共查询到20条相似文献,搜索用时 31 毫秒
1.
A transformation and regeneration system has been developed for Nicotiana alata, a plant which is being intensively studied as a model of gametophytic self-incompatibility. Plantlets can be regenerated efficiently from seedling hypocotyls. Kanamycin-resistant, transformed plants have been obtained by cocultivation of regenerating hypocotyls with Agrobacterium tumefaciens strain LBA4404 containing a binary vector. The transformation frequency was low with <1% of tissue explants regenerating transformed plants. The transformed plants contained from one to three copies of the introduced DNA. In most cases, the kanamycin resistance phenotype was transmitted to the offspring as a normal Mendelian factor. In one unusual case, none of the offspring inherited the kanamycin resistance of the transformed maternal parent. This plant may have been chimeric or the kanamycin resistance gene may have been inactivated. 相似文献
2.
Bao-Hong Zhang Fang Liu Zhi-Hong Liu Hong-Mei Wang Chang-Bing Yao 《Plant Growth Regulation》2001,33(2):137-149
The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed. 相似文献
3.
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
4.
E. B. Swanson L. R. Erickson 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1989,78(6):831-835
Summary Microspore-derived embryos of Brassica napus were transformed using the disarmed octopine-producing LBA4404 strain of Agrobacterium tumefaciens containing the binary vector pBin19. Octopine-producing strains have previously been reported to be ineffective in transforming Brassica. Four actively growing yellow/ green sectors were selected from the embryos on 50 mg/l kanamycin and plants regenerated. Analysis for NPT-II activity in these young plants initially indicated no expression of the bacterial NPT-II gene. The plants were nevertheless grown to maturity, selfed and S1 seed was collected. Three of the S1 plants produced microspores which were from 4 to 20 times more tolerant to kanamycin than the original parent. Southern analysis revealed that one plant (EC-1) had a single site of insertion and the other two plants (EC-2 and EC-6) had two sites of insertion with sequence homology to the bacterial NPT-II gene. Microspores from the EC-2 and EC-6 transgenics produced embryos on approximately five times the level of kanamycin tolerated by microspores from untransformed plants, while the EC-1 transgenic produced microspores with more than 20 times the tolerance to kanamycin. Analysis of S1 progeny of the EC-1 transgenic indicated that 100% of the progeny exhibited the trait through both Southern analysis and by expressing tolerance to kanamycin in microspore-derived embryos. 相似文献
5.
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
6.
An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine
(BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration
medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l
BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots
were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong
β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear
genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective
transformation and direct regeneration of E. tereticornis Sm. 相似文献
7.
Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings 总被引:5,自引:0,他引:5
Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation
was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants
from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic
embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%)
from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into
plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated
from single embryos of E. senticosus were acclimatized in a greenhouse.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
8.
Zhiqiang Pan Jingkun Ho Qin Feng Dao-Shun Huang Ralph A. Backhaus 《Plant Cell, Tissue and Organ Culture》1996,46(2):143-150
In vitro grown shoot tissue of facultative apomictic lines of guayule (Parthenium argentatum Gray), a rubber producing desert shrub, were transformed by Agrobacterium-mediated DNA transfer and regenerated into complete plants. Guayule shoots of lines 11591, UC101 and UC104 were inoculated with A. tumefaciens strains LBA4404 or PC2760 harboring the binary vector pCGN1557. Axillary shoots were regenerated from transformed cells and rooted in vitro in the presence of kanamycin. Genetic transformation in all cases was verified by Southern blot analysis. Transgenic plants were grown to maturity in the greenhouse and, as predicted for apomictic species, all seed produced possessed kanamycin resistance. Because apomicts have limitations for gene transfer by normal sexual crosses, this method offers a new means of transferring genes into this species.Abbreviations BA
benzyladenine
- EDTA
ethylene diamine tetraacetate
- kanR
kanamycin resistance
- MS salts
salts of Murashige and Skoog medium (1962)
- NAA
naphthalene acetic acid
- NPT-II
neomycin phosphotransferase
- SDS
sodium dodecyl sulfate 相似文献
9.
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
10.
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
11.
Somatic embryos differentiated from hypocotyl explant in cotton (Gossypium hirsutum L.) exhibited very divergent morphologies. Six different types of somatic embryos based on cotyledon development were observed.
