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1.
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
2.
Summary A reproducible system has been developed for the production of transgenic plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for -glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg/l) was used as a selectable agent. Inclusion of acetosyringone (50M) in the Agrobacterium suspension and co-culture media proved to be indispensable for successful transformation. Transformation efficiency of Basmati 370 was 22% which was as high as reported in japonica rice and dicots. A large number of morphologically normal, fertile transgenic plants were obtained. Integration of foreign genes into the genome of transgenic plants was confirmed by Southern blot analysis. GUS and HygR genes were inherited and expressed in R1 progeny. Mendelian segregation was observed in some R1 progeny.Abbreviations GUS
ß-glucuronidase
- HygR
hygromycin-resistance
- AS
acetosyringone 相似文献
3.
Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens
Ming Cheng Robert L. Jarret Zhijian Li Aiqiu Xing James W. Demski 《Plant cell reports》1996,15(9):653-657
Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediated transformation system. Leaf section explants were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBI121 containing the genes for -glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Approximately 10% of the shoots regenerated on selection medium were GUS-positive. Five independent transformation events resulted in the production of 52 fertile transgenic peanut plants. On average, 240 d were required between seed germination for explant preparation and the production of mature t1 seed by T0 plants. Molecular analysis of transgenic plants confirmed the stable integration of the transgenes into the peanut genome. GUS expression segregated in a 31 Mendelian ratio in most T1 generation plants.Abbreviations GUS
-glucuronidase
- NPTII
neomycin-phosphotransferase II
- MS
medium, Murashige and Skoog medium (1962)
- BA
N6, enzyladenine
- NAA
1-naphthaleneacetic acid 相似文献
4.
E. C. Ulian J. M. Magill R. H. Smith 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1994,88(3-4):433-440
Transgenic petunia (Petunia hybrida Vilm.) plants were obtained from Agrobacterium-mediated shoot apex transformation. Studies at the phenotypic as well as molecular level established both the presence of the NPT II (neomycin phosphotransferase II) and GUS (-glucuronidase) genes and their level of activity. Twenty-nine primary transformed plants showed varying patterns of phenotype expression of both genes. NPT II and GUS expression in 7 primary plants over a 4-month interval showed varying levels of gene expression within and among individual plants. All primary transgenic plants were self-pollinated and backcrossed to establish the inheritance patterns of both genes. Mendelian and non-Mendelian inheritance patterns for both genes were observed. Analysis of the progeny showed poor transmission of the foreign genes through the pollen especially when two or more bands were present in the Southern hybridization. Most plants whose progeny segregated in Mendelian ratios for either the NPT II or GUS gene had just one copy of the gene. In this study where both foreign genes were examined in both self and test crosses, no transgenic plant showed Mendelian patterns of inheritance for both foreign traits.Department of Plant Pathology and Microbiology 相似文献
5.
K. Judith Webb Margaret J. Gibbs Sue Mizen Leif Skøt John A. Gatehouse 《Transgenic research》1996,5(5):303-312
The herbage legume,Lotus corniculatus (bird's-foot trefoil), was transformed using the disarmedAgrobacterium tumefaciens strain LBA4404 (pAL4404) carrying a binary construct, pJit73. This plasmid carries two antibiotic resistance genes,aphIV andnptII encoding resistance to hygromycin and kanamycin respectively, and the easily detectable reporter gene,uidA encoding the enzyme -glucuronidase (GUS). Transgenic plants were regenerated from two separate co-cultivations of leaves withA. tumefaciens either with or without an acetosyringone pretreatment. A total of 110 putative transformants were regenerated, 52 (47%) of which grew on selection media containing hygromycin. Twenty-five plants were analysed further for morphological variation and presence of transgenes and were used to study the inheritance of expression of the transgenes in the T1 generation. Expression patterns of the transgenes in the T1 progeny generated from these 25 plants differed. In the majority of plant linesaphIV anduidA transgenes segregated together, but the apparent number of copies of the transgenes varied. No expression of either transgene was detected in the progeny from three plants, while the progeny from six other plants were resistant to hygromycin but had no GUS expression. Progeny of all of the remaining 16 plants had GUS activity. For three plants, inheritance data were consistent with more than one dose ofuidA andaphIV; another two plants yielded data expected for exactly one dose of both transgenes. In the progeny of the remaining 11 plants, the percentage of seedlings expressing bothuidA andaphIV was lower than expected. 相似文献
6.
Jeffrey L. Carpenter Steven D. Kopczak D. Peter Snustad Carolyn D. Silflow 《Plant molecular biology》1993,21(5):937-942
In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active. 相似文献
7.
M.-J. Cho H. W. Choi B. B. Buchanan P. G. Lemaux 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(8):1253-1262
Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during
seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B1- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance
gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B1- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T0 leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T1 progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1
for GUS, one expressed bar alone, one lacked transmission of either gene to T1 progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T5 progeny in one line, T4 progeny in one line, T3 progeny in three lines and T2 or T1 progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous
lines the expression of the GUS protein, driven by either the B1- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T1 progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter
was gradually lost in T2 or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by
the hordein promoters in T2 or later generations. We conclude that the B1- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley
and potentially to modify barley seeds through genetic engineering.
Received: 28 May 1998 / Accepted: 19 December 1998 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.
Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants 总被引:3,自引:0,他引:3
Limanton-Grevet A. Jullien M. 《Molecular breeding : new strategies in plant improvement》2001,7(2):141-150
Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T1 progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T2 progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations. 相似文献
10.
Directed excision of a transgene from the plant genome 总被引:40,自引:0,他引:40
Sandra H. Russell Joyce L. Hoopes Joan T. Odell 《Molecular & general genetics : MGG》1992,234(1):49-59
Summary The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, -glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALSr), were integrated into the genome of tobacco and Arabidopsis. The ALSr gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALST gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The -glucuronidase gene remained active. The excision efficiency varied in F1 progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F1 progeny were chimeric and some F2 progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations. 相似文献