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1.
Yamada Tsuyoshi Teraishi Masayoshi Hattori Kazumi Ishimoto Masao 《Plant Cell, Tissue and Organ Culture》2001,67(1):47-54
Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the -glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 M acetosyringone and 10 mg l–1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l–1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l–1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes. 相似文献
2.
Teresa K. Orlikowska Harwood J. Cranston William E. Dyer 《Plant Cell, Tissue and Organ Culture》1995,40(1):85-91
The effects of co-cultivation conditions on transformation efficiency and direct shoot regeneration from seedling explants of safflower cv. Centennial were examined. Agrobacterium tumefaciens strain EHA105/p35SGUSInt was more infective than LBA4404/pBI121 as determined by numbers of sectors expressing -glucuronidase activity. Compared to nontransformed controls, efficiency of direct shoot regeneration was markedly decreased by co-cultivation with EHA105 and the decrease exacerbated by addition of acetosyringone, indicating that a hypersensitive response to bacterial infection may reduce organogenetic potential. Likewise exposure of co-cultivated explants to kanamycin or geneticin in selective media reduced regeneration efficiency. Addition of 500 mg l-1 carbenicillin slightly increased numbers of regenerating shoots. Tranfformed shoots were obtained only when kanamycin selection was initiated 1 or 2 days after co-cultivation. Presence of transgenes in geneticin-resistant shoots was confirmed using polymerase chain reaction and Southern hybridization assays.Abbreviations AS
acetosyringone
- GUS
-glucuronidase
- MS
Murashige and Skoog (1962)
- NAA
naphthaleneacetic acid
- NPTII
neomycin phosphotransferase II
- PCR
polymerase chain reaction
- TDZ
thidiazuron 相似文献
3.
Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis 总被引:3,自引:0,他引:3
Slavica Ninković Jovanka Miljuš-Djukić Mirjana Nešković 《Plant Cell, Tissue and Organ Culture》1995,42(3):255-260
Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l–1 sucrose, 1 g l–1 yeast extract and 0.05 mg l–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA
6-benzyladenine
- GUS
-glucuronidase
- Km
kanamycin
- NPTII
neomycin phosphotransferase II
- X-gluc
5-bromo-4-chloro-3-indolyl--glucuronic acid
- BM
basal medium 相似文献
4.
Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA
6-benzyladenine
- GUS
-glucuronidase
- IAA
indole-3-acetic acid
- NAA
-naphthaleneacetic acid
- NOS
nopaline synthase
- NPT II
neomycin phosphotransferase II
- SDS
Lauryl sulfate 相似文献
5.
Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV
Cauliflower Mosaic Virus
- Cf
Cefotaxime
- GUS
-glucuronidase
- Km
Kanamycin
- MS
Murashige and Skoog
- NOS
nopaline synthase
- NPTII
neomycin phosphotransferase II
- PCR
polymerase chain reaction 相似文献
6.
Alvarez R Alonso P Cortizo M Celestino C Hernández I Toribio M Ordás RJ 《Plant cell reports》2004,23(4):218-223
A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and -glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.Abbreviations BA
N6-Benzyladenine
- GUS
-Glucuronidase
- MSSH
Expression-proliferation medium
- NAA
-Naphthaleneacetic acid
- nptII
Neomycin phosphotransferase gene
- uidA
-Glucuronidase gene 相似文献
7.
H. P. Hong J. L. Gerster R. S. S. Datla D. Albani G. Scoles W. Keller L. S. Robert 《Plant cell reports》1997,16(6):373-378
A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS
-Glucuronidase 相似文献
8.
