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1.
Summary An efficient transformation system for Chinese cabbage cotyledon explants was developed using Agrobacterium tumefaciens strains LBA4404 harbouring the plasmid pMOG 411 and the plasmid pBinΩSCK respectively. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings, growth conditions and status of Agrobacterium suspension, preculture of explants, cocultivation time, ratio between Agrobacterium and explants, acetosyringone and concentration of kanamycin had a significant influence on transformation frequency and plant regeneration. The presences of the antibacterial peptide gene and the cowpea trypsin inhibitor gene in those selected shoots in kanamycin medium were confirmed by PCR, Southern blotting and Northern blotting, The frequency of regeneration from cotyledons was analyzed in eight parental lines of Chinese cabbage and five lines were suitable for obtaining transformed plants, among which Qingmaye C and Qingmaye D were more responsive than other lines. 相似文献
2.
Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) 总被引：1，自引：0，他引：1
An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic
resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium
219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive
plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining
17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern
blot analysis.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
3.
选用鲁花14与花育23花生品种, 通过农杆菌介导开展了轮状病毒抗原蛋白VP4基因遗传转化研究, 从转化植株中随机选取26株表现Kan抗性植株进行npt II基因的PCR检测, 结果有22株能扩增出620 bp左右的npt II基因条带, 阳性率约为84.62%。提取npt II基因显示为阳性的植株叶片DNA作模板, 用VP4基因特异引物进行PCR扩增, 经琼脂糖凝胶电泳分析, 所有npt II基因阳性的植株均扩增出了约2 350 bp的特异性条带, 而野生型没有。对部分转基因植株进一步进行PCR-Southern杂交和Southern杂交分析, 发现转基因植株中出现了阳性杂交信号, 表明VP4基因的确已经整合到花生的基因组中, 并且是1~2个拷贝。用RT-PCR分析了11株转G1VP4基因的植株, 证明插入鲁花14中的VP4基因已经正常转录, 利用Western blot方法检测筛选到的4株转基因花生, 分别提取其蛋白, 在30 kD处出现特异性蛋白条带, 这为获得转基因创新种质材料奠定了基础。 相似文献
4.
John L. Norelli Herb S. Aldwinckle Luis Destéfano-Beltrán Jesse M. Jaynes 《Euphytica》1994,77(1-2):123-128
Summary Apple (Malus domestica) transgenic T1 was obtained byAgrobacterium tumefaciens-mediated transformation of Malling 26 rootstock using the plasmid binary vector pLDB 15. pLDB 15 contains within its T -DNA
a gene encoding the lytic protein attacin E. The integration of the attacin E gene into the apple genome was confirmed by
Southern analysis. Northern analysis indicated the presence of an attacin E mRNA in plants inoculated withErwinia amylovora. After inoculation ofin vitro grown plants of T1, Malling 26, and Malling 7 (resistant control) withE. amylovora, the loglo of the inoculum concentration lethal to 50% of the plants was 5.4, 4.4, and 5.6, respectively. In greenhouse trials
for resistance to fire blight, T1 was significantly more resistant than ‘Mailing 26’. 相似文献
5.
农杆菌介导加幼苗直接形成将Bt-cry1A(b)基因转入印度棉花 总被引：2，自引：0，他引：2
利用农杆菌介导法用Bt-cry1A(b)基因对印度栽培棉种进行非基因依赖型遗传转化和植株再生.将印度栽培棉品种Anjali(LRK-516)和LRA-5166与携带Bt-cry1A(b)+nptⅡ基因的根癌农杆菌LBA4404共培养,在含100tμg@ml-1卡那霉素的筛选培养基上获得了可能的转基因直接再生苗.细菌浓度、共培养时间、感染组织的阶段和大小、标记筛选浓度、培养基成分和激素等都影响转化效果和效率.对程序进行了优化.经PCR、Southern杂交、ELISA等方法分别检测,证实了Bt-cry基因的插入和表达.Southern分析表明转化植株存在3～5个基因拷贝,但CRY蛋白表达量非常低(为叶蛋白的0.003%～0.004%)且生化抗虫性很弱或基本不影响鳞翅目昆虫.尽管如此,该方案仍适用于其它CRY基因或重要经济型基因进行非基因依赖型遗传转化和再生,产生转基因棉花. 相似文献
6.
