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1.
Yancheva SD Shlizerman LA Golubowicz S Yabloviz Z Perl A Hanania U Flaishman MA 《Plant cell reports》2006,25(3):183-189
An efficient and reproducible system for Agrobacterium-mediated transformation of the pear (Pyrus communis L.) cultivar Spadona was developed. Leaf explants of in vitro propagated plants were cocultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504, carrying the uidA-intron and nptII genes. Under selective conditions, 5% of the plantlets regenerated and were positively stained for GUS. However, most of
the GUS-positive plants re-callused and subsequently died, leaving only 0.3–0.8% of these plantlets to reach maturity. In
order to identify transformed shoots at early stages of regeneration, we introduced the green fluorescent protein (GFP) into
the pear cultivar Spadona using the plasmid PZP carrying the nuclear-targeted GFP and nptII genes. High expression levels of GFP were detected in transgenic cells as early as 7 days after transformation. GFP marked-callii
and transformed plants were observed after 14 and 24 days, respectively. Fluorescence microscopy screening of transformed
plant material, under the selection of kanamycin, increased the transformation frequency to 3.0–4.0%. We conclude that the
introduction of GFP improves the selection of transformed plants of Spadona pear. 相似文献
2.
Summary Stable genetic transformation of embryogenic cultures of Abies nordmanniana (Nordmann fir or Caucasian fir) was achieved using the Biolistic? transformation technology, followed by regeneration of transgenic plants. Selection of the transgenic tissue was based on
the antibiotic resistance induced by the neomycin phosphotransferase II gene (npt II), in combination with the antibiotic geneticin. Six transclones were recovered from a total of 215 bombardments. Expression
of the β-glucuronidase gene (uidA) was confirmed by histochemical analysis, and expression of npt II was verified by quantification of NPTII protein by enzyme linked immunosorbent assay (ELISA). Both genes were still expressed
in the embryogenic tissue after 5 yr of in vitro culture and in mature somatic embryos and plants produced from these cultures. The integration of npt II was confirmed by Southern hybridization in embryogenic tissue after 5 yr of culture. After 5 yr of growth, uidA was still expressed in needles from the transformed trees. 相似文献
3.
C. Walter L. J. Grace A. Wagner D. W. R. White A. R. Walden S. S. Donaldson H. Hinton R. C. Gardner D. R. Smith 《Plant cell reports》1998,17(6-7):460-468
A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered
was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature
somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both
genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones.
Received: 9 May 1997 / Revision received: 18 September 1997 / Accepted: 18 October 1997 相似文献
4.
M. Sujatha S. Vijay S. Vasavi P. Veera Reddy S. Chander Rao 《Plant Cell, Tissue and Organ Culture》2012,110(2):275-287
Lack of a genotype-independent transformation protocol for sunflower is a major bottleneck in improving this important oilseed crop. An efficient Agrobacterium-mediated transformation protocol is described, which was adaptable across a broad range of sunflower genotypes. The improved transformation approach used cotyledons from mature seeds vertically split through the embryo axis. The LBA 4404 Agrobacterium strain was used, which carried pCAMBIA 2301 plasmid containing UidA as the reporter gene and nptII as the selectable marker for transformants on kanamycin. Bacterial titer, cotyledon type, acetosyringone concentration and vacuum application enhanced the transformation efficiency. Wounding, enzyme pretreatment and sonication significantly reduced the transformation frequency. Putative transgenic shoots were obtained through both axillary proliferation and adventitious shoot regeneration. Following two and three cycles of selection on kanamycin for axillary and adventitious shoots, respectively, putative transformed shoots were obtained at an average frequency of 3.0?%. Reporter gene histochemical assay and molecular characterization of primary and T1 transgenic plants revealed stable transgene integration, expression and monogenic inheritance. The standardized procedure was tested on 28 genotypes comprising sets of inbred, maintainer, restorer and hybrid lines. Transformation was successful in all genotypes albeit with variable frequency in all except the hybrid lines wherein it was stable around 4.0?%. The procedure opens possibilities of directly improving any commercial genotypes of sunflower. 相似文献
5.
