共查询到20条相似文献,搜索用时 484 毫秒
1.
The US Department of Energy recently released a 6.8X draft of the genome sequence for Nisqually-1, a genotype of black cottonwood
(Populus trichocarpa). To improve its utility for functional genomics research, having an efficient means for transformation and regeneration
is necessary. To examine several parameters known to affect the transformation rate, we cocultivated leaf disc and stem explants
with a strain ofAgrobacterium tumefaciens harboring a binary plasmid vector containing genes for both neomycin phosphotransferase (NPTII) and β-glucuronidase (GUS). Shoot regeneration from stem explants was observed in the presence of kanamycin when thidiazuron was incorporated in the
selection medium. Transformation efficiency was influenced by the level of thidiazuron to which explants were exposed during
the early stages of shoot induction. Histochemical assays revealed expression of theGUS gene in leaf, stem, and root tissues of transgenic plants. Polymerase chain reaction confirmed the presence of both selectable
marker and reporter genes in all lines that stained positive for β-glucuronidase activity. By use of our modified protocol,
transgenic plants were recovered within 6 mo at an efficiency of 6%, adequate to produce a large number of transgenic events
with modest effort. 相似文献
2.
Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through
callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The
highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence
of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive
transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed
normal phenotype under in vitro and field conditions. 相似文献
3.
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
4.
Swati Jagga-Chugh Sumita Kachhwaha Manju Sharma Aditi Kothari-Chajer S. L. Kothari 《Plant Cell, Tissue and Organ Culture》2012,109(3):401-410
Microprojectile bombardment mediated genetic transformation parameters have been standardized for seed derived callus of Eleusine coracana. Plasmid pCAMBIA 1381 harboring hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (gus A) as reporter gene, was used for the optimization of gene transfer conditions. The transient GUS expression and survival
of putative transformants were taken into consideration for the assessment of parameters. Optimum conditions for the microprojectile
bombardment mediated genetic transformation of finger millet were 1,100 psi rupture disk pressure with 3 cm distance from
rupture disk to macrocarrier and 12 cm microprojectile travel distance. Double bombardment with gold particles of 1.0 μm size
provided maximum transient GUS expression and transformation efficiency. Osmotic treatment of callus with 0.4 M sorbitol enhanced
efficiency of particle bombardment mediated genetic transformation. Regenerative calli were bombarded at optimum conditions
of bombardment and placed on regeneration medium with hygromycin to obtain transformed plants. The integration of hptII and gus A genes was confirmed with PCR amplification of 684 and 634 bp sizes of the bands respectively from putative transformants
and Southern blot hybridization using PCR amplified DIG labeled hptII gene as probe. PCR analysis with hptII gene specific primers indicated the presence of transgene in T1 generation plants. Thus a successful genetic transformation system was developed using particle bombardment in E. coracana with 45.3% transformation efficiency. The protocol will be helpful for the introgression of desired genes into E. coracana. 相似文献
5.
Fabienne Mourgues Elisabeth Chevreau Claudie Lambert An de Bondt 《Plant cell reports》1996,16(3-4):245-249
An efficient and reproducible method was established for genetic transformation of one pear variety (Conferénce) usingAgrobacterium tumefaciens-mediated gene transfer. Wounded leaves of in vitro micropropagated plants were cocultivated with the disarmed strain EHA101 harbouring the binary vector pFAJ3000 carrying the chimaericnptII andgus genes. The protocol included a 3–6 month dark period on a regeneration medium solidified with gelrite, which contained 100 mg/l kanamycin. Up to 42% of inoculated leaves produced transformed buds or bud clusters. Expression, presence and integration of transgenes was confirmed by a histochemical test, polymerase chain reaction and Southern blot hybridisation, respectively. The transgenec plants could be successfully acclimatized in the glasshouse. This transformation procedure was also successfully applied to two other pear varieties, namely Doyenné du Cornice and Passe-Crassane, albeit at much lower transformation rates. 相似文献
6.
London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering
could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots
from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene β-glucuronidase (GUS), we have followed the
transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of
leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD600 of 0.8–1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer.
Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established.
By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained
the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines,
the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome. 相似文献
7.
8.
A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from
Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166
and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM
2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin
(Km) were then recovered on CIM + 25 mg l−1 Km + 250 mg l−1 timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per
explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM
α-naphthaleneacetic acid (NAA) + 25 mg l−1 Km + 250 mg l−1 timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and
18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration
of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot
analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production
of transgenic celery plants. 相似文献
9.
Sergei F. Krasnyanski Jagdeep Sandhu Leslie L. Domier Dennis E. Buetow Schuyler S. Korban 《In vitro cellular & developmental biology. Plant》2001,37(4):427-433
Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa
mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their
effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable
regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation
of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed
the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T0 and T1 plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic
plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative
tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression
of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than
when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T1 plants segregated in a 3∶1 Mendelian ratio. 相似文献
10.
Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants,
leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types
began first with culture on 50 mg l−1 of Km for 6 wk and then transfer to 30 mg l−1 of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl−1 cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m−2s−1. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%,
and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment
for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four. 相似文献
11.
Y. Y. Wu Q. J. Chen X. H. Cui H. Chen J. Chen X. C. Wang 《Russian Journal of Plant Physiology》2007,54(4):524-529
We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were
tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest
callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine.
Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized
regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin,
a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques,
we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results
indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and
transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596.
The text was submitted by the authors in English. 相似文献
12.
Pramila Shah N. K. Singh Neeraj Khare Meenal Rathore S. Anandhan M. Arif Rupesh Kumar Singh S. C. Das Z. Ahmed Narendra Kumar 《Plant Cell, Tissue and Organ Culture》2008,95(3):363-371
Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore,
experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation.
6-benzylaminopurine at 0.05 mg l−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets
in indole-3-aceticacid 0.5 mg l−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after
co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by
polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent. 相似文献
13.
Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted
plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration
of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration
system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine
(BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis
with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box
genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed
in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two
MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation. 相似文献
14.
Antonella Furini Csaba Koncz Francesco Salamini Dorothea Bartels 《Plant cell reports》1994,14(2-3):102-106
Summary An efficient procedure for Agrobacterium tumefaciens- mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum has been developed. Leaf explants were inoculated with A. tumefaciens strain GV3101 carrying the gene for kanamycin- or hygromycin-resistance and the ßglucuronidase reporter gene. Parameters which affected the transformation efficiency were the age of the explant, the degree of wounding and the presence of an antioxidant in the medium. Under optimal conditions, calli originated in more than 80% of leaf explants. Transformed plants were obtained from more than 50% of the cultured calli during regeneration in the presence of a suitable antibiotic. The stable integration of T-DNA was confirmed by Southern blot analysis and its expression by assays for ß-glucuronidase activity.Abbreviations GUS
ß-glucuronidase
- MUG
4-methyl-umbelliferyl ß-D-glucuronide
- ABA
abscisic acid
- NPTII
neomycin phosphotransferase II
- CaMV
cauliflower mosaic virus
- MSAR
modified MS medium
- MS
Murashige and Skoog 相似文献
15.
16.
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
17.
Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants 总被引:6,自引:0,他引:6
C.-K. Ho S.-H. Chang J.-Y. Tsay C.-J. Tsai V. L. Chiang Z.-Z. Chen 《Plant cell reports》1998,17(9):675-680
An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments
with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and
regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more
than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant
to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants.
Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis,
further confirming the integration and expression of T-DNA in these plants.
Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998 相似文献
18.
Osvaldo A. Castellanos-Hernández Araceli Rodríguez-Sahagún Gustavo J. Acevedo-Hernández Benjamín Rodríguez-Garay José Luis Cabrera-Ponce Luis Rafael Herrera-Estrella 《Plant Cell, Tissue and Organ Culture》2009,99(2):175-181
A biolistic protocol for the stable genetic transformation of the hardwood tree Paulownia elongata was developed. Leaf explants were bombarded using the PDS-1000/He system with plasmid pBI121. The introduced DNA contained
the β-glucuronidase (GUS) reporter gene and neomycin phosphotransferase (nptII) as a selection marker. Transformed calli were induced and selected on medium supplemented with 50 mg L−1 kanamycin, and transgenic plants were regenerated through indirect organogenesis. Complete plants were successfully transferred
to soil and established under greenhouse conditions. Different helium pressures and explant positions were used and the transformation
frequency was calculated. Optimal conditions for genetic transformation were bombardment of the abaxial leaf surface at a
pressure of 450 psi. The integration of the transgenes in the plant genome and their stable expression was demonstrated by
fluorometric GUS assay, determination of NPTII activity and PCR analysis. This method allows the production of transgenic
trees of P. elongata in a relatively short time. 相似文献
19.
R. Dibax C. Deschamps J. C. Bespalhok Filho L. G. E. Vieira H. B. C. Molinari M. K. F. De Campos M. Quoirin 《Biologia Plantarum》2010,54(1):6-12
The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced
on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots
were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to
pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing
kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied.
The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline
content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots
was similar in both types of plants. 相似文献
20.
Transgenic garlic (Allium sativum) plants have been recovered directly from immature leaf material by selective culture following Agrobacterium-mediated transformation. This method involved the use of a binary vector containing the mgfp-ER reporter gene and hpt selectable marker, and followed a similar protocol developed previously for the transformation of immature onion embryos.
The choice of tissue and post-transformation selection procedure resulted in a large increase in recovery of transgenic plants
compared with previously confirmed allium transformation protocols. The presence of transgenes in the genome of the plants
was confirmed using Southern analysis. This improvement in frequency and the use of clonal commercial “Printanor” germplasm
now makes possible the integration of useful agronomic and quality traits into this crop. 相似文献