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1.
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
2.
Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of
``escape' shoots produced. Although the use of a wild-type ``shooty' Agrobacterium strain allowed relatively high frequencies of β-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type T-DNA and would not root.
Both use of a liquid medium/kanamycin overlay and horizontal placement of stem segments increased the efficiency of kanamycin
selection. Wounding via particle bombardment prior to Agrobacterium inoculation did not increase transformation frequencies. The concentration of benzyladenine (BA) in the regeneration/selection
medium inversely influenced the numbers of shoots that regenerated and the subsequent ability of the shoots to root. Regeneration
in the presence of kanamycin also influenced the ability of shoots to root. Many of the shoots that regenerated on selection
medium were chimeric for GUS expression, and plants established from such shoots ranged from non-staining to solidly staining
for GUS. However, solidly transformed plants with integrated T-DNA were obtained, and these plants have maintained the expression
of transgenes over several years. The transgenic plants include ones of sour orange (C. aurantium L.) and Key lime (C. aurantifolia (Christm.) Swing.), two species not previously transformed, and have integrated and express the coat protein gene of citrus
tristeza virus. This is the first report of a potentially agriculturally important transgene being expressed in Citrus.
Received: 8 October 1996 / Revision received: 1 April 1997 / Accepted: 18 April 1997 相似文献
3.
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
4.
Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants 总被引:1,自引:0,他引:1
G. A. Moore C. C. Jacono J. L. Neidigh S. D. Lawrence K. Cline 《Plant cell reports》1992,11(5-6):238-242
Summary A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage ( 5%) of the shoots regenerated in the presence of 100 g/ml kanamycin were GUS+. Polymerase chain reaction (PCR) analysis confirmed that GUS+ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis. 相似文献
5.
Development of insect-resistant transgenic cauliflower plants expressing the trypsin inhibitor gene isolated from local sweet potato 总被引:7,自引:0,他引:7
Agrobacterium-mediated transformation was used to introduce a trypsin inhibitor gene into Taiwan cauliflower (Brassica oleracea var. botrytis L.) cultivars. The TI gene was isolated from a well-adapted Taiwan sweet potato cultivar and was expected to be especially
effective in combating local pests. In vitro regeneration studies indicated that 4-day-old cauliflower seedling hypocotyl
segments, pretreated with 2,4-dichlorophenoxyacetic acid for 3 days and incubated on a silver-ion-containing shoot induction
medium, gave regeneration rates greater than 95%. Optimum transformation conditions were determined. G418 selection at 15
mg/l was initiated 1 week after cocultivation, and the dose was doubled 1 week later. Over 100 putative transgenic plants
were produced. Transgenic status was confirmed by in vitro TI activity, and Southern and Western hybridization assays. The
transgenic plants demonstrated in planta resistance to local insects to which the control plants were vulnerable.
Received: 21 July 1997 / Revision received: 27 February 1998 / Accepted: 16 March 1998 相似文献
6.
S. Krasnyanski R. A. May A. Loskutov T. M. Ball K. C. Sink 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(3-4):676-682
Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint
plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis
through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts,
they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced
with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both
transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types
of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations.
Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert
during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived
both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone,
low-menthol, high-menthofuran and –pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly,
around 1.2%, in transgenic plants produced by both methods.
Received: 22 November 1998 / Accepted: 4 Januar 1999 相似文献
7.
Summary A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) was developed using epicotyl segments. The segments were infected with Agrobacterium harboring the binary vector pBI121 or pBI101-O12-p1. Both vectors contained the neomycin phosphotransferase II (NPTII) and the -glucuronidase (GUS) genes. In the plasmid pBI101-O12-p1, the GUS gene was directed to the promoter region of ORF12 (rolC) of the Ri plasmid. On a selection medium containing 100 or 200 g/ml kanamycin, adventitious shoots were formed from 21.7–44.6% of the segments. Histochemical GUS assay showed that 55.4–87.7% of the shoots expressed the GUS gene. The stable integration of this gene was also confirmed by polymerase chain reaction (PCR) analysis and by Southern blot analysis. When the pBI101-O12-p1 plasmid was used, the GUS activity was found to be located in phloem cells of leaf, stem and root. More than 100 transformed plants were obtained using this method within 2–3 months. 相似文献
8.
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
9.
Robinia pseudoacacia (black locust) plants were regenerated after co-cultivation of stem and leaf segments with Agrobacterium tumefaciens strain GV3101 (pMP90) that harbored a binary vector that included genes for β-glucuronidase (GUS) and hygromycin phosphotransferase. Successful transformation was confirmed by the ability of stem and
leaf segments to produce calli in the presence of hygromycin, by histochemical and fluorometric assays of GUS activity in
plant tissues, and by Southern blotting analysis. In this transformation system, about 2 months were required for regeneration
of transgenic plants from stem and leaf segments. The frequency of transformation from stem segments was approximately 24%,
and the morphology of regenerated plants resembled that of the original parental strain.
