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分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3:1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒(SMV)并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。 相似文献
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To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T8 plant, having uidA (or gus) driven by the barley endosperm-specific B1-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T4 plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H).
Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F1 progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F2 seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental
plants. FISH was used to screen F2 plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH
results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating
that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F3 and F4 generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably
expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such
stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies. 相似文献
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Narender S. Nehra Ravindra N. Chibbar Nick Leung Karen Caswell Cliff Mallard Lee Steinhauer Monica Baga Kutty K. Kartha 《The Plant journal : for cell and molecular biology》1994,5(2):285-297
A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny. 相似文献
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Expression and inheritance of foreign genes in transgenic peanut plants generated byAgrobacterium-mediated transformation 总被引:11,自引:0,他引:11
To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T2 plants gave either 100% or 3:1 expression of the -glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T1 plants. Fluorometric GUS assay in T1 and T2 generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.Abbreviations
GUS
-Glucuronidase
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MS
Murashige and Skoog
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MU
4-Methylumbelliferone
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NPTII
Neomycin phosphotransferase II 相似文献
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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
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Asad S Mukhtar Z Nazir F Hashmi JA Mansoor S Zafar Y Arshad M 《Molecular biotechnology》2008,40(2):161-169
A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for
cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were
treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and
selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic
callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene
copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1
AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is
first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate
fertile cotton transformants. 相似文献
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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
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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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The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants 总被引:10,自引:0,他引:10
James VA Avart C Worland B Snape JW Vain P 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(4):553-561
Segregating T1, T2 and T3 transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in
order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected
β-glucuronidase (gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression
level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene
expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation
events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene
silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors
influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering
molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing
the likelihood of an additive effect on transgene expression level is discussed.
Received: 21 March 2001 / Accepted: 29 June 2001 相似文献
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To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium
tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD600 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion
of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding
explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea,
binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T0 transgenic plants were generated, representing 3.6% transformation frequency. T0 transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T1 progeny. PCR, RT-PCR, and Southern hybridization analysis of T0 and T1 transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein
in T0 and T1 transgenic chickpea plants was achieved maximum up to 116 ng mg−1 of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea
for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development
of chickpea plants with novel traits. 相似文献