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     

高山红景天愈伤组织的诱导及培养研究

分别以高山红景天根、茎、叶及种子萌发的子叶为外植体诱导出５种愈伤组织，并比较了这５种愈伤组织的生长速率及红景天甙的积累能力。通过对茎诱导愈伤组织的继代培养，初步选择了适宜的激素配比为３ｍｇ／Ｌ ＢＡＡ＋０．３ｍｇ／ＬＮＡＡ，最适温度范围为２１－２５℃，ｐＨ值为５．８。光照对愈伤组织生长影响不显著，但不利于红景天甙的合成。     

欧洲红豆杉细胞培养的研究

从欧洲红豆杉（Ｔａｘｕｓｂａｃｃａｔａ）的嫩茎及什叶诱导出愈伤组织，并对之进行了愈伤组织培养及细胞悬浮培养研究；利用ＨＰＬＣ方法测定了培养物合成紫杉醇的能力．探索了提高培养细胞生长率及紫杉醇含量的一些措施．结果表明，欧洲红豆杉愈伤组织及悬浮培养细胞的生长速率已分别达到０．２７ｇ·Ｌ－１·ｄ－１和０．３５ｇ·Ｌ－１·ｄ－１，紫杉醇含量分别为０．００３１％和０．０１７％。     

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     

鞑靼荞麦离体再生体系的建立

研究了苗龄、外植体、激素配比对鞑靼荞麦(Fagopyrum tataricum Gaertn.)离体培养的影响,初步建立了鞑靼荞麦离体再生体系.结果表明,鞑靼荞麦离体再生的最佳取材时间为苗龄6～8 d;诱导愈伤组织的最适培养基为Ms+2.0 mg/L 2,4.D+1.5 mg/L 6-BA,子叶诱愈率达75%左右,下胚轴诱愈率可高达86.62%;愈伤组织分化的最适培养基为MS+0.1 mg/L IAA+2.0 mg/L 6-BA+1.0 mg/L K1.+0.5 mg/L TDZ,来自下胚轴愈伤组织的分化率可达9.52%.下胚轴的诱愈率与分化率均高于子叶,更适于离体再生培养.培养基巾加入AgNO_3后,能有效降低褐化率.生根最适培养基为含有0.5 mg/L NAA的1/2MS培养基,生根率在50%左右.TDZ在诱导鞑靼荞麦的愈伤组织分化出芽的过程中起到明显的促进作用,可提高分化率约20%.表3图2参23     

3种镉超富集植物毛状根体系对镉胁迫响应的比较

近年来,转基因毛状根组织被越来越多地应用于重金属和有机污染物的植物修复技术研究中,已成为进行污染物毒性响应机制研究的便捷的实验室工具。为了探究龙葵、油菜、芥菜3种镉(cadmium,Cd)超富集植物对Cd毒性胁迫响应的差异,以诱导出的3种植物毛状根为研究材料,从毛状根的生长状态、富集Cd的能力、根组织细胞的凋亡程度和抗氧化酶活性等方面进行了探讨。结果表明:Cd浓度为0~50μmol·L-1时,龙葵、油菜、芥菜毛状根受Cd毒害的影响都不明显;Cd浓度为75~100μmol·L-1时,龙葵、油菜、芥菜毛状根均表现出对Cd胁迫的防御响应。在较高的Cd浓度(100μmol·L-1)下,龙葵毛状根的生物量受Cd毒害的影响最小,芥菜次之,油菜受影响最大;同时龙葵毛状根富集的Cd含量最高(745.0μg·g-1),芥菜次之(681.4μg·g-1),油菜最差(505.2μg·g-1)。龙葵、油菜、芥菜毛状根在Cd胁迫下的细胞凋亡水平均随Cd浓度的升高而升高,当Cd浓度为100μmol·L-1时,龙葵毛状根比油菜和芥菜毛状根的细胞凋亡程度均低。同时3种植物毛状根在不同浓度Cd处理下抗氧化酶活性的变化有一定差异。从上述结果综合来看,龙葵毛状根受Cd毒害的影响最小、富集Cd的能力最好,是进一步开展Cd超富集植物转基因改造研究的较好的实验室载体。     

长期继代培养马铃薯愈伤组织的植株再生

对影响马铃薯愈伤组织发生、生长和长期继代培养的愈伤组织再生植株的几种因素进行了研究。马铃薯茎段的愈伤组织发生频率高于叶片的愈伤组织发生频率。Ｄｉｃａｍｂａ诱导茎段和叶片的愈伤组织发生频率在９０％以上；而２，４－Ｄ的诱导效果不佳并诱发外植体生根。再生培养其中加入０．５ｇ／Ｌ的酶解酷蛋白是必需的；蔗糖的浓度以２％－４％为宜；附加ＺＴ２．０ｍｇ／Ｌ、ＧＡ０．１ｍｇ／Ｌ可使长期继代的愈伤组织的植株再生频率     

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.     

