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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 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
3.
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
4.
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
5.
Qi Zhu Fengtao Wu Feng Ding Dong Ye Yongqin Chen Yi Li Yang Zhifan 《Plant Cell, Tissue and Organ Culture》2009,96(3):317-324
Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important
steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and
female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive
to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation
medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three
days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for
formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh
selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of
the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to
produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated
from initial infected callus explants. 相似文献
6.
Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 260C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP
6-benzylamino purine
- 2,4-D
2,4dichlorophenoxy acetic acid
- IAA
Indole acetic acid
- IBA
Indole butaric acid
- NAA
Naphthalene acetic acid 相似文献
7.
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
8.
Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia 总被引:3,自引:0,他引:3
Sergio G. Nebauer Isabel Arrillaga Lucas del Castillo-Agudo Juan Segura 《Molecular breeding : new strategies in plant improvement》2000,6(6):539-552
Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line. 相似文献
9.
<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis> 总被引:3,自引:0,他引:3
A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies. 相似文献
10.
Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants 总被引:3,自引:0,他引:3
Gruchała Agnieszka Korbin Małgorzata Żurawicz Edward 《Plant Cell, Tissue and Organ Culture》2004,79(2):153-160
Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA4404 strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l –1 IBA and 1.8 mg l–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency. 相似文献
11.
Genetic transformation of strawberry: Stable integration of a gene to control biosynthesis of ethylene 总被引:7,自引:0,他引:7
Helena Mathews W. Wagoner J. Kellogg R. Bestwick 《In vitro cellular & developmental biology. Plant》1995,31(1):36-43
Summary Efficient methods ofAgrobacterium-mediated transformation are described for two Pacific Northwest cultivars of strawberry (Fragaria ×ananassa), Tristar and Totem. We report stable incorporation of a gene for control of ethylene biosynthesis, into strawberry (cultivar
Totem) for the first time. Cultivar Tristar was transformed with disarmed strains ofAgrobacterium tumefaciens (A. tumefaciens), LBA4404 or EHA101, containing a binary vector with marker genesuidA andnptII. Cultivar Totem was transformed withA. tumefaciens strains EHA101 or EHA105 harboring binary vectors with selectable marker genesnptII orhpt and with a gene for S-adenosylmethionine hydrolase (SAMase) for control of ethylene biosynthesis. The frequency of transgenic
shoots ranged from 12.5% to 58.8% of the original treated explants when using plasmids containing the gene encoding SAMase.
Primary shoot regenerants obtained on selection medium were subjected to several iterations of tissue isolation and reculture
on higher stringency selection medium for recovering uniformly transformed plantlets. Transgenic plants were confirmed by
their ability to undergo rooting on medium with selection at 60 mg/liter kanamycin or 10 mg/liter hygromycin. About 95–100%
of the transformation events from different experiments were capable of profuse rooting in the presence of selection. Insertion
of the SAMase gene and its integration into the strawberry genome were confirmed by Southern hybridization. About 500 plants
from 250 independent transgenic events have been successfully transferred to soil for further evaluation. 相似文献
12.
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
13.
Antara Ghosh T. R. Ganapathi Pravendra Nath V. A. Bapat 《Plant Cell, Tissue and Organ Culture》2009,97(2):131-139
Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation
of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation
of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male
flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained
was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50 mg settled cell volume (SCV)
was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried
out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression
of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration
of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation
with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The
present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of
disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist
in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana. 相似文献
14.
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
15.
We have achieved high-frequency shoot regeneration in radish(Raphanus sativus). Cotyledon explants from four-day-old seedlings were suitable for the effective induction of shoots on Murashige and Skoog’s
(MS) medium containing 3.0 mg/L kinetin. We also determined that it was essential to include 1- to 2-ram petiole segments
with the cotyledons for efficient induction. When the regenerated shoots were transferred to an MS liquid medium containing
0.1 mg/L NAA, roots formed within four weeks, and normal plant development ensued. We established a transformation protocol
using anAgrobacterium binary vector that carries the GUS reporter gene. Preculturing the explants for I d in an MS medium containing 3.0 mg/L kinetin
also increased efficiency. Five days of cocultivation proved best for delivering T-DNA into radish. Transformation frequencies
of up to 52% were obtained in shoot induction media that contained 3.0 mg/L kinetin. 相似文献
16.
