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1.
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
2.
Khanna Harjeet Becker Doug Kleidon Jennifer Dale James 《Molecular breeding : new strategies in plant improvement》2004,14(3):239-252
Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group (AAA) cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars (Cavendish and Lady Finger) and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) and gusA (-glucuronidase) or nptII (neomycin phosphotransferase) and a modified gfp (green fluorescent protein) gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome. T-DNA:banana DNA boundary sequences were amplified and sequenced in order to study the integration profile. 相似文献
3.
An improved protocol for genetic transformation of juvenile explants of Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.), Duncan (Citrus paradisi Macf.), Hamlin (Citrus sinensis (L.) Osbeck) and Mexican Lime (Citrus aurantifolia Swingle) cultivars using a vector containing a bifunctional egfp-nptII fusion gene is described. Several parameters were investigated to optimize genetic transformation of these four cultivars.
It was determined that a short preincubation in hormone rich liquid medium and subculture of Agrobacterium for 3 h in YEP medium containing 100 μM acetosyringone were required for improvement of transformation efficiency. Co-cultivation
duration as well as addition of acetosyringone to co-cultivation medium also played an important role in transformation efficiency
as did OD600 value of the Agrobacterium suspension used for transformation. We regenerated numerous EGFP expressing transgenic lines from all four cultivars. Based
on these results, we conclude that genetic transformation of citrus is cultivar specific and optimization of conditions for
maximum transgenic production are required for each individual cultivar. 相似文献
4.
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
5.
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
6.
Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants 总被引:11,自引:0,他引:11
R. Ghorbel J. Juárez L. Navarro L. Peña 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(1-2):350-358
The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration
of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital
marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed
from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus,
most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium
is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy
discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating
the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots
have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors
and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which
is currently a topic of much discussion for the commercialization of transgenic plants.
Received: 28 October 1998 / Accepted: 28 November 1998 相似文献
7.
Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.) 总被引:1,自引:0,他引:1
Nadolska-Orczyk Anna Orczyk Waclaw 《Molecular breeding : new strategies in plant improvement》2000,6(2):185-194
Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation. 相似文献
8.
Transformation of cereals via Agrobacterium and the pollen pathway: a critical assessment 总被引:1,自引:0,他引:1
Peter Langridge Reinhold Brettschneider Paul Lazzeri † Horst Lörz 《The Plant journal : for cell and molecular biology》1992,2(4):631-638
It has been proposed that transgenic plants of cereals can be generated by inoculating florets with Agrobacterium at or near anthesis. This procedure is shown to lead to the production of embryos of wheat and barley with enhanced resistance to antibiotic selection. It has also been possible to recover plants of wheat, barley and maize that gave positive hybridization signals with probes produced from within the T-DNA of the Agrobacterium vector. However, no evidence was found for transmission of the bands detected by hybridization in the progeny of the putative transgenic plants nor could enzyme activity associated with the resistance genes be found in plant extracts. Furthermore, undigested genomic DNA from the plants that were positive when probed with the T-DNA, showed hybridization to bands smaller than the genomic DNA. It is suggested that the apparent transformation is an artifact of the procedure and does not reflect transformation of the plant nuclear genome. 相似文献
9.
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
10.
Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems 总被引:3,自引:0,他引:3
K. Datta Z. Koukolíková-Nicola N. Baisakh N. Oliva S.K. Datta 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(6):832-839
A concise T-DNA element was engineered containing the rice class-I chitinase gene expressed under the control of CaMV35S and the hygromycin phosphotransferase gene (hph) as a selectable marker. The binary plasmid vector pNO1 with the T-DNA element containing these genes of interest was mobilized
to Agrobacterium
tumefaciens strain LBA4404 to act as an efficient donor of T-DNA in the transformation of three different indica rice cultivars from
different ecosystems. Many morphologically normal, fertile transgenic plants from these rice cultivars were generated after
Agrobacterium-mediated transformation using 3-week-old scutella calli as initial explants. Stable integration, inheritance and expression
of the chimeric chitinase gene were demonstrated by Southern blot and Western blot analysis of the transformants. Bioassay data showed that transgenic
plants can restrict the growth of the sheath blight pathogen Rhizoctonia solani. Bioassay results were correlated with the molecular analysis. Although we obtained similar results upon DNA-mediated transformation,
this report shows the potential of the cost-effective, simple Agrobacterium system for genetic manipulation of rice cultivars with a pathogenesis-related (PR) gene.
Received: 26 July 1999 / Accepted: 27 August 1999 相似文献
11.
Jaindra Nath Tripathi Abubaker Muwonge Leena Tripathi 《In vitro cellular & developmental biology. Plant》2012,48(2):216-224
In banana and plantain research, it is essential to establish embryogenic cell suspensions together with a highly efficient
regeneration and transformation system. This article describes the development of an embryogenic cell suspension (ECS), regeneration,
and transformation for plantain cv. “Gonja manjaya”. ECS was established using highly proliferative multiple buds. The frequency
of embryogenic friable callus formation was 56.8% of the cultured explants. Friable embryogenic calli with many translucent
proembryos were transferred to liquid medium and homogenous cell suspensions were established within 3–4 mo. Approximately
25,000 to 30,000 plants per 1.0 ml of settled cell volume were regenerated in approximately 13–14 mo. ECSs were transformed
using Agrobacterium strain EHA 105 harboring the binary vector pBI121. About 50–60 transgenic plants per 0.5 ml settled cell volume were regenerated
on selective medium containing 100 mg l−1 kanamycin. Histochemical GUS assays using different tissues of putatively transformed plants demonstrated stable expression
of uidA gene. The presence and integration of the uidA gene were confirmed by PCR and Southern blot analysis, respectively. This is the first report showing establishment of embryogenic
cell suspension cultures and Agrobacterium-mediated transformation of an important plantain cultivar, “Gonja manjaya”. This study shows the huge potential for genetic
transformation of plantains for disease or pest resistance, as well as tolerance to abiotic factors such as drought stress
using this robust regeneration and transformation protocol. 相似文献
12.
