共查询到20条相似文献,搜索用时 453 毫秒
1.
Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after
applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency
2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with
the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume
of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed
colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos,
on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer
onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos
solution at 10.0 mg/l. 相似文献
2.
K. V. Krishnamurthy K. Suhasini A. P. Sagare M. Meixner A. de Kathen T. Pickardt O. Schieder 《Plant cell reports》2000,19(3):235-240
Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene
is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection
pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression
of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots
were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by
Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that
none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction.
Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999 相似文献
3.
Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N6–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase
II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l).
The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc
solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated
small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin
for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated
by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the
polymerase chain reaction.
Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999 相似文献
4.
High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng 总被引:4,自引:0,他引:4
W. Tang 《Plant cell reports》2000,19(7):727-732
The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l–1 6-benzyladenine (BA), and 500 mg l–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l–1 α-naphthaleneacetic acid, 0.1 mg l–1 BA, and 500 mg l–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot
organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic
callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos
per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious
shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious
shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed
in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic
embryogenesis.
Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999 相似文献
5.
E. M. Southgate M. R. Davey J. B. Power R. J. Westcott 《In vitro cellular & developmental biology. Plant》1998,34(3):218-224
Summary Techniques for transforming intact tissues of cereals were evaluated for their efficacy in transforming immature embryos and
Type II callus of maize (Zea mays L.). The techniques used were particle bombardment, tissue electroporation, tissue electrophoresis, and silicon carbide fibers.
Each method was assessed in terms of transient β-glucuronidase (GUS) expression. High levels of GUS expression were observed
in A188 Type II callus using both tissue electroporation and particle bombardment, with means of 417.8 and 954.5 blue expression
units (beu) per g fresh weight (FW) callus, respectively. Only particle bombardment resulted in high transient gene expression
in immature embryos, with a mean transformation frequency of 34.8 b.e.u. per embryo. Very low levels of GUS expression were
achieved with silicon carbide-mediated gene transfer, even when employing tissues used in the original publication (Black
Mexican Sweet suspension cells). GUS expression was not obtained following tissue electrophoretic gene delivery. 相似文献
6.
Four antibiotics were evaluated for their effects on eliminating the hypervirulent Agrobacterium tumefaciens strain C58C1 ATHV RifR (pEHA101)/p35-gus-intron from walnut somatic embryos and on the production of secondary somatic embryos and the transformed somatic embryos.
Exposure to 100–1000 mg l−1 of ampicillin, carbenicillin or cefotaxime respectively for up to 60 days did not eliminate the A. tumefaciens while timentin at 500–1000 mg l−1 eradicated it from somatic embryos. One-hour acidified medium treatments and the addition of 100 mg l−1 kanamycin to 500 mg l−1 ampicillin, carbenicillin, cefotaxime or timentin were of little help in eliminating the Agrobacterium. All four antibiotics reduced somatic embryo production, carbenicillin minimally and cefotaxime maximally, especially at
higher concentrations, in comparison with antibiotic-free medium. Putative transformed embryos were selected for continued
proliferation on a 100 mg l−1 kanamycin-containing medium. Histochemical assessments indicated that more gus-positive somatic embryos, particularly fully gus-positive embryos, regenerated from timentin-containing medium than from other antibiotic-containing media under equivalent
conditions. Transformed embryos have been grown and converted into plants and gus activity was observed in whole plants.
Received: 13 July 1999 / Revision received: 2 December 1999 / Accepted: 6 December 1999 相似文献
7.
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
8.
