影响发根农杆菌对结野葛遗传转化效率的因素

研究了发根农杆菌R1601对野葛(Puerariafobata)进行遗传转化的影响因素.结果表明叶片是转化效率较高的外植体,经发根农杆菌R160l感染12d后可获得毛状根;降低YEB活化液的pH值,或以肌醇(10g/L)代替酵母提取成分,可明显提高菌种的致根性;叶片预培养2～3d可提前3～4d出根.表5参14     

影响发根农杆菌对野葛遗传转化效率的因素

研究了发根农杆菌R1601对野葛（Pueraria lobata)进行遗传转化的影响因素，结果表明：叶片是转化效率较高的外植体，经发根农杆菌R1601感染12d后可获得毛状根；降低YEB活化液的pH值，或以肌醇（10g/L)代替醇母提取成分，可明显提高菌种的致根性，叶片预培养2－3d可提前3－4d出根。     

发根农杆菌转化海边香豌豆及转化体的体细胞胚胎发生

将海边香豌豆无功苗的子叶和下胚轴切段在附加ρ（2,4-D)=1mgL^-1,ρ（6BA)=0.5mgL^-1的MS培养基上预培养3d，然后与发根农杆菌A4菌液共培养30min。洗涤之后，在附加羧苄青霉素的无激素的MS培养基上培养。7－10d后，外植体切面处长出许多毛状根。继代培养时生长旺盛，且产生许多侧根和分枝。志状根的诱导频率与无菌苗培养天数和外植体来源部位有关。子叶切块的诱导率明显高于下胚轴，16d龄无菌苗的子叶切块诱导毛状根的频率最高。乙酰丁香酮处理菌液可以有效提高子叶外植体的毛状根诱导频率。将毛状根切段培养在含有ρ（2，4－D）＝1.0mgL^-1的MS培养基上可诱导出愈伤组织。该种愈伤组织转移到含有ρ（2，4－D）＝0.3mgL^-1的MS培养基上培养时，诱导出了许多早期体细胞胚。检测的转化组织均含有农杆碱和甘露醇。图2表1参19     

发根农杆菌转化青蒿影响因素的研究

对叶盘法转化过程中各种影响因素进行研究．结果表明：发根农杆菌种类、基本培养基和青蒿株系对转化率有明显影响,而培养基ｐＨ在５．２～５．８对转化率没有明显影响;发根农杆菌ＡＴＣＣ１５８３４的转化率最高;基本培养基中ＭＳ效果最好;青蒿株系０２５对发根农杆菌最敏感;处于对数生长期的发根农杆菌（Ｄ６６０ｎｍ＝０．７５）稀释５倍后用来转化效果最好;幼嫩的叶片的转化率比成熟叶片高;向共培养培养基中加入乙酰丁香酮等酚类物质对转化没有促进作用．在最适的转化条件下,发根农杆菌ＡＴＣＣ１５８３４对青蒿株系０２５的转化率可达到１００％．ＰＣＲ检测证明,青蒿发根基因组中含有发根农杆菌Ｒｉ质粒ＴＤＮＡ上的ｒｏｌＣ片段．     

