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1.
We report here an in planta method to produce transgenic Brassica napus plants. The procedure included Agrobacterium-mediated inoculation of plants at various development stages along with a vacuum infiltration step. The flowering stage appeared to be the most receptive stage for transformation and production of transgenic plants. In some cases, the flowering stage was induced either by cold treatment or by high density planting. Molecular and genetic analysis revealed that single and multiple copy events were generated and that the transgenes were transmitted to the T1 and T2 progeny in a Mendelian fashion.Abbreviations AFP Adult flowering plants - ELISA Enzyme linked immunosorbent assay - GS Germinating seedlings - GUS -Glucuronidase - ISFP Induced small flowering plants - MS Murashige and Skoog - PPO Protoporphyrinogen oxidase - TE Tris-EDTA buffer - YEP Yeast extract-peptone mediumCommunicated by W.A. Parrott 相似文献
2.
An early flowering mutant plant of Eucalyptus grandis with normal vegetative growth was found in a nursery in northern Brazil. This mutant plant flowers at approximately 90 days
from germination. A cross between a wild-type (normal flowering) tree and the mutant was carried out, generating a progeny
of 88 individuals where early flowering segregated in an approximate 1:1 ratio. A genome scan with 100 microsatellite markers
distributed across the genome was carried out using bulk segregant analysis (BSA) on two contrasting bulks of 15 plants each.
Linkages (LOD>3.0) with a major effect early flowering quantitative trait locus (QTL) were detected and confirmed by a full
scale cosegregation analysis for markers EMBRA27, EMBRA60, EMBRA164, EMBRA158, EMBRA91, and EMBRA65. A localized linkage map
involving the six loci and the early flowering QTL named Eucalyptus early flowering 1 (Eef1) was constructed belonging to linkage group #2 in the existing microsatellite reference map. The Eef1 locus was mapped between markers EMBRA27 and EMBRA164, with distances of 21.8 and 6.4 cM, respectively. In introgression
experiments, these two markers could be successfully used with an expected precision of 98% to select plants carrying the
Eef1 mutant allele, assuming no recombination interference in the genomic segment. Early flowering could be a very useful trait
both in breeding as well as experimental genetics of Eucalyptus. 相似文献
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<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>) 总被引:1,自引:1,他引:0
Byoung-Kyu?Lee Seung-Hee?Yu Yul-Ho?Kim Byung-Ohg?Ahn Han-Sun?Hur Sang-Chul?Lee Zhanyuan?Zhang Jang-Yong?Lee
Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l−1 phosphinothricin (PPT) and 150 mg l−1 kanamycin, respectively, for shoot induction and 1 mg l−1 PPT and 125 mg l−1 kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T0 and T1 plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T0 and T1 transgenic plants. 相似文献
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Olga A. Shulga Tatyana Yu Mitiouchkina Anna V. Shchennikova Konstantin G. Skryabin Sergey V. Dolgov 《In vitro cellular & developmental biology. Plant》2011,47(5):553-560
Chrysanthemum is one of the most important commercial cut flowers in the world. Early-flowering cultivars are required to
produce quality chrysanthemum flowers with a lower cost of production. To shorten the vegetative growth phase of chrysanthemum,
three AP1-like genes from Asteraceae were constitutively overexpressed in 80 independent transgenic chrysanthemum lines. All lines were characterized by PCR and
RT-PCR and demonstrated that overexpression of compositae AP1-homologs in transgenic chrysanthemum under long-day conditions had no effect on plant development compared to non-transgenic
controls. Conversely, under short-day conditions, transgenic plants commenced bud initiation 2 wk earlier than non-transgenic
chrysanthemum plants. Subsequently, transgenic chrysanthemum flowers showed color earlier and resulted in full opening of
inflorescences 3 wk prior to non-transgenic control plants. These results open new possibilities for genetic improvement and
breeding of chrysanthemum cultivars. 相似文献
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Szucs P Skinner JS Karsai I Cuesta-Marcos A Haggard KG Corey AE Chen TH Hayes PM 《Molecular genetics and genomics : MGG》2007,277(3):249-261
The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis,
we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F2 progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the
two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny
with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F2 populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model
explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive
genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter
vs spring type” alleles at the VRN-H loci. 相似文献
9.
Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R1 transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants. 相似文献
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Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification
of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic
plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis
thaliana
AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type
plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants
exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations
of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those
of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin
synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does
not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants. 相似文献
12.
The role of cytokinins in the promotion of flowering in the endangered species Kniphofia leucocephala Baijnath. was investigated using shoots maintained in culture for 3 years. The highest percentage flowering (65%) was obtained on media containing 20 μM benzyladenine (BA). The inclusion of isopentenyladenine and zeatin in the media also resulted in flowering, but these treatments were less effective than BA in inducing flowering. The effect of cytokinins on flowering was dose-dependent, with high concentrations of BA inhibiting flower formation. Treatments that resulted in rooting of explants produced no flowers. The resulting inflorescences in all treatments did not mature and senesced prematurely, even when gibberellic acid (GA3) was applied post-flower-emergence. 相似文献
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This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three
cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation
of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and
from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79%
of the selected plants proved to be transgenic; however, only 14.3% of the T0 plants and 27.5% of the T1 showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene
expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined
with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T0 plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic
and non-transgenic individuals. Southern blot analysis of T0 and T1 showed simple integration pattern with the low copy number of the introduced transgenes. 相似文献
15.
