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1.
Ana Sofia Duque Susana de Sousa Araújo Matilde Ataíde Cordeiro Dulce Maria Santos Manuel Pedro Fevereiro 《Plant Cell, Tissue and Organ Culture》2007,90(3):325-330
We developed an alternative methodology for in vitro selection of transgenic Medicago truncatula cv. Jemalong plants using a bifunctional construct in which the coding sequences for the green fluorescent protein (GFP)
and the β-glucuronidase protein (GUS) are fused. An Agrobacterium-mediated transformation protocol was used followed by regeneration via somatic embryogenesis in the dark, to avoid the synthesis and the consequent autofluorescence of chlorophyll. This method
is a clear advantage over antibiotic and herbicide selection in which survival of non-transformed tissue is commonly reported,
with the reassurance that all the somatic embryos selected as GFP positive are transformed. This was subsequently corroborated
by the detection of GUS activity in leaves, stems and roots of the regenerated plants. Without antibiotic selection, and performing
the embryo induction in the dark, it was possible to attest the advantage of using GFP as an in vivo detectable reporter for
early embryo selection. The fusion with the GUS coding sequence provided additional evidence for the transformation of the
previously selected embryos. 相似文献
2.
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
3.
The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits
within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated
with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut
into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid
(NAA), 5 μM N6-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated
after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant
genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed
in T1 generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment
of this transformation system is invaluable for investigating fruit-tree-specific phenomena. 相似文献
4.
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
5.
Silvia Flores-Benítez Juan F. Jiménez-Bremont Sergio Rosales-Mendoza Gerardo R. Argüello-Astorga Rosalba Castillo-Collazo Ángel Gabriel Alpuche-Solís 《Plant Cell, Tissue and Organ Culture》2007,91(3):215-224
Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and
embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants
was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving
a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive
with the GUS assay after 14 months on selective medium while still undergoing regeneration. 相似文献
6.
Xiuping Zou Demou Li Xiaoying Luo Keming Luo Yan Pei 《In vitro cellular & developmental biology. Plant》2008,44(3):169-177
Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected
with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable
marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation
efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic
acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy
was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied
on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency
and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically
reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical
assay, PCR, and Southern blot analysis. 相似文献
7.
Hai Ping Hong Hongyi Zhang Paula Olhoft Steve Hill Hunt Wiley Effie Toren Helke Hillebrand Todd Jones Ming Cheng 《In vitro cellular & developmental biology. Plant》2007,43(6):558-568
A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes
as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced
on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with
various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect
on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a
low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside,
and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction
medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected
calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been
regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including
leaf, stem, and roots. Southern and T1 plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy
number ranging from 1–5 copies. 相似文献
8.
A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues
for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance
was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM)
and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l−1 in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of
transgenic cells, compared to 50 or 75 mg l−1, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered
after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization.
Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots,
inflorescences and the embryo and endosperm of developing berries. 相似文献
9.
An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum
infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the
highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water
deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by
drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney
bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney
bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. 相似文献
10.
Summary An efficient protocol has been developed for the regeneration of plantlets from leaf explants of witloof chicory (Cichorium intybus L.). Regeneration via callus was obtained on modified Murashige and Skoog semisolid medium (MS) containing 2.0 μM indole-3-acetic acid +5.0 μM 6-furfurylaminopurine (kinetin), and 1000 mgl−1 casein hydrolyzate. At least five or more shoots regenerated from each callus. The shoots were rooted on MS +0.2 μM indole-3-butyric acid. The plantlets thus obtained were successfully established in soil after bardening. Esculin accumulation
was recorded in plant tissues at different stages of differentiation in in vitro cultures and compared with in vivo-grown, plants. The esculin accumulation was higher in in vitro plants. 相似文献
11.
Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve
this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration
from rice shoot apical meristem. By use of MS medium containing 4 mg L−1 thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage.
All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil
within 5–8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either
250 mg L−1 carbenicillin or 250 mg L−1 cefotaxime to kill the rice shoot apical meristem was 50 mg L−1 and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency
of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient
GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T0 transformant plantlets. 相似文献
12.
13.
Rakosy-Tican E Aurori CM Dijkstra C Thieme R Aurori A Davey MR 《Plant cell reports》2007,26(5):661-671
Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish
a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using
Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy,
PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best
performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. ‘Baltica’, respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the
effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras
in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of
tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed
to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation. 相似文献
14.
Tileye Feyissa Li-Hua Zhu Legesse Negash Margareta Welander 《Plant Cell, Tissue and Organ Culture》2007,88(3):277-288
The effects of growth regulators, wounding and antibiotics on regeneration of Hagenia abyssinica were investigated and the rolB gene was introduced into this species by Agrobacterium-mediated transformation. Regeneration was affected by type of growth regulators, wounding and antibiotics. Up to 100% regeneration
could be obtained. Three transformed clones (T1, T2.1, T2.2), confirmed by PCR and Southern blot, were obtained only by excluding
kanamycin from the selection medium 6 weeks after culture, followed by selection during shoot multiplication. RT-PCR revealed
strong expression of rolB gene in shoots and roots of all the transgenic clones, but from leaf samples, it was detected only in T1. Rooting frequency
was 77% (T1), 50% (T2.1), 57% (T2.2) and 0% for control shoots on growth regulator-free rooting medium. 相似文献
15.
The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages
than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze–thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally,
transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study
suggests a potential industrial application of AGAP expressed in plants. 相似文献
16.
Summary Despite intensive efforts, a reproducible and reliable method for transformation of sugarbeet plants is still lacking. Having
examined several explants, we found that cells around the main vein of leaves of plantlets reared from tissue-cultured apical
meristems are sufficiently competent for transformation and subsequent regeneration. A transformation protocol was designed
by evaluating alterations in several parameters such as plant genotype, Agrobacterium strain, antibiotics, darkness and duration of co-culture period. An average transformation rate of 6.2% transformed shoots
per explant was achieved as judged by Southern blotting. Consistent inactivation of reporter genes was correlated to multiple
copies of transgenes present in some transformants. The necessary steps for rooting and planting of transformed shoots were
also established. 相似文献
17.
E. A. Popowich A. P. Firsov T. Y. Mitiouchkina V. L. Filipenya S. V. Dolgov V. N. Reshetnikov 《Plant Cell, Tissue and Organ Culture》2007,90(3):237-244
Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP
and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning
preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l−1 kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l−1 kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants
expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines
were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same
time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation. 相似文献
18.
Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented
with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot
induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot
developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil
and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age. 相似文献
19.
<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
20.
An efficient and reproducible transformation method of sonication- assisted Agrobacterium-mediated transformation (SAAT) was developed for chickpea (Cicer arietinum L.). Agrobacterium tumefaciens (LBA4404) harboring pCAMBIA1305.2 was used to transform decapitated embryo explants of two cultivars of chickpeas. By using
a series of co-cultivation, callus induction, shoot initiation and root inducing media, a large number of transgenic plants
were recovered. Transient expressions of GUS gene were detected by X-Gluc histochemical assay in transformed tissues. DNA analysis of T0 and T1 plants by PCR and Southern
hybridization confirmed the integration of transgenes in initial and next generation transformants in different transgenic
lines. The transformation efficiency was more than two times higher in SAAT treatment than simple Agrobacterium without sonication. 相似文献