南美蟛蜞菊毛状根的离体培养及其提取液对种子萌发的影响

采用溶液培养法研究培养基的碳源、氮源和无机磷浓度对南美蟛蜞菊(Wedelia trilobata)毛状根生长以及其提取物对水稻、鬼针草、马唐种子萌发的影响.结果表明,当培养基含糖量小于5%时,以葡萄糖为碳源促进南美蟛蜞菊毛状根生长的效果最好,蔗糖次之;而大于5%时仅麦芽糖仍能促进其生长,而当麦芽糖浓度大于7%时,其生长也逐渐被抑制.培养基中添加(NO_3)~--N(263.1μg/mL)和(NH_4)~+-N(577.2μg/mL)组合时,或无机磷含量为MS培养基无机磷用量的2～3倍时对毛状根生长的促进效果最佳;培养基缺Ca2+时,毛状根变得纤细,分枝多,生长旺盛;但随着培养基Ca~(2+)浓度的增加,其生长受到抑制.毛状根的乙醇提取液比水提取液对水稻(Oryza sativaL.)、鬼针草(Bidens pilosaL.)、马唐(Common crabgrass herb)种子的萌发抑制作用强,其水提取液处理的水稻、鬼针草、马唐种子的萌发率分别是84%、93%、20%,而乙醇提取液处理的马唐种子萌发率只有3%,水稻和鬼针草种子则完全不萌发.与野生根的乙醇提取液相比,毛状根的乙醇提取液对水稻、鬼针草、马唐种子的萌发抑制作用更强.暗培养毛状根的乙醇提取液对水稻、鬼针草、马唐种子萌发的抑制作用要比光照培养的毛状根乙醇提取液的抑制作用强,光照培养的毛状根乙醇提取液处理的水稻、鬼针草、马唐种子的萌发率分别是28.7%、3.9%、18.7%,暗培养毛状根的乙醇提取液处理的水稻、马唐种子萌发率只有4%和1%,鬼针草则完全不萌发.本实验为今后规模培养南美蟛蜞菊毛状根选择合适的培养条件及将南美蟛蜞菊毛状根中的次生代谢产物应用于农业生产奠定了实验与技术基础.图9表1参19     

发根农杆菌T—DNA基因对3种葛属植物毛状根形态和葛根素含量的影响

野葛、山葛和三裂叶葛的离体叶片与发展根农杆菌R1601直接感染诱导出毛状根并能够合成葛根素，毛状根离体培养后均出现两种形态：一种是有较高的葛根素含量的典型毛状根，另一种是葛根素含量低但生长速度较快的愈伤组织化毛状根，两类毛状根均有rol/B基因存在，表明rolB基因对所有毛状根的诱导和生长是必需的，aux1存在于所有愈伤组织化毛状根，而在典型毛状根中的检出率为20％－50％，表明aux1基因有诱导毛状根产生愈作组织的作用，含有ags基因的毛状根葛根素含量低于不含ags基因的毛状葛根素含量，表明ags基因不利于次生代谢合成，表3参12     

镉胁迫下油菜毛状根的生理响应及铁钾含量

利用液体悬浮培养法研究不同镉(Cd)浓度(0、25、50、100、200和400μmol/L)胁迫下油菜毛状根的生理响应及对铁钾含量的影响,探究油菜毛状根对镉胁迫的耐受与富集能力.结果显示:(1)低镉浓度(100μmol/L以下)对毛状根的生长无显著影响,高镉浓度(100μmol/L以上)下毛状根的生长则受到明显的抑制,25μmol/L镉胁迫7 d时毛状根的鲜重最大(4.34 g).(2)油菜毛状根中活性氧(ROS)的含量随着镉浓度的增加而上升,根中主要抗氧化酶(超氧化物酶SOD、过氧化物酶POD、过氧化氢酶CAT)的活性在镉胁迫1 d时,表现出先降低后升高的趋势;镉胁迫7 d时,表现出先升高后降低的趋势.(3)碘化丙啶PI染色与丙二醛MDA分析表明,根细胞的损伤随着镉浓度的增加越发严重.(4)油菜毛状根中的镉含量随着培养基中镉浓度的增加而增加,400μmol/L、7 d时,达到最大值2.97 mg/g.毛状根中的铁含量在镉胁迫1 d时随着镉浓度的增加显著上升,最大值达到14.52 mg/g;镉胁迫7 d时没有明显变化.镉胁迫7 d时毛状根中的钾含量是镉胁迫1 d时(15.73 mg/g)的1.6倍.本研究结果表明,油菜毛状根对镉胁迫的生理响应变化与镉的作用浓度和时间相关;同时镉胁迫造成了毛状根中铁、钾元素代谢紊乱,但油菜毛状根对镉有较好的富集效果.     

根癌农杆菌介导工业化产甘油假丝酵母的遗传转化

通过构建质粒pCAM3300-zeocin,将其电击转化到根癌农杆菌LBA4404中.将含有目的质粒pCAM3300-zeocin的根癌农杆菌和工业化产甘油假丝酵母(Candida glycerinogenes)WL2002-5共培养,利用zeocin抗性基因为筛选标记,实现了pCAM3300-zeocin对产甘油假丝酵母的转化.并根据产甘油假丝酵母的生长特性,对转化条件进行了优化,在共培养时间为24h,产甘油假丝酵母和根癌农杆菌的细胞比例为1∶(500～1000)时最高转化率达到2个转化子/104个酵母细胞.初步建立了根癌农杆菌介导转化产甘油假丝酵母的转化方法.     

