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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.
Eliane R. Santarém John J. Finer 《In vitro cellular & developmental biology. Plant》1999,35(6):451-455
Summary Transgenic soybean can be efficiently produced by particle bombardment of embryogenic suspension culture material. Unfortunately,
the time required to obtain a transformation-competent soybean suspension culture line is often lengthy and can result in
reduced fertility of regenerated plants. In addition, establishment and maintenance of embryogenic suspension cultures can
be very difficult. The objective of this work was to minimize the time required to obtain transformation-competent embryogenic
tissue and optimize DNA delivery into that tissue. Somatic embryos were induced from immature cotyledons of soybean [Glycine max (L.) Merrill cv ‘Jack’] by placement of cotyledons, adaxial side up, on a MS-based induction medium containing 40 mg (181
μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1 and 6% sucrose. Embryogenic tissues, which formed from the surface of the cotyledons
within 2–4 wk, were transferred to an embryo proliferation medium containing 20 mg (90 μM) 2,4-D per 1 and 3% sucrose. After 4 wk, proliferative embryogenic tissue could be used for transformation via particle bombardment.
Desiccation of target tissue, period of subculture prior to bombardment, and the number of bombardments per target tissue
were evaluated for enhancement of transient β-glucuronidase (GUS) expression. The highest number of blue foci was observed
when the target tissue was desiccated for 10 min in an uncovered Petri plate containing proliferation medium, subcultured
on the same day of bombardment, and bombarded three times on a single day. For stable transformation, selection was started
20 d after bombardment using 9 mg hygromycin per 1 for 4 wk, and 18 mg per 1 thereafter. Stably transformed clones were obtained
from tissue bombarded once and twice on a single day. GUS assays and Southern hybridization analysis of DNA from putative
clones confirmed stable integration of the introduced genes. Fertile transgenic plants were obtained in 11–12 mo following
culture initiation. 相似文献
3.
Stacy Winfield Rodrick Lawton Henry Daniell Sarwan K. Dhir 《In vitro cellular & developmental biology. Plant》2001,37(5):648-653
Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from
leaf segments for particle bombardment experiments. A buffer solution containing 1×106 protoplasts ml−1 was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm−1. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell
walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells
continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed
after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024
cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were
observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing
sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene
expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation
efficiency and obtain transgenic sweet potato plants. 相似文献
4.
Z. T. Li S. Dhekney M. Dutt M. van Aman J. Tattersall K. T. Kelley D. J. Gray 《In vitro cellular & developmental biology. Plant》2006,42(3):220-227
Summary A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes
was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single
promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating
potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S)
promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic
plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos,
and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation
parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl−1 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments
did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic
lines than did the control. A 48h co-cultivation period combined with 75 mgl−1 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome:
63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized
procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole
plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr. 相似文献
5.
A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues
for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance
was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM)
and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l−1 in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of
transgenic cells, compared to 50 or 75 mg l−1, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered
after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization.
Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots,
inflorescences and the embryo and endosperm of developing berries. 相似文献
6.
Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants 总被引:11,自引:0,他引:11
R. Ghorbel J. Juárez L. Navarro L. Peña 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(1-2):350-358
The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration
of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital
marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed
from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus,
most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium
is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy
discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating
the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots
have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors
and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which
is currently a topic of much discussion for the commercialization of transgenic plants.
Received: 28 October 1998 / Accepted: 28 November 1998 相似文献
7.
Roderick Lawton Stacy Winfield Hanery Daniell Ajmer S. Bhagsari Sarwan K. Dhir 《Plant Molecular Biology Reporter》2000,18(2):139-139
Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was
analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated
mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks
of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing
GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic
calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato
tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection
of transformed sweet potato plants. 相似文献
8.
A soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) polyubiquitin promoter gives strong constitutive expression in transgenic soybean 总被引:1,自引:0,他引:1
Carlos M. Hernandez-Garcia Adriana P. Martinelli Robert A. Bouchard John J. Finer 《Plant cell reports》2009,28(5):837-849
The success of plant genetic transformation relies greatly on the strength and specificity of the promoters used to drive
genes of interest. In this study, we analyzed gfp gene expression mediated by a polyubiquitin promoter (Gmubi) from soybean (Glycine max) in stably transformed soybean tissues. Strong GFP expression was observed in stably transformed proliferative embryogenic
tissues. In whole transgenic plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules
in young plants as well as in leaf veins, petioles, flower petals, pollen, pods and developing seeds in mature plants. GFP
expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. Introduction of an intron-less
version of the Gmubi promoter (Gmupri) displayed almost the same GFP expression pattern albeit at lower intensities. The Gmubi
promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression
in soybean. 相似文献
9.
Summary Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following
germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 μM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 μM) kinetin per 1, 1 mg of both 2,4-D (4.5 μM) and kinetin (4.7 μM per 1, or 2 mg of both 2,4-D (9.1 μM) and kinetin (9.3 μM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and
kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in
liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were
inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 μM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ
per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant
tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment,
four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic
via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos
occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred
to fresh medium for germination. 相似文献
10.
We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin
for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic
embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7
d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in
regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L
giberellic acid (GA3). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern
hybridization to confirm gene integration. 相似文献
11.
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
12.
Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent
cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with
emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region
of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis
on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm
in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation
period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants
or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An
optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl−1 hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl−1 hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature
cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols
for transformation of soybean should be established under both Agrobacterium and selection conditions. 相似文献
13.
An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene.
In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5′ flanking regions of the Japanese
flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence
protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a
result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement
component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills.
Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and
heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide,
GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns
in transgenic zebrafish. 相似文献
14.
H. A. Richards V. A. Rudas H. Sun J. K. McDaniel Z. Tomaszewski B. V. Conger 《Plant cell reports》2001,20(1):48-54
A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived
embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from
the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l–1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red
to yellow. Basta tolerance was observed in T1 plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene.
Received: 11 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000 相似文献
15.
Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.) 总被引:4,自引:0,他引:4
Zhang CL Chen DF McCormac AC Scott NW Elliott MC Slater A 《Molecular biotechnology》2001,17(2):109-117
Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely
upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic
plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that
was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged
that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes”
and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance
genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for
enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet
via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several
days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained
at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic
calli were regularly monitored under the microscope with blue light when they were transferred to media without selective
agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has
potential for the development of selectable marker-free transgenic sugar beet. 相似文献
16.
Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe). 相似文献
17.
M. Ashfaq Farooqui A. V. Rao T. Jayasree A. Sadanandam 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,95(4):702-705
The effects of atrazine on cotyledon cultures of Solanum melongena were investigated with a view to establishing a system for in vitro selection of resistant mutants. At herbicide levels producing
little growth inhibition some chlorophyll loss occurred associated with the production of albino shoots. At 15 mg/l bleaching
was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated
with severe suppression of growth. Mutagenesis with EMS resulted in herbicide-resistant mutants based on the embryogenic ability
of mutagenised explants placed on medium containing selective levels of sucrose (0.2%) and atrazine (15 mg/l). Different morphogenetic
responses were observed when the levels of sucrose (0.2–5%) were altered. Somatic embryogenesis was observed at low sucrose
concentrations (0.2–0.5%). Both embryogenesis and shoot regeneration occurred in 1% sucrose. Shoot regeneration was maximum
in 2% sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3–5%). However, lowering
of sucrose concentration from 2% to 0.2% caused complete bleaching, permitting the selection of herbicide-resistant mutants.
Received: 26 November 1996 / Accepted: 20 December 1996 相似文献
18.
19.
Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids. 相似文献
20.
Y. Y. Wu Q. J. Chen X. H. Cui H. Chen J. Chen X. C. Wang 《Russian Journal of Plant Physiology》2007,54(4):524-529
We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were
tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest
callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine.
Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized
regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin,
a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques,
we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results
indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and
transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596.
The text was submitted by the authors in English. 相似文献