Received: 2 September 1999 / Revision received: 30 November 1999 / Accepted: 4 December 1999 相似文献
10.
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
11.
Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 °C on kanamycin free shoot
regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm−3 kanamycin and 300 mg dm−3 cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm−3 kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl
explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival
rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on
transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved. 相似文献
12.
Perumal Venkatachalam Natesan Geetha Narayanasamipillai Jayabalan Saravanababu Lakshmi Sita 《Journal of plant research》1998,111(4):565-572
Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants
followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot
regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before
transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting
transformation efficiency are discussed. 相似文献
13.
Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues 总被引:3,自引:0,他引:3
Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured
with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with
8 g ⋅ l–1 agar, 7.5 mg ⋅ l–1 6-benzylaminopurine, 1.0 mg ⋅ l–1 -naphthaleneacetic acid, 300 mg ⋅ l–1 cefotaxime and 80 mg ⋅ l–1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants
produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically
transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem
segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments
produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident
in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican
lime transgenic plants were obtained, 60 of which were adapted to growing in soil.
Received: 15 March 1997 / Revision received: 30 December 1997 / Accepted: 19 January 1998 相似文献
14.
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
15.
Ufuk Celikkol Akcay M. Mahmoudian H. Kamci M. Yucel H. A. Oktem 《Plant cell reports》2009,28(3):407-417
A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found
to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application
of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection
regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with
Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced
with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS
assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to
soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T0, T1, T2 and T3 plants. PCR assays of T1, T2 and T3 progenies confirmed the stable transmission of the transgene to the next generations. 相似文献
16.
17.
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
18.
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
19.
Procedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of eight different citrus species [’Milam’ Rough lemon (Citrus jambhiri Lush), ‘Volkamer’ lemon (Citrus volkameriana L), Rangpur lime (Citrus limonia L), ‘Hamlin’ sweet orange (Citrus sinensis L Osbeck), ‘Duncan’ grapefruit (’Citrus paradisi’ Macf), Sour orange (Citrus aurantium L), ‘Cleopatra’ mandarin (Citrus reticulata Blanco) and Carrizo citrange (Citrus sinensis L Osbeck x Poncirus trifoliata L Raf) ] were plasmolyzed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v) ] prior to Agrobacterium inoculation. Plasmolyzed epicotyl explants were cocultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agl-1 (carrying pCAMBIA1303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and β-glucuronidase (GUS) genes. The binary vector, pCAMBIA1303 also contained a fused mGFP5 gene at the 3’ end of GUS gene as a reporter. Epicotyl explants of Rangpur lime, Rough and ‘Volkamer’ lemons plasmolyzed in 9–12 % maltose showed transient GUS gene expression comprising up to 95 % of the cut surface of explants, while Carrizo citrange showed 80 % expression when they were plasmolyzed in 6–10 % sucrose. On the other hand, epicotyl explants of ‘Hamlin’ sweet orange, Grapefruit, Sour orange and ‘Cleopatra’ mandarin showed transient GUS expession in 80–90 % of explants with 6–10 % sucrose. Basal portions of the regenerated putative transgenic shoots harvested from the cut surface of epicotyl explants within 2–3 months, were assayed for GUS, and apical portions were shoot-tip grafted in vivo for the production of whole plants. The transformation efficiencies in different species obtained are the highest so far reported for citrus. 相似文献
20.
Gaurab Gangopadhyay Subhash Kanti Roy Sangita Basu Gangopadhyay Kalyan Kumar Mukherjee 《Plant Cell, Tissue and Organ Culture》2009,97(3):295-302
A protocol for Agrobacterium-mediated transformation of a local ‘elite’ Indian variety (Queen) of pineapple [Ananus comosus (L.) Merr, family Bromeliaceae] has been established using a standard transformation vector (pCAMBIA 1304). High transformation
efficiency, expressed as the mean percentage of transgenic micro-shoots regenerated from initial callus explants (20.6%) was
achieved using a novel encapsulation-based, antibiotic selection procedure. The Agrobacterium-infected micro-shoots derived from callus explants survived selection in high concentration of hygromycin (60 mg l−1 and beyond) in encapsulated alginate beads. The integration of transgene in hygromycin-resistant shoots and plants was confirmed
by histochemical GUS assay, PCR amplification and Southern hybridization. It is possible to eliminate false antibiotic positives
in pineapple transformation program to a large extent following this procedure. 相似文献