根癌农杆菌介导的高赖氨酸蛋白基因转化叶用莴苣的研究

构建了植物表达载体pLBI，该载体携带35S启动子、高赖氨酸蛋白基因(LRP)、NPTⅡ基因和NOS终止子，对影响根癌农杆菌(Agrobacterium tumefaciens L.)转化的多种因素进行探索后，建立了一种农杆菌介导的稳定的叶用莴苣遗传转化系统．选择目前生产上大量推广应用的叶用莴苣品种，以其无菌苗子叶为外植体，经农杆菌LBA4404(含质粒pLBI)感染，在含ρ(Kan)／mgL^-1=100的芽分化培养基上筛选转化芽，转入含相同浓度抗生素的生根培养基上进行生根筛选，直至得到完整的再生植株．PCR扩增及Southern blot的检测结果均表明，高赖氨酸蛋白基因已整合到叶用莴苣基因组中．图4表4参6     

青蒿毛状根分支生长的动力学模型

近年来，用m质粒转化药用植物诱导毛状根生产有用代谢产物的研究日益增多，转化产生的毛状根不仅生长迅速，分支多，而且遗传性和生理、生化特性稳定，具有较强的次生代谢合成能力，因此可望应用于大规模生产有用的次级代谢产物［‘－’］．青蒿毛状根可产生大量的次生代谢产物     

外源Mo降低As(Ⅲ)和As(Ⅴ)对水稻的毒性及As的积累

通过溶液培养试验,研究外源添加Mo对2种价态砷(As(Ⅲ)和As(Ⅴ))胁迫下水稻吸收积累Mo和As的影响。结果表明,这2种价态的As对水稻生长均有抑制作用,As(Ⅲ)比As(Ⅴ)对水稻毒害更明显,添加Mo可缓解As对水稻的毒害。As添加可影响水稻根系和茎叶对Mo的吸收积累,但是不同价态As对Mo积累量的影响不一致。同时,Mo的添加也可以显著地降低水稻根系和茎叶对2个价态As的吸收积累。在100μmol·L~(-1)As(Ⅲ)处理下,添加0.1和0.5 mg·L~(-1)的Mo可导致水稻根系As积累量分别比对照处理降低38.8%和52.8%,茎叶As积累量分别降低5.1%和10.6%;当As(V)浓度为100μmol·L~(-1)时,添加0.1和0.5 mg·L~(-1)的Mo可导致水稻根系As积累量分别比对照处理降低15.4%和62.4%,茎叶As积累量分别降低11.9%和23.7%。Mo的添加还能显著地降低2种价态As在水稻根系和茎叶中的富集系数。因此,通过施用适量的Mo肥可以用来防治农田As污染,降低As对人体健康的危害。     

Induced alkaloid defence inAtropa acuminata in response to mechanical and herbivore leaf damage

Summary Measurement of tropane alkaloid content in leaves ofAtropa acuminata after mechanical damage showed a maximum increase to 153% of the control 8 days later. There were no changes in the root or stem after similar damage. The plant responded to repeated mechanical damage by doubling its alkaloid content at 11 days after the initial wounding. But on further treatments, there was a slight decrease in alkaloid content with time. Mollusc feeding produced an increase of 164% in alkaloid content after 4 days. These results indicate that induced defence systems in angiosperms can vary considerably from plant species to plant species and that the effects of mechanical damage may differ in different parts of the same plant.     

