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1.
An efficient and reproducible Agrobacterium-mediated transformation system via repetitive secondary somatic embryogenesis was developed for Rosa rugosa ‘Bao white’. Somatic embryogenesis was induced from in vitro-derived unexpanded leaflet explants on MS medium supplemented with 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L Kinetin and 30 g/L glucose. Secondary somatic embryos were successfully proliferated via cyclic secondary somatic embryogenesis on MS medium containing 1.0 mg/L 2,4-D, 0.01 mg/L 6-benzyladenine and 45 g/L glucose under light intensity of 500–1,000 lux. The highest germination rate (86.33 %) of somatic embryos was observed on 1/2-strength MS medium containing 1.0 mg/L BA. Relying on the repetitive secondary somatic embryogenesis and A. tumefaciens strain EHA105 harboring the binary vector pBI121, a stable and effective Agrobacterium-mediated transformation pattern was developed. The presented transformation protocol, in which somatic embryo clumps at globular stage (0.02–0.04 g) were infected by Agrobacterium for 60 min and co-cultivated for 2 days, and then selected under a procedure of 3 steps, were confirmed to be optional by GUS histochemical assay and Southern blot analysis. The procedure described here will be very useful for the introgression of desired genes into R. rugosa ‘Bao white’ and the molecular analysis of gene function. 相似文献
2.
Summary Transgenic grape plants were regenerated from somatic embryos derived from immature zygotic embryos of seedless grape (Vitis vinifera L.) selections. Somatic embryos were bombarded twice with 1 m gold particles using the Biolistic PDS-1000/He device (Bio-Rad Laboratories) and then exposed to Agrobacterium tumefaciens strain C58/Z707 containing the binary plasmid pGA482GG or pCGN7314. Following cocultivation, secondary embryos were allowed to proliferate on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 20–40 g/ml kanamycin (kan). Transgenic embryos were identified after 3–5 months under selection and allowed to germinate and develop into rooted plants on Woody Plant Medium containing 1 M 6-benzylaminopurine (BAP), 1.5% sucrose, 0.3% activated charcoal and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kan, positive GUS and PCR assays, and Southern analysis. 相似文献
3.
Wenting Liu Zongsuo Liang Changjuan Shan Frederic Marsolais Lining Tian 《In vitro cellular & developmental biology. Plant》2013,49(1):17-23
A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines. 相似文献
4.
Chandra I. Franklin Tony N. Trieu Brandt G. Cassidy Richard A. Dixon Richard S. Nelson 《Plant cell reports》1993,12(2):74-79
Summary Kanamycin resistant callus was produced from leaf disc or hypocotyl expiants of green bean (Phaseolus vulgaris L.) when cultured on a defined medium containing 50 mg/l kanamycin after 4 days of co-cultivation with Agrobacterium tumefaciens strain EHA101 containing the binary vector pKYLX71GUS. The presence of neomycin phosphotransferase II (NPT-II) in crude cellular extracts from the kanamycin resistant callus was confirmed by ELISA. The expression of the ß-glucuronidase (GUS) reporter gene was confirmed by histochemical and fluorimetric analyses. Southern blot border analysis confirmed the integration of the foreign DNA. In addition to the evidence obtained from Southern analysis, the absence of Agrobacterium in the transformed callus cultures was confirmed by microscopic observation and through test cultures. Using the above protocol, bean callus cultures were also transformed with a bean chalcone synthase promoter-GUS fusion. These cultures, when treated with the elicitor glutathione, showed higher levels of GUS expression than the unelicited callus clumps. 相似文献
5.
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
6.
Agrobacterium-mediated transformation and plant regeneration from hypocotyl segments of Japanese persimmon (Diospyros kaki Thunb) 总被引:1,自引:0,他引:1
Hypocotyl segments from the seeds of Japanese persimmon (Diospyros kaki Thunb) were cultured on a modified Murashige and Skoog medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea, zeatin
or 6-benzylaminopurine. The highest frequency of shoot regeneration was observed when the segments were cultured on medium
containing 2 mg/l of zeatin. This culture system was adapted to Agrobacterium-mediated transformation. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the β-glucuronidase gene. Regenerated shoots were selected on a medium containing kanamycin. Histochemical GUS assay showed that
the shoots regenerated from the segments inoculated with EHA101/pSMAK251 expressed the gus gene. The presence and integration of the gus gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The regeneration frequency of transformed
shoot was 11.1%. The transgenic shoots were rooted and developed into whole plants within 4–5 months.
