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1.
Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi 总被引:1,自引:0,他引:1
An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were
inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase
(HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified
MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were
cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting
putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant
transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant
calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants
was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis. 相似文献
2.
Slavica Ninković Tatjana Djordjević Branka Vinterhalter Branka Uzelac Aleksandar Cingel Jelena Savić Svetlana Radović 《Plant Cell, Tissue and Organ Culture》2010,103(1):81-91
Agrobacterium rhizogenes A4M70GUS-mediated transformation of two local breeding lines of sugar beet was obtained using 4-week-old seedlings. Root
formation efficiency was 61.54% for SBa genotype and 36.36% for SBb genotype. Five highly proliferated hairy root lines have
been established in liquid hormone-free MS medium. Transgenic nature of the hairy root clones was evaluated by GUS assay,
PCR and RT-PCR analyses. Hairy root-derived calli were induced using different plant growth regulators (PGRs): auxin, auxin/cytokinin
and cytokinin. The best callus induction response was achieved on MS medium containing both 1 mg/l 2,4-dichlorophenoxyacetic
acid (2,4-D) and 1 mg/l thidiazuron (TDZ). Globular embryo-like structures were observed in friable callus after its prolonged
cultivation on MS medium supplemented with TDZ and giberellic acid (GA3) at 1 mg/l each, followed by growth on MS medium containing 1% glucose and 0.5 mg/l 2,3,5-triiodobenzoic acid (TIBA). Histological
analysis revealed somatic embryos at different stages of development in hairy root-derived callus of sugar beet. 相似文献
3.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
4.
Mingjia Yang Xiangming Xie Caixia Zheng Fangqiu Zhang Xiaoqing He Zhiru Li 《Plant Cell, Tissue and Organ Culture》2008,95(2):141-147
A protocol was developed for Agroacterium-mediated genetic transformation of Acacia crassicarpa via organogenesis by using in vitro phyllode (leaf) as the explant. Phyllode (leaf) explants were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI101 (harboring antisense Pt4CL1 with respect to the Pt4CL1P promoter). The selection for transgenic shoots was performed through two consecutive steps on
Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators and antibiotics in the
following order: 0.5 mg/l thidiazuron (TDZ), 0.5 mg/l α-naphthaleneacetic acid (NAA), 300 mg/l carbenicillin (Car) and 20 mg/l
kanamycin (Km) for 10 days; 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days; 0.5 mg/l indole-3-butyric acid (IBA), 100 mg/l
Car and 20 mg/l Km 50 days. 21.7% of nodules produced multiple adventitious shoot buds, of which 27.7% survived in initial
selection. The shoot buds were subjected to repeated selection on MS medium supplemented with 0.1 mg/l TDZ, 200 mg/l Car and
20 mg/l Km for 60 days. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.5 mg/l
IBA, 100 mg/l Car 20 mg/l Km 50 days. Genomic PCR analysis confirmed the incorporation of the antisense Pt4CL1 with respect to the Pt4CL1P promoter fragment into the host genome. 相似文献
5.
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
6.
S. Zhihui M. Tzitzikas K. Raemakers M. Zhengqiang R. Visser 《In vitro cellular & developmental biology. Plant》2009,45(6):776-782
Pea (Pisum sativum L. cv. Espace) seeds directly cultured on thidiazuron (TDZ)-containing medium formed high numbers of shoots. The number of
shoots per seedling depended on the concentration and duration of the TDZ treatment. The best treatment was 12-wk incubation
on MS medium supplemented with 4 mg/l TDZ followed by 4-wk culture on MS medium supplemented with 0.5 mg/l benzylaminopurine
(BA) and produced more than 400 shoots/seedling. Isolated shoots rooted at a high frequency on MS medium containing 2–3 mg/l
indole-3-butyric acid and 2 mg/l α-naphthalene acetic acid. In addition to the formation of shoots, bud-containing tissues
(BCT) were formed at the cotyledonary nodes, shoot nodes, tendrils, stipules, and internodes. The BCT from the cotyledonary
nodes and the shoot nodes was maintained in its pure state on MS medium supplemented with 4 mg/l TDZ by repeated culture.
