共查询到20条相似文献,搜索用时 15 毫秒
1.
Maria I. C. S. Gama Rui P. Leite Jr. Antonio R. Cordeiro Daniel J. Cantliffe 《Plant Cell, Tissue and Organ Culture》1996,46(3):237-244
Stable expression of foreign genes was achieved in sweet potato (Ipomoea batatas (L.) Lam) plants using anAgrobacterium tumefaciens mediated system. Embryogenic calluses produced from apical meristems of cultivar White Star were multiplied and cocultivated withA. tumefaciens strain EHA101 harboring a binary vector containing the -glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes. The calluses were transferred to selective regeneration medium and kanamycin resistant embryos were recovered which developed into morphologically normal plants. Histochemical and fluorimetric GUS assays of plants developed from the kanamycin resistant embryos were positive. Amplified DNA fragments were produced in polymerase chain reactions using GUS-specific primers and DNA from these plants. Transformation was confirmed by Southern analysis of the GUS gene. With the developed method, transgenic sweet potato plants were obtained within 7 weeks. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.Florida Agricultural Experiment Station Journal Series N-02231. This research was partially supported by CNPq/RHAE (Brazil). 相似文献
2.
Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants 总被引:9,自引:0,他引:9
Almond (Prunus dulcis Mill.) leaves were transformed with the marker genes gusA (β-glucuronidase) and nptII (neomycin phosphotransferase II) via Agrobacterium-mediated transformation. Bacterial strains and preculture of explants affected efficiency of gene transfer evaluated by transient
expression assays. Following transformation, shoots were induced from primary explants on medium without kanamycin and exposed
to selection 20 days after cocultivation. From 1419 original leaves, four shoots (A, B, C and D) were obtained that showed
amplification of the predicted DNA fragments by polymerase chain reaction (PCR). After micropropagation of these shoots, only
those cloned from shoot D gave consistently positive results in histochemical GUS detection and PCR amplification. Southern
blot hybridisation confirmed stable transgene integration in clone D, which was also negative in PCR amplification of an Agrobacterium gene. Additional molecular analysis suggested that the remaining three shoots (A, B and C) were chimeric.
Received: 28 March 1998 / Revision received: 18 April 1998 / Accepted: 12 May 1998 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.
The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene
transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous
efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all
the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears
to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce
stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of
the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general
guidelines for the development of a transformation protocol are discussed. 相似文献
5.
Vengadesan G Amutha S Muruganantham M Anand RP Ganapathi A 《Plant cell reports》2006,25(11):1174-1180
Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l−1 activated charcoal, 1.5 mg l−1 phosphinothricin, and 300 mg l−1 cefotaxime. Phosphinothricin at 1.5 mg l−1 was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l−1 phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l−1 phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta. 相似文献
6.
Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses,
because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic
calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken
up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was
used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks
of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic
nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and
GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable
tool for functionality tests of candidate genes in forage and turf grasses. 相似文献
7.
Transgenic plants of ramie (Boehmeria nivea Gaud.) obtained by Agrobacterium mediated transformation
Diva Maria A. Dusi Manuel Dubald Elionor Rita P. de Almeida Linda S. Caldas Eugen S. Gander 《Plant cell reports》1993,12(11):625-628
A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg et al. 1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker gene. 相似文献
8.
A highly efficient and fast Agrobacterium-mediated leaf disc transformation system for the Arabidopsis thaliana L. genotype C24 was developed. This protocol is also amenable to other ecotypes - as could be shown for Landsberg erecta and Wassllewskija. Besides the hygromycin selection also the G418 and kanamycin selection were established. Furthermore the described procedure is appliable not only to leaf explants but also to expanded cotyledons which proved to be an excellent alternative as explant source for transformation experiments.Abbreviations BAP
6-Benzylaminopurine
- CIM
Callus induction medium
- 2.4D
2,4-Dichlorophenoxyacetic acid
- GA3
Gibberellic acid
- IAA
Indole-3-acetic acid
- IBA
Indole-3-butyric acid
- 2-IP
N6-,-Dimethylallyladenosine
- HPT
Hygromycin phosphotransferase
- NAA
-Naphthaleneacetic acid
- NPT II
Neomycin phosphotransferase type II
- SEM
Shoot elongation medium
- SIM
Shoot induction medium
- RIM
Root induction medium 相似文献
9.
Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N6-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA
-naphthaleneacetic acid
- BA
6-benzyladenine
- GA3
gibberellic acid
- 2iP
N6-2-isopentenyl adenine
- KT-30
N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU)
- TDZ
N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron) 相似文献
10.
Transgenic carnations obtained byAgrobacterium tumefciens-mediated transformation of leaf explants 总被引:1,自引:0,他引:1
Anne-Claire van Altvorst Tjitske Riksen Herma Koehorst Hans J. M. Dons 《Transgenic research》1995,4(2):105-113
For the development of anAgrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing -glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenic shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulentAgrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way. 相似文献
11.
Bhalla Prem L. Smith Nicole 《Molecular breeding : new strategies in plant improvement》1998,4(6):531-541
We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct. 相似文献
12.
Mahmoud A. Hassan Harry Jan Swartz Gordon Inamine Phillip Mullineaux 《Plant Cell, Tissue and Organ Culture》1993,33(1):9-17
Experiments in shoot regeneration and virulentAgrobacterium tumefaciens-mediated transformation were used to develop a protocol forRubus transformation. This protocol was then used to produce transformedRubus plants fromin vitro internodes inoculated with anAgrobacterium tumefaciens encoding neomycin phosphotransferase on its disarmed T-DNA. Two transformed plants were selected from 800 inoculations on a medium containing 10 µg ml–1 kanamycin. Results indicated that this level of kanamycin successfully selected against non-transformed cells but did not reduce the number of transformed, kanamycin-resistant, shoots formed. Enzyme assays and Southern blot analysis verified the presence of the -glucuronidase gene in the plant genome. 相似文献
13.
An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO3 and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All Ro transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R1 seed progeny and showed co-segregation.Abbreviations BA
benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- NAA
naphthaleneacetic acid
- NPTII
neomycin phosphotransferase type II
- GUS
-glucuronidase
- CaMV
cauliflower mosaic virus
- MS
Murashige and Skoog
- X-Gluc
5-bromo-4-chloro-3-indolyl-D--glucuronic acid
- IBA
indolebutyric acid
- SDS
sodium dodecyl sulfate 相似文献
14.
Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants 总被引:6,自引:0,他引:6
C.-K. Ho S.-H. Chang J.-Y. Tsay C.-J. Tsai V. L. Chiang Z.-Z. Chen 《Plant cell reports》1998,17(9):675-680
An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments
with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and
regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more
than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant
to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants.
Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis,
further confirming the integration and expression of T-DNA in these plants.
Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998 相似文献
15.
A protocol for Agrobacterium tumefaciens-mediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9–12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous growth phenotype were obtained by extended culture on media containing 600 mg l−1 kanamycin. After 9 months of a stringent selection pressure, the removal of kanamycin from the final medium together with the culture of the transformed calluses under nutritional stress led to the formation of several transgenic adventitious shoots. Transformation was confirmed by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for the nptII gene). With this approach, a transformation efficiency of 22.7% was achieved. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for this cactus species. 相似文献
16.
Robinia pseudoacacia (black locust) plants were regenerated after co-cultivation of stem and leaf segments with Agrobacterium tumefaciens strain GV3101 (pMP90) that harbored a binary vector that included genes for β-glucuronidase (GUS) and hygromycin phosphotransferase. Successful transformation was confirmed by the ability of stem and
leaf segments to produce calli in the presence of hygromycin, by histochemical and fluorometric assays of GUS activity in
plant tissues, and by Southern blotting analysis. In this transformation system, about 2 months were required for regeneration
of transgenic plants from stem and leaf segments. The frequency of transformation from stem segments was approximately 24%,
and the morphology of regenerated plants resembled that of the original parental strain.