Factors affecting Agrobacterium tumefaciens-mediated transformation in several black poplar clones 总被引:4,自引:0,他引:4
M. Confalonieri A. Balestrazzi S. Bisoffi R. Cella 《Plant Cell, Tissue and Organ Culture》1995,43(3):215-222
Transient expression of the uidA reporter gene was used in preliminary experiments with two oncogenic and two disarmed Agrobacterium tumefaciens strains in order to test the efficiency of T-DNA transfer to N084 x Populus nigra and N107 x P. nigra clones. The oncogenic strain A281 pKIWI105 produced the highest average number of GUS spots per leaf disc. In order to optimize the production of transgenic plantlets from different P. nigra clones (San Giorgio, Jean Pourtet, N084 x P. nigra and N107 x P. nigra, respectively), two A. tumefaciens strains (GV2260 p35S GUS, A281pKIWI105) and bacterial concentrations (7×108; 1.2×09 bacteria ml-1) were used. Following co-cultivation with A281 pKIWI105, the frequency of leaf discs producing kanamycin-resistant calli was not significantly different between the clones and bacteria concentrations used. Transformed shoots were regenerated from all clones, except for Jean Pourtet. Co-cultivation of leaf discs with GV2260 p35S GUS produced very few calli which died when transferred to selective regeneration medium. In addition, the effects of acetosyringone and leaf wounding were evaluated for the San Giorgio and Jean Pourtet clones, using the same strains. Factors which significantly affected the transformation efficiency of leaf explants were the P. nigra clone, the A. tumefaciens strain, and the presence of acetosyringone. Genetic transformation of calli and regenerated plantlets was confirmed by their ability to grow and root on Woody Plant Medium containing kanamycin, by histochemical -glucuronidase assays, and Southern blot hybridization analyses.Abbreviations BA
benzyladenine
- GUS
-glucuronidase
- IBA
indolebutyric acid
- MS
Murashige and Skoog
- NAA
-naphthaleneacetic acid
-
nptII
neomycin phosphotransferase II gene
-
uidA
-glucuronidase gene
- WPM
Woody Plant Medium 相似文献
9.
Summary A high frequency shoot regeneration (80%) was developed from callus of leaf discs and stem internodes of Moricandia arvensis. Leaf discs were shown to be a preferable starting material for transformation experiments. Agrobacterium tumefaciens strain GV3101/pMP90 used in this study contained a binary vector with genes for kanamycin resistance, hygromycin resistance and -glucuronidase (GUS). Maximum transformation efficiency (10.3%) was achieved by using kanamycin at the rate of 200 mg/l as a selection agent. Presence of tobacco suspension culture during co-cultivation and a pre-selection period of seven days after co-cultivation was essential for successful transformation. Transgenic plants grew to maturity and exhibited flowering in a glasshouse. GUS activity was evident in all parts of leaf and the presence of GUS gene in plant gemone was confirmed by PCR analysis.Abbreviations GUS
-glucuronidase 相似文献
10.
11.
Procedures were developed for rapid and prolific adventitious shoot regeneration of Arabidopsis thaliana (L.) Heynh ecotypes Landsberg erect and C24 from cotyledon explants at 90–100% efficiency. Immature cotyledons had the highest shoot regeneration efficiency. Prolific regeneration was achieved in Murashige and Skoog medium (MS) supplemented with 0.1–0.4 mg–1 naphthalene acetic acid (NAA) and 1.0 mg–1 6-benzylaminopurine (BAP) within 2 weeks. The regenerated plants had a normal phenotype and produced fertile flowers and set seeds. The above regeneration protocol was used to develop a transformation method using disarmed strains of Agrobacterium tumefaciens strains pGV3850::pH1121 based on kanamycin (Km) selection. Transgenic shoots were produced within 2–3 weeks after inoculation. Transformation of shoots was confirmed by GUS histochemical assay, as well as southern blot hybridization.Abbreviations NAA
1-Naphthalene acetic acid
- BAP
6-Benzylaminopurine
- Km
Kanamycin
- MSO
Murashigea and Skoog medium [18] without hormonal supplements
- GUS
-Glucuronidase
- NB
Nutrient broth
- NPT-II
Neomycin phosphotransferase II
- X-Gluc
5-Bromo-4-chloro-3-indolyl glucuronide
- CTAB
Cetyl triethylammonium bromide 相似文献
12.
E. Nenz F. Pupilli F. Paolocci F. Damiani C. A. Cenci S. Arcioni 《Plant Cell, Tissue and Organ Culture》1996,45(2):145-152
Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid with kinetin, N6 – 2 or benzyladenine in different combinations and concentrations. Only those calli induced in presence of -naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.Abbreviations BA
benzyladenine
- 2,4-d
2,4-dichlorophenoxyacetic acid
- 1AA
indole-3-acetic acid
- NAA
-naphthaleneacetic acid
- 2iP
N6 – 2
- PA
proanthocyanidins
- NOS
nopaline synthase
- NI TII
neomycin phosphotransferase
- GUS
-glucuronidase
- CaMV
cauliflower mosaic virus 相似文献
13.