E. A. Popowich A. P. Firsov T. Y. Mitiouchkina V. L. Filipenya S. V. Dolgov V. N. Reshetnikov 《Plant Cell, Tissue and Organ Culture》2007,90(3):237-244
Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP
and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning
preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l−1 kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l−1 kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants
expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines
were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same
time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation. 相似文献
17.
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
18.
Shen-Jie Wu Hai-Hai Wang Fei-Fei Li Tian-Zi Chen Jie Zhang Yan-Jie Jiang Yezhang Ding Wang-Zhen Guo Tian-Zhen Zhang 《Plant Molecular Biology Reporter》2008,26(3):174-185
Agrobacterium-tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency
of coculture, selection cultivation, and plant regeneration. After 48-h cocultivation, the number of β-glucuronidase (GUS)-positive
calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse-granule
EC. It indicated that efficiency of transient transformation was affected by EC morphology. And transient transformation efficiency
was also improved by cocultivation on the medium adding 50 mg l−1 acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density was beneficial to production
of more kanamycin-resistant (Km-R) calli lines. From an original 0.3-g EC, an average of 20 Km-R calli lines were obtained
from a selection dish and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated
on the differentiation medium with dehydration treatments and the GUS-positive rate of regeneration plants was about 72.60%.
Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase
II gene and uidA. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly
with the low copy number, has been integrated into the cotton genome.
Shen-Jie Wu and Hai-Hai Wang should be considered as joint first authors 相似文献
19.
Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation 总被引:3,自引:0,他引:3
Estopà Montserrat Marfà V. Melé E. Messeguer J. 《Plant Cell, Tissue and Organ Culture》2001,65(3):211-220
The effect of several β-lactam antibiotics on shoot regeneration, growth and rooting of carnation (Dianthus caryophyllus L.), and their use in combination with kanamycin in Agrobacterium-mediated genetic transformation studies, was determined. Carbenicillin, cefotaxime and ticarcillin increased the regeneration
rate when added alone in non-inoculated explants; whereas, with inoculated explants, this effect was only observed in ticarcillin-containing
medium. Cefotaxime inhibited root growth in both transgenic and non-transgenic shoots. Rooting of non-transgenic shoots was
completely inhibited in all culture media containing kanamycin. The different antibiotics used, alone or in combination, did
not prevent the occurrence of false positive shoots, but it was possible to select transgenic shoots when rooting was induced
in a kanamycin + ticarcillin-containing medium. Regenerated transformed shoots were free of Agrobacterium after culturing in rooting medium, as was proven by the PCR analysis for the nptI gene, the antibiogram and the culture of tissue pieces of transgenic shoots on LB broth. The use of kanamycin and timentin
with or without carbenicillin, was very useful in the transformation procedure, for the elimination of Agrobacterium in regenerated shoots before their transfer to greenhouse conditions and also in the selection of transgenic versus false-positive
shoots.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
20.
Chen Y Lu L Deng W Yang X McAvoy R Zhao D Pei Y Luo K Duan H Smith W Thammina C Zheng X Ellis D Li Y 《Plant cell reports》2006,25(10):1043-1051
An in vitro plant regeneration method and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Euonymus alatus. More than 60% of cotyledon and 70% of hypocotyl sections from 10-day-old seedlings of E. alatus produced 2–4 shoots on woody plant medium (WPM) supplemented with 5.0 mg/l 6-benzylaminopurine (BA) plus 0.2 mg/l α-naphthalene acetic acid (NAA), and 77% of shoots produced roots on WPM medium with 0.3 mg/l NAA and 0.5 mg/l Indole-3-butyricacid (IBA). On infection with Agrobacterium tumefaciens strain EHA105 harboring a gusplus gene that contained a plant recognizable intron from the castor bean catalase gene to ensure plant-specific β-glucuronidase (GUS) expression, 16% of cotyledon and 15% of hypocotyl explants produced transgenic shoots using kanamycin as a selection agent, and 67% of these shoots rooted. Stable insertion of T-DNA into the host genome was determined with organ- and tissue-specific expression of the gusplus gene and further confirmed with a PCR-based molecular analysis. 相似文献