共查询到20条相似文献,搜索用时 31 毫秒
1.
Chen Long-Fang O. Huang Jia-Yuan Wang Yung-Hsiang Chen Yu-Ting Shaw Jei-Fu 《Molecular breeding : new strategies in plant improvement》2004,14(3):199-213
A mutant broccoli ers (ethylene-response-sensor, boers) gene was obtained through site directed mutagenesis by replacing the isoleucine with phenylanine at the 62th residue. Two plasmids were constructed with this mutant gene regulated by the CaMV 35S promoter together with the nptII (kanamycin resistance gene) coding sequence and hpt (hygromycin resistance gene), respectively, for the pBI-mERSI62F and pSM1H-mERSI62F plasmids. Genetic transformation of the above two constructs via A. tumefaciens has been conducted to evaluate their effects on floret yellowing of harvested broccoli. Over a hundred transformants have been obtained on the selected cotyledon and hypocotyl explants. PCR and Southern analysis demonstrated integration of the transgenes in the transformants. However, through Southern hybridization, we determined that multi-site integration and DNA rearrangements had occurred in most transformants. Morphological and characteristic alternation such as slower plant growth, shorter plant height, easy branching, late bolting, and relative higher mortality in comparison with other transgenes were noted in some transformants. Transgenic lines showing delayed senescence in leaves and floral heads were obtained. The expression of transgene was confirmed by Northern blot analysis. The transformed progenies also showed ethylene insensitivity in seed germination, detached leaves and harvested florets. Nevertheless, in most lines, the yellowing was only delayed 1–2 days. 相似文献
2.
Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens 相似文献
3.
Mao-Sen LiuHui-Chun Li You-Min ChangMin-Tze Wu Long-Fang Oliver Chen 《Plant science》2011,181(3):288-299
Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days’ post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence. 相似文献
4.
L. Scaramelli A. Balestrazzi M. Bonadei E. Piano D. Carbonera M. Confalonieri 《Plant cell reports》2009,28(2):197-211
Expression of the uidA reporter gene was tested in transformation experiments of barrel medic (Medicago truncatula Gaertn.) with the ipt-type control vectors pIPT5, pIPT10 and pIPT20 and distinct in vitro culture conditions. The highest GUS expression levels
were obtained with the pIPT10 construct carrying the ipt gene under the control of the native ipt promoter and using kanamycin as selective agent. The ipt-shooty transformants, characterized by the absence of both rooting ability and apical dominance associated with vitrification,
were easily identified by visual selection. Using only the ipt gene as selectable marker, we obtained a stable transformation frequency of 9.8% with pIPT10 construct. The ipt-type MAT vector pEXM2 was then used to monitor the excision events mediated by the yeast Recombinase and the consequent production
of ipt marker-free transgenic plants. Transgenic ipt-shooty lines were recovered at a frequency of 7.9% in the absence of kanamycin-based selection. The ipt-shooty phenotype was maintained in all the transgenic lines and no reversion to the normal phenotype occurred. PCR analysis
revealed the presence of the ‘hit and run’ cassette in the genome of all the regenerated ipt-shooty lines while RT-PCR experiments confirmed the expression of the R gene, encoding the yeast Recombinase. A detailed molecular investigation, carried out to verify the integrity of the RS sites,
revealed that these regions were intact in most cases. Our results with barrel medic suggest that the MAT system must be carefully
evaluated and discussed on a case by case basis.
L. Scaramelli, A. Balestrazzi and M. Confalonieri have contributed equally to this work. 相似文献
5.
An efficient system for Agrobacterium tumefaciens-mediated transformation of Solanum gilo was established. The marker genes for kanamycin resistance and ß-glucuronidase expression were introduced. A comparison between cotyledon and hypocotyl explants showed that while regeneration was better from hypocotyl explants, cotyledon explants gave better transformation efficiency (46% vs. 32%). Four levels of kanamycin selection (100, 150, 200 and 250 mg/l) were tested for effect on transformation efficiency with each type of explant. Lower levels of kanamycin worked better using cotyledon explants, while higher levels of kanamycin worked better for hypocotyl explants. All nine t0 plants tested for expression of the kan
r
gene were positive. The progeny of three of these plants showed a pattern of classical Mendelian inheritance (3 to 1) for both the kan
r
and the ß-glucuronidase genes.Abbreviations MS
Murashige and Skoog (1962) medium
- 2,4-D
2,4-Dichlorophenoxyacetic acid
- NPTII
neomycin phosphotransferase
- GUS
ß-glucuronidase 相似文献
6.
Xin-Hua Feng Shyam K. Dube Paul J. Bottino Shain-dow Kung 《Plant molecular biology》1990,15(3):407-420
The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids. 相似文献
7.