Sridevy Sriskandarajah Peter B. Goodwin Jim Speirs 《Plant Cell, Tissue and Organ Culture》1994,36(3):317-329
Transformed shoots of the major apple scion cultivar Delicious (Malus × domestica Borkh.) were obtained by cocultivation withAgrobacterium tumefaciens carrying disarmed plasmids. The transformation efficiency was influenced by the type of plasmid and by the inoculation temperature. Initial selection involved a callus stage followed by shoot regeneration. Shoot regeneration occurred only in the dark. Shoots grew in the light and were rooted in the presence of 100 mg l–1 kanamycin. Of the range of plasmids tested, the cointegrates pGV 3850::1103neo and pGV 3850::1103gus gave a higher frequency of transformation than the binary vector pGV 3111 × pKIWI. Elongation of transformed shoots was enhanced by culture in a mixture of the cytokinins 6(--dimethylallylamino)purine and 6-benzyladenine. Up to 60% of the elongated shoots rooted in 100 mg l–1 kanamycin. Transformation was indicated by kanamycin resistance, -glucuronidase assay, nopaline synthesis, and by integration of the T-DNA as judged by Southern analysis. 相似文献
14.
P. D. S. Caligari Y. M. H. B. Yapabandara E. M. Paul J. Perret P. Roger J. M. Dunwell 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,86(7):875-879
Two inbred cultivars of Nicotiana tabacum (tobacco), Samsun and Xanthi, were transformed with the plasmid pBI 121 using Bin 19 in Agrobacterium tumefaciens. The plasmid carries the nptII gene conferring kanamycin resistance and the uidA gene encoding -glucuronidase (GUS). Progeny carrying the genes in the homozygous condition were identified and selfed over several generations. One line homozygous for the introduced genes and one untransformed control from each cultivar were then selected and crossed reciprocally to give four families per cultivar. Seeds from each family were grown in a replicated field trial and all plants scored for a range of morphological and agronomic characters. In addition, leaf samples were taken and GUS activity measured. In the Samsun material, which contained one copy of the introduced gene at a single locus and showed high levels of GUS expression, the transformed homozygote showed twice the level of GUS activity as the hemizygotes, wheareas in the Xanthi line, which had a lower level of GUS, the hemizygotes showed the same level of GUS activity as the transformed homozygote. The agronomic data showed differences between the families, but the source of such differences could not be ascribed unambiguously. The results are discussed in the light of related information on gene expression and field performance from other transgenic material. 相似文献
15.
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
16.
Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l–1 thidiazuron, 0.5 mg l–1 -naphthaleneacetic, 10 mg l–1 kanamycin (Km), and 250 mg l–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m–2 s–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy. 相似文献
17.
Men Shuzhen Ming Xiaotian Liu Rongwei Wei Chunhong Li Yi 《Plant Cell, Tissue and Organ Culture》2003,75(1):63-71
A protocol was developed to obtain stable transgenic orchids (Dendrobium nobile) via Agrobacterium-mediated transformation of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strains AGL1 and EHA105 were used, with each containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing -glucuronidase gene (gus-int) as a reporter gene. PLBs were co-cultivated with A. tumefaciens, which had been activated with 100 M acetosyringone (AS), for 2–3 days until the growth of A. tumefaciens was observed on co-cultivation medium containing 100 M AS. Following co-cultivation, PLBs were cultured on selective medium containing 30 mg l–1 hygromycin and 250 mg l–1 cefotaxime. Proliferating PLBs were repeatedly selected for hygromycin resistance. A high efficiency of transformation (18%) was obtained with a total of 73 stably transformed lines produced. Incorporation and expression of the transgenes were confirmed by Southern blot analysis and GUS histochemical assay. 相似文献
18.
Transient expression of -glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B5 vitamins, 0.1 mg dm–3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm–3 -naphthaleneacetic acid, 3 % glucose + 50 mg dm–3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures. 相似文献
19.
K.M. Khawar S. Gulbitti-Onarici S. Çöçü S. Erisen C. Sancak S. Özcan 《Biologia Plantarum》2004,48(3):441-444
Transformation of fenugreek (Trigonella foenumgraecum) was carried out with A281 oncogenic strain of Agrobacterium tumefaciens using root, cotyledon and hypocotyl explants excised from 1-week-old seedlings, which showed that the plant was highly susceptible to transformation. Tumors (calli) were selected on 50 mg dm–3 kanamycin. They were analyzed for -glucuronidase (GUS) expression. Presence of uidA (gus) gene, was confirmed by polymerase chain reaction (PCR) amplification. 相似文献
20.
A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD6000.6 and diluted to a cell density of 109 cells ml–1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 g ml–1). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l–1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T1 seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1. 相似文献