Vitamins E and C reduce oxidative stress and mitochondrial permeability transition caused by camptothecin – an in vitro study

Camptothecin (CPT), a broad spectrum antineoplastic agent, is known to induce oxidative stress and mitochondria are among the main sources of intracellular reactive oxygen species (ROS). We investigated the merit of vitamins E and C supplementation on CPT-induced mitochondrial alterations in vitro. Following treatment of isolated liver mitochondria with CPT, we assessed the mitochondrial membrane permeability transition (MPT), concentration of malondialdehyde, antioxidants and activities of the enzymes of the respiratory chain and Krebs cycle. Our results provide evidence that CPT caused mitochondrial swelling, increased lipid peroxidation and transition of mitochondrial permeability. The CPT lowered the levels of reduced mitochondrial thiols suggesting that thiol oxidation is the mechanism underlying CPT-induced MPT. Identical experiments were also performed after preincubating the mitochondria with vitamins E and C. It was found that vitamins E and C pretreatment inhibited the deleterious effects of CPT and loss of enzyme activity was restored by antioxidant supplementation. Our results suggest that the toxicity of CPT was mediated by an increase in ROS production by mitochondria. However, the addition of vitamins E or C ameliorated the oxidative stress. We propose that an attempt to counteract the deleterious consequences of chemotherapy with nutritional therapies may be a rational approach in superior patient care especially in a disease like cancer.     

White locoweed toxicity is facilitated by a fungal endophyte and nitrogen-fixing bacteria

Mutualistic interactions with fungal endophytes and dinitrogen-fixing bacteria are known to exert key biological influences on the host plant. The influence of a fungal endophyte alkaloid on the toxicity of a plant has been documented in Oxytropis sericea. Oxytropis sericea is a perennial legume responsible for livestock poisoning in western North America. Livestock poisoning is attributed to the alkaloid swainsonine, which is synthesized inside the plant by the fungal endophyte Embellisia sp. In this study, the ability of Oxytropis sericea to form a dinitrogen-fixing symbiosis with Rhizobium and the effects of this symbiosis on the production of swainsonine by Embellisia sp. were evaluated in a greenhouse environment. Seeds of O. sericea were grown in plastic containers. Twenty-week-old O. sericea seedlings were inoculated with four strains of Rhizobium. Twenty weeks after inoculation, plant growth and root nodulation by Rhizobium were measured. Dinitrogen fixation was confirmed using an acetylene reduction assay (ARA) on excised root nodules. Dry leaves were analyzed for swainsonine content. A second set of plants was treated with fungicide to evaluate the effect of reduced fungal endophyte infection on plant growth and swainsonine production. All inoculated plants produced indeterminate nodules. The ARA indicated that 98% of the excised nodules were fixing dinitrogen. Rhizobium-treated plants had greater swainsonine concentrations than the non-inoculated controls. Plants that established from seeds treated with fungicide had lower biomass than non-fungicide-treated controls and plants treated with foliar fungicide. Plants treated with foliar fungicide and the controls had greater swainsonine concentrations than the plants that received seed fungicide. This greenhouse study is the first report of nodulation and dinitrogen fixation in O. sericea. It also demonstrates that dinitrogen fixation increases the production of swainsonine in O. sericea plants infected with Embellisia sp. Results from this study suggest that dinitrogen fixation affects swainsonine production and has the potential to support the symbiosis between Embellisia sp. and O. sericea when soil nitrogen is limited. Oxytropis sericea competitiveness appears to be facilitated by an ability to simultaneously associate with Rhizobium and a fungal symbiont.     

Effect of nitrogen fertilization on caffeine production in coffee (Coffea arabica)

Nitrogen (N) based secondary metabolite production is thought to be costly to plants because N is required for growth, as well as, the synthesis of these compounds. Therefore, variation in N availability may result in variation in N-based secondary metabolite production. Here, we determine the effect of N fertilization on caffeine (N-based alkaloid) production in coffee (Coffea arabica) seedlings. A growth chamber experiment was performed with three N treatments applied to seedlings. N fertilization increased plant growth, leaf biomass, and plant N. Caffeine concentration in phloem exudates was greater in high-N fertilized plants relative to intermediate- and low-N plants. However, leaf, stem, root, and total overall caffeine concentration and content did not differ across N treatments. These results suggest caffeine in coffee is strongly regulated by genetic factors, and environment is likely less important to caffeine phenotype. This is among the first studies to investigate the effect of N fertilization on caffeine within the phloem, which has important implications for herbivores that are sensitive to caffeine and plant N and feed from the phloem of coffee.     

