根癌农杆菌对单子叶植物竹子的细胞转化

本实验证明:具有重要经济价值的禾本科植物——竹子(bamboo)可被根癌农杆菌转化。在经有毒性的根癌农杆菌C_(580、A_(348)感染的竹子材料中,分别测到了专性的胭脂碱和章鱼碱,而对照处理及无毒性的根癌农杆菌VirA~-_(211)mx、NTI感染的竹子材料中,测不到冠瘿碱。     

Agrobacterium and plant genetic engineering

Erratum

Agrobacterium and plant genetic engineering     

Agrobacterium rhizogenes mediated induction of apparently untransformed roots and callus in chrysanthemum

The agropine type Agrobacterium rhizogenes strain LBA9402 induced callus and roots on stems of greenhouse grown plants and on leaf disks of in vitro grown plantlets of chrysanthemum (Dendranthema grandiflora Tzvel.). In this callus and roots no opines were detected, nor were any of the other features of the hairy root syndrome observed. Experiments aimed to identify the nature of the tumour-like growth revealed that induction was correlated with the presence of the TR-DNA on the Ri-plasmid. Root induction was probably the result of auxin synthesis following transient expression of iaaM and iaaH genes, present on the TR-DNA. The chrysanthemum cultivar used, cv. Parliament, showed a high auxin sensitivity compared to tobacco. Analysis of early transformation events using the GUSintron reporter gene revealed that low efficiency gene transfer and transient gene expression took place, but most probably without stable integration of the T-DNA in the plant genome. The results presented here stress the fact that callus formation or root induction as measures for transformation efficiency should be used with caution.     

Modeling growth and succinoglucan production by Agrobacterium radiobacter NCIB 9042 in batch cultures

Wild-type Agrobacterium radiobacter NCIB 9042 has been cultivated in batch cultures on a synthetic medium which was adapted for growth and succinoglucan production. Experiments were carried out in a 4-L stirred-tank aerated reactor. Glucose, biomass, polysaccharide, protein, and inorganic- and organic-nitrogen concentrations were measured, and oxygen consumption and CO(2) production rates were obtained by a gas-balance technique. Nitrogen balance shows that inorganic nitrogen is entirely recovered into proteins. The carbon balance is satisfied with in +/-5%. Stoichiometric equations for biomass growth and succinoglucan synthesis were established. The biosyntheticpolymer pathways including ATP and cofactor consumption were investigated. From previous studies, a (P/O) value of 1.66 is selected for oxygen sufficient cultures. The actual ATP requirements of 25.4 mmol ATP/g succinoglucan (38.5 mol ATP/mol succinoglucan), determined by a metabolic analysis, is 2.39 times the stoichiometric value. Experimental results were modeled by a system of differential equations. The exponential growth phase was described by a nitrogen-limited Monod equation. Subsequent succinoglucan synthesis followed a slightly modified Luedeking-Piret relation partitioning internal and external polysaccharide. Experimentally determined coefficients are compared with published results for continuous culture of A. radiobacter NCIB 11883.     

Monitoring hairy-root growth by image analysis

Hairy roots, incited byAgrobacterium rhizogenes, form a useful system for analysing the expression and phenotypic effects of foreign genes in plant root tissue. Image analysis offers a non-invasive method of describing their growth in culture. Images of pea (coarse) andBrassica (fine) hairy roots were captured, processed and analysed without difficulty using a commercially available image analysis system. The value of this method in monitoring intermediate changes in growth pattern was illustrated by following the changes in five putatively chlorsulfuron-resistantBrassicc hairy-root lines cultured with and without a selective level of chlorsulfuron. Areas of hairy-root research where this technique will be particularly useful are discussed.     

Broad-host-range cloning vectors derived from the W-plasmid Sa

Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (M_r 5.6?7.2 × 10⁶), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI,     

Agrobacterium tumefaciens ferritins play an important role in full virulence through regulating iron homeostasis and oxidative stress survival