Morad Jafari Peyman Norouzi Mohammad Ali Malboobi Behzad Ghareyazie Mostafa Valizadeh Seyed Aboulghasem Mohammadi Mozhgan Mousavi 《Euphytica》2009,165(2):333-344
Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation.
PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment
with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot
blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis.
The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic
plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance
of the inserted transgene was confirmed in F1 plants obtained through crossing of T0 plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected
to further bioassay analyses against other lepidopteran pests of sugar beet. 相似文献
7.
利用黄籽甘蓝型油菜自交系建立和优化了遗传转化系统。首先构建了由质粒pCNR与Δ6脂肪酸脱氢酶基因插入到植物的高效表达载体pCAMBIA2301G。利用在Murashige和Skoog培养基(含有200 mmol L–1乙酰丁香酮)培养5~7 d的下胚轴外植体与农杆菌株LBA4404共培养63~69 h (pCNR),再于芽诱导培养基上培养3个月诱导芽再生。在最佳条件下,平均转化效率约为1.3%。转化植株的GUS分析和PCR分析结果表明,外源基因成功导入甘蓝型油菜。Southern杂交表明,这些转化子含有目标基因1~2个拷贝。用气相色谱分析转基因植物种子的脂肪酸,γ-亚麻酸含量达8.2%。 相似文献
8.
Molecular genetic mapping of peach 总被引：17，自引：0，他引：17
Summary A project to develop a linkage map of the peach (Prunus persica) genome is underway using an F2 population segregating for several morphological characters and pest resistance e.g., nectarine
(g), weeping shape (pl) and aphid resistance (Rml). The RAPID technique was used to analyse 270 plants. Linkage analysis of the F2 population was performed using the MAPMAKER
software. Eight linkage groups were established and RAPID markers flanking thepl gene were found. 相似文献
9.
Daniel Zohary 《Euphytica》1992,60(1):75-77
Summary The mode of origin of the hexaploid (6x=48) European plum Prunus domestica has been re-examined. It is argued that the evidence in support of an allo-polyploid origin and the participation of 4x P. spinosa in the formation of this fruit tree is far from being satisfactory. Instead, the morphological affinities between P. domestica, P. spinosa and P. cerasifera (= P. divaricata) and the available cytogenetic evidence seem to implicate 2x, 4x, 6x P. cerasifera as the sole wild stock from which the cultivated 6x European plum could have evolved. 相似文献
10.
Summary Rhizomania is a disease of sugar beet caused by the furovirus beet necrotic yellow vein virus (BNYVV). Coat protein mediated resistance has been reported for a number of viral diseases. This approach to virus resistance was therefore attempted for control of rhizomania. Two constructs of the coat protein gene of BNYVV were introduced into sugar beet by Agrobacterium-mediated transformation. The mRNA level was estimated to be 0.01% of the poly A+ RNA. Expression of the coat protein gene was under the detection limit of our western blotting protocol i.e. below 0.01 g/50 g (0.02% of the total soluble protein). One transformation event per construct was tested in a greenhouse assay and in rhizomania infested soil in a field trial. In the greenhouse assay, transgenic plants showed a strong reduction of virus multiplication when compared to non-transgenic plants. This result was confirmed in the field trial, where a significant difference in virus multiplication was shown between plants with and without the coat protein gene. 相似文献
11.
为建立一个稳定的谷子遗传转化体系,对216份谷子种质资源进行筛选,确定材料178成熟胚为受体材料,从植物激素浓度、防褐化剂、农杆菌菌液浓度、侵染时间、乙酰丁香酮（AS）浓度等方面对谷子再生及转化体系进行优化。结果表明,在MS培养基中加入生长素2,4-D 9μmol/L、细胞分裂素Kinetin 4μmol/L、硝酸银8mg/L,愈伤组织诱导率最高且能有效防止褐化。在侵染液和共培养基中加入100μmol/L AS,gus表达效果最好。农杆菌溶液OD600为0.3~0.5,侵染15min,共培养3d时,抗性愈伤组织率最高。获得的转基因植株,经PCR检测及gus染色分析,确定外源bar基因已成功整合到谷子基因组中。初步建立了一套谷子遗传转化体系,为今后谷子遗传转化提供一些参考。 相似文献
12.