Received: 2 September 1999 / Revision received: 30 November 1999 / Accepted: 4 December 1999 相似文献
17.
Belladonna or deadly nightshade (Atropa belladonna L.) is an important medicinal plant in the family Solanaceae. It is a model plant for studying plant alkaloid biosynthesis. In this study, a reliable protocol for efficient transformation of A. belladonna using Agrobacterium tumefaciens was developed. Hypocotyl and cotyledon explants were co-cultivated with three opine-type Agrobacterium strains (LBA4404: pBISN1, GV3101: pBISN1, and EHA105: pBISN1). Selection and regeneration of transformed cells were conducted on two regeneration media; RM1 (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium (MS) salts, Gamborg B5 vitamins (Gamborg et al. in Exp Cell Res 50:151–158, 1968), 4.56 μM zeatin, and 2.9 μM indole-3-acetic acid (IAA)] and RM2 (MS salts, B5 vitamins, 4.65 μM kinetin, and 1.14 μM IAA), each containing 100 mg l?1 kanamycin and 250 mg l?1 timentin. Both regeneration media and type of explant had significant effects on frequencies of transformation. Using an optimal regeneration medium and regardless of the strain of Agrobacterium used, over 80 % of hypocotyl explants and 60 % of cotyledons developed at least one transformed shoot after 2–3 months of selection. Most transformants exhibited a normal phenotype while growing in the greenhouse. Southern blot analysis confirmed the stable integration of the nptII transgene in T1 plants. 相似文献
18.
Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP
6-benzylaminopurine
- Cf
ClaforanR
- 2.4D
2,4-dichlorophenoxy acetic acid
- Km
kanamycin
- MS
Murashige and Skoog (1962)
- NAA
-naphthalene acetic acid
- NPT II
neomycinphosphotransferase
- npt II
neomycinphosphotransferase II gene
- NOS
nopaline synthase
- nos
nopaline synthase gene
- ocs
octopine synthase gene
- IAA
indole-3-acetic acid 相似文献
19.
X. Niu X. Li P. Veronese R. A. Bressan S. C. Weller P. M. Hasegawa 《Plant cell reports》2000,19(3):304-310
Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent
of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications
were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic
evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted
in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher
concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration
medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the
number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average
transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein,
osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is
a viable alternative to plant breeding and clonal selection for improvement of this crop.
Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999 相似文献
20.
Annemieke Vergauwe Ronny Cammaert Dirk Vandenberghe Christiane Genetello Dirk Inze Marc Van Montagu Elfride Van den Eeckhout 《Plant cell reports》1996,15(12):929-933
Summary A transformation system was developed for Artemisia annua L. plants. Leaf explants from in vitro grown plants developed callus and shoots on medium with 0.05 mg/L naphthaleneacetic acid and 0.5 mg/L N6-benzyladenine after transformation with the C58C1 RifR (pGV2260) (pTJK136) Agrobacterium tumefaciens strain. A concentration of 20 mg/L kanamycin was added in order to select transformed tissue. Kanamycin resistant shoots were rooted on naphthaleneacetic acid 0.1 mg/L. Polymerase chain reactions and DNA sequencing of the amplification products revealed that 75% of the regenerants contained the foreign genes. 94% of the transgenic plants showed a -glucuronidase-positive response.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- BA
N6-benzyladenine
- GM
germination medium
- GMVIT
germination medium with vitamins
- GUS
-glucuronidase
- Kin
kinetin (N6-furfurylaminopurine)
- NAA
-naphthaleneacetic acid
- NPT II
neomycin phosphotransferase II
- PCR
Polymerase Chain Reaction
- T-DNA
transfer-DNA
- X-glucuronide
5-bromo-4-chloro-3-indolyl -D-glucuronide 相似文献