Ri质粒诱导喜树毛状根培养基中喜树碱的研究

喜树(Camptotheca acuminate)可产生具有抗癌作用的喜树碱,有许多用细胞培养方法制备喜树碱的尝试,但都因极低的产率而无从深入。植物毛状根培养的应用将成为一种生物碱生产的替代方法。以发根农杆菌(Agrobacterium rhizogenes)A41.2556感染喜树诱导产生毛状根,建立喜树碱毛状根产生体系。甘露碱检测及PCR结果证实发根农杆菌Ri质粒的rolB基因已在喜树毛状根基因组中整合并得到表达。确定了用RP-HPLC测定发根培养基中喜树碱的的方法,比较了不同的吸附树脂对培养基中喜树碱的富集能力,从培养30d的喜树培养基中获得喜树碱干质量分数为0.14mg·g-1。     

OsZEP2基因沉默对2,2',5-三氯联苯胁迫下水稻抗氧化反应的影响

载脂蛋白基因OsZEP2沉默突变株(mutation type, MT)和野生型(wide type, WT)的水稻愈伤组织被暴露于10、50、100μg·mL~(-1)的2,2′,5-三氯联苯(PCB18)的培养基3 d后,通过比较两种水稻愈伤组织的生长、PCB18积累和抗氧化反应的变化情况,来探究水稻在多氯联苯胁迫下OsZEP2基因对水稻解毒响应机制的影响.实验发现,PCB18可以抑制WT和MT的生长,但PCB18的中浓度胁迫对两种愈伤组织生长的促进作用十分明显,即有"hormesis"效应.PCB18对MT生长的抑制高于WT,低浓度CB18(10μg·mL~(-1))胁迫抑制MT生长(4.5%),中浓度PCB18(50μg·mL~(-1))促进MT生长(8.3%),高浓度PCB18(100μg·mL~(-1))抑制MT生长(9.9%).OsZEP2基因沉默后促进了PCB18在愈伤组织的积累,并导致培养基中的PCB18去除效率提高.OsZEP2基因沉默降低类胡萝卜素含量,导致MT本身的氧化胁迫增强;并且在不同浓度PCB18胁迫下,MT的类胡萝卜素含量稳定,MT的抗氧化反应活性低与WT.在PCB18胁迫下,MT体内的SOD和CAT酶的活性都明显低于WT,这可能是导致膜脂过氧化程度显著升高的重要原因之一.另外,两种嫩弱的愈伤组织中的POD酶抗氧化活性较弱,但OsZEP2沉默可以促进POD酶活性升高.因此,OsZEP2基因沉默导致植物体内的PCB18积累增多,抗氧化反应减弱,不利于植物对PCB18毒性的抵抗.     

Preparation and assay of C-glucosyltransferase from roots of Pueraria lobata

C-glucosyltransferase (EC 2.4.1.X) is one of the key enzymes for the biosynthesis of puerarin. This paper describes the methodology in purification and assay of the enzyme for the first time in Puerarin lobata (Wild.) Ohwi. C-glucosyltransferase from roots of P. lobata was extracted and partially purified by (NH4)2SO4 saturation. The effects of pH, temperature, and substrate concentration on the activity of the enzyme were investigated. The properties of the puerarin produced by C-glucosyltransferase were studied by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The peak activity of C-glucosyltransferase was detected in fraction of by 80% saturation of (NH4)2SO4 and the optimal conditions for enzymatic reaction were 35.5 micromol l(-1) of isoliquiritigenin and 560 micromol l(-1) of UDP-G at pH 8.1, 28 degrees C for 1 h. Mn2+ at 1 mmol l(-1) and Al3+ at 1 mmol l(-1) increased the enzyme activity, while Mg2+ inhibited its activity. The enzyme activity in Nicotiana tabacum and P. lobata were detected under the above assay conditions. Higher activity was found in roots than in leaves and stems of P. lobata, while no enzyme activity was detected in leaves of N. tabacum. It was the first time that activity of C-glucosyltransferase, which transforms isoliquiritigenin to puerarin, was detected in P. lobata.     