Received: 18 August 1997 / Revision received: 8 October 1997 / Accepted: 11 November 1997 相似文献
7.
Summary Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25g/ml kanamycin and 500g/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes. 相似文献
8.
Artur da Câmara Machado Monika Puschmann Helene Pühringer Robert Kremen Hermann Katinger Margit Laimer da Câmara Machado 《Plant cell reports》1995,14(6):335-340
Embryogenic lines of Prunus subhirtella autumno rosa were established on a modified MS medium supplemented with 1 mg/l NAA, 0.06 mg/l IBA and 0.04 mg/l BA from petioles of axenically grown shoots of adult origin. To induce normal development of plantlets we compared a range of approaches on solid culture media as well as in suspension cultures including treatments with ABA, GA3, zeatin, darkness, and cold. A series of experiments were conducted to follow the temporal pattern of somatic embryo development.Separation of embryos at different stages of development was carried out by sieving the suspension cultures through nylon nets. While the embryogenic masses were used for further subcultures, well formed embryos were used for germination experiments.Transformed Prunus subhirtella plants were regenerated from somatic embryos by inoculating an embryogenic callus with Agrobacterium strain LBA 4404 containing the ß-glucuronidase (GUS) gene on plasmid pBinGUSint. Several putative transformed embryogenic calli were selected for continued proliferation on kanamycin containing media. Finally transgenic plants were regenerated on shoot multiplication medium containing kanamycin. Embryos and plants were shown to express the GUS gene by histochemical assays and northern blot analysis. With a yield of 110 transgenic lines from a single transformation experiment this approach appears ideal for the study of the influence on level of expression caused by different copy number, site of insertion etc. This will be helpful in establishing parameters according to which the best transgenic line for a chosen purpose should be selected.Abbreviations BA
6-benzylaminopurine
- IBA
3-indolebutyric acid
- GA3
gibberellic acid
- NAA
1-naphthylacetic acid
- ABA
abscisic acid
- GUS
ß-glucuronidase
- NPTII
neomycin phosphotransferase II
- SDS
sodium dodecyl sulphate
- SSC
standard saline citrate
- PEM
proembryogenic masses
Dedicated to Franticek Novak 相似文献
9.
Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which β-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed
by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation.
Received: 22 October 1997 / Revision received: 7 January 1998 / Accepted: 2 February 1998 相似文献
10.
Thankaraj Salammal Mariashibu Kondeti Subramanyam Muthukrishnan Arun Subramanian Mayavan Manoharan Rajesh Jeevaraj Theboral Markandan Manickavasagam Andy Ganapathi 《Acta Physiologiae Plantarum》2013,35(1):41-54
For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l?1 citric acid, 100 µM acetosyringone, and 100 mg l?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed. 相似文献
11.
Zhiqiang Pan Jingkun Ho Qin Feng Dao-Shun Huang Ralph A. Backhaus 《Plant Cell, Tissue and Organ Culture》1996,46(2):143-150
In vitro grown shoot tissue of facultative apomictic lines of guayule (Parthenium argentatum Gray), a rubber producing desert shrub, were transformed by Agrobacterium-mediated DNA transfer and regenerated into complete plants. Guayule shoots of lines 11591, UC101 and UC104 were inoculated with A. tumefaciens strains LBA4404 or PC2760 harboring the binary vector pCGN1557. Axillary shoots were regenerated from transformed cells and rooted in vitro in the presence of kanamycin. Genetic transformation in all cases was verified by Southern blot analysis. Transgenic plants were grown to maturity in the greenhouse and, as predicted for apomictic species, all seed produced possessed kanamycin resistance. Because apomicts have limitations for gene transfer by normal sexual crosses, this method offers a new means of transferring genes into this species.Abbreviations BA
benzyladenine
- EDTA
ethylene diamine tetraacetate
- kanR
kanamycin resistance
- MS salts
salts of Murashige and Skoog medium (1962)
- NAA
naphthalene acetic acid
- NPT-II
neomycin phosphotransferase
- SDS
sodium dodecyl sulfate 相似文献
12.
Zuker Amir Ahroni Asaph Tzfira Tzvi Ben-Meir Hagit Vainstein Alexander 《Molecular breeding : new strategies in plant improvement》1999,5(4):367-375
Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T0 and T1 generations confirmed the transgenic nature of the selected plants. 相似文献
13.