Ferritins are a large family of iron storage proteins, which are used by bacteria and other organisms to avoid iron toxicity and as a safe iron source in the cytosol. Agrobacterium tumefaciens, a phytopathogen, has two ferritin-encoding genes: atu2771 and atu2477. Atu2771 is annotated as a Bfr-encoding gene (Bacterioferritin, Bfr) and atu2477 as a Dps-encoding gene (D NA binding p rotein from s tarved cells, Dps). Three deletion mutants (Δbfr, Δdps, and bfr-dps double-deletion mutant ΔbdF) of these two ferritin-encoding genes were constructed to investigate the effects of ferritin deficiency on the iron homeostasis, oxidative stress resistance, and pathogenicity of A. tumefaciens. Deficiency of two ferritins affects the growth of A. tumefaciens under iron starvation and excess. When supplied with moderate iron, the growth of A. tumefaciens is not affected by the deficiency of ferritin. Deficiency of ferritin significantly reduces iron accumulation in the cells of A. tumefaciens, but the effect of Bfr deficiency on iron accumulation is severer than Dps deficiency and the double mutant ΔbdF has the least intracellular iron content. All three ferritin-deficient mutants showed a decreased tolerance to 3 mM H₂O₂ in comparison with the wild type. The tumour induced by each of three ferritin-deficient mutants is less than that of the wild type. Complementation reversed the effects of ferritin deficiency on the growth, iron homeostasis, oxidative stress resistance, and tumorigenicity of A. tumefaciens. Therefore, ferritin plays an important role in the pathogenesis of A. tumefaciens through regulating iron homeostasis and oxidative stress survival.     

Agrobacterium as a tool for gene transfer in woody species: The state of the art and practical perspectives. A review

Abstract

The Agrobacterium-mediated ability to transfer genes into organisms without sexual crossing provides breeders with new opportunities to improve the efficiency of plant production. Gene transfer offers advantages over classical genetic manipulation since plants are improved without disruption of the integrity of their genomes. Several useful genes isolated from microrganisms and affecting pest resistance, rooting ability, hormonal metabolism etc., are now available. These genes can be easily cloned into suitable Ti and Ri derived plasmid vectors and transferred into woody species. The scarce ability of most fruit trees to regenerate the whole plant from in vitro-cultured cells remains the main obstacle to a wider use of gene transfer technology.     

农杆菌介导的日本落叶松胚性细胞遗传转化研究

基于体细胞胚胎发生技术平台,利用携带pSuper1300+质粒,以潮霉素为筛选标记基因的农杆菌GV3101介导日本落叶松遗传转化,对植物受体材料生理状态、农杆菌浓度和浸染时间以及共培养时间等影响因素进行了研究、分析和讨论.结果表明:综合优化各影响因素,生长旺盛的日本落叶松胚性细胞,经浓度为0.4(OD600)的农杆菌浸染10min,共培养2d,再用含400mg/L的头孢霉素的液体培养基清洗脱菌,然后在含400mg/L的头孢霉素固体培养基上恢复培养,并置于含5mg/L潮霉素的固体培养基上多次筛选,最终共获得54个抗性细胞系,转化率平均为0.94个/g.PCR检测鉴定,所有抗性细胞系均为阳性转化体,并排除了农杆菌污染导致的假阳性.研究建立并优化了农杆菌介导的日本落叶松遗传转化技术,为进行遗传改良和基因功能鉴定提供有利平台.     

Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil

Dunaliella bardawil, a unicellular microalga, grows in relatively high concentrations of salt and has so far been refractory to Agrobacterium-mediated transformation. An inverse relationship between salt concentration and hygromycin resistance was observed. Co-cultivation at 0.2?M NaCl allowed growth of both D. bardawil and A. tumefaciens. Lowering salt concentrations also enabled the use of lower concentrations of hygromycin, the selection agent. Cells resistant to 100?mg?l^?1 hygromycin were selected and growth of Agrobacterium was completely eliminated in these cells using cefotaxime/potassium clavulanate. The concentration of sodium chloride was gradually increased to 1.0?M with simultaneous reduction of hygromycin concentration for better growth of D. bardawil. Agrobacterium was unable to survive in the growth medium used for Dunaliella. Expression of β-glucuronidase (uidA), green fluorescent protein (GFP) and hygromycin phosphotransferase (hpt) in the hygromycin-resistant culture was detected using X-gluc as substrate and Western blotting using GFP antibodies and RT-PCR respectively. Cells growing in 1.0?M NaCl (in the absence of hygromycin) retained their ability to grow in hygromycin even after 18 months of cultivation. These cells expressed GFP and PCR for hpt gene was positive. The stability of the integrated transgene and resistance to hygromycin in three different transformation events were ascertained periodically. Southern blotting of DNA extracted from hygromycin resistant cells (HRC) that were 15–18 months old established the presence of the integrated transgene in the DNA of D. bardawil. Results of the present study substantiate A. tumefaciens-mediated transformation of the unicellular marine alga D. bardawil. Agrobacterium tumefaciens-mediated transgene integration along with the massive outdoor cultivation methods used for D. bardawil may allow the commercial synthesis of secondary metabolites and heterologous proteins.