The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their
public acceptance and future commercialization. In addition, their elimination may allow gene stacking by the same selection
strategy. In apricot, selection using the selectable marker gene nptII, that confers resistance to aminoglycoside antibiotics, is relatively effective. An attractive alternative is offered by
the MAT system (multi-auto-transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with an MAT vector has been attempted
in the apricot cultivar ‘Helena’. Regeneration from infected leaves with Agrobacterium harboring a plasmid containing the ipt gene was significantly higher than that from non-transformed controls in a non-selective medium. In addition, transformation
efficiencies were much higher than those previously reported using antibiotic selection, probably due to the integration of
the regeneration-promoting ipt gene. However, the lack of an ipt expression-induced differential phenotype in apricot made difficult in detecting the marker genes excision and plants had
to be evaluated at different times. PCR analysis showed that cassette excision start occurring after 6 months approximately
and 1 year in culture was necessary for complete elimination of the cassette in all the transgenic lines. Excision was confirmed
by Southern blot analysis. We report here for the first time in a temperate fruit tree that the MAT vector system improves
regeneration and transformation efficiency and would allow complete elimination of marker genes from transgenic apricot plants
by site-specific recombination. 相似文献
13.
Gunvant Patil Amit Deokar P. K. Jain R. J. Thengane R. Srinivasan 《Plant cell reports》2009,28(11):1669-1676
To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose
isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented
medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed
using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of
20 g l−1 mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection
pressure of 15 g l−1 mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been
transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into
the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker
genes. 相似文献
14.
Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R1 progeny.Abbreviations BAP
6-benzylaminopurine
- NAA
1-naphthaleneacetic acid
- NPT
neomycin phosphotransferase 相似文献
15.
Emily Wu Brian Lenderts Kimberly Glassman Maya Berezowska-Kaniewska Heather Christensen Tracy Asmus Shifu Zhen Uyen Chu Myeong-Je Cho Zuo-Yu Zhao 《In vitro cellular & developmental biology. Plant》2014,50(1):9-18
Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum. 相似文献
16.
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
17.
Antonio-José López-Pérez Leonardo Velasco María Pazos-Navarro Mercedes Dabauza 《Plant Cell, Tissue and Organ Culture》2008,94(2):189-199
Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless
grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD600) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression
of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher
with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation.
However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD600 being more effective for the transformation of Crimson Seedless and 0.2 OD600 for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone
and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration
of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot. 相似文献
18.
Lei Sun Lin Zhou Miao Lu Ming CAI Xi-Wang Jiang Qi-Xiang Zhang 《Plant Molecular Biology Reporter》2009,27(1):102-108
In this study, a superbinary vector was constructed to evaluate the potential of a twin T-DNA system for generating selectable
marker-free transgenic chrysanthemum plants. The first T-DNA of the pCAMBIA 1300 vector contained the hygromycin phosphotransferase
(hpt) selectable marker gene, while the second T-DNA carried the β-glucuronidase gene (uidA) and featuring the gene of interest. The two T-DNA regions were placed adjacent to each other with no intervening region.
This vector was then used to transform transversal thin cell layers (1–2 mm thick) of internodal stem segments of chrysanthemum
via Agrobacterium-mediated transfer. Putative transgenic plants were obtained and analyzed for presence and integration of the transgene using
polymerase chain reaction amplification and Southern blotting. The primary cotransformation frequency was calculated at 38.4%.
A total of 17 hpt-resistant/gus-positive T0 plants were evaluated for segregation in the next generation (T1), and among those
approximately 15.7% carried the transgene. Overall, the two T-DNA system appeared to be a useful approach to generate marker-free
transgenic chrysanthemum plants, thereby eliminating public concerns regarding proliferation of antibiotic and herbicide resistance
genes into the environment. 相似文献
19.
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
20.
Agrobacterium-mediated transformation (AMT) of sugarcane has been limited by low transformation efficiency, high variability between experiments
and genotype specificity. We tested combinations of parameters that have been useful in other recalcitrant plant systems,
aiming to develop an efficient and reproducible method. Applied to elite sugarcane cultivar Q117, key parameters were (i)
minimal handling of callus near the time of co-cultivation, (ii) use of a super-binary helper vector with additional virB,C,G gene copies, and (iii) use of Agrobacterium strain AGL1. Transformation efficiency was in the range 0.5 to 3.5 stably transformed, embryogenic-callus-forming lines per
gram fresh weight of co-cultivated callus, over six independent callus batches. Addition of 5 μM copper sulphate to the callus-growth
medium appeared beneficial in a single further test. Following selection for aminoglycoside resistance conferred by PUbi-aphA, 87 % of transformed lines that formed embryogenic callus were regenerable to plants. Southern blot analysis of 24 transgenic
lines showed 21 % with a single-copy insertion of an intact T-DNA without vector backbone, and a mean transgene copy number
of 2.5. Over multiple batches, the AMT protocol approached the transformation efficiency from our routine conditions for particle
bombardment of Q117. However, the same parameters were ineffective for AMT of cultivars Q208 and Q172, and yielded a lower
transformation efficiency (0.02) with KQ228. As experienced in other systems such as rice, high-efficiency transformation
of one recipient genotype may provide useful starting parameters for work towards AMT of additional genotypes. 相似文献