Summary Semilooper resistant transgenic castor plants were produced through Agrobacterium-mediated genetic transformation method. Two castor cultivars, Jyothi and VP1 were transformed using the super-binary vector
pTOK233 carrying gus A and hpt genes. Putative transformants were regenerated following selection on the hygromycin containing medium. GUS positive primary
transformants, when subjected to Southern analysis, revealed stable integration of gus A into their genomes. In the T1 generation, a monogenic segregation ratio of 3 GUS positive: 1 GUS negative plants was observed. Furthermore, transformation
experiments were carried out with the Agrobacterium pSB111 super-binary vector carrying a synthetic delta endotoxin gene cryIAb and the herbicide resistance gene bar both driven by cauliflower mosaic virus 35S promoter. Putative transformants were regenerated through selection on the phosphinothricin
containing medium and Basta tolerant transformants were subjected to molecular analysis. PCR analysis revealed the presence
of both bar and cryIAb genes in the Basta tolerant primary transformants. Southern analysis of PCR positive plants with cryIAb probe showed a 3 Kb band upon HindIII digestion and a > 6 Kb band with BamHI digestion, thus suggesting stable integration of cryIAb intact expression cassette and independent nature of the transformants. The primary transformants subjected to ELISA disclosed
varied levels of Cry protein. These transgenics expressing cryIAb – when bioassayed against freshly hatched semilooper larvae – induced substantial (> 88%) insect mortality. Southern analysis
of 2T1 plants revealed the presence of cryIAb gene, indicating stable inheritance of the transgene into the next generation. In T1, all the Southern-positive plants for cryIAb invariably exhibited tolerance to Basta, denoting co-segregation of both bar and cryIAb genes. Transgenics, expressing cryIAb exhibited ample resistance against the castor semilooper. 相似文献
13.
14.
花叶病毒(soybean mosaic virus, SMV)病是大豆主要病害之一,生产上常采用种植抗性品种方法来防治。本研究以RNA干扰花叶病毒衣壳蛋白(coat protein, CP)基因为表达载体,Bar基因作为筛选标记基因,成熟子叶节为外植体,采用农杆菌介导法获得了22株T0代转基因大豆生根苗,经草丁膦涂抹、Bar试纸条和PCR法鉴定,获得RNAi CP转基因植株18株;对转基因植株T1代的遗传分析表明,外源基因能够稳定遗传到下一代且符合孟德尔遗传规律;T1代Southern杂交表明,导入的干扰片段为单拷贝;花叶病毒摩擦接种表明RNAi CP转基因大豆植株具有抗花叶病毒特性;摩擦接种后3周,DAS-ELISA检测进一步表明,RNAi CP转基因植株花叶病毒检出率仅为7.69%,而非转基因植株为100%。这表明RNAi花叶病毒CP基因可用于抗大豆花叶病毒的研究。 相似文献
15.
Summary The almond of commerce (Prunus dulcis (Mill.) D.A. Webb) is self-incompatible (SI) and requires honey-bees to effect the transfer of pollen among cultivars that flower simultaneously. Four year old trees from the F2 generation of several peach x almond hybrids were studied to determine whether self-compatibility (SC) and the potentiality for natural, i.e., abiotic, self pollination (NSP) are genetically related or are inherited independently. Both SC and the high potentiality for NSP are characteristic of peach (Prunus persica (L.) Batsch) but not almond. Forty percent of SC genotypes exhibited adequate NSP (SC, +NSP) for good fertility i.e., without insect-mediated pollination. The remaining 60% of SC genotypes (SC,-NSP) exhibited an average 61% reduction in fruit set on limbs bagged to exclude honeybees during anthesis relative to fruit set on open pollinated limbs. Our data are consistent with the concept that fertility is dependent upon the load of compatible pollen deposited on the stigma. Fruit set reduction on bagged limbs, compared with bagged and self-pollinated limbs, was presumably due to a) lack of/insufficient pollination for fertilization and/or b) post-zygotic abortion of genetically inferior recombinants. Selection following manual self-pollination may result in SC genotypes with or without the capacity for NSP. In contrast, significant fruit set on limbs enclosed during pistil receptivity necessitates that the genotype selected express both SC and the potentiality for NSP. 相似文献
16.