Key message
The development of transgenic citrus plants by the biolistic method.Abstract
A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.6.
Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture
originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical
meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature
axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured.
These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the
apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All
five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure.
The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively.
There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either
the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination
of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material
available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane. 相似文献
7.
An effective method has been developed for the stable transformation and regeneration of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' by microprojectile bombardment. Embryogenic cell suspensions were initiated using immature
male flowers as the explant. Cells were co-bombarded with the neomycin phosphotransferase (nptII) selectable marker gene under the control of a banana bunchy top virus (BBTV) promoter or the CaMV 35S promoter, and either
the β-glucuronidase (uidA) reporter gene or BBTV genes under the control of the maize polyubiquitin promoter. Plants were regenerated, under selection
with kanamycin, that were co-transformed with nptII and either the uidA or BBTV genes. Molecular characterisation of transformants demonstrated that the transgenes had been stably integrated into
the banana genome.
Received: 22 June 1998 / Revision received: 29 March 1999 / Accepted 1 May 1999 相似文献
8.
The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l−1. Resistant embryogenic tissue recovery required 10–12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained. 相似文献
9.
Summary Linseed flax (Linum usitatissimum L.) was transformed by bombarding hypocotyl tissues with gold particles coated with plasmid DNA carrying the β-glucuronidase
(GUS) (uid-A) and neomycin phosphotransferase II (npt-II) genes. Transient expression of the introduced β-glucuronidase gene was used to study factors influencing the DNA delivery,
while progeny analyses confirmed stable transformation. The efficiency of DNA delivery, uptake and expression was significantly
affected by the duration of hypocotyl preculture, bombardment distances, the level of chamber vacuum, the quantity of DNA,
and the size of particles. Nineteen independent GUS-positive shoots were recovered and regenerated into whole plants, from
which 10 plants successfully produced viable seeds. Analysis of T1 and T2 self pollinated progeny for histochemical and fluorometric GUS assays and polymerase chain reaction (PCR) analyses for uid-A, plus npt-II PCR and germination assays in progeny plants demonstrated that the transgenes were expressed in selected plants and transmitted
to progeny, usually via a single Mendelian locus. The results show that particle bombardment can be used to produce transgenic
Linum plants. The system is rapid, simple and offers an alternative to Agrobacterium methods. 相似文献
10.
A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with
five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4–10 mg) were inoculated with EHA105:p35SGUSINT
bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed
masses appeared after 20–30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern
blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old
tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic
plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed
somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens
and/or biotic stresses. 相似文献
11.
Wenhao Dai Zong-Ming Cheng Wayne Sargent 《In vitro cellular & developmental biology. Plant》2003,39(1):6-11
Summary An efficient regeneration and transformation system was developed for two elite aspen hybrid clones (Populus canescens × P. grandidentada and P. tremuloides × P. davidiana). Callus was induced from in vitro leaf explants on modified Murashige and Skoog medium (MSA) and woody plant medium (WPM) containing four different combinations
of cytokinins and auxins. Callus tissues regenerated into shoots on WPM medium supplemented with 2.0 mgl−1 (9.12 μM) zeatin or 0.01 mgl−1 (0.045 μM) thidiazuron. P. canescens × P. grandidentata exhibited the higher callus and shoot production. In vitro leaf explants from the two hybrid clones were cocultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uidA gene encoding for β-glucuronidase (GUS) and the npt II gene encoding for neomycin phosphotransferase II. Transformation was confirmed by GUS assays, polymerase chain reaction,
and Southern blot analyses. Agrobacterium concentration, acetosyringone, and pH of the cocultivation medium were evaluated for enhancing transformation efficiency
with the clone P. canescens × P. grandidentata. 相似文献
12.
Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 °C on kanamycin free shoot
regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm−3 kanamycin and 300 mg dm−3 cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm−3 kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl
explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival
rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on
transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved. 相似文献
13.
