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1.
Success in plant genetic transformation depends on the efficiency of explant regeneration and transgene integration. Whereas
the former one depends on explant totipotency, the latter depends on the activity of host DNA repair and chromatin organisation
factors. We analyzed whether factors that result in an increase in recombination frequency can also increase transformation
efficiency. Here, we report that a threefold increase in the concentration of NH4NO3 in the growth medium results in more than a threefold increase in the Agrobacterium tumefaciens-mediated transformation frequency of Nicotiana tabacum plants. Regeneration of calli without selection showed that the increase in transformation frequency was primarily due to
the increase in transgene integration efficiency rather than in tissue regeneration efficiency. PCR analysis of insertion
sites showed a decrease in the frequency of truncations of the T-DNA right border and an increase on the left border. We hypothesize
that exposure to ammonium nitrate modifies the activity of host factors leading to higher frequency of transgene integrations
and possibly to the shift in the mechanism of transgene integrations.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
2.
An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical -glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis. 相似文献
3.
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
4.
Asad S Mukhtar Z Nazir F Hashmi JA Mansoor S Zafar Y Arshad M 《Molecular biotechnology》2008,40(2):161-169
A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for
cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were
treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and
selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic
callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene
copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1
AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is
first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate
fertile cotton transformants. 相似文献
5.
6.
Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.) 总被引:1,自引:0,他引:1
Nathalie Pawlicki Rajbir S. Sangwan Brigitte S. Sangwan-Norreel 《Plant Cell, Tissue and Organ Culture》1992,31(2):129-139
To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of transformation of petiole explants of the variety Nanco was greater than 45%. This procedure does not require acetosyringone or prolonged precultivation period. Using kanamycin (100 mg l-1) for selection, a large number of transgenic plantlets developed from the embryogenic calli within 8–10 weeks of culture on hormone-free medium. Transformation was confirmed by histochemical detection of -glucuronidase activity in the transformed cells, by the ability of petiole segments to produce embryogenic calli in presence of kanamycin, and by Southern hybridization analyses. 相似文献
7.
In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic
auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence
that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and
somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic
embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth
day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos
auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly
displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal
cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged
in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected. 相似文献
8.
AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at
27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L
and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic
embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR
amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus
far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants. 相似文献
9.
A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic
cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0–490 transgenic plants per
0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation
frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at
100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration
gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments
carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated
that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in
other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar,
and was at least 1 month faster for regenerating transgenic plants. 相似文献
10.
Leelavathi S Sunnichan VG Kumria R Vijaykanth GP Bhatnagar RK Reddy VS 《Plant cell reports》2004,22(7):465-470
A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3–6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.V.G. Sunnichan and R. Kumria contributed equally to this investigation 相似文献
11.
Antonio-José López-Pérez Leonardo Velasco María Pazos-Navarro Mercedes Dabauza 《Plant Cell, Tissue and Organ Culture》2008,94(2):189-199
Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless
grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD600) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression
of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher
with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation.
However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD600 being more effective for the transformation of Crimson Seedless and 0.2 OD600 for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone
and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration
of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot. 相似文献
12.
Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens 相似文献
13.
Ragnhild Lyngved Jenny Renaut Jean-François Hausman Tor-Henning Iversen Anne Kathrine Hvoslef-Eide 《Journal of Plant Growth Regulation》2008,27(4):353-369
Somatic embryogenesis can be used to produce artificial seeds of Cyclamen persicum, one of the most important ornamental plants for the European market, both as a potted plant in northern Europe and a bedding
plant in the cool winters in southern Europe. The aim of this study was to obtain new insights into the molecular biology
of somatic embryogenesis, which in turn can be useful for the improvement of tissue culture methodology. Total proteins were
characterized from two isogenic cell lines of Cyclamen persicum, one that was embryogenic and one that never has shown any embryogenic capacity. The extracted proteins were separated by
two-dimensional differential gel electrophoresis (2-D DIGE) and selected proteins were treated using the ETTAN Dalt Spot Handling
Workstation. Protein identification was performed using MALDI-TOF-MS. More than 1200 Cyclamen proteins were detected; 943 proteins were common to both lines. The different protein patterns of the embryogenic and non-embryogenic
cell lines were obvious: One hundred eight proteins were more abundant in the embryogenic cells, and 97 proteins in the non-embryogenic
cells. Among the differentially expressed proteins, 128 were identified. MALDI-TOF-MS analysis enabled 27 spots to be proposed
as candidates for embryo-specific proteins, as they were unique to the embryogenic cell line. The proteins identified are
involved in a variety of cellular processes, including cell proliferation, protein processing, signal transduction, stress
response, metabolism, and energy state, but the majority are involved in protein processing and metabolism. The main functions
of the putative embryo-specific proteins have been discussed in proportion to their role in the somatic embryogenesis process.
Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
R. Lyngved and J. Renaut contributed equally to this work. 相似文献
14.
Grégory Guirimand Vincent Burlat Audrey Oudin Arnaud Lanoue Benoit St-Pierre Vincent Courdavault 《Plant cell reports》2009,28(8):1215-1234
The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits
a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of
organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize
MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized
to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus
to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular
localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol
10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation
of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids,
i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement
with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H
is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both
necessary and sufficient to drive the ER anchoring.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
The influence of light regime, explant position and orientation on direct embryo formation from leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were investigated to optimize the protocol for regenerating of this orchid. When explants were cultured in light,
direct embryogenesis was retarded in both species. Embryos showed whitish to pale green in color and larger size than those
cultured in darkness. Furthermore, light regime induced explant browning, embryo necrosis and eventually low plantlet conversion
rate. Sixty days of culture in darkness is the most suitable duration for direct embryo induction. Explant orientation also
significantly affected direct embryo formation, and explants placed adaxial-side-up on culture medium had higher embryogenic
response than abaxial-side-up orientation. In both species, the cut end had highest embryogenic competence than other parts
of the explant. Moreover, when the leaf explant was cut transversely into two segments, the leaf basal segment had higher
embryogenic competence than the leaf tip segment. 相似文献
16.
Rubén álvarez José M. álvarez Jaime M. Humara ángeles Revilla Ricardo J. Ordás 《Biotechnology letters》2009,31(9):1477-1483
The bar gene was introduced into the cork oak genome. Cork oak embryogenic masses were transformed using the Agrobacterium strain AGL1 which carried the plasmid pBINUbiBar. This vector harbours the genes, nptII and bar, the latter under control of the maize ubiquitin promoter. The transgenic embryogenic lines were cryopreserved. Varying activities
of phosphinothricin acetyl transferase were detected among the lines, which carried 1–4 copies of the insert. Molecular and
biochemical assays confirmed the stability and expression of the transgenes 3 months after thawing the cultures. These results
demonstrate genetic engineering of herbicide tolerance in Quercus spp.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Rubén álvarez, Ricardo J. Ordás are contributed equally. 相似文献
17.
Réjean Desgagnés Serge Laberge Guy Allard Habib Khoudi Yves Castonguay Jacques Lapointe Réal Michaud Louis-P. Vézina 《Plant Cell, Tissue and Organ Culture》1995,42(2):129-140
Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 g ml-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity. 相似文献
18.
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
19.
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
20.
Samaj J Salaj T Matúsová R Salaj J Takác T Samajová O Volkmann D 《Plant cell reports》2008,27(2):221-229
Arabinogalactan proteins (AGPs) are important proteoglycans regulating somatic embryogenesis in diverse plant species. Embryogenic
cells of somatic embryos are covered by special extracellular cell wall layer called extracellular surface matrix network
(ECMSN) at their early developmental stages. Here we show that highly embryogenic cell line AC78 of hybrid fir (Abies alba × Abies cephalonica) differs from very low-embryogenic cell line AC77 in the abundance, subcellular localization and deposition of subset of
secreted AGPs. A specific AGP epitope containing Gal residues and reacting to Gal4 antibody is secreted and deposited into
ECMSN, which covers the surface of the embryogenic cells showing high embryogenic and regeneration capacity in the cell line
AC78. On the other hand, this Gal4 AGP epitope was not secreted and/or found on the surface of meristematic cells showing
low embryogenic and regeneration capacity in the cell line AC77, as well as on the surface of non-embryogenic suspensor cells
and callus cells in both cell lines AC77 and AC78. As a positive control, we have used another AGP epitope LM2 (containing
glucuronic acid) showing no significant differences in these two Abies hybrid lines. This study defines specific AGPs containing β-(1→6)-galactotetraosyl group as a first molecular component of
ECMSN covering embryogenic cells in gymnosperms.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献