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We present a simple and rapid method for screening second-generation transgenic rice plants (T1) to identify homozygous plants. The plasmid (pfd11) used for rice transformation contains a partially deleted cytochrome
c gene (cyc) for comparing with the endogenous cyc for copy number. After polymerase chain reaction (PCR) amplification of a segment
of the cyc in transgenic rice DNA followed by agarose gel electrophoresis, two specific bands are obtained. The upper band
represents the endogenous cyc, and the lower band represents the partially deleted cyc in the transgene. The first-generation
plants (T0) that harbor a single copy of the transgene are selected based on the fact that the density of the lower band is half as
dense as the upper band. Next, only plants harboring a single copy of the transgene are advanced to the second generation
(T1). The same PCR procedure is used again, and homozygous T1 plants are easily identified from samples in which the intensity of the two bands is the same. 相似文献
3.
A cytokinin biosynthetic gene encoding isopentenyl transferase (ipt) was cloned with its native promoter from Agrobacterium tumefaciens and introduced into tobacco plants. Indolebutyric acid was applied in rooting medium and morphologically normal transgenic
tobacco plants were regenerated. Genetic analysis of self-fertilized progeny showed that a single copy of intact ipt gene had been integrated, and T2 progeny had become homozygous for the transgene. Stable inheritance of the intact ipt gene in T2 progeny was verified by Southern hybridization. Northern blot hybridization revealed that the expression of this ipt gene was confined in leaves and stems but undetectable in roots of the transgenic plants. Endogenous cytokinin levels in
the leaves and stems of the transgenic tobaccos were two to threefold higher than that of control, but in roots, both the
transgenic and control tobaccos had similar cytokinin levels. The elevated cytokinin levels in the transgenic tobacco leaves
resulted in delayed leaf senescence in terms of chlorophyll content without affecting the net photosynthetic rate. The root
growth and morphology of the plant were not affected in the transgenic tobacco. 相似文献
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5.
M. V. Ramana Rao K. S. Behera N. Baisakh S. K. Datta G. J. N. Rao 《Plant Cell, Tissue and Organ Culture》2009,99(3):277-285
Transgenic rice was developed from ‘Swarna’, the most popular indica rice cultivar (Oryza sativa L.) in South East Asia, with a potato chymotrypsin inhibitor gene (pin2) through Agrobacterium-mediated transformation. Four out of nine primary transgenic plants had a single-copy T-DNA insertion while other five plants
had two copies. Mendelian pattern of inheritance of the transgene (pin2) was observed in the T1 generation progeny plants. Whole plant bioassays conducted at both vegetative and reproductive stages and cut stem assays
showed enhanced levels of resistance of transgenic rice against yellow stem borer. The transgenic rice lines with plant derived
proteinase inhibitor genes would develop into resistant cultivars to fit into resistance breeding strategies as an important
component of integrated pest management in rice. 相似文献
6.
Chidambaram Parameswari Rajasekaran Sripriya Karuppannan Veluthambi 《In vitro cellular & developmental biology. Plant》2010,46(5):395-402
In an analysis of 339 independent T
0 transgenic rice lines generated by Agrobacterium-mediated transformation, albino plants appeared in the T
1 generation in two single-copy transgenic lines, O54 and O36 and in one double-copy transgenic line, C18. While the T
0 plants of these three lines were green, albino and green plants emerged in a 1:3 ratio in the T
1 generation. The albino phenotype segregated as a monogenic recessive trait. Southern blot analysis of the green and albino
plants in the T
1 generation confirmed that the albino trait and the T-DNA insertion events were unlinked. Segregation of the albino trait
from the transgenic trait in the lines O54 and O36 was confirmed in T
2 and T
3 generations, respectively. Homozygous transgenic plants free from the albino trait were also identified. In the double-copy
transgenic line C18, we genetically separated the two transgenic loci, out-segregated the albino locus from both transgene
loci, and identified homozygous plants for each of the transgenic events by Southern blot analysis in the T
1 generation itself. Thus, we demonstrate that when an albino trait appears in the T
1 generation and is unlinked to a transgene locus, the albino locus can be segregated from the transgene locus and homozygous
transgenic lines free from albinos can be established. 相似文献
7.
Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies 总被引:5,自引:0,他引:5
A. Beaujean R. S. Sangwan M. Hodges B. S. Sangwan-Norreel 《Molecular & general genetics : MGG》1998,260(4):362-371
Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants. Here, we report differential
transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines,
thus demonstrating a gene dosage effect. Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and β-glucuronidase (uidA/ GUS), driven respectively by the mas 1′ and mas 2′ promoters, we have generated more than 150 independent transgenic (R0) Nicotiana tabacum plants containing one or more T-DNA copies. Transgene analyses of these R0, their selfed R1 lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome)
on uidA expression. However, transgene (GUS) expression levels were not proportional to transgene copy number. More than 150 haploids
and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R0 plants containing single and multiple copies of the uidA gene. We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental
haploid plants. This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number.
Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression.
In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R0 plants) to the homozygous (DH) state: e.g. an increase of 50% was observed in the homozygous DH as compared to the original
heterozygous diploid plants. We propose that ploidy coupled with homozygosity can result in a new type of gene activation,
creating differences in gene expression patterns.
Received: 27 April 1998 / Accepted: 12 August 1998 相似文献
8.
Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts 总被引:26,自引:0,他引:26
We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The To plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T1 and T2 progenies. Enzyme analyses on T1 progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of To plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants. 相似文献
9.
Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T-DNA borders 总被引:3,自引:0,他引:3
Madan K. Bhattacharyya Bruce A. Stermer Richard A. Dixon 《The Plant journal : for cell and molecular biology》1994,6(6):957-968
A new binary vector for Agrobacterium-mediated plant transformation was constructed, in which two selectable markers, for kanamycin and hygromycin resistance, were placed next to the right and left T-DNA borders, respectively, and a CaMV 35S promoter-driven β-glucuronidase (GUS) gene was placed between these markers as a reporter gene (transgene). Using double antibiotic selection, all transgenic tobacco plants carrying at least one intact copy of the T-DNA expressed the transgene, and this population exhibited reduced variability in transgene expression as compared with that obtained from the parent vector pBI121. Absence of the intact transgene was the major reason for transgenic plants with little or no transgene expression. Integration of truncated T-DNAs was also observed among transgenic plants that expressed the transgene and carried multiple T-DNA inserts. The copy number of fully integrated T-DNAs was positively associated with transgene expression levels in R0 plants and R1 progeny populations. Variability due to position effect was determined among 17 plants carrying a single T-DNA insert. The coefficient of variability among these plants was only 35.5%, indicating a minor role for position effects in causing transgene variability. The new binary vector reported here can therefore be used to obtain transgenic populations with reduced variability in transgene expression. 相似文献
10.
Takuma Ishizaki Takashi Kumashiro 《In vitro cellular & developmental biology. Plant》2011,47(3):339-347
In the use of genetic transformation in breeding, there are several possible problems including multiple copy insertion of
transgene, sterility caused by somaclonal variation and gene silencing. In this study, we characterized transgenic New Rice
for Africa (NERICA) produced by Agrobacterium-mediated methods with respect to copy number of transgene, fertility, and expression level of an introduced GUS gene. Southern
blot analysis of primary transformants demonstrated that about half of the events carried a single copy of the transgene regardless
of the cell density of Agrobacerium for inoculation. We examined ten procedures, consisting of different time periods and times of subculture for callus formation
and the starting times of hygromycin-based selection of transformed cells, for transformation of NERICA cultivars to produce
transformants within a short culture period at high frequency. A new culture method developed in this study required only
about 1.5 mo from the beginning of tissue culture to transformants, whereas a standard protocol we developed previously needed
about 2 mo of culture; however, it did not significantly reduce percentages of sterile plants. Fertile T0 plants produced fertile T1 plants at higher frequency. However, fertility was not inherited in a simple fashion: both fertile and partially sterile
T0 plants produced fertile, partially sterile and sterile T1 plants. Expression assay of an introduced GUS gene revealed position effects in seven independent homozygous transformed
lines carrying one copy of the transgene. Points to pay attention to in the use of genetic transformation in breeding are
discussed. 相似文献
11.
Protoporphyrinogen oxidase (Protox) in the porphyrin pathway is the target site of the peroxidizing herbicides such as carfentrazone-ethyl and oxyfluorfen. In an attempt to develop herbicide-resistant plants, transgenic rice plants were generated via expression of herbicide-insensitive Bacillus subtilis Protox gene fused to the transit sequence for targeting to the plastid using Agrobacterium-mediated gene transformation. Homozygous transgenic rice lines of T3 generation selected by hygromycin resistance test were examined if they are resistant to the herbicides carfentrazone-ethyl and oxyfluorfen. The homozygous transgenic lines had single copy insertion of B. subtilis Protox gene into their genomes and express its mRNA. Compared to wild-type rice, the transgenic lines were less susceptible to the herbicides when examined with respect to growth, electrolyte leakage, chlorophyll loss and lipid peroxidation. The in vitro Protox activities in transgenic lines were about 56 % higher than those in wild-type rice. With 10 µM concentration of the herbicides in the enzyme assays, Protox activities in transgenic lines were similar to those in non-inhibited wild-type rice. Less amount of protoporphyrin IX was accumulated in transgenic lines than in wild-type rice upon the treatment of the herbicides at 10 µM concentration. Our results indicated that expression of B. subtilis Protox gene was stably transmitted into T3 rice plants and reduced their sensitivity to carfentrazone-ethyl and oxyfluorfen.This work was supported by Ministry of Agriculture and Forestry of Korea and Agricultural Plant Stress Research Center (grant No. R11-2001-09203000-0) funded by Korea Science and Engineering Foundation. 相似文献
12.
Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny 总被引:17,自引:1,他引:16
Philippe Vain Barbara Worland Ajay Kohli John W. Snape Paul Christou George C. Allen William F. Thompson 《The Plant journal : for cell and molecular biology》1999,18(3):233-242
To investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing a gusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T0), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non-expressing lines compared to the population of plants without MARs. However, variation in β-glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T1), transgene expression levels were significantly correlated with those of the T0 parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals. 相似文献
13.
Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker 总被引:13,自引:0,他引:13
W. P. Chen X. Gu G. H. Liang S. Muthukrishnan P. D. Chen D. J. Liu B. S. Gill 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(8):1296-1306
Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression
of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was
to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter
and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded
with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration
medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T0) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected
35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal
and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T0 and T1 transgenic plants. Mendelian segregation of herbicide resistance was observed in some T1 progenies. Interestingly, a majority of the T1 progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression
under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated
in T1 transgenic wheat plants.
Received: 12 May 1998 / Accepted: 15 May 1998 相似文献
14.
Effective selection and regeneration of transgenic rice plants with mannose as selective agent 总被引:22,自引:0,他引:22
Lucca Paola Ye Xudong Potrykus Ingo 《Molecular breeding : new strategies in plant improvement》2001,7(1):43-49
A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T1 generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%. 相似文献
15.
J. Tu I. Ona Q. Zhang T. W. Mew G. S. Khush S. K. Datta 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(1-2):31-36
An elite indica rice variety, ‘IR72’, was transformed with a cloned gene, Xa21, through particle bombardment. Molecular analysis of transgenic plants revealed the presence of a 3.8-kb EcoRV-digested DNA fragment corresponding to most of the Xa21 coding region and its complete intron sequence, indicating the integration of Xa21 into the genome of ‘IR72’. In the T1 generation, the transgene was inherited and segregated in a 3:1 ratio. After inoculation with the prevalent races 4 and 6
of Xanthomonas oryzae pv. oryzae (Xoo), T1 plants positive for the transgene were found to be resistant to bacterial blight (BB). We also observed that the level of
resistance to race 4 of Xoo was higher due to the pyramiding of Xa21 and Xa4 present in ‘IR72’. Since the inactivation of the transgene Xa21 occurred in the two transgenic T1 plants, a larger progeny should be obtained for selecting homozygous line with a consistently higher level of resistance
to the BB pathogen.
Received: 13 October 1997 / Accepted: 21 October 1997 相似文献
16.
G. Sridevi N. Sabapathi P. Meena R. Nandakumar R. Samiyappan S. Muthukrishnan K. Veluthambi 《Journal of plant biochemistry and biotechnology.》2003,12(2):93-101
Agrobacterium-mediated transformation of an elite indica rice variety, Pusa Basmati 1, was performed using LBA4404 (pSB1, pMKU-RF2) that harbours a rice chitinase gene (chi11) under the control of the maize ubiquitin (Ubi1) promoter-intron. Right border (gus) and left border (hph) flanking sequences and the transgene (chi11) in the middle of the T-DNA were used as probes in Southern analysis. Out of eleven independent T0 plants regenerated, three had single copy T-DNA insertions and eight had multiple T-DNA insertions. Nine T0 plants carried the complete T-DNA with the chitinase transgene. Two T0 plants did not carry chi11, though they had other T-DNA portions. Three plants harbouring single copy insertions and one plant harbouring two inserted copies were analyzed in detail. A segregation ratio of 3:1, reflecting T-DNA insertion at a single locus, was observed in the progeny of all the four T0 plants. Northern and western blot analyses of T1 plants revealed constitutive expression of chitinase at high levels. Bioassays of T1 plants indicated enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, in comparison to control plants. A homozygous transgenic line was established from one T0 line, which exhibited the maximum resistance to R. solani. 相似文献
17.
S. Zhang M.-J. Cho T. Koprek R. Yun P. Bregitzer P. G. Lemaux 《Plant cell reports》1999,18(12):959-966
Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial
varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and
bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic
lines produced T0 plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T1 seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T1 and T2 progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T1 and T2 progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T1 and T2 progeny tested.
Received: 4 January 1999 / Accepted: 14 January 1999 相似文献
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19.
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
20.
Inheritance of gusA and neo genes in transgenic rice 总被引:21,自引:0,他引:21
Jianying Peng Fujiang Wen Richard L. Lister Thomas K. Hodges 《Plant molecular biology》1995,27(1):91-104
Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T0) transformants and their progeny plants. T0 plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T1 to T2 plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T2 generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice. 相似文献