Shoot development was accomplished when the BCT were left on MS medium supplemented with 4 mg/l TDZ without subculture prior
to transfer onto MS medium supplemented with 0.5 mg/l BA. 相似文献
7.
Effect of sorbitol concentration on regeneration of embryogenic calli in upland rice varieties (Oryza sativa L.) 总被引:1,自引:0,他引:1
Pengpeng Geng Honggui La Huaqi Wang Elliot J. C. Stevens 《Plant Cell, Tissue and Organ Culture》2008,92(3):303-313
This study describes the impact of sorbitol on plantlets regeneration frequency (PRF) of four rice cultivars (japonica, Oryza sativa L.) both of which mature and immature embryo-derived calli were investigated. The variance analysis results showed that PRF
of the three elite upland rice cultivars, Handao297 (HD297), Handao502 (HD502), Handao65 (HD65) and one lowland rice cultivar
Zhongzuo93 (ZZ93) were significantly increased with addition of appropriate amount of sorbitol in culture media. Supplementing
appropriate sorbitol in the media of a continous culture from induction and maintenance to regeneration for mature embryo-derived
calli could improve PRF dramatically, originally from 27.6% up to a maximum of 71.8%. Especially to low regenerative capacity
(LRC) cultivar HD65, the PRF was increased over 7-fold (from 9.7% to 74.0%). The optimum concentrations of sorbitol for calli
induction, subculture and differentiation were 5, 20 and 40 g/l, respectively. Adding sorbitol, only in maintenance media
at concentration of 20 g/l, also enhanced the PRF greatly in all the cultivars from 27.6% to 43.3%. Similar results were observed
when incorporating with maltose in regenerating media both in immature and mature embryo-derived calli. The optimal concentration
was 25 g/l sorbitol + 20 g/l maltose and 20 g/l sorbitol + 25 g/l maltose, respectively. HD297 appeared to be the most responsive
genotype compared to other cultivars in PRF, 99.2% in immature embryo-derived calli and 76.8% in mature embryo-derived calli.
The results and relevant conclusions might be valuable to establish an efficient plant regeneration system from somatic embryogenesis
culture in upland rice. 相似文献
8.
Xiang Liu Chun-Miao Feng Robert Franks Rongda Qu De-Yu Xie Qiu-Yun Jenny Xiang 《Plant cell reports》2013,32(1):77-87
Key message
Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species.Abstract
Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants. 相似文献9.
Zhao-Hong Meng Ai-Hua Liang Wei-Cai Yang 《In vitro cellular & developmental biology. Plant》2007,43(2):111-118
We have evaluated the effects of the antibiotic hygromycin B on cotton (Gossypium hirsutum L.) callus induction, callus proliferation, and seed germination. Nontransgenic cotyledon and hypocotyl showed obvious variance
in tolerance to hygromycin. Cotyledons were more sensitive to hygromycin than hypocotyls. Hygromycin at 7.5 and 20 mg l−1 completely inhibited callus initiation from cotyledon and hypocotyl explants, respectively. Nontransformed calli did not
grow on media supplemented with 10 mg l−1 hygromycin and were killed at 15 mg l−1. In seed germination assay, the presence of 20 mg l−1 hygromycin significantly suppressed shoot and root elongation of seedlings. This hygromycin concentration was applied to
select regenerated transgenic plantlets and their progenies. Based on these results, we developed an efficient hygromycin
selection protocol for Agrobacterium-mediated cotton transformation and regeneration. 相似文献
10.
Wenyan Wang Chenggang Wang Bang-Lian Huang Bangquan Huang 《Plant Cell, Tissue and Organ Culture》2008,92(2):165-171
A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l−1 BA, 1 mg l−1 NAA and 1 mg l−1 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l−1 BA, 0.2 mg l−1 NAA and 100 μmol l−1As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l−1 BA, 0.2 mg l−1NAA, 500 mg l−1 Cef and 10 mg l−1 hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l−1 on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l−1BA, 0.2 mg l−1 NAA, 500 mg l−1 Cef and 25 mg l−1 hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical
assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli
was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate
that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species. 相似文献
11.
