Endophytic colonisation of tobacco,corn, wheat and soybeans by the fungal entomopathogen Beauveria bassiana (Ascomycota,Hypocreales)

We demonstrate the effectiveness of three inoculation methods (foliar spray, seed immersion and root immersion) in establishing fungal the entomopathogen Beauveria bassiana as an endophyte in tobacco, corn, wheat and soybean. Colonisation of leaves by B. bassiana was assessed 7, 14, 21 and 28 days post-inoculation. There were significant differences (p < 0.001) in endophytic colonisation among the different inoculation techniques.     

Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460     

Disposable bioreactor for cell culture using wave-induced agitation

This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

大豆fad基因反义表达载体构建及转化烟草研究

使用PCR方法从大豆基因组DNA中扩增出大豆油酸脱饱和酶基因fad2-1,连接到pMD18-T载体中,转化大肠杆菌JM109菌株.测序后,用DNAstar软件进行同源性比对.然后将正确的序列反向克隆到表达载体pBt,并转化农杆菌菌株LBA4404,经双酶切鉴定和PCR扩增检测,获得具有该基因反向序列的农杆菌工程菌,转化...     

Bioreactor operation for transgenic Nicotiana tabacum cell cultures and continuous production of recombinant human granulocyte-macrophage colony-stimulating factor by perfusion culture

The production of hGM-CSF was investigated in both a flask and a 5-l bioreactor, using transgenic Nicotiana tabacum suspension cells. While the maximum cell density and secreted hGM-CSF in the flask were 15.4 g l⁻¹ and 6.5 μg l⁻¹, respectively, those in the bioreactor were 15.6 g l⁻¹ and 7.6 μg l⁻¹. No detectable growth inhibition, shorter production of hGM-CSF and reduced cell viability in the batch bioreactor were observed under the specific conditions used compared with the flask culture. To improve the productivity, a perfusion culture was carried out in the bioreactor, with three different perfusion rates (0.5, 1.0 and 2.0 day⁻¹). In all cases, the hGM-CSF in the medium was significantly increased during the overall culture period (16 days), with maximum values 3.0-, 9.4- and 6.0-fold higher than those obtained in the batch cultures, respectively, even though the intracellular hGM-CSF content was not significantly varied by the perfusion rate. In terms of the total amount of hGM-CSF secreted, 205.5, 1073.2 and 1246.3 μg accumulated in the perfusate within 16 days at the perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. It was concluded that the beneficial effect of perfusion on the production of hGM-CSF originated from the reduced proteolytic degradation due to the lower protease activity caused by the perfusion. Additionally, the cell growth and physiology in the perfusion culture were somewhat negatively affected by the increased perfusion rate, although the dry cell density steadily increased, and as a result, 19.4, 22.4 and 22.9 g l⁻¹ of maximum cells were obtained with perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. This work highlighted the importance of proteolytic degradation in plant cell cultures for the production of secretory proteins and the feasibility of perfusion strategies for the continuous production of foreign proteins by the prevention of protein loss due to proteolytic enzymes.     

烤烟‘K326’的组织培养与快速繁殖

为了提供市场所需的大量种苗,适应云南烤烟(Nicotiana tabacum)规模化生产的需要,以田间烤烟的叶片、茎段、培养瓶内种子萌发的叶片和茎段为外植体进行组织培养实验.实验比较了5种培养基,筛选出烤烟的最佳愈伤组织诱导和愈伤组织不定芽分化培养基为MS 6-BA 2 mg·L-1 NAA0.2 mg·L-1 Sugar30 g·L-1 Agar6 g·L-1;生根培养基为MS Sugar30g·L-1 Agar6 g·L-1,生根率为100%;用营养土移栽后,存活率为100%.     

一个高频率的活体-离体胚胎发生研究系统

通过对受精后烟草(Nicotiana tabacumL.)胚珠的体外培养,建立了一个简便、实用、高频率的胚胎发生研究系统。采用MS培养基附加6%蔗糖,然后换以2%蔗糖对烟草胚珠进行培养,结果受精后胚珠在体外可以完成正常的胚胎发生并可直接萌发成幼苗。追踪观察表明,合子体外胚胎发生过程中关键发育事件,如合子休眠期间的定向生长、一次不对称分裂的完成、胚柄的形成和解体、胚的分化等过程均可在体外重现。体外培养形成的胚珠及胚胎在形态上与自然状态下形成的胚珠及胚胎几乎没有差异。此体系操作简单,稳定性好,且关键发育事件的时空调控与自然胚胎发育进程相吻合。     

Characterization of a tobacco extensin gene and regulation of its gene family in healthy plants and under various stress conditions

A genomic clone (Ext 1.4) encoding an extensin was isolated from a Nicotiana tabacum genomic library. The encoded polypeptide showed features characteristic of extensins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of one Tyr-Leu-Tyr-Lys motif suggests the possibility for one intramolecular isodityrosine cross-link whereas numerous Val-Tyr-Lys motifs may participate in intermolecular cross-links. This extensin appears to be close to an extensin already characterized in N. tabacum but very different from the Ext 1.2 extensin of N. sylvestris. The analysis of genomic DNA gel blots using probes spanning different parts of the gene showed that the Ext 1.4 gene belongs to a complex multigene family having one member originating from N. sylvestris and three members from N. tomentosiformis. The Ext 1.4 specific probe found a 1.4 kb mRNA in stems, roots, ovaries and germinating seeds of healthy plants. The Ext 1.4 gene family is strongly induced in actively dividing cell suspension cultures and after wounding of leaves or stems in conditions where root formation occurs. On the contrary, it is not induced in leaves in response to a hyperensitive reaction to a viral infection or after elicitor treatment.     

