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1.
Jayashree R Rekha K Venkatachalam P Uratsu SL Dandekar AM Kumari Jayasree P Kala RG Priya P Sushma Kumari S Sobha S Ashokan MP Sethuraj MR Thulaseedharan A 《Plant cell reports》2003,22(3):201-209
Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The -glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l-1 kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l-1 spermine and 0.1 mg l-1 abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l-1 gibberellic acid, 0.2 mg l-1 kinetin (KIN) and 0.1 mg l-1 indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.Communicated by L. Peña 相似文献
2.
Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and -glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.Abbreviations BAP
6-benzylaminopurine
- CaMV
cauliflower mosaic virus
- GUS
-glucuronidase
- IBA
indole-3-butyric acid
- IM
infection medium
- NAA
1-naphthalene acetic acid
-
neo
gene encoding NPTII
- NPTII
neomycin phosphotransferase
- RIM
root-inducing medium
- SEM
shoot-elongation medium
- SIM
shoot-inducing medium
- t-nos
polyadenylation site of the nopaline synthase gene
-
uidA
gene encoding GUS
- WM
wash medium
- X-Gluc
5-bromo-4-chloro-3-indolyl -D-glucuronide 相似文献
3.
Zhijian T. Li S. A. Dhekney M. Dutt D. J. Gray 《Plant Cell, Tissue and Organ Culture》2008,93(3):311-321
An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP
and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing
period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin
added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic
plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on
MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic
plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated
to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’. 相似文献
4.
Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants 总被引:1,自引:6,他引:1
Leandro Peña Magdalena Cervera José Juárez Antonio Navarro José A. Pina Nuria Durán-Vila Luis Navarro 《Plant cell reports》1995,14(10):616-619
Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA
benzyladenine
- CaMV
cauliflowermosaic virus
- GUS
ß-glucuronidase
- LB
Luria Broth
- MS
Murashige and Skoog
- NAA
naphthalenacetic acid
- NOS
nopaline synthase
- NPT II
neomycin phosphotransferase II
- PEG
polyethylene glycol
- RM
rooting medium
- SRM
shoot regeneration medium 相似文献
5.
Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS
ß-glucuronidase
- X-Gluc
5-bromo-4-chloro-3indolyl ß-D-glucuronic acid
- NPT II
neomycin phosphotransferase II 相似文献
6.
Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola. 相似文献
7.
Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants 总被引:2,自引:0,他引:2
Ebrahim Firoozabady David L. DeBoer Donald J. Merlo Edward L. Halk Lorraine N. Amerson Kay E. Rashka Elizabeth E. Murray 《Plant molecular biology》1987,10(2):105-116
Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton. 相似文献
8.
An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar Blue Uniform was more responsive than White Uniform. Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by -glucuronidase and ELISA analyses, respectively.Abbreviations BAP 6-Benzylaminopurine - NAA -Naphthalene acetic acid - TDZ Thidiazuron - BU Blue Uniform - WU White Uniform 相似文献
9.
Véronique Guellec Chantal David Michel Branchard Jacques Tempé 《Plant Cell, Tissue and Organ Culture》1990,20(3):211-215
Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP
benzylaminopurine
- IAA
indoleacetic acid
- NAA
naphtaleneacetic acid
- NPT II
neomycin phosphostransferase II
- EDTA
ethylenediaminetetraacetic acid 相似文献
10.
Perumal Venkatachalam Natesan Geetha Narayanasamipillai Jayabalan Saravanababu Lakshmi Sita 《Journal of plant research》1998,111(4):565-572
Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants
followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot
regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before
transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting
transformation efficiency are discussed. 相似文献
11.
Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants 总被引:3,自引:0,他引:3
Limanton-Grevet A. Jullien M. 《Molecular breeding : new strategies in plant improvement》2001,7(2):141-150
Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T1 progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T2 progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations. 相似文献
12.
M.-L. Wang G. Uruu L. Xiong X. He C. Nagai K. T. Cheah J. S. Hu G.-L. Nan B. S. Sipes H. J. Atkinson P. H. Moore K. G. Rohrbach R. E. Paull 《In vitro cellular & developmental biology. Plant》2009,45(2):112-121
A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from
Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing
of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from
two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided
the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated
by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated
from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent
lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic
evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement. 相似文献
13.
A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis. 相似文献
14.
Lucas Olivier Kallerhoff Jean Alibert Gilbert 《Molecular breeding : new strategies in plant improvement》2000,6(5):479-487
Split embryonic axes of 21-day old immature sunflower (Helianthus annuus L.) embryos were bombarded by microparticles and then co-cultured with disarmed Agrobacterium tumefaciens strain EHA105 bearing a binary vector carrying nptII and uidA genes. Apical shoot bud development and organogenesis induced on the explants led T0 transgenic plants. Southern blot analysis revealed complex integration patterns in T0 plants. The uidA gene segregated as a dominant trait and single-insertion events were observed in T1 plants. Patterns similar to those of T1 plants were observed in T2 progeny. 相似文献
15.
Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants 总被引:1,自引:0,他引:1
G. A. Moore C. C. Jacono J. L. Neidigh S. D. Lawrence K. Cline 《Plant cell reports》1992,11(5-6):238-242
Summary A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage ( 5%) of the shoots regenerated in the presence of 100 g/ml kanamycin were GUS+. Polymerase chain reaction (PCR) analysis confirmed that GUS+ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis. 相似文献
16.
S. A. Dhekney R. E. Litz D. A. Moraga Amador A. K. Yadav 《In vitro cellular & developmental biology. Plant》2007,43(3):195-202
Papaya (Carica papaya L.) production is affected by low temperatures that occur periodically in the subtropics. The C-repeat binding factor (CBF)
gene family is known to induce the cold acclimation pathway in Arabidopsis thaliana. Embryogenic papaya cultures were induced from hypocotyls of “Sunrise Solo” zygotic embryos on semisolid induction medium.
The CBF 1/CBF 3 genes along with the neomycin phosphotransferase (NPT II) gene were placed under the control of the CaMV 35
S promoter and introduced into a binary vector pGA 643. Embryogenic cultures were transformed with Agrobacterium strain GV 3101 harboring pGA 643. After selection of transformed embryogenic cultures for resistance to 300 mg l−1 kanamycin, somatic embryo development was initiated and transgenic plants were regenerated. The presence of the CBF transgenes
in regenerated plants was confirmed by Southern blot hybridization. The papaya and the related cold-tolerant Vasconcella genomes were probed for the presence of cold inducible sequences using polymerase chain reaction (PCR). Possible cold inducible
sequences were present in the Vasconcella genome but were absent in the Carica genome. 相似文献
17.
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
18.
A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba × A. cephalonica (cell lines AC2, AC78) and Abies alba × A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l−1). The T-DNA of plant transformation vector contained the β-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium
containing 10 mg geneticin l−1. GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of
embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been
obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings. 相似文献
19.
Jovanka Miljuš-Djukić Mirjana Nešković Slavica Ninković Radomir Crkvenjakov 《Plant Cell, Tissue and Organ Culture》1992,29(2):101-108
Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R2 seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP
6-benzylaminopurine
- 2,4-D
dichloro-phenoxyacetic acid
- 2iP
6-(, ,-dimethylallyl-amino)-purine
- IBA
indole-3-butyric acid
- IAA
indole-3-acetic acid
- Km
kanamycin
- NPTII
neomycin phosphotransferase II 相似文献