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1.
Summary The ability to recover male gametophyte derived plants, which is necessary to get transformed haploid plants, was verified for a hybrid of maize. Using the isolated microspore culture technique, a 9 × 10–5 plant regeneration frequency was obtained. Maize microspores were bombarded with tungsten particles using a PDS He/1000 apparatus. GUS expression in the microspores was maximum with 1.1 m diameter tungsten microprojectiles for 1100 and 1350 psi helium pressures at a 6 cm distance between the launch point and the target cells. Increasing the amount of DNA coated on the microparticles from 1.66 to 4 g DNA/mg of particles allowed a two-fold and four-fold increase of the GUS-expressing microspore frequency for 1100 and 1350 psi helium pressure bombardment, respectively. Optimal concentration of solidifying agent in the bombardment support culture medium was found to be 1%. Cell density ranging from 25000 microspores/bombardment to 100000 microspores/bombardment did not affect the frequency of GUS-expressing microspores. Using these optimal conditions, the maximum frequency of GUS-expressing microspores was found to be about 9 × 10–4, while maintaining an embryo formation frequency about 5 × 10–4.Abbreviations GUS
-glucuronidase
- PEG
polyethylene glycol 相似文献
2.
Since its development in the mid-1980s, microprojectile bombardment has been widely employed as a method for direct gene transfer into a wide range of plants, including the previously difficult-to-transform monocotyledonous species. Although the numerous instruments available for microprojectile-mediated gene delivery and their applications have been widely discussed, less attention has been paid to the critical factors which affect the efficiency of this method of gene delivery. In this review we do not wish to describe the array of devices used for microprojectile delivery or their uses which have already been definitively described, but instead wish to report on research developments investigating the factors which affect microprojectile-mediated transformation of plants. 相似文献
3.
A novel protocol, based on biolistics and regeneration via organogenesis, was developed for genetic transformation of cassava
(Manihot esculenta Crantz). The in vitro performance of cassava cultivars CMC40, MPer183 and MCol22 was evaluated, and the regeneration protocol was modified to improve
shoot production from explants for transformation experiments. Somatic cotyledons were used as a target tissue in the transformation
experiments using the Particle Inflow Gun and a plasmid containing the uidA gene in transient assays. The effect of different parameters for particle bombardment efficiency, including the amount of
DNA used, the flying distance of the projectiles and the pre- and post-plasmolysis time of the target tissue, was evaluated
and the conditions were partially optimised. Stably transformed cassava plants of cvs. MCol22 and TMS60444 were produced using
the partially optimised conditions and two different vector constructs carrying the hpt gene as the selectable marker. The selection protocol was optimised further, and a rooting test was developed for screening
the regenerants for antibiotic resistance to reduce the number of escapes obtained after primary selection. The production
of stably transformed cassava lines and the expression of the transgenes was verified by Southern blot analysis and RT-PCR.
Received: 10 December 1999 / Revision received: 12 January 2000 / Accepted: 7 February 2000 相似文献
4.
玉米优良自交系愈伤组织基因枪转化的研究 总被引:12,自引:0,他引:12
利用基因枪将Bt基因导入玉米E28幼胚愈合组织中,在潮霉素浓度为5mg/L选择培养基上,经过35天的继代和分化2次选择及45天继代继续3次选择后分化再生成植株,PCR及Southern分子杂交结果表明,潮霉素基因及Bt基因已导入并整合到玉米基因组中,E282次选择的转化频率达到7.8%,显著地高于3次选择2.9%的转化频率,选择至再生的时间缩短25天;自交系Pal372次选择的转化频率为13.3% 相似文献
5.
Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems.
Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol
Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant
to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato. 相似文献
6.
Jason Arsenault Andras Nagy Jeffrey T. Henderson John A. O'Brien 《Journal of visualized experiments : JoVE》2014,(92)
Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.New modifications to the accelerator chamber have enhanced the gene gun''s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene''s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun. 相似文献
7.
本文报道了用基因枪法将雪花莲外源凝集素基因(gna)转移到优良籼型杂交稻保持系D297B中。通过PCR、Southern blotting和Western blotting等分子检测方法证明,外源基因已整合到受体材料的基因组中并得到表达。蛋白活性测定结果显示转基因在受体中表达的蛋白具有生物活性。潮霉素抗性分析表明外源基因多数是以单位点形式整合并以3∶1方式遗传。另外,PCR证据也说明抗虫基因与选择标记基因基本上是共整合和共遗传的 相似文献
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9.
The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l(-1) thidiazuron (TDZ) and subjected to bombardment after 5-7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding beta-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 mum gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l(-1) BA and hygromycin (20, 40 and 60 mg l(-1)) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T(0) and T(1) plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system. 相似文献
10.