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1.
Transformation of soybean via particle bombardment of embryogenic suspension culture tissue 总被引:18,自引:0,他引:18
John J. Finer Michael D. McMullen 《In vitro cellular & developmental biology. Plant》1991,27(4):175-182
Summary Embryogenic suspension culture tissue of soybean (Glycine max Merrill.) was bombarded with particles coated with plasmid DNAs encoding hygromycin resistance andβ-glucuronidase (GUS). One to two weeks after bombardment, embryogenic tissue was placed in a liquid proliferation medium containing
hygromycin. Four to six weeks after bombardment, lobes of yellow-green, hygromycin-resistant tissue, which began as outgrowths
on brown clumps of hygromycin-sensitive tissue, were isolated and cultured to give rise to clones of transgenic embryogenic
material. In vivo GUS assays of hygromycin-resistant clones showed that the early outgrowths could be negative, sectored,
or positive for GUS activity. Transgenic, fertile plants could be routinely produced from the proliferating transgenic embryogenic
clones. Southern hybridization analyses confirmed stable transformation and indicated that both copy number and integration
pattern of the introduced DNA varied among independently transformed clones. Hybridization analysis of DNA from progeny plants
showed genetic linkage of multiple copies of introduced DNA. An average of three transgenic clones were obtained per bombardment
making this procedure very suitable for transformation of soybean. 相似文献
2.
A protocol for consistent, large-scale production of fertile transgenic rice plants 总被引:10,自引:0,他引:10
A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment
of embryogenic tissues (Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines
carrying the hygromycin phosphotransferase (hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates,
when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure,
transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0
plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model
for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies.
Received: 12 March 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998 相似文献
3.
An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested. 相似文献
4.
Wenqi Cai Carol Gonsalves Paula Tennant Gustavo Fermin Manoel Souza Jr. Nonglak Sarindu Fuh-Jyh Jan Hai-Ying Zhu Dennis Gonsalves 《In vitro cellular & developmental biology. Plant》1999,35(1):61-69
Summary A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors
in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos,
followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of
kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media
containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants
from germination to the root development medium only after the explants had elongating root initials, had at least two green
true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat
protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic
embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents
an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated
this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars
with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should
be useful for the routine transformation and regeneration of papaya.
Based in part on a presentation at the 1997 SIVB Congress on In Vitro Biology held in Washington, DC, June 14–18, 1997. 相似文献
5.
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
6.
Yidong Ran Nicola Patron Qin Yu Suzan Georges John Mason German Spangenberg 《Plant Cell, Tissue and Organ Culture》2014,118(1):67-75
Lolium rigidum Gaud. is an annual grass grown for forage but also an economically damaging crop weed. A single genotype somatic embryogenic callus line, VLR1-60, was identified from a herbicide susceptible L. rigidum population, VLR1, and proved to be amenable to Agrobacterium tumefaciens-mediated transformation. Somatic embryogenic calli were continuously induced from the meristematic region of VLR1-60 plants multiplied in vitro and the basic tolerance level of VLR1-60 to hygromycin B was determined. A hygromycin phosphotransferase gene was used as a selectable marker for hygromycin B selection. Somatic embryogenic calli derived from in vitro grown vegetative tillers were co-cultivated with the A. tumefaciens strain EHA105 harbouring binary vector carrying reporter genes and selectable marker in the presence of acetosyringone for 3 days. Inoculated calli were recovered on callus proliferation medium containing Timentin? but lacking hygromycin and were then subcultured onto media with hygromycin concentrations increased progressively through time for selection of transformed plant cells. Putative transgenic plants were recovered and integration of transgenes was confirmed by Southern hybridization analysis and by detection of DsRed or GUS activity in transgenic plants. The frequency of plant transformation was 1.3 %. The ability to transform L. rigidum will provide opportunities for functional characterization of genes to improve forage quality and increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make L. rigidum a damaging crop weed. 相似文献
7.
8.
Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method 总被引:7,自引:0,他引:7
José L. Cabrera-Ponce Ariadne Vegas-Garcia Luis Herrera-Estrella 《Plant cell reports》1995,15(1-2):1-7
A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus
-glucuronidase
- NPTII
neomycin phophotransferase II
-
bar
phophinothricin acetyl transferase gene
- Pat
phosphinothricin acetyl transferase
- PPT
phosphinothricin
- Km
kanamycin
- 2,4-D
2,4-dichlorophenoxyacetic acid
- K
kinetin
- BAP
benzylaminopurine
- IBA
indolbutyric acid 相似文献
9.
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
10.
