factors were evaluated
including combinations of arginine, mannopine, acetosyringone and the use of feeder cell layers. The new protocol includes
the use of 200 μm acetosyringone in LB medium for bacterial growth, the use of a Brassica campestris feeder cell layer, 10 mm mannopine and 50 μm acetosyringone in the co-cultivation medium and 1 mm arginine in the selection medium. The use of this optimised protocol produced transformation rates of 33% in preliminary
experiments transforming broccoli with the antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene from pTOM13.
Received: 2 July 1998 / Revision received: 9 February 2000 / Accepted: 17 February 2000 相似文献
2.
Upadhyaya CP Venkatesh J Gururani MA Asnin L Sharma K Ajappala H Park SW 《Biotechnology letters》2011,33(11):2297-2307
Salt-tolerance was studied in transgenic potato. It was conferred by overexpression of ascorbate pathway enzyme (d-galacturonic acid reductase, GalUR). As genetic engineering of the GalUR gene in potato enhances its ascorbic acid content (l-AsA), and subsequently plants suffered minimal oxidative stress-induced damage, we now report on the comprehensive aptness
of this engineering approach for enhanced salt tolerance in transgenic potato (Solanum tuberosum L. cv. Taedong Valley). Potatoes overexpressing GalUR grew and tuberized in continuous presence of 200 mM of NaCl. The transgenic plants maintained a higher reduced to oxidized
glutathione (GSH:GSSG) ratio together with enhanced activity of glutathione dependent antioxidative and glyoxalase enzymes
under salinity stress. The transgenics resisted an increase in methylglyoxal that increased radically in untransformed control
plants under salinity stress. This is the first report of genetic engineering of ascorbate pathway gene in maintaining higher
level of GSH homeostasis along with higher glyoxalase activity inhibiting the accumulation in methylglyoxal (a potent cytotoxic
compound) under salt stress. These results suggested the engineering of ascorbate pathway enzymes as a major step towards
developing salinity tolerant crop plants. 相似文献
3.
Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.) 总被引:3,自引:3,他引:0
An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar
Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months
from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made
possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of
geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis.
Received: 12 October 1998 / Revision received: 17 May 1999 Accepted: 14 June 1999 相似文献
4.
Xiuping Zou Demou Li Xiaoying Luo Keming Luo Yan Pei 《In vitro cellular & developmental biology. Plant》2008,44(3):169-177
Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected
with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable
marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation
efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic
acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy
was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied
on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency
and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically
reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical
assay, PCR, and Southern blot analysis. 相似文献
5.
Transgenic yellow lupin (Lupinus luteus L.) plants have been generated by meristem co-cultivation with Agrobacterium tumefaciens. The binary plasmid pPZBNIa contains the bar gene under the control of a CaMV 35 S promoter. The transformation method involves inoculation of embryonic axis explants
with A. tumefaciens, flooding the meristem with glufosinate, and initial culture on non-selective medium. Shoots were transferred to culture
medium containing 20 mg/l glufosinate. Following subculture, shoots were grafted onto non-transgenic narrow-leafed lupin (L. angustifolius L.) seedling rootstocks, or rooted in vitro. The overall transformation efficiency, as determined at the T1 generation, was 0.05%–0.75%. The transgenic nature of plants grown to the T6 generation was confirmed by phosphinothricin acetyl transferase, PCR and Southern analyses.
Received: 20 March 1999 / Revision received: 17 July 1999 / Accepted: 17 August 1999 相似文献
6.
7.
A procedure for producing transgenic Chinese cabbage plants by inoculating cotyledonary explants with Agrobacterium tumefaciens strain EHA101 carrying a binary vector pIG121Hm, which contains kanamycin-resistance and hygromycin-resistance genes and
the GUS reporter gene, is described. Infection was most effective (highest infection frequency) when explants were infected
with Agrobacterium for 15 min and co-cultivated for 3 days in co-cultivation medium at pH 5.2 supplemented with 10 mg/l acetosyringone. Transgenic
plants of all three cultivars used were obtained with frequencies of 1.6–2.7% when the explants were regenerated in shoot
regeneration medium solidified with 1.6% agar. A histochemical GUS assay and PCR and Southern blot analyses confirmed that
transformation had occurred. Genetic analysis of T1 progeny showed that the transgenes were inherited in a Mendelian fashion.
Received: 15 December 1998 / Revision received: 2 July 1999 · Accepted: 8 July 1999 相似文献
8.
We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the
recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the
rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear
DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 × 106-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 × 103 genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil
samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification
(e.g. by PCR).
Received: 20 May 1997 / Accepted: 17 October 1997 相似文献
9.
Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m−2 s−1) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized
xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content,
together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the
recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry
(Fv/Fm). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast
to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (Fv′/Fm′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed
for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the
degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover,
a higher content of ascorbate appeared to increase the requirement for reduced glutathione.
Received: 20 May 1999 / Accepted: 29 October 1999 相似文献
10.
Plant regeneration via somatic embryogenesis was achieved from leaf petioles of Pelargonium sp. `Frensham' cultured on Murashige and Skoog medium containing 15 μM N6-benzyladenine, and 5 μM α-naphthaleneacetic acid (NAA). More than 80% of these somatic embryos converted into plants when
isolated and cultured on Murashige and Skoog medium supplemented with 15 μM NAA. Stable transgenic plants were obtained by
co-cultivation of the petioles (prior to culture) with Agrobacterium tumefaciens strains LBA4404 (harbouring a binary vector pBI121 carrying the nptII and gus genes) and LBG66 (harbouring a binary plasmid pJQ418 carrying the gus/int:nptII fusion gene). Transformants were selected using kanamycin and transformation was verified by β-glucuronidase histochemical
assay and polymerase chain reaction. Southern analysis further confirmed the integration of these genes into the genome of
transgenic plants. We report here for the first time, an Agrobacterium-mediated model transformation system coupled with regeneration via somatic embryogenesis for production of transgenics in
Pelargonium sp.
Received: 20 September 1996 / Accepted: 13 November 1996 相似文献
11.
Shunhua Ding Qingtao Lu Yan Zhang Zhipan Yang Xiaogang Wen Lixin Zhang Congming Lu 《Plant molecular biology》2009,69(5):577-592
To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic
tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated
the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses
of CO2 assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated
that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease
in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type
plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio
than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities
of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced
an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of
H2O2 in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration
of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate
redox state. 相似文献
12.
A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions.
Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus
induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed
on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM l-cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet
genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions.
Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed
in four R1 progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R1 progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes
into the genome of finger millet in the future. 相似文献
13.
Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance.
The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from
the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l
sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell
clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed
from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free
NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l
abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS
assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant
phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates.
Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999 相似文献
14.
15.
An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar ‘Florex Gold’) is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l−1 kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six
of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic
plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay.
This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation. 相似文献
16.
17.
Takashi Nemoto Taisuke Watanabe Yutaka Mizogami Jun-ichi Maruyama Katsuhiko Kitamoto 《Applied microbiology and biotechnology》2009,82(6):1105-1114
The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l−1 h−1, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition
strategy, which reached 8.46 ± 0.31 g l−1, 41.7 ± 1.4%, and 0.18 ± 0.01 g l−1 h−1 with the best continuous feeding strategy (0.2 g l−1 h−1), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L−1 h−1) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway
were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when
the l-Met feeding rate reached 0.5 g l−1 h−1. 相似文献
18.
Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation 总被引:1,自引:0,他引:1
Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4–5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10–12 weeks after Agrobacterium-mediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass. 相似文献
19.
In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation
efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l−1) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone.
The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced
more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with
3 mg l−1 hygromycin for 10 days, 5 mg l−1 hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l−1 hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants
in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression
and inheritance of the transgenes were confirmed by molecular and genetic analysis. T1 plants were analyzed and transmission of transgenes to the T1 generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation. 相似文献
20.
An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration 总被引:9,自引:0,他引:9
L. Zhang J. J. Rybczynski W. G. Langenberg A. Mitra R. French 《Plant cell reports》2000,19(3):241-250
A method for producing large numbers of transgenic wheat plants has been developed. With this approach, an average of 9.7%
of immature embryo explants were transformed and generated multiple self-fertile, independently transformed plants. No untransformed
plants, or escapes, were regenerated. This transformation procedure uses morphogenic calli derived from scutellum tissue of
immature embryos of Triticum aestivum cv. Bobwhite co-bombarded with separate plasmids carrying a selectable marker gene (bar) and a gene of interest, respectively. Transformed wheat calli with a vigorous growth phenotype were obtained by extended
culture on media containing 5.0 mg/l bialaphos. These calli retained morphogenic potential and were competent for plant regeneration
for as long as 11 months. The bar gene and the gene of interest were co-expressed in T0 progeny plants. This wheat transformation protocol may facilitate quantitative
production of multiple transgenic plants and significantly reduce the cost and labor otherwise required for screening out
untransformed escapes.
Received: 15 June 1998 / Revision received: 6 April 1999 / Accepted: 26 April 1999 相似文献