Silvia Flores-Benítez Juan F. Jiménez-Bremont Sergio Rosales-Mendoza Gerardo R. Argüello-Astorga Rosalba Castillo-Collazo Ángel Gabriel Alpuche-Solís 《Plant Cell, Tissue and Organ Culture》2007,91(3):215-224
Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and
embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants
was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving
a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive
with the GUS assay after 14 months on selective medium while still undergoing regeneration. 相似文献
14.
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
15.
Anber Hassanein Latifa Hamama Karine Loridon Noëlle Dorion 《Plant cell reports》2009,28(10):1521-1530
Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses
from a 33-μF capacitor in a 250-V cm−1 electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient
expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast
suspension conductivity of >1,500 μS cm−1 allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing
uidA and nptII genes. When selection (50 mg l−1 kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l−1 kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant
rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated
plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones. 相似文献
16.
Pramila Shah N. K. Singh Neeraj Khare Meenal Rathore S. Anandhan M. Arif Rupesh Kumar Singh S. C. Das Z. Ahmed Narendra Kumar 《Plant Cell, Tissue and Organ Culture》2008,95(3):363-371
Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore,
experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation.
6-benzylaminopurine at 0.05 mg l−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets
in indole-3-aceticacid 0.5 mg l−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after
co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by
polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent. 相似文献
17.
Taskin Kemal Melik Turgut Kenan Gulhan Ercan A. Scott Rod J. 《Plant Cell, Tissue and Organ Culture》2003,72(2):173-180
An efficient method for adventitious shoot regeneration for Arabis drummondii and a transformation protocol for A. gunnisoniana from hypocotyl explants are described. Hypocotyl explants from 7-day-old aseptically grown seedlings were cultured on MS medium containing plant growth regulators (6-benzylaminopurine, 1-phelyl-3- (1,2,3-thiadiazol-5-yl) urea, -naphthaleneacetic acid and 2,4-dichlorophenoxy-acetic acid). After 4 weeks in culture, high frequency of adventitious shoot regeneration was observed. Regenerated shoots were rooted on half-strength MS basal medium supplemented 1% (w/v) sucrose, with or without NAA. This protocol was then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS 21 medium supplemented with 50 mg l kanamycin. PCR analysis verified the presence of the nptII gene in the plant DNA isolated from kanamycin resistant shoots. 相似文献
18.
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
19.
Cryopreservation for eradication of Jujube witches' broom phytoplasma from Chinese jujube (Ziziphus jujuba)
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R.R. Wang H.Q. Mou X.X. Gao L. Chen M.F. Li Q.C. Wang 《The Annals of applied biology》2015,166(2):218-228
Here, we report efficient eradication of Jujube witches' broom phytoplasma (Candidatus Phytoplasma ziziphi) from Chinese jujube (Ziziphus jujuba) by cryopreservation. Shoot tips (1.0 mm in size) with 5–6 leaf primordia (LPs) excised from diseased in vitro stock shoots were subject to droplet‐vitrification cryopreservation. Shoot tips following cryopreservation were post‐cultured on a recovery medium for survival. Plantlet regeneration was obtained by micrografting of surviving shoot tips upon in vitro rootstocks. With this protocol, 85% of shoot tips survived following cryopreservation, among which 75% regenerated into whole plantlets and all of them were free of phytoplasma, regardless of the sizes used for cryopreservation. Ultrastructural studies demonstrated that phytoplasma was absent in the apical dome, and leaf primordia (LPs) 1 and 2, while abundance of phytoplasma was present in the lower parts of shoot tips, leaf primordium 3 and older tissues. Histological observations showed that much more damage was found in cells located in the lower part of apical dome, leaf primordium 3 and older tissues than in those at the upper part of apical dome and in the LPs 1 and 2. These cells were most likely to survive and regenerate into phytoplasma‐free plantlets following cryopreservation and micrografting. Ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreservation followed by micrografting. Results reported here would provide technical support for production of phytoplasma‐free plants and for long‐term storage of germplasm of Chinese jujube. 相似文献
20.
E. Firoozabady Y. Moy W. Tucker K. Robinson N. Gutterson 《Molecular breeding : new strategies in plant improvement》1995,1(3):283-293
We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis. 相似文献