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1.
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
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Konstantin V. Kiselev Anna V. Turlenko Yuri N. Zhuravlev 《Plant Cell, Tissue and Organ Culture》2010,103(2):197-204
Calcium-dependent protein kinases (CDPKs) are proposed to play an essential role in plant defense responses. In this study,
we aimed to define the full sequence of a CDPK gene of Panax ginseng and analyze its expression in roots, leaves, and cell cultures of P. ginseng, one of the most valuable Chinese traditional medicinal herbs. We isolated the full-length cDNA of a P. ginseng CDPK gene, which was designated PgCDPK1a. PgCDPK1a shares high sequence identity at the amino acidic level with previously reported CDPK sequences for other plant species. We analyzed PgCDPK1a expression in the leaves of wild-growing P. ginseng plants, and in the roots and leaves of cultivated P. ginseng plants growing in an open experimental nursery at a natural ginseng habitat. PgCDPK1a was more actively expressed in the young leaves of cultivated P. ginseng plants than in that of wild-growing ones. Finally, we analyzed the expression of the gene in control GV and five rolC and rolB transgenic callus cultures of P. ginseng with different levels of fresh biomass accumulation, pathogen-related gene expression, and ginsenoside production. We observed
a strong positive correlation between fresh biomass accumulation of P. ginseng cell cultures and expression of the PgCDPK1a gene. There was a less clear negative correlation between the expression of pathogen-related genes and the content of ginsenosides
with the PgCDPK1a expression in cell cultures of P. ginseng. Perhaps, PgCDPK1a is involved in ginseng growth, as a positive regulator. 相似文献
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Fei-Fei Li Shen-Jie Wu Tian-Zi Chen Jie Zhang Hai-Hai Wang Wang-Zhen Guo Tian-Zhen Zhang 《Plant Cell, Tissue and Organ Culture》2009,97(3):225-235
Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes.
However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different
gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than
80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot
analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied
from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis
of T1 plants demonstrated that multiple transgenes were inherited and expressed in progenies.
Fei-Fei Li and Shen-Jie Wu are joint first authors. 相似文献
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Y. V. Inyushkina K. V. Kiselev V. P. Bulgakov Yu. N. Zhuravlev 《Biochemistry. Biokhimii?a》2009,74(8):917-924
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A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were
transformed in the presence of 100 μM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l−1 was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with
13.3 μM 6-benzylaminopurine, 4.5 μM thidiazuron, 50 mg l−1 adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls
were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse.
This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash. 相似文献
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Luciana Delgado-Benarroch Barry Causier Julia Weiss Marcos Egea-Cortines 《Planta》2009,229(6):1219-1229
Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is
perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in
organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified
the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation
is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general
and local gene networks.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
Y. Bao P. Dharmawardhana R. Arias M. B. Allen C. Ma Steven H. Strauss 《Plant cell reports》2009,28(6):947-962
We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs
to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common
in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems
as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were
placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary
growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response
regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events
recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended
to stimulate meristem development to promote clonal propagation and genetic transformation.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Michal Moyal Ben Zvi Amir Zuker Marianna Ovadis Elena Shklarman Hagit Ben-Meir Shamir Zenvirt Alexander Vainstein 《Molecular breeding : new strategies in plant improvement》2008,22(4):543-553
As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility
of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment
of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA3-pretreated mother plants and a two-step selection scheme. The GA3 treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated
explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing
shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila
was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes
rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could
be beneficial to the ornamental industry. 相似文献
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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
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Suk Young Oh Chun Hua Wu Elena Popova Eun Joo Hahn Kee Yoeup Paek 《Journal of Plant Biology》2009,52(4):348-354
We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm
root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently
observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation
when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root
tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated
cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ
between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors,
a mean value of 12.5 g dw L−1 was recorded for post-freeze-regenerated cultures versus 9.1 g dw L−1 for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation,
by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of
P. ginseng adventitious roots. 相似文献
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Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR)
tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay
showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving
the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation. 相似文献
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Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships
were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Cotton transgenics for resistance against cotton leaf curl disease using antisense movement protein gene (AV2) were developed in an Indian variety (F846) via Agrobacterium-mediated transformation using the protocol developed previously. A binary vector pPZP carrying the antisense AV2 (350 bp) gene along with the nptII gene was used. Transgenic nature of the putative transgenics was confirmed by molecular analysis. Shoots were induced on selection medium and subcultured on rooting medium containing IBA and 75 mg l–1 kanamycin. Transgenic plants were recovered in 12–16 weeks from the time of gene transfer to establishment in pots. Preliminary analysis of the field-established plantlets was conducted by PCR. T1 plants were obtained from T0 seeds, the presence of the AV2 and nptIIgenes in the transgenic plants was verified by PCR and integration of T-DNA with AV2 into the plant genome of putative transgenics was further confirmed by Southern blot analysis. Several T1 lines were maintained in the greenhouse. Progeny analysis of these plants by PCR analysis showed a classical Mendelian pattern of inheritance. 相似文献
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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