Samanta Zelasco Valentina Ressegotti Massimo Confalonieri Daniela Carbonera Paolo Calligari Martina Bonadei Stefano Bisoffi Keiko Yamada Alma Balestrazzi 《Plant Cell, Tissue and Organ Culture》2007,91(1):61-72
Genetic transformation of an elite white poplar genotype (Populus alba L., cv. ‘Villafranca’) was performed with MAT vectors carrying the ipt and rol genes from Agrobacterium spp. as morphological markers. The effects associated with the use of different gene promoters and distinct in vitro regeneration
protocols were evaluated. Poplar plantlets showing abnormal ipt and rol phenotypes were produced only in the presence of exogenous growth regulators. The occurrence of abnormal ipt and rol phenotypes allowed the visual selection of transformants. The ipt-type MAT vector pEXM2 was used to monitor the activity of the yeast site-specific recombination R/RS system in the transformed
white poplar cells. Results from these experiments demonstrated that recombinase-mediated excision events occurred during
the early stages of in vitro culture, thus causing the direct production of ipt marker-free transgenic plants with normal phenotype at an estimated frequency of 36.4%. Beside this unexpected finding, transgenic
ipt-shooty plants were obtained at a frequency of 63.6% and normal shoots were subsequently recovered after a prolonged period
of in vitro culture. Although the transformation efficiency observed in this study, using both ipt and nptII genes as selection markers, was similar to that previously reported with standard vectors carrying only the nptII gene, the easy identification of ipt transformants, the early recombinase-mediated excision events and finally the relatively short time period required to produce
ipt marker-free transgenic plants support for the choice of MAT vectors as a reliable strategy for the future production of marker-free
GM poplars. 相似文献
8.
Root explants ofArabidopsis thaliana ecotype C24 were bombarded with the plasmid pCH harboring the hygromycin phosphotransferase gene (hpt). A selection condition with post-bombardment culture of 3 days followed by culture with 20 mgl−1 hygromycin gave the highest yield of transformants. More than 44% of explant clumps formed transformant shoots. 相似文献
9.
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
10.
Géraldine Bonnard Bruno Tinland François Paulus Ernö Szegedi Léon Otten 《Molecular & general genetics : MGG》1989,216(2-3):428-438
Summary A DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3 non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3 coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.Abbreviations LHR
limited host range
- WHR
wide host range
-
onc
oncogenicity genes
-
iaaH
indoleacetamide hydrolase gene
-
iaaM
tryptophan monooxygenase gene
-
ipt
isopentenyl transferase gene
-
tzs
transzeatin secretion gene
- NAA
-naphthalene acetic acid
- BAP
6-benzylaminopurine
- Km
kanamycin
- Neo
neomycin
- Cm
chloramphenicol 相似文献
11.
We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large
number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by
nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both
histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency
in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks
associated with the use of these traditional selection marker genes can be eliminated. 相似文献
12.
A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.Communicated by P. Debergh 相似文献
13.
Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter
kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed
6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed
cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was
obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression
of the NOS gene in the selfed R1 progeny. NPTII activity was observed in the R2 generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this
procedure were unsuccessful. 相似文献
14.
Rita Gandill Satish C. Matheshwari Jitendra P. Khurana Paramjit Khurana 《In vitro cellular & developmental biology. Plant》2001,37(5):629-637
Summary
Agrobacterium-mediated transformation of Arabidopsis, ecotype ‘Estland’, was established from root explants using kanamycin selection. Continuous light during callus and shoot
induction phases was promotive for shoot regeneration, as compared to light/dark cycles. Use of optimized conditions for transformation
led to the formation of kanamycin-resistant calluses (up to 77%) and transformed plantlets at a frequency of up to 45%. Southern
analysis showed the presence of 1.2. or more T-DNA inserts in 33%, 50%, and 17% of the primary transformants, respectively.
Mendelian, as well as non-Mendelian, inheritance patterns were obtained upon screening the progeny (T1) of various transformants
for the expression of gus and nptII genes; the analysis of some of these transformants at the molecular level also corroborated the Mendelian inheritance pattern.
Moreover, genotypes of the T1 progeny could be predicted on the basis of T2 progeny analysis. 相似文献
15.
Summary Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes. 相似文献
16.
Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent
cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with
emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region
of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis
on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm
in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation
period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants
or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An
optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl−1 hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl−1 hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature
cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols
for transformation of soybean should be established under both Agrobacterium and selection conditions. 相似文献
17.
A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization
of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed
for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression
of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results
indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the
problems related to the use of antibiotics diminished.
Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000 相似文献
18.
A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 μM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols. 相似文献
19.
O. Fedorowicz G. Bartoszewski P. Stoeva K. Niemirowicz-Szczytt 《Acta Physiologiae Plantarum》2000,22(3):277-281
Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we
have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation
of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing
kanamycin. The preliminary analysis indicated that 18 plants are putative transformants. 相似文献