The growth hormones (2,4-dichlorophenoxyacetic acid and kinetin) used in induction and maintenance media did not affect embryo
rooting and germination. The 95 % conversion of normal embryos (with two cotyledons) was achieved, while an overall conversion
was only 38 %. Horn shaped embryos failed to exhibit shoot growth. Poorly developed apical meristems were responsible for
lower conversion percentages in some of embryo classes. However, regenerated plants phenotypically resembled to seed grown
control plants regardless of somatic embryo morphology. 相似文献
12.
Bao-Hong Zhang Hong-Mei Wang Fang Liu Yun-Hai Li Zheng-De Liu 《In vitro cellular & developmental biology. Plant》2001,37(2):300-304
Summary 2,4-Dichlorophenoxyacetic acid (2,4-D) resistant plants of transgenic cotton (Gossypium hirsutum L.) were produced using Agrobacterium tumefaciens containing a plasmid carrying the neomycin phosphotransferase II (npt II) and 2,4-D monooxygenase (tfd A) genes. An in vitro assay was performed to determine the sensitivity of seed germination, and the growth of seedlings of transgenic and non-transgenic
cotton to various concentrations of kanamycin and 2,4-D. The results indicated that kanamycin caused the cotyledons of non-transgenic
plants to turn white, but transgenic plants grew normally. Seed germination and seedling growth of non-transgenic plants were
strongly inhibited by 2,4-D, but only slightly for transgenic plants. Transgenic plants and non-transgenic plants can be clearly
distinguished by the use of 2 mg l−1 2,4-D in seed germination medium. There was a high correlation between the response of seed germination and the growth of
seedlings to kanamycin or 2,4-D, based on the germination ration, albino ratio, dry weight or fresh weight. On this basis,
we development a rapid method for identifying transgenic plants that has been verified in the field. These findings will allow
identification of cotton transformants at an early stage of plant development, saving time and improving cultivars containing
the 2,4-D resistance trait. 相似文献
13.
J. Puonti-Kaerlas T. Eriksson P. Engström 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,80(2):246-252
Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis. 相似文献
14.
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
15.
Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding. 相似文献
16.
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
17.
A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation. 相似文献
18.
Zhen Zhu Karen Woodbury Hughes Leaf Huang 《In vitro cellular & developmental biology. Plant》1991,27(2):77-83
Summary Protoplasts ofNicotiana tabacum var. Xanthi were incubated with liposomes containing the plasmid plGVneo23 encoding kanamycin resistance. Transformed protoplasts
and calli and plants derived from transformed protoplasts were treated with the demethylating agent 5-azacytidine. Three lines
of evidence indicate that 5-azacytidine can increase NPT II activity in transformed cell lines and plants: a) Addition of
azacytidine to the protoplast medium increased the proportion of kanamycin-resistant transformants recovered. b) NPT II activity
could not be detected in approximately 50% of calli derived from transformed protoplasts although such calli grew slowly on
medium containing kanamycin. Treatment of NPT-negative calli with 5-azacytidine restored detectable gene activity and increased
the growth rate of the callus in the presence of kanamycin. c) Shoot tips regenerated from transformed calli were either NPT-positive
or NPT-negative. When shoots were NPT-negative, treatment with 5-azacytidine restored detectable gene activity and improved
growth in the presence of kanamycin. 相似文献
19.
Plants of a diploid wild cotton species (G. klotzschianum A.) were efficiently regenerated from protoplasts isolated from immature somatic embryos and suspension cultures by studying various factors affecting regeneration. Purified protoplasts were cultured with the density of 2–10×105 ml−1, and the medium was k3 inorganic salts with modified KM8P organic compositions, supplemented with several combinations of PGRs. Calluses were formed from protoplasts of suspension cultures and immature somatic embryos. The influences of carbon sources and GA3 on callus differentiation and somatic embryo germination were analyzed. Somatic embryos germinated normally and formed regenerated plantlets. Regenerated plantlets were transferred to the soil and seeds were obtained. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed 23 primers that gave 74 clear reproducible bands, with amplification products being monomorphic for 14 tested plantlets. A total of 1036 bands obtained exhibited no aberration in RAPD banding patterns in the 14 plants. Plants regenerated via somatic embryogenesis from the diploid cotton protoplasts have genetic homogeneity. 相似文献
20.
H. Kisaka T. Kameya 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1994,88(1):75-80
Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids. 相似文献