Langzeitmonitoring möglicher Auswirkungen gentechnisch veränderter Pflanzen auf Pflanzengesellschaften Konzeptentwicklung am Beispiel von Raps (Brassica napus)

Ecological monitoring of genetically engineered plants (GEPs) is being developed in the Bavarian Environmental Protection Agency. Focusing on oilseed rape (Brassica napus, Cruciferae), a method is being established to measure the impact of the commercial release of GEPs on plant communities. Oilseed rape is known to escape from cultures and to establish outside the fields. It also produces hybrid offspring with wild relatives of the European flora. The study site is located in Bavaria (Germany) and comprises an area of 5700 ha. It is characterized by a mosaic of protected natural habitats in close vicinity to intensively cultivated farmland. In this region species composition and population density of Cruciferae is documented prior to a possible commercial release of GEPs. Furthermore Cruciferae of the study site are tested for the presence of transgenes. Therefore, plant material of selected Cruciferae is randomly sampled. We also test oilseed rape honey from the study site and pollen from pollen traps for the presence of hybrid pollen. The molecular method used is the ‘real-time PCR’. In addition, an intensive monitoring program is established in two types of biotopes, which are located in the study site. Multivariate statistics are being used to correlate plant community data with soil parameters.     

A Possible Relationship Between Enteromorpha (Chlorophyta) and Melosira nummuloides (Chrysophyta: Bacillariales)

While investigating the effect of fluoride on the green alga, Enteromorpha, healthy chains of the centric diatom, Melosira nummuloides appeared in cultures containing a small piece of Enteromorpha, nutrient medium and all concentrations of fluoride, as Na₂SiF₆ to 160 p.p.m. Both the Melosira alone and the Enteromorpha, accompanied by Melosira, were transferred into a culture medium (germ intercepting filtrate, S=30g./kg. to which 0.4 ml./1. of the plant nutrient 'Kerigrow' was added) with a normal fluoride content; after three weeks the Melosira alone was present only in short, broken chains, whereas that in the presence of Enteromorpha had formed long, healthy chains.     

Precopulatory assessment of male quality in an arctiid moth (Utetheisa ornatrix): hydroxydanaidal is the only criterion of choice

Females of the moth Utetheisa ornatrix (Lepidoptera: Arctiidae) mate preferentially with males that excel in three quantitatively correlated attributes: body mass, systemic content of defensive pyrrolizidine alkaloid (derived from the larval diet), and glandular content of the courtship pheromone hydroxydanaidal (derived from the alkaloid). By so choosing, the females obtain direct phenotypic benefits (alkaloid and nutrient received with the spermatophore), and indirect genetic benefits (genes for large size, a heritable trait). We asked whether the female appraises the courting male on the basis of all three attributes, or whether, as had been postulated, she does so on the basis of the intensity of the pheromonal scent alone. We present data indicating that male possession of hydroxydanaidal is indeed the sole criterion of choice. Females fail to differentiate between males that differ in body mass or alkaloid content if the males lack hydroxydanaidal, but choose between males that are size-matched and alkaloid-free if one of the males has been experimentally endowed with hydroxydanaidal. We show moreover that females are able to differentiate between males that contain unequal quantities of hydroxydanaidal. Females abide by these criteria whether or not they themselves contain alkaloid. Their choice was also unaffected by whether they were confined singly with 2 males in small mating chambers, or were in groups of 10 with 20 males in large flight cages.     

麻黄愈伤组织细胞的悬浮培养

用中麻黄幼苗的３种外植体进行了离体培养。不同外植体诱导愈伤组织的结果表明：最佳培养基为ＭＳ＋２ｍｇ／Ｌ２，４－Ｄ＋１ｍｇ／Ｌ６－ＢＡ；最佳外植体为下胚轴；用源于下胚轴的愈伤组织进行悬浮培养，从第３代开始建立起稳定的悬浮系。愈伤组织、悬浮培养细胞及培养液中都含有麻黄碱，悬浮培养液中的麻黄碱含量高于愈伤组织和悬浮培养细胞。表２参１２