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1.
The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles,
from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness
on medium supplemented with thidiazuron (8.4 μm). Shoot formation occurred at sites of excision on the leaf blade and petiole either directly from cells of the explant or
via a primary callus. Analyses of transient GUS activity data indicated that DNA delivery by microprojectile bombardment was
more effective than Agrobacterium infection. However, no transgenic plants were obtained from over 22,000 leaf explants after particle bombardment. Cocultivation
of leaf explants with Agrobacterium strain EHA 105 and kanamycin selection produced transgenic plants. Greater transient and stable -glucuronidase (GUS) activities
were detected in explants or propagules transformed with the construct where gusA was driven by the pBISN1 promoter rather than a CaMV 35S promoter. Eight plants were subsequently regenerated and verified
as transgenic based on detection of the nptII transgene by PCR and Southern blot analyses. The Southern analyses indicated that the plants were derived from eight unique
transformation events. All transgenic plants appeared morphologically normal. Analyses of GUS activities in leaves sampled
from different portions of these transgenic plants, 10 months after transfer to the greenhouse, indicated that six out of
the eight original regenerants were uniformly transformed, i.e., did not exhibit chimeric sectors.
Received: 12 December 1997 / Revision received: 3 June 1997 / Accepted: 18 July 1997 相似文献
2.
3.
Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment 总被引:5,自引:0,他引:5
Dai Shunhong Zheng Ping Marmey Philippe Zhang Shiping Tian Wenzhong Chen Shouyi Beachy Roger N. Fauquet Claude 《Molecular breeding : new strategies in plant improvement》2001,7(1):25-33
We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T0, T1 and T2 transgenic plants. Oryza
sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hygr), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T1 and T2 generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T1 lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression. 相似文献
4.
Pooja Jha Shashi Anjana Rustagi Pankaj Kumar Agnihotri Vishvas M. Kulkarni Vishnu Bhat 《Plant Cell, Tissue and Organ Culture》2011,107(3):501-512
A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions
for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system,
without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin
phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A
number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration,
co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The
highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30 min with Agrobacterium O.D.600 = 1.2 under a negative pressure of 0.5 × 105 Pa and co-cultivated for 3 days in medium containing 400 μM acetosyringone. Histochemical GUS assay and polymerase chain
reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of
the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid
and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed
protocol will facilitate the insertion of desirable genes of useful traits into pearl millet. 相似文献
5.
Anindita Banerjee Sharmila Chattopadhyay 《In vitro cellular & developmental biology. Plant》2009,45(1):57-64
Phyllanthus amarus Schum & Thonn. is a source of various pharmacologically active compounds such as phyllanthin, hypophyllanthin, gallic acid,
catechin, and nirurin, a flavone glycoside. A genetic transformation method using Agrobacterium tumefaciens was developed for this plant species for the first time. Shoot tips of full grown plants were used as explants for Agrobacterium-mediated transformation. Transgenic plants were obtained by co-cultivation of shoot tips explants and A. tumefaciens strain LBA4404 containing the pCAMBIA 2301 plasmid harboring neomycin phosphotransferase II (NPT II) and β-glucuronidase
encoding (GUS) genes in the T-DNA region in the presence of 200 μM acetosyringone. Integration of the NPT II gene into the
genome of transgenic plants was verified by PCR and Southern blot analyses. Expression of the NPT II gene was confirmed by
RT-PCR analysis. An average of 25 explants was used, out of which an average of 19 explants produced kanamycin-resistant shoots,
which rooted to produce 13 complete transgenic plants. 相似文献
6.
F. D. Espasandin M. M. Collavino C. V. Luna R. C. Paz J. R. Tarragó O. A. Ruiz L. A. Mroginski P. A. Sansberro 《Plant Cell, Tissue and Organ Culture》2010,102(2):181-189
A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which
carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented
with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml−1) and cefotaxime (400 μg ml−1) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during
the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots
per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS
strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and
Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines
were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90%
of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase
from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter
showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the
same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the
cloned materials was established. 相似文献
7.
Nadolska-Orczyk A Pietrusinska A Binka-Wyrwa A Kuc D Orczyk W 《Cellular & molecular biology letters》2007,12(2):206-219
This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression.
In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based
on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%)
was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and,
depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants
where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein. 相似文献
8.
Isabel M.G. Padilla Agnieszka Golis Adele Gentile Carmine Damiano Ralph Scorza 《Plant Cell, Tissue and Organ Culture》2006,84(3):309-314
To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds
and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression
than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an
interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes
(56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing
GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants
but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes
were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes,
respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation
of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize
expression of this marker gene in peach. 相似文献
9.
A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions.
Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus
induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed
on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM l-cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet
genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions.
Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed
in four R1 progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R1 progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes
into the genome of finger millet in the future. 相似文献
10.
Thomas Altmann Brigitte Damm Wolf B. Frommer Thomas Martin Peter C. Morris Dieter Schweizer Lothar Willmitzer Renate Schmidt 《Plant cell reports》1994,13(11):652-656
Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM
Arabidopsis medium
- ANOVA
analysis of variance
- DAPI
4,6-Diamidino-2-phenylindole
- PEG
polyethyleneglycol 相似文献
11.
Puddephat I.J. Robinson H.T. Fenning T.M. Barbara D.J. Morton A. Pink D.A.C. 《Molecular breeding : new strategies in plant improvement》2001,7(3):229-242
In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers TL sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T0 plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T1 progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri TL-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of TL-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli. 相似文献
12.
Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia 总被引:3,自引:0,他引:3
Sergio G. Nebauer Isabel Arrillaga Lucas del Castillo-Agudo Juan Segura 《Molecular breeding : new strategies in plant improvement》2000,6(6):539-552
Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line. 相似文献
13.
