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1.
Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue 总被引:12,自引:0,他引:12
Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue.
Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension
cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were
inoculated with 1 ml of diluted (OD600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT.
When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic
tissue confirmed T-DNA integration.
Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997 相似文献
2.
Agrobacterium‐mediated transformation of Vitis Cv. Monastrell suspension‐cultured cells: Determination of critical parameters
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Mingyu Chu Carmen Quiñonero Hülya Akdemir Nuria Alburquerque María Ángeles Pedreño Lorenzo Burgos 《Biotechnology progress》2016,32(3):725-734
Although some works have explored the transformation of differentiated, embryogenic suspension‐cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication‐assisted Agrobacterium‐mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp‐expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725–734, 2016 相似文献
3.
Factors affecting soybean cotyledonary node transformation 总被引:21,自引:0,他引:21
Cotyledonary node transformation efficiency was evaluated using a sonication assisted Agrobacterium-mediated transformation (SAAT) protocol, three dissimilar A. tumefaciens strains, and explants derived from 28 diverse cultivars and/or genotypes of soybean [Glycine max (L.) Merr.]. The explants were evaluated at 10 and 45 days after co-cultivation for transformation with a binary vector containing
both a GUS-intron gene and an NPTII selectable marker. The best overall strain of A. tumefaciens was determined to be KYRT1 based on stable GUS transformation of soybean cotyledonary node explants measured at the terminal
45 day evaluation point. SAAT did not increase stable transformation at 45 days post co-cultivation. SAAT was determined to
significantly decrease shoot proliferation of some genotypes, but it is unclear what effect this may have on the recovery
of transformed shoots. Significant differences were also detected between genotypes for transformation and shoot proliferation
frequency.
Received: 9 March 1998 / Revision received: 9 July 1998 / Accepted: 28 July 1998 相似文献
4.
5.
Martina Beranová Slavomír Rakouský Zuzana Vávrová Tomáš Skalický 《Plant Cell, Tissue and Organ Culture》2008,94(3):253-259
A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and
cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO4 solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus
(35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium
to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of
microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used
to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria
elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound
facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends
on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency
in flax. 相似文献
6.
Maria Luiza Peixoto de Oliveira Vicente J. Febres Marcio Gilberto Cardoso Costa Gloria A. Moore Wagner Campos Otoni 《Plant cell reports》2009,28(3):387-395
An improved method for the Agrobacterium infiltration of epicotyl segments of ‘Pineapple’ sweet orange [Citrus sinensis (L.) Osbeck] and ‘Swingle’ citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional
Agrobacterium-mediated inoculation method (‘dipping’ method). It was observed that the morphogenic potential of the epicotyl segments decreased
as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments,
but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained
when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern
blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4%
for ‘Pineapple’ sweet orange and 11.2% for ‘Swingle’ citrumelo) are the highest ones reported for both cultivars. 相似文献
7.
Summary Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following
germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 μM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 μM) kinetin per 1, 1 mg of both 2,4-D (4.5 μM) and kinetin (4.7 μM per 1, or 2 mg of both 2,4-D (9.1 μM) and kinetin (9.3 μM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and
kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in
liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were
inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 μM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ
per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant
tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment,
four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic
via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos
occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred
to fresh medium for germination. 相似文献
8.
Tree legumes as feedstock for sustainable biofuel production: Opportunities and challenges 总被引:1,自引:0,他引:1
Concerns about future fossil fuel supplies and the environmental effects of their consumption have prompted the search for alternative sources of liquid fuels, specifically biofuels. However, it is important that the sources of such biofuel have minimal impact on global food supplies, land use, and commodity prices. Many legume trees can be grown on so-called marginal land with beneficial effects to the environment through their symbiotic interaction with “Rhizobia” and the associated process of root nodule development and biological nitrogen fixation. Once established legume trees can live for many years and some produce an annual yield of oil-rich seeds. For example, the tropical and sub-tropical legume tree Pongamia pinnata produces large seeds (∼1.5-2 g) that contain about 40% oil, the quality and composition of which is regarded as highly desirable for sustainable biofuel production. Here we consider the benefits of legume trees as future energy crops, particularly in relation to their impact on nitrogen inputs and the net energy balance for biofuel production, and also ways in which these as yet fully domesticated species may be further improved for optimal use as biofuel feedstock. 相似文献
9.
Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression 总被引:23,自引:0,他引:23
Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the efficiency of Agrobacterium infection by introducing large numbers of microwounds into the target plant tissue. Using immature cotyledons of soybean
as explants, we evaluated the effects of the following parameters on transient β-glucuronidase (GUS) activity: cultivars, binary vectors, optical density of Agrobacterium during infection, duration of sonication treatment, co-culture conditions, length of explant preculture and addition of acetosyringone
during co-culture. The extent of tissue disruption caused by sonication was also determined. The highest GUS expression was
obtained when immature cotyledons were sonicated for 2 s in the presence of Agrobacterium (0.11 OD600nm) followed by co-cultivation with the abaxial side of the explant in contact with the culture medium for 3 days at 27°C. The
addition of acetosyringone to the co-culture medium enhanced transient expression. No differences were observed when different
cultivars or binary vectors were used. Cotyledons sonicated for 2 s had moderate tissue disruption, while the longer treatments
resulted in more extensive damage.
Received: 1 October 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998 相似文献
10.
SAAT: sonication-assisted Agrobacterium-mediated transformation 总被引:18,自引:0,他引:18
Plant transformation via Agrobacterium can be limited by both host specificity and the inability of Agrobacterium to reach the proper cells in the target tissue. Described here is a new and efficient Agrobacterium-based transformation technology that overcomes these barriers and enhances DNA transfer in such diverse plant groups as dicots, monocots, and gymnosperms. This new technology, called sonication-assisted Agrobacterium-mediated transformation (SAAT), involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. Scanning electron and light microscopy reveal that SAAT treatment produces small and uniform fissures and channels throughout the tissue allowing the Agrobacterium easy access to internal plant tissues. Unlike other transformation methods, this system has the potential to transform meristematic tissue buried under several cell layers. SAAT increases transient transformation efficiency in several different plant tissues including leaf tissue, immature cotyledons, somatic and zygotic embryos, roots, stems, shoot apices, embryogenic suspension cells and whole seedlings. A 100- to 1400-fold increase in transient - glucuronid ase expression has been demonstrated in various tissues of soybean, Ohio buckeye, cowpea, white spruce, wheat and maize. Stable transformation of both soybean and Ohio buckeye has been obtained using SAAT of embryogenic suspension culture tissues. For soybean, SAAT treatment was necessary to obtain stable transformation with this tissue 相似文献