Agrobacterium Cell Density Research Articles

A simple and effective system for foreign gene expression in plants via root absorption of agrobacterial suspension

Due to the laborious and scale-up limitation we have developed a simple system named “root absorption” to express foreign proteins in plants successfully. It has been shown that GFP was expressed in tobacco plants by root absorbing the Agrobacterium suspension containing TMV-based P35S-30B-GFP vector. Various factors influencing the gene expression were studied including Agrobacterium cell density, seedling age, plant materials and inoculation conditions. This system has the special advantages as simple and convenient work process, ease to scale-up and higher level of expression than leaf infiltration. Interestingly, GFP was expressed at 24 h post-absorption. We assume that the root absorption system will facilitate the large-scale production of the recombinant pharmaceutical proteins in plants by means of transient expression.

Factors influencing T-DNA transferring into pollen of lily in vitro

Direct pollen transformation method improves the classical transformation procedures because some tissue culture steps and subsequent regeneration can be avoided. A critical step in the development of Agrobacterium-mediated transformation is the establishment of optimum conditions for T-DNA delivery into tissue. The pollen grains of David lily (Lilium davidii Duchartre) are transformable by Agrobacterium during their germination, and extremely high GUS expression frequency of pollen had been achieved (92.7 ± 2.7%), but not for the ungerminated pollen. The culture medium, Agrobacterium cell density, duration of co-cultivation, and the combination of bacterial strains and plasmids should be optimized to get the highest transformation frequency. Thus, a method for pollen monocotyledonous species reproductive tissues transformation by Agrobacterium in monocots has been successfully developed.

이탈리안 라이그래스의 형질전환에 미치는 몇 가지 요인의 영향

ABSTRACT A system for the production of transgenic plants has been developed for Italian ryegrass( Lolium multiflorum Lam.) via Agrobacterium -mediated transformation of embryogenic callus. Mature seed-derived calli wereinfected and co-cultured with Agrobacterium EHA101 carrying standard binary vector pIG121Hm encoding thehygromycin phosphotransferase(HPT), neomycin phosphotransferase II (NPTII) and intron-containing - βglucuronidase(intron-GUS) genes in the T-DNA region. The effects of several factors on transformation and theexpression of the GUS gene were investigated. Inclusion of 200 M acetosyringone(AS) in inoculation andμco-cultivation media lead to a significant increase in stable transformation efficiency. Increasing Agrobacterium cell density up to 1.0 in OD 600 during infection increased transformation efficiency of embryogenic calli. Thehighest transformation efficiency was obtained when embryogenic calli were incoulated with Agrobacterium inthe presence of 0.1% Tween20 and 200 M AS. Hygromycin resistant calli were developed into complete plantsμvia somatic embryogenesis. GUS histochemical assay and PCR analysis of transgenic plants demonstrated thattransgenes were integrated into the genome of Italian ryegrass.(

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps

The factors influencing transfer of an intron - containing β-glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of β-glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of β-glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of β-glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.