Giovanna S. Fontana Luigi Santini Sofia Caretto Giovanna Frugis Domenico Mariotti 《Plant cell reports》1993,12(4):194-198
In the grain legume Cicer arietinum L. (chickpea), the seed-derived embryo axes deprived of the apical meristem were able to regenerate adventitious shoots on Murashige and Skoog (1962) medium supplemented with kinetin. This protocol was suitable for Agrobacterium-mediated gene transfer by the co-cultivation technique. Chickpea transgenic plants showed neomycin phosphotransferase II and ß-glucuronidase activities and the presence in their genome of integrated bacterial DNA.Abbreviations 6-BAP
6-benzyl-aminopurine
- CaMV
cauliflower mosaic virus
- GUS
ß-glucuronidase
- IAA
indole-3-acetic acid
- Kn
kanamycin
- MU
methyl umbelliferone
- NAA
naphthaleneacetic acid
- NPTII
neomycin phosphotransferase II 相似文献
14.
A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis. 相似文献
15.
Manoharan Rajesh Murugaraj Jeyaraj Ganeshan Sivanandhan Kondeti Subramanyam Thankaraj Salammal Mariashibu Subramanian Mayavan Gnanajothi Kapil Dev Vasudevan Ramesh Anbazhagan Markandan Manickavasagam Andy Ganapathi 《Plant Cell, Tissue and Organ Culture》2013,114(1):71-82
An efficient Agrobacterium-mediated genetic transformation method has been developed for the medicinal plant Podophyllum hexandrum Royle, an important source of the anticancer agent podophyllotoxin. Highly proliferating embryogenic cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA 2301, which contains npt II and gusA as selection marker and reporter genes, respectively. The transformed somatic embryos and plantlets were selected on Murashige and Skoog (MS) basal medium containing kanamycin and germination medium, respectively. GUS histochemical analysis, polymerase chain reaction and Southern blot hybridisation confirmed that gusA was successfully integrated and expressed in the P. hexandrum genome. Compared with cefotaxime, 200 mg l?1 timentin completely arrested Agrobacterium growth and favoured somatic embryo development from embryogenic cells. Among the different Agrobacterium strains, acetosyringone concentrations and co-cultivation durations tested, embryogenic callus infected with A. tumefaciens EHA 105 and co-cultivated for 3 days on MS basal medium containing 100 μM acetosyringone proved to be optimal and produced a transformation efficiency of 29.64 % with respect to germinated GUS-positive plantlets. The Agrobacterium-mediated genetic transformation method developed in the present study facilitates the transference of desirable genes into P. hexandrum to improve the podophyllotoxin content and to enhance other useful traits. 相似文献
16.
A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with
five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4–10 mg) were inoculated with EHA105:p35SGUSINT
bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed
masses appeared after 20–30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern
blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old
tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic
plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed
somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens
and/or biotic stresses. 相似文献
17.
Maria I. C. S. Gama Rui P. Leite Jr. Antonio R. Cordeiro Daniel J. Cantliffe 《Plant Cell, Tissue and Organ Culture》1996,46(3):237-244
Stable expression of foreign genes was achieved in sweet potato (Ipomoea batatas (L.) Lam) plants using anAgrobacterium tumefaciens mediated system. Embryogenic calluses produced from apical meristems of cultivar White Star were multiplied and cocultivated withA. tumefaciens strain EHA101 harboring a binary vector containing the -glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes. The calluses were transferred to selective regeneration medium and kanamycin resistant embryos were recovered which developed into morphologically normal plants. Histochemical and fluorimetric GUS assays of plants developed from the kanamycin resistant embryos were positive. Amplified DNA fragments were produced in polymerase chain reactions using GUS-specific primers and DNA from these plants. Transformation was confirmed by Southern analysis of the GUS gene. With the developed method, transgenic sweet potato plants were obtained within 7 weeks. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.Florida Agricultural Experiment Station Journal Series N-02231. This research was partially supported by CNPq/RHAE (Brazil). 相似文献
18.
Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The
PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L−1 gellan gum-solidified NDM containing 10 g L−1 sucrose, 20 mg L−1 hygromycin and 40 mg L−1 meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The
highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 μM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different
sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization. 相似文献
19.
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
20.
A. Nadolska-Orczyk A. Przetakiewicz K. Kopera A. Binka W. Orczyk 《Journal of Plant Growth Regulation》2005,24(1):2-10
Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two
of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes
the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin
pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation
with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants
obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was
examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T0 transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed
Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian
and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species
are discussed. 相似文献