An efficient Agrobacterium-mediated protocol for the stable genetic transformation of Eschscholzia californica Cham. (California poppy) via somatic embryogenesis is reported. Excised cotyledons were co-cultivated with A. tumefaciens strain GV3101 carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l−1 paromomycin as the selective agent and 200 mg l−1 timentin to eliminate the Agrobacterium. Four to five weeks after infection, paromomycin-resistant calli grew on 80% of explants in the presence of 2.0 mg l−1 1-naphthaleneacetic acid (NAA) and 0.1 mg l−1 6-benzylaminopurine (BAP). Calli were cultured on somatic embryogenesis induction medium containing 1.0 mg l−1 NAA and 0.5 mg l−1 BAP, and somatic embryos were visible on 30% of the paromomycin-resistant calli within 3–4 weeks. Three to four weeks after
the somatic embryos were transferred to phytohormone-free plant regeneration medium, 32% converted to paromomycin-resistant
plants. Detection of the neomycin phosphotransferase gene and high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all plant tissues
confirmed the integrative transformation of the regenerated plants. The normal alkaloid profile of California poppy was unaffected
by the transformation process; thus, the reported protocol could serve as a valuable tool to investigate the molecular and
metabolic regulation of the benzophenanthridine alkaloid pathway.
Received: 27 October 1999 / Revision received: 6 December 1999 / Accepted: 11 January 2000 相似文献
9.
For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation
buffer containing 2% cellulase and filtered through 550, 400, 250 and 100 μm stainless mesh. Filtered embryogenic microcolonies
of 100–250 μm with pBI121 were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed
calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA
among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance
calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic
rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and
root apex. 相似文献
10.
Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige
and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation.
Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l).
After the germination of somatic embryos on medium with 10 mg/l GA3, the embryos were transferred to 1/2-MS medium without GA3. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration
of transformed ginseng plants might be valuable character for increasing root yield.
Received: 27 March 1999 / Revision received: 18 May 1999 / Accepted 8 July 1999 相似文献
11.
Cotyledonary-stage somatic embryos (3–5 mm in length) originating from nucellar explants of Mangifera indica L. cv. Amrapali were encapsulated individually in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully
on 0.6% agar-gelled medium containing B5 macrosalts (half strength), Murashige and Skoog microsalts (full strength), 3% sucrose
and 2.9 μM gibberellic acid. The percentage of germination of ESEs was higher than that of naked somatic embryos of the same
size on the same medium. The germinability of ESEs was increased (73.61±7.08%) when the medium was supplemented with full-strength
B5 macrosalts. Of the germinating ESEs, 45.83±3.40% developed into plantlets. Abscisic acid at 0.004 and 0.02 μM had no significant
influence on germination and plantlet development, but caused a 3-week delay in germination. Well-developed plantlets regenerated
from ESEs have been successfully established in soil.
Received: 9 February 1998 / Accepted: 22 March 1999 相似文献
12.
The effects of 11 different auxins and one cytokinin-like compound were tested at four concentrations for their ability to
induce primary and repetitive somatic embryos from mature, dry peanut (Arachis hypogaea L.) epicotyls of genotype AT120. Treatment with picloram and centrophenoxine at 83.0 and 124.4 μm resulted in the greatest number of embryos per explant and the highest percentage of explants responding. In a follow-up
experiment, picloram, centrophenoxine, and dicamba were tested at 83.0 and 124.4 μm on four peanut genotypes (AT120, 59-4144, GK7, and VC1). Picloram and centrophenoxine induced similar numbers of globular-stage
and total embryos from each genotype, while dicamba was less effective. Similar results were observed with percentage of responding
axes. Genotypes AT120 and VC1 yielded more clusters of repetitive embryos than GK7 and 59-4144. After 5 months, embryos derived
from repetitive embryogenic cultures were converted into mature plants.
Received: 8 February 1999 / Revision received: 9 June 1999 / Accepted: 30 June 1999 相似文献
13.
Gene transfer into isolated and cultured tobacco zygotes by a specially designed device for electroporation 总被引:4,自引:0,他引:4
We have established a technique for isolating, culturing and transforming tobacco zygotes. Zygotes were isolated by microdissection
or enzymatic maceration from fertilized embryo sacs. Viable zygotes cocultured with mesophyll protoplasts underwent first
division after 3 days of culture. Zygotes isolated by microdissection underwent a higher frequency of first division (61.2%)
than those isolated by enzymatic maceration (30.5%). Globular embryos were formed only from microdissected zygotes, at a frequency
of 8.7% after 1–2 weeks in culture. An efficient millicell device for the electroporation of DNA into zygotes was established.