烟草中央细胞体内与离体发育中的细胞化学变化

应用金霉素（ＣＴＣ）ｆｌｕｐｈｅｎａｚｉｎｅ（ＦＰＺ）荧光增白剂（ＣＷ）,ＤＡＰＩ等荧光探针,标记由大叶烟草体内直接分离和经过离体培养的中央细胞及卵器细胞,观察膜结合钙及钙调素的分布与细胞僻才细胞核的动态,用碘－碘化钾,苏丹Ⅲ等染色鉴定中央细胞与胚乳细胞内的淀粉与油脂,探讨了中央细胞等在受精前后与培养前后的细胞化学变化。     

烟草薄层培养器官发生的控制及细胞学观察

普通烟草(Nicotiana tabacum)花梗表皮薄层组织在不同生长素和细胞分裂素配比的MS培养基上及不同的培养条件下,可分别诱导,得到直接发生的营养芽和花芽,以及根和不发生器官分化的愈伤组织。组织间的相互联系,影响器官发育潜能的发挥。细胞学观察发现,直接发生的营养芽和花芽起源于薄层组织的亚表皮细胞层。     

Cytokinin-like effects of caffeine in bioassays

Cytokinin-like effects of pure caffeine were tested in bioassays specific for this hormonal activity [radish cotyledon growth and chlorophyll (Chl) biosynthesis in cucumber cotyledon and tobacco cell suspension] and in cell elongation bioassays [elongation of segments from soybean internode and internode elongation in dwarf cultivars of guandu (Cajanus cajan) and mucuna (Mucuna deeringiana)]. 6-Benzyl-aminopurine and kinetin (KIN) were used for comparison with caffeine. Although weaker than those given by cytokinins, positive responses were observed in all specific bioassays and in elongation of soybean internodes. A remarkable synergistic effect between caffeine and KIN was observed for the synthesis of Chl in the tobacco cell suspension bioassay, in which different concentrations of the alkaloid were combined with a single concentration of KIN. The hormone-like effect of caffeine might be related to the resemblance between caffeine and adenine derivatives. This revised version was published online in August 2006 with corrections to the Cover Date.     

Leghaemoglobin biosynthesis in a new single cell system from soybean root nodules

Single cells were effectively released from 35–45-day-old soybean ( Glycine max L. cv. Yaefusanari) nodules by treatment with an enzymic solution containing 1 mg/ml maceration enzyme (Pectolyase Y-23), 0.5 M mannitol, 2% (w/v) sucrose and 0.5% (w/v) potassium dextran sulfate. Bacteroid-containing cells were purified by Percoll density gradient centrifugation. Electron microscopic observation showed that these cells were protoplasts enclosed by a thin wall and with well preserved internal structures including bacteroids. The single cells obtained were stable against centrifugation and vigorous pipetting. The cells retained the ability to synthesize proteins including leghaemoglobin. The ratio of leghaemoglobin components synthesized in the single cells was similar to that of components synthesized in the nodules. The bacteroidal cell fraction was further separated into three fractions by a Percoll density gradient centrifugation. Comparison of the absolute and relative leghaemoglobin content, the activity of glutamine synthetase in the cytoplasm and the activity of 3-hydroxybutyrate dehydrogenase in the bacteroid suggests that these fractions contained cells in different stages of symbiosis. This new single cell system should provide a useful experimental system for analyzing events in the root nodule.     

刺激剂 (elicitor) 在植物细胞培养次生代谢物生产中的应用

刺激剂（ｅｌｉｃｉｔｏｒ）在植物细胞培养中被用来作为提高次生代谢物产量的手段。文中概括介绍了微生物、寡聚糖、蛋白质、第二信使及其他物质作为刺激剂在植物细胞培养中的应用及其研究成果。     

A mechanistic model for gas–liquid mass transfer prediction in a rocking disposable bioreactor

Rocking disposable bioreactors are a newer approach to smaller-scale cell growth that use a cyclic rocking motion to induce mixing and oxygen transfer from the headspace gas into the liquid. Compared with traditional stirred-tank and pneumatic bioreactors, rocking bioreactors operate in a very different physical mode and in this study the oxygen transfer pathways are reassessed to develop a fundamental mass transfer (k_La) model that is compared with experimental data. The model combines two mechanisms, namely surface aeration and oxygenation via a breaking wave with air entrainment, borrowing concepts from ocean wave models. Experimental data for across the range of possible operating conditions (rocking speed, angle, and liquid volume) confirms the validity of the modeling approach, with most predictions falling within ±20% of the experimental values. At low speeds (up to 20 rpm) the surface aeration mechanism is shown to be dominant with a of around 3.5 hr⁻¹, while at high speeds (40 rpm) and angles the breaking wave mechanism contributes up to 91% of the overall (65 hr⁻¹). This model provides an improved fundamental basis for understanding gas–liquid mass transfer for the operation, scale-up, and potential design improvements for rocking bioreactors.