Most potato transgenic research has focused on development of resistance to pathogens and modification of potato physiology.
Many transgenes, particularly those conferring pathogen resistance, could substantially lower potato production costs in developing
countries. However, transgenes have not been reported in sexually propagated 4x-2x potato hybrids commonly grown in developing countries. Two transgenes,the Bacillus thuringiensis cry3Aa endotoxin protein gene and the PVY°coat protein gene, were engineered intodiploid and tetraploid potato using Agrobacterium tumefaciens-mediated transformation. Cry3Aa was produced at high levels in several lines while the PVY° coat protein was not expressed.
Diplandroid and tetraploid genotypes were crossed to produce transgenic 4x-2x hybrids. Genetic transformation had no discernable effect on fertility ofthe primary transformants, germination of4x-2x seed derived from the transformants and agronomic performance(tuber set, average tuber weight and total tuber yield) of the
4x-2xhybrids. The transgenic 4x-2xhybrids produced non-viable pollen and could only be crossed as female parents. Results suggest that transgenes, such ascry3Aa, could be expressed in 4x-2x hybrids to lower costs of production with no significant effect on plant phenotype.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
17.
Summary Pollen grain germination inside anthers has so far been known in only eleven chasmogamous angiospermous species. The discovery of this phenomenon in some varieties of Prunus amygdalus (almond) and Malus pumila (apple) is therefore significant. The anomaly appears to be genetically controlled, the gene expression occurring under specific environmental conditions. 相似文献
18.
改良大豆子叶节再生体系的研究 总被引：15，自引：0，他引：15
采用大豆种子萌发5~6 d后的子叶节作外植体，对农杆菌介导的大豆遗传转化系统及其影响因素进行了研究。结果表明，将子叶节与农杆菌共培养60 h后放入含卡那霉素50 mg/L的诱芽培养基上，待苗长至4~5 cm后放入生根培养基中进行生根，最后移栽到培养土中，效果较好。对大豆遗传转化再生的主要因素，如大豆基因型、诱导出芽所需激素浓度、卡那霉素筛选压力等条件做了一系列的研究，建立了一个比较好的遗传转化系统，其转化率达到2.8%。 相似文献
19.
M. Hill K. Launis C. Bowman K. McPherson J. Dawson J. Watkins M. Koziel M. S. Wright 《Euphytica》1955,85(1-3):119-123
Summary A synthetic Bt gene encoding a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis was successfully introduced into elite maize using microprojectile bombardment of immature embryos. The method used to initiate and identify transformation events is described. We describe the detailed parameters used for the Biolistics device as well as the plasmids used for the transformations. The plasmids contained the synthetic Bt gene driven by either the 35S CaMV promoter or a combination of two tissue-specific promoters, leaf and pollen, derived from maize. Specific conditions for the culture of Type I callus from immature embryos, the phosphinothricin (PPT) selection protocol, and the regeneration of plants are discussed. T0 and T1 plants were initially identified using the pH-dependent chlorophenol red test and/or the histochemical -glucuronidase (GUS) assay. PCR and Southern data confirm the presence of the 35S CaMV promoter and the synthetic Bt gene. 相似文献
20.
选用鲁花14与花育23花生品种, 通过农杆菌介导开展了轮状病毒抗原蛋白VP4基因遗传转化研究, 从转化植株中随机选取26株表现Kan抗性植株进行npt II基因的PCR检测, 结果有22株能扩增出620 bp左右的npt II基因条带, 阳性率约为84.62%。提取npt II基因显示为阳性的植株叶片DNA作模板, 用VP4基因特异引物进行PCR扩增, 经琼脂糖凝胶电泳分析, 所有npt II基因阳性的植株均扩增出了约2 350 bp的特异性条带, 而野生型没有。对部分转基因植株进一步进行PCR-Southern杂交和Southern杂交分析, 发现转基因植株中出现了阳性杂交信号, 表明VP4基因的确已经整合到花生的基因组中, 并且是1~2个拷贝。用RT-PCR分析了11株转G1VP4基因的植株, 证明插入鲁花14中的VP4基因已经正常转录, 利用Western blot方法检测筛选到的4株转基因花生, 分别提取其蛋白, 在30 kD处出现特异性蛋白条带, 这为获得转基因创新种质材料奠定了基础。 相似文献