Porphyra katadae Miura var. hemiphylla Tseng et T. J. Chang, a species distributed around the Liaodong and Shandong Peninsulas of China, produces gametophytes from late winter to early spring. These are monoecious with male and female reproductive tissues in distinct halves or sectors. Vegetative tissues from sectors expected to differentiate into sexual tissue were cultured in the laboratory. Male and female reproductive organs, as well as conchocelis and blades, were differentiated from these tissues. The male and female reproductive tissues were in patches and mixed on the cultured tissue pieces. This was quite different from the wild-type sectored individuals. The F1 conchospore germlings also produced monospores, carposporangia, spermatangia and conchocelis. These carposporangia and spermatangia were in patches and were mixed on the F1 fronds. The results imply that P. katadae var. hemiphylla is possibly sex-differentiated rather than sex-determined. This is the first report of such a dimorphic life history in the genus Porphyra. 相似文献
16.
Tasawar Sultana Farah Deeba Farah Naz Ray J. Rose S. M. Saqlan Naqvi 《Acta Physiologiae Plantarum》2016,38(11):255
To evaluate the effectiveness of a germin-like protein (GLP) in legumes against the serious soil-borne pathogen Fusarium oxysporum f. sp. lentis, an Oryza sativa root-expressed GLP (OsRGLP1) was expressed in the model legume Medicago truncatula using the recombinant vector pCOsRGLP1. The transgene was highly expressed in M. truncatula transformed lines as assessed by RT-qPCR. Consistent with the active status of the transgene there was an elevated accumulation of H2O2 in transformed progeny. Enzymatic characterization of T1 transgenic progeny showed increased superoxide dismutase (SOD) activity. The additional SOD activity in transgenic lines was insensitive to potassium cyanide and sensitive to H2O2 indicating its resemblance to FeSOD. The effectiveness of the OsRGLP1 gene was tested by monitoring the root disease after infection of wild-type and transgenic lines. Wild-type plants were greatly affected by the pathogen infection showing a percent disease index value of 50 compared to 10–18 for the transgenic lines. The tolerance of the transgenic lines leads to recovery in fresh weight and pod production to an almost normal level. Analysis of defense-related genes downstream of hydrogen peroxide (H2O2) in transgenic plants showed induction of salicylic acid and jasmonate signaling pathways and increased expression of some pathogenesis-related-1 (PR-1) genes and a plant defensin gene. Overall, the findings suggest that OsRGLP1 provides protection against the fungal pathogen F. oxysporum that may involve the direct influence of H2O2 on signaling pathways leading to the activation of defense-related genes. 相似文献
17.
Qi Zhu Fengtao Wu Feng Ding Dong Ye Yongqin Chen Yi Li Yang Zhifan 《Plant Cell, Tissue and Organ Culture》2009,96(3):317-324
Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important
steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and
female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive
to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation
medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three
days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for
formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh
selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of
the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to
produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated
from initial infected callus explants. 相似文献
18.
Indrajit Dutta Prasenjit Saha Sampa Das 《In vitro cellular & developmental biology. Plant》2008,44(5):401-411
Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige
and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver
nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency
of stable transformation was found to be approximately 19% in the T
0 generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern
blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of
T
1 seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency
over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea. 相似文献
19.
Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated
as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression
profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level
in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one
to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that
the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly
improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure
analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant
transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes.
Rugang Chen and Hanxia Li are contributed equally to this work. 相似文献
20.
To ensure that the initiation of flowering occurs at the correct time of year, plants need to integrate a diverse range of
external and internal signals. In Arabidopsis, the photoperiodic flowering pathway is controlled by a set of regulators that include CONSTANS (CO). In addition, Arabidopsis plants also have a family of genes with homologies to CO known as CO-LIKE (COL) about which relatively little is known. In this paper, we describe the regulation and interactions of a novel member of
the family, COL5. The expression of COL5 is under circadian and diurnal regulation, but COL5 itself does not appear to affect circadian rhythms. COL5, like CO, is regulated by GIGANTEA. Furthermore, COL5 is expressed in the vascular tissue. Using COL5 over-expressing lines we show that, under short days, constitutive expression of COL5 affects flowering time and the expression of the floral integrator genes, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO 1. Constitutive expression of COL5 partially suppresses the late flowering phenotype of the co-mutant plants. However, plants with loss of COL5 function do not show altered flowering. Taken together, our results suggest that COL5 has COL activity, but may either not
have a role in regulating flowering in wild-type plants or may act redundantly with other flowering regulators.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献