Yoichiro Hoshino Yukiko Kashihara Tomonari Hirano Naho Murata Koichi Shinoda 《Plant Cell, Tissue and Organ Culture》2008,94(1):45-54
Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 days after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators
(PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the
calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloropyridine-2-carboxylic acid
(picloram). Adventitious embryos developed from the suspension cells on half-strength MS medium supplemented with 0.5 mg l−1 of both α-naphthaleneacetic acid and N6-benzylaminopurine; they grew into plantlets on the same medium. The plantlets formed rhizomes following transfer to half-strength
MS medium without PGRs, and acclimatized plants were easily established. Subsequently, Agrobacterium-mediated transformation system was applied. The suspension cells were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which contain neomycin phosphotransferase II, hygromycin phosphotransferase
and intron-containing ?-glucuronidase (intron-GUS) genes. Seven days after co-cultivation, the cells were subjected to GUS assay; staining was most pronounced in the cells
subcultured in a picloram-containing liquid medium and co-cultivated with EHA101/pIG121Hm. The co-cultivated cells were transferred
to the MS medium containing picloram and 20 mg l−1 hygromycin; 1 month later, several hygromycin-resistant callus lines showing GUS activity were obtained. Transgenic plants
were obtained through our plant regeneration system, and foreign gene insertion into the regenerated plants was confirmed
by polymerase chain reaction. 相似文献
12.
Rangaraj Nandakumar Li Chen Suzanne M. D. Rogers 《In vitro cellular & developmental biology. Plant》2007,43(3):187-194
A highly reproducible Agrobacterium-mediated transformation system was developed for the wetland monocot Juncus accuminatus. Three Agrobacterium tumefaciens binary plasmid vectors, LBA4404/pTOK233, EHA105/pCAMBIA1201, and EHA105/pCAMBIA1301 were used. All vectors contained the
35SCaMV promoter driven, intron containing, β-glucuronidase (gus), and hygromycin phosphotransferase (hptII) genes within their T-DNA. After 48 h of cocultivation, 21-d-old seedling derived calli were placed on medium containing
timentin at 400 mg l−1, to eliminate the bacteria. Calli were selected on MS medium containing 40 or 80 mg l−1 hygromycin, for 3 mo. Resistant calli were regenerated and rooted on MS medium containing hygromycin, 5 mg l−1(22.2 μM) of 6-benzylamino-purine (BA) and 0.1 mg l−1(0.54 μM) of alpha-naphthaleneacetic acid (NAA), respectively. Seventy-one transgenic cell culture lines were obtained and
39 plant lines were established in the greenhouse. All the plants were fertile, phenotypically normal, and set viable seed.
Both transient and stable expression of the gus gene were demonstrated by histochemical GUS assays of resistant calli, transgenic leaf, root, inflorescence, seeds, and whole
plants. The integration of gus and hptII genes were confirmed by polymerase chain reaction (PCR) and Southern analysis of both F0 and F1 progenies. The integrated genes segregated to the subsequent generation in Mendelian pattern. To our knowledge, this is the
first report of the generation of transgenic J. accuminatus plants. 相似文献
13.
In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation
efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l−1) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone.
The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced
more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with
3 mg l−1 hygromycin for 10 days, 5 mg l−1 hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l−1 hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants
in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression
and inheritance of the transgenes were confirmed by molecular and genetic analysis. T1 plants were analyzed and transmission of transgenes to the T1 generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation. 相似文献
14.
We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin
for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic
embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7
d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in
regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L
giberellic acid (GA3). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern
hybridization to confirm gene integration. 相似文献
15.
Meiru Li Hongqing Li Huawu Jiang Guojiang Wu 《Plant Cell, Tissue and Organ Culture》2008,93(3):249-255
Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful
genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with
a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively.
For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473,
1962) (MS) medium supplemented with 1.5 mg l−1 benzyladenine (BA) and 0.05 mg l−1 indole-3-butyric acid (IBA) (CI medium) containing 5 mg l−1 hygromycin and 500 mg l−1 cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots
were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l−1 BA, 0.05 mg l−1 IBA, and 0.5 mg l−1 gibberellic acid (GA3) (SI medium), 5 mg l−1 hygromycin and 250 mg l−1 cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l−1 hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay.
Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as
44%. 相似文献
16.
Myoung K. Kim Harry E. Sommer Jeffrey F. D. Dean Scott A. Merkle 《In vitro cellular & developmental biology. Plant》1999,35(1):37-42
Summary A sweetgum (Liquidambar styraciflua) nodule culture system was developed and integrated with genetic transformation by microprojectile bombardment. Nodule cultures
were established from seedling hypocotyls and proliferated in liquid medium containing 0.1 mg (0.45 μM) thidiazuron (TDZ) per 1 and 0.01 mg (0.045 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1. Shoots differentiated from the nodules in liquid media containing (per 1)
1 mg (4.4 μM) benzyladenine (BA), 0.5 mg (2.2 μM) BA, and 0.01 mg (0.054 μM) naphthaleneacetic acid (NAA), or 0.5 mg BA, 0.01 mg NAA, and 0.05 mg (0.23 μM) TDZ under the light. Differentiating shoots required 4 wk of dark treatment for further development on semisolid medium
containing 1 mg BA per 1. Elongated shoots were harvested and the basal ends were soaked in a solution containing 10 mg (49.2
μM) indole-3-butyric acid (IBA) per 1 before being planted in potting mix for ex vitro rooting. Roots formed and leaves expanded
in 2 wk. Sweetgum nodules were stably transformed by microprojectile bombardment with a 7.4-kb plasmid, pTRA 140, harboring
CaMV 35S-HPH and CaMV 35S-GUS. Evidence that nodules growing in the presence of hygromycin B were stably transformed was provided
by polymerase chain reaction analysis and β-glucuronidase activity. Sweetgum shoots differentiated in liquid medium in the
presence of hygromycin B. Shoots transferred to solid medium lacking hygromycin B elongated and displayed β-glucuronidase
activity in their expanding leaves and stems. Southern analysis confirmed the presence of the GUS gene in nodules and shoots.
Transgenic shoots initiated roots and showed leaf expansion 2 wk after being planted in potting mix. 相似文献
17.
Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the
Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium
with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by
twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on
the MS medium supplemented with 1 mg l−1 indole-3-butyric acid and 15 mg l−1 hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for β-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at
a rate of 54.6% with an average of 3.9 ± 0.39 transgenic plantlets per explant was achieved in the present transformation
system. It took only 2–3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient
and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering
studies in this medicinally important plant. 相似文献
18.
Reproducible and high-frequency transgenic plant regeneration from callus and embryo axes of four different genotypes of chickpea
(Cicer arietinum) was achieved after Agrobacterium-mediated transformation. Three different strains of Agrobacterium (EHA105, AGL1 and LBA4404) harboring the binary vector pCAMBIA1301 containing β-glucuronidase (GUS) and hygromycin phosphotransferase (hpt) genes under the control of a CaMV35S promoter were used. The highest number of transgenic plants was obtained from cotyledonary
node-derived calli of genotype Pusa-256. A highly efficient rooting was achieved on Murashige and Skoog medium supplemented
with indole-3-butyric acid. The stable integration of the gene was confirmed by molecular analyses of the transformed plants.
Inheritance of GUS and hpt gene was followed through two generations and they showed the expected 3:1 inheritance. 相似文献
19.
Pejman Azadi Dong Poh Chin Kiyo Kuroda Raham Sher Khan Masahiro Mii 《Plant Cell, Tissue and Organ Culture》2010,101(2):201-209
A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase,
and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency
of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this
modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv.
Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium.
Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses. 相似文献
20.
Bo Wang Lijun Liu Xuxia Wang Jinyu Yang Zhenxia Sun Na Zhang Shimei Gao Xiulong Xing Dingxiang Peng 《Plant cell reports》2009,28(9):1319-1327
In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable
transformation were evaluated. Agrobacterium at a concentration of OD = 0.5–0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L
during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20°C, in comparison to 15, 25 and
28°C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was
optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation
efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation
rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed
by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient
Agrobacterium-mediated transformation of ramie.
An erratum to this article can be found at 相似文献