The electroporated zygotes divided in vitro at a frequency of 54.6% and developed into proembryos. Introduced GFP gene constructs
showed transient expression in about 2.6% of the electroporated tobacco zygotes.
Received: 2 February 2000 / Revision received: 6 April 2000 / Accepted: 24 May 2000 相似文献
14.
Pierre J. Charest Yvonne Devantier Denis Lachance 《In vitro cellular & developmental biology. Plant》1996,32(2):91-99
Stable genetic transformation ofPicea mariana (black spruce) was obtained via particle bombardment into two target tissues, mature cotyledonary somatic embryos and suspensions
from embryonal masses, with the Biolistic PDS-1000/He device. Seven transgenic embryogenic cell line were obtained from the
mature cotyledonary somatic embryos after secondary somatic embryogenesis from two different cell lines (R4F14 and 119794-014).
The suspension culture from embryonal masses produced five transgenic cell lines from one cell line (R4F14). Expression of
the introduced β-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II) genes was detected by histochemistry and
fluorometry, and by ELISA in 10 of the lines. Two lines showed only NPT II gene expression. Four of the five lines obtained
after bombardment of suspensions of embryonal masses showed lower levels of expression of GUS and NPT II. The integration
of the foreign genes was confirmed by polymerase chain reaction analyses and Southern hybridization for GUS and NPT II, and
complex hybridization patterns were observed. The 12 transgenic lines obtained had a typical embryogenic morphology and were
capable of maturation and germination. Over 40 transgenic trees were regenerated from one of the transgenic lines, and they
have a normal phenotype. 相似文献
15.
High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode 总被引:8,自引:0,他引:8
Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective
medium containing 200 μg ml−1 kanamycin and 500 μg ml−1 carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene
incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the
Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for
the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode
(Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could
complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for
testing genes that might impart resistance to soybean cyst nematode.
Received: 13 July 1999 / Accepted: 8 August 1999 相似文献
16.
Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells 总被引:7,自引:0,他引:7
This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium
scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient
expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated
tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS
activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings
the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently
deliver the plasmid DNA into intact plant cells.
Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998 相似文献
17.
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
18.
F. Bekkaoui R. S. S. Datla M. Pilon T. E. Tautorus W. L. Crosby D. I. Dunstan 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,79(3):353-359
Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498 相似文献
19.
Summary Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet
organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm−1) vector DNA concentration (100 μgml−1), carrier DNA concentration (100 μgml−1), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid
vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and
GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when
optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg−1 (protein) min−1. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108.
Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot
analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted
excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S
promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly
except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.
Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the US Department of Agriculture
and does not imply its approval to the exclusion of other products or veudors that may also be suitable. 相似文献
20.
V. L. M. De Pádua M. C. Pestana M. Margis-Pinheiro D. E. De Oliveira E. Mansur 《In vitro cellular & developmental biology. Plant》2000,36(5):374-378
Summary Direct gene transfer into peanut intact embryonic leaflets was performed through electroporation. In transient β-glucuronidase
expression assays, maximal expression was obtained by using pulses of 625 V cm−1 in EPRm (modified electroporation) buffer supplemented with 75 μM NaCl. Kanamycin-resistant plants were obtained, and the presence of the nptII gene was demonstrated by PCR analysis. The positive effect of electroporation on the efficiency of in vitro regeneration was demonstrated. Explants submitted to field strengths between 500 and 625 V cm−1 displayed a significantly increased number of shoots and originated faster growing calluses relative to control explants.
Whereas in control explants callus formation occurred only at the petiolule, electroporated leaflets developed additional
organogenic calluses on the foliar lamina.
These authors have contributed equally to this work. 相似文献