E. Corredoira M. C. San-José A. M. Vieitez A. Ballester 《Plant Cell, Tissue and Organ Culture》2007,91(3):281-288
The aim of the present work was to study the effect of the developmental stage of the somatic embryos and of the genotype
on the genetic transformation of embryogenic lines of European chestnut (Castanea sativa Mill.) and the cryopreservation of the embryogenic lines that are generated. As an initial source of explants in the transformation
experiments, it was found that the use of somatic embryos isolated in the globular stage or clumps of 2–3 embryos in globular/heart-shaped
stages was more effective (30%) than when embryos at the cotyledonary stage were used (6.7%). All of the seven genotypes tested
were transformed, and transformation efficiency was clearly genotype dependent. Three transgenic lines were successfully cryopreserved
using the vitrification procedure, and the stable integration of the uidA gene into the transgenic chestnut plants that were regenerated subsequent to cryopreservation was demonstrated. 相似文献
11.
Bo Yu Hong Zhai Yuping Wang Ning Zang Shaozhen He Qingchang Liu 《Plant Cell, Tissue and Organ Culture》2007,90(3):265-273
Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l−1 hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis.
β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed
that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic
plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed
in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in
quantity of independent events per transformation experiment in sweetpotato. 相似文献
12.
Transgenic radiata pine (Pinus radiata D. Don) plants containing a Bacillus thuringiensis (Bt) toxin gene, crylAc, were produced by means of biolistic transformation of embryogenic tissue. Using the selectable marker gene nptII and corresponding geneticin selection, 20 independent transgenic lines from five genotypes were established. Over 200 plants regenerated from ten transgenic lines were successfully transferred to soil. The integration and expression of the introduced genes in transgenic tissue and/or plants were confirmed by PCR, Southern hybridisation and neomycin phosphotransferase II (NPTII) and Bt ELISA assays. Bioassays with larvae of the painted apple moth, Teia anartoides, demonstrated that transgenic plants displayed variable levels of resistance to insect damage, with one transgenic line being highly resistant to feeding damage. 相似文献
13.
Whisker-mediated transformation of embryogenic callus of maize 总被引:4,自引:0,他引:4
The present study was designed to establish embryogenic callus as a target tissue for whisker-mediated transformation of
maize (Zea mays L.). Silicon carbide whiskers were used to deliver the bar and uidA (GUS) genes into embryogenic maize callus. Samples of osmotically-treated Type II callus were vigorously agitated in the
presence of whiskers and plasmid DNA using a standard laboratory vortex or a modified dental amalgamator. On average, three
transgenic callus lines were obtained per 100 samples treated. Plants were regenerated from several GUS-expressing callus
lines and DNA analyses confirmed stable integration and inheritance. As with other direct DNA delivery methods involving embryogenic
maize callus, integration patterns of the inserted DNA appeared to be complex. Although currently less efficient than microparticle
bombardment on a per target basis, whisker-mediated transformation of embryogenic callus represents a viable method for transgenic
maize production.
Received: 14 May 1999 / Revision received: 11 October 1999 / Accepted: 11 October 1999 相似文献
14.
Background
Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered. 相似文献15.
Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue 总被引:12,自引:0,他引:12
Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue.
Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension
cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were
inoculated with 1 ml of diluted (OD600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT.
When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic
tissue confirmed T-DNA integration.
Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997 相似文献
16.
Transgenic sugarcane plants via microprojectile bombardment 总被引:16,自引:1,他引:15
Transgenic sugarcane plants were produced by bombardment of embryogenic callus with high-velocity DNA-coated microprojectiles, followed by a selection and regeneration procedure designed for this target tissue. Optimal bombardment conditions for embryogenic callus required microprojectile velocities higher than those previously found effective for sugarcane suspension culture cells. Bombardment of target tissues twice increased the number of transiently expressing cells in regenerable callus regions, to more than 300 per treated plate. Stable transformants were obtained following bombardment with the neomycin phosphotransferase (npt-II) gene under the control of the Emu strong monocot promoter. Stepped increases in antibiotic concentration during selection and regeneration allowed recovery of actively growing callus and plants on media containing geneticin concentrations completely inhibitory to untransformed controls. NPT-II levels in transformed plants were 20–50 times the background levels in control plants in ELISA assays, and Southern analysis revealed integration of one to three copies of the introduced gene in the sugarcane genome. The procedures described yield one to three transgenic plants per treated plate within 16 weeks of bombardment and provide a simple, efficient and broadly applicable system for genetic transformation of sugarcane. A similar approach should be applicable to other members of the Poaceae able to form embryogenic callus. 相似文献
17.
Krystyna Klimaszewska Denis Lachance Gervais Pelletier Marie-Anne Lelu Armand Séguin 《In vitro cellular & developmental biology. Plant》2001,37(6):748-755
Summary Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation
procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed
by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic
plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos. 相似文献
18.
A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing #-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l-1 cefotaxime for 1 week followed by a medium containing 75 mg l-1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resistant (Hygr) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hygr cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 µM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 µM AS. Hygr embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies. 相似文献
19.
A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic
cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0–490 transgenic plants per
0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation
frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at
100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration
gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments
carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated
that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in
other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar,
and was at least 1 month faster for regenerating transgenic plants. 相似文献
20.
Wenting Liu Zongsuo Liang Changjuan Shan Frederic Marsolais Lining Tian 《In vitro cellular & developmental biology. Plant》2013,49(1):17-23
A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines. 相似文献