We have generated putative promoter tagged transgenic lines inArachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated byAgrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4 mg/l in combination with 0.1 mg/l α-napthaleneacetic
acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters
enhancing genetic transformation viz. seedling age,Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation
with CN explants from 6-day-old seedlings co-cultivated withAgrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation
frequency was achieved with p35S GUSINT in Β-glucuronidase (GUS) assays. Among thein vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS
percentage. We have generated over 141 putative T0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the
green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase
chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression
or blue spots in at least one plant part. The progeny of 15 T0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression
patterns were more or less similar in both T0 and corresponding T1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic
lines in the present communication. 相似文献
14.
Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation
efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature
zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons
exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons,
at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a
higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic
potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant
survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival
of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement
of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and
subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from
initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments.
Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron
gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited
as a single Mendelian locus.
Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000 相似文献
15.
Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter
kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed
6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed
cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was
obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression
of the NOS gene in the selfed R1 progeny. NPTII activity was observed in the R2 generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this
procedure were unsuccessful. 相似文献
16.
An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA3 Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz 相似文献
17.
《Plant science》2002,162(5):697-703
We report a method for Agrobacterium-mediated transformation of Elatior Begonia (Begonia×hiemalis Fotsch). Young leaf discs were infected with Agrobacterium tumefaciens strains AGL0 and LBA4404. Each strain has a binary vector plasmid, pIG121Hm that includes the β-glucuronidase (GUS) gene with an intron as a reporter gene, and both the neomycin phosphotransferase II and the hygromycin phosphotransferase genes as selection markers. Explants were cultured on modified MS medium supplemented with 1.0 mg/l BA, 0.5 mg/l IAA, 300 mg/l ticarcillin, and either 100 mg/l kanamycin and 5 mg/l hygromycin, or 300 mg/l kanamycin for selection and regeneration. Out of 500 explants infected with AGL0, 16 plantlets were regenerated, and out of 628 explants infected with LBA4404, two plantlets were regenerated after 4 months of culture. Transformation was confirmed by Southern blot analysis of the GUS gene and by histochemical assays of GUS activity in plant tissues. Ten in vitro transgenic plants were obtained from AGL0 infected explants only. 相似文献
18.
An efficient and reproducible Agrobacterium-mediated genetic transformation of Withania coagulans was achieved using leaf explants of in vitro multiple shoot culture. The Agrobacterium strain LBA4404 harboring the binary vector pIG121Hm containing β-glucuronidase gene (gusA) under the control of CaMV35S promoter was used in the development of transformation protocol. The optimal conditions for the Agrobacterium-mediated transformation of W. coagulans were found to be the co-cultivation of leaf explants for 20 min to agrobacterial inoculum (O.D. 0.4) followed by 3 days of co-cultivation on medium supplemented with 100 μM acetosyringone. Shoot bud induction as well as differentiation occurred on Murashige and Skoog medium supplemented with 10.0 μM 6-benzylaminopurine, 8.0 μM indole 3-acetic acid, and 50.0 mgl?1 kanamycin after three consecutive cycles of selection. Elongated shoots were rooted using a two-step procedure involving root induction in a medium containing 2.5 μM indole 3-butyric acid for 1 week and then transferred to hormone free one-half MS basal for 2 weeks. We were successful in achieving 100 % frequency of transient GUS expression with 5 % stable transformation efficiency using optimized conditions. PCR analysis of T0 transgenic plants showed the presence of gusA and nptII genes confirming the transgenic event. Histochemical GUS expression was observed in the putative transgenic W. coagulans plants. Thin layer chromatography showed the presence of similar type of withanolides in the transgenic and non-transgenic regenerated plants. A. tumefaciens mediated transformation system via leaf explants developed in this study will be useful for pathway manipulation using metabolic engineering for bioactive withanolides in W. coagulans, an important medicinal plant. 相似文献
19.
Summary A rapid transformation and regeneration system has been developed forM. truncatula cv Jemalong (barrel medic) by which it is possible to obtain transgenic plants within 2.5 months. The procedure involvesAgrobacterium-mediated transformation of cotyledon explants coupled with the regeneration of transformed plants via direct organogenesis. To develop the procedure,M. truncatula explants were transformed with the binary plasmid pSLJ525 which carries thebar gene. Thebar gene encodes phosphinothricin acetyl transferase, and transformed plants were selected on media containing phosphinothricin (Ignite, AgrEvo). Transformed plants show phosphinothricin acetyl transferase activity and Southern blot analysis indicates that they carry thebar gene integrated into their genomes. The resistance to phosphinothricin is stable and is inherited by the R1 progeny as a single dominant Mendelian trait. The transgenic plants are highly resistant to the broad spectrum herbicide, Ignite and therefore may also have commercial applications. 相似文献
20.
Summary Freshly isolated cell suspension protoplasts ofSolanum dulcamara were mixed withAgrobacterium rhizogenes, allowed to settle for 2 h, exposed to electrical pulses and further incubated for 2h. Two pulses of 600 V cm–1 for 2 msec separated by 15 sec produced transformed colonies at relative and absolute transformation frequencies which were 3–4 and 10 fold greater than those obtained by co-cultivation of 3 days old protoplast-derived cells with bacteria. Transformed colonies were not produced when freshly isolated protoplasts were mixed withAgrobacterium but not electroporated. Biochemical analysis confirmed the transgenic nature of plants regenerated from protoplast-derived tissues.Abbreviations MS
Murashige and Skoog (1962)
- NPTII
Neomycin phosphotransferase II
- SDS
Sodium dodecylsulphate 相似文献