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1.
Summary A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding
line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (gusA) reporter gene under the control of either the osmotin or proteinase inhibitor II gene promoter, and the npt II selectable marker gene. Transient uidA expression was visualized as 500–4000 blue units per 200 mg of bombarded cells 2 d after bombardment. Stably-transformed
calluses were recovered on both kanamycin and paromomycin media. The greatest number of GUS (+) calluses was obtained when
50 or 100 mgl−1 of kanamycin was applied 2 d after transformation for 3–5 wk, followed by either no selection or reduced levels of the antibiotic.
PCR analyses of the GUS (+) callus lines revealed the expected size fragment for uidA and npt II genes. Stable incorporation of the uidA gene into the genome was confirmed by Southern blot analyses. Several transformed embryos were detected by histochemical
β-glucuronidase (GUS) staining. 相似文献
2.
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
3.
Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotypes 总被引:5,自引:0,他引:5
Eighteen of 20 short-season soybean (Glycine max (L.) Merrill) genotypes (maturity group 0 and 00) screened for proliferative embryogenic capacity formed secondary globular
embryos, at rates of 1–70% of cultured immature cotyledons. Five genotypes produced embryogenic cultures which were proliferative
for at least 6 months. Proliferative embryogenic cultures of AC Colibri and X2650–7–2–3 were bombarded using a Bio-Rad PDS-1000/He
particle gun. Co-bombardments with plasmid pairs pHygr (encoding a type IV aminoglycoside phosphotransferase;aphIV) and pRD300pat (encoding a phosphinothricin N-acetyltransferase;pat) or pRD300pat and pFF19G (β-glucuronidase;uidA or gus) resulted, respectively, in 12 hygromycin-selected lines with multiple insertions of aphIV and pat, and two l-phosphinothricin-selected lines plus three β-glucuronidase-positive lines recovered without selection. Although
fertile plants were recovered from young proliferative cultures, transgenic plants, which were derived from cultures 12–14
months of age, were sterile.
Received: 8 January 1998 / Revision received: 12 January 1999 / Accepted: 12 July 1999 相似文献
4.
Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic
plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic
plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved.
The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines,
the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated
one or two copies of the uidA gene.
C. Gao and J. Liu contributed equally to the work. 相似文献
5.
Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean 总被引:4,自引:0,他引:4
Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have
constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the
coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean.
Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS
constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression
of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed. 相似文献
6.
Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic
embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated
Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension
cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented
with 0.1 mg l–1 picloram and 50 mg l–1 kanamycin sulfate for 2–4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for
2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium
(without picloram) followed by transfer to maturation medium with 100 mg l–1 kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction,
and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively.
Received: 2 June 1997 / Revision received: 26 September 1997 / Accepted: 11 October 1997 相似文献
7.
Embryogenic tissues of tea were cocultivated withAgrobacterium tumefaciens LBA4404. The plasmid pBi121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA) reporter gene, was used as binary vector. The highest transformation frequency (12 transformants/g fresh weight [FW] of
treated embryogenic tissue) was obtained with 5-day-old tissues grown in liquid medium and cocultivated withAgrobacterium for 2 d in the same medium but containing 50 μM acetosyringone. There was improvement in the recovery of kanamycin-resistant
tissues when tissues were first grown for 10 d on a medium containing 350 mg/L Timentin to prevent bacterial overgrowth, before
application of the selection pressure. Resistant tissues obtained after 6 wk on kanamycin-selection medium showed stableuidA expression. Polymerase chain reaction demonstrated the presence of the transgenes, while Southern hybridization confirmed
their integration into the genome. Transgenic plants were regenerated from transformed tissues within 4 mo after coculture. 相似文献
8.
U. K. S. Shekhawat T. R. Ganapathi L. Srinivas V. A. Bapat T. S. Rathore 《Plant Cell, Tissue and Organ Culture》2008,92(3):261-271
An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium
tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin
(5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase
in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed
suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg
of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent
media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed
embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first
report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album.
U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work. 相似文献
9.
Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz) 总被引:4,自引:0,他引:4
In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants. 相似文献
10.
Tissue derived from embryogenic suspension cultures of cassava was bombarded with microparticles coated with a plasmid containing theuidA gene, which codes for-glucuronidase (GUS). After 3 days, the effect of different bombardment parameters was evaluated by comparing the numbers of blue spots that resulted from histological GUS assays. Counting of blue spots was performed using a system comprised of a black and white video camera, a stereoscope and a personal computer. A reproducible counting method was established by optimizing GUS assay conditions, preparation of tissue samples and acquisition of video images in view of attaining the highest possible contrast between the blue spots and the surrounding tissue. The effects of bombardment pressure, microparticle size, number of bombardments, and osmotic pretreatment on GUS expression were investigated. Optimal transient expression of theuidA gene was observed after bombardment at 1100 psi, with a particle size of 1 µm, an osmotic pretreatment and two bombardments per sample. The highest number of blue spots observed was 2400 per square centimeter of bombarded tissue. 相似文献
11.
Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues 总被引:29,自引:0,他引:29
Highly regenerative tissues of tall fescue and red fescue produced from mature seed-derived embryogenic callus were induced
and proliferated on medium containing 2,4-dichlorophenoxyacetic acid (4.5 or 9.0 μM), 6-benzylaminopurine (0, 0.044, 0.44
or 2.2 μM) and cupric sulfate (0.1 or 5.0 μM) under dim-light conditions (10 to 30 μE m–2 s–1, 16 h light). Tall fescue tissues were transformed with three plasmids containing the genes for hygromycin phosphotransferase
(hpt), phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA;gus), and red fescue with three plasmids containing hpt, uidA and a synthetic green fluorescent protein gene [sgfp(S65T)]. DNA from T0 plants of eight independently transformed lines from tall fescue and 11 from red fescue were analyzed by PCR and DNA blot
hybridization. The co-expression frequency of all three transgenes [hpt/bar/uidA or hpt/uidA/sgfp(S65T)] in transgenic tall fescue and red fescue plants was 25–27%; for two transgenes [hpt/bar or hpt/uidA for tall fescue and hpt/uidA or hpt/sgfp(S65T) for red fescue], the co-expression frequency was 50–75%.
Received: 28 September 1999 / Revision received: 13 March 2000 / Accepted: 16 March 2000 相似文献
12.
High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures 总被引:6,自引:0,他引:6
K. Rajasekaran R. L. Hudspeth J. W. Cary D. M. Anderson T. E. Cleveland 《Plant cell reports》2000,19(6):539-545
Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and
stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment,
was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable
expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed
cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum
transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures
less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with
abnormal morphology and a high degree of sterility.
Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999 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.
Cerda Francisca Aquea Felipe Gebauer Marlene Medina Consuelo Arce-Johnson Patricio 《Plant Cell, Tissue and Organ Culture》2002,70(3):251-257
Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation. 相似文献
15.
A novel protocol, based on biolistics and regeneration via organogenesis, was developed for genetic transformation of cassava
(Manihot esculenta Crantz). The in vitro performance of cassava cultivars CMC40, MPer183 and MCol22 was evaluated, and the regeneration protocol was modified to improve
shoot production from explants for transformation experiments. Somatic cotyledons were used as a target tissue in the transformation
experiments using the Particle Inflow Gun and a plasmid containing the uidA gene in transient assays. The effect of different parameters for particle bombardment efficiency, including the amount of
DNA used, the flying distance of the projectiles and the pre- and post-plasmolysis time of the target tissue, was evaluated
and the conditions were partially optimised. Stably transformed cassava plants of cvs. MCol22 and TMS60444 were produced using
the partially optimised conditions and two different vector constructs carrying the hpt gene as the selectable marker. The selection protocol was optimised further, and a rooting test was developed for screening
the regenerants for antibiotic resistance to reduce the number of escapes obtained after primary selection. The production
of stably transformed cassava lines and the expression of the transgenes was verified by Southern blot analysis and RT-PCR.
Received: 10 December 1999 / Revision received: 12 January 2000 / Accepted: 7 February 2000 相似文献
16.
17.
A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba × A. cephalonica (cell lines AC2, AC78) and Abies alba × A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l−1). The T-DNA of plant transformation vector contained the β-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium
containing 10 mg geneticin l−1. GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of
embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been
obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings. 相似文献
18.
Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l−1 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l−1 hygromycin and 200 mg l−1 cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato. 相似文献
19.
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
20.
M.-J. Cho H. W. Choi B. B. Buchanan P. G. Lemaux 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(8):1253-1262
Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during
seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B1- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance
gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B1- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T0 leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T1 progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1
for GUS, one expressed bar alone, one lacked transmission of either gene to T1 progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T5 progeny in one line, T4 progeny in one line, T3 progeny in three lines and T2 or T1 progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous
lines the expression of the GUS protein, driven by either the B1- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T1 progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter
was gradually lost in T2 or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by
the hordein promoters in T2 or later generations. We conclude that the B1- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley
and potentially to modify barley seeds through genetic engineering.
Received: 28 May 1998 / Accepted: 19 December 1998 相似文献