Nutrient consumption and production of isoflavones in bioreactor cultures of Pueraria lobata (Willd)

This paper reports the successful culture of Pueraria lobata (Willd.) suspension cells in a bioreactor. In vitro culture of this Chinese herb has potential as an alternative production method for industrial applications. Calli of P. lobata obtained from leaf explants were cultured in a 5.0 L bioreactor for two weeks. During this period, the pH of the medium declined from 5.8 to 4.5. By the end of the run about 70% of the sugars and reducing sugars and about 50% of nitrate was consumed. Almost 70% of inorganic phosphate and about 80% of the iron was exhausted. The bioreactor results indicated an isoflavone yield of 328.9 microg/ml, with an increase of about 1.77 fold. The yield of puerarin increased about 2.42 fold and reached 73.4 microg/ml in the bioreactor culture.     

Ri质粒转化烟草的影响因素及豌豆凝集素基因在烟草发根中的表达

对Ri质粒转化烟草的影响因素进行研究,以期获得较为理想的实验系统,用发根农杆菌介导,将豌豆凝集素基因导入烟草,获得转基因的烟草发根,GUS组织化学染色,蛋白免疫原位杂交证明,gus报告基因和豌豆凝集素基因已在转基因烟草发根中得到了转译表达,本结构将为瘤菌能否扩大宿主范围,为研究烟草与豌豆根瘤菌相互作用,结瘤固氮的可能性奠定基础。     

Production of puerarin and isoflavones in cell suspension cultures of Pueraria lobata (Willd.): effects of medium supplementation with casein hydrolysate and coconut milk

Callus induced from leaf explants of Pueraria lobata seedlings were suspended in Gamborg B5 medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid, 1 mg l(-1) naphthalene acetic acid, 0.5 mg l(-1) kinetin and 30 g l(-1) sucrose. The effects of coconut milk and casein hydrolysate (CH) on cell growth and yields of puerarin and isoflavones in cells suspension were studied. The contents of total isoflavones and puerarin in suspension cultures were determined by spectrophotometry and HPLC. Coconut milk (10%, filter sterilized) decreased the growth of cell cultures and the accumulation of total isoflavones, while 0.2% CH promoted the growth of cell cultures and the accumulation and release of puerarin and total isoflavones. The total yield of puerarin and isoflavones were 34% and 40.8% higher than in the control, respectively. The optimum medium for cell cultures of leaves of P. lobata seedlings was B5 liquid medium supplemented with 2% sucrose, 1.0 mg l(-1) 2,4-D, 1.0 mg l(-1) NAA, 0.5 mg l(-1) kinetin and 20 mg l(-1) CH. The procedure use is a potentially useful for the production of isoflavones.     

发根农杆菌Ri质粒rol基因矮化性状表达研究进展

rol基因的表达能诱导被感染植物产生发根,再培养后所形成的植株具有一系列的如矮化,生根力强等表现型,国内外学者对rol基因这一特性作了深入的研究.对rol基因产生的矮化性状及表达进行了综述,并探讨了rol基因矮化性状研究面临的问题和可能的解决途径.     

利用GFP和抗性双类型标记监测联合固氮菌在玉米根际的定殖

将来自质粒ｐＫＲＰ１０、ｐＫＲＰ１１和ｐＫＲＰ１２的氯霉素、卡那霉素和四环素抗性基因分别插入质粒ＧＦＰｍｕｔ２中ｇｆｐ基因下游的ＰｓｔＩ位点,得到ｇｆｐ和不同抗性基因共存的重组质粒,转化Ｅｎｔｅｒｏｂａｃｔｅｒ ｇｅｒｇｏｖｉａｅ ５７－７野生型菌株和耐铵工程菌Ｅ７后,得到既有抗生素抗性又在蓝光下呈现亮绿荧光的菌株。用它们接种玉米后,利用这两种选择标记双重筛选重新分离到的细菌确定了接种菌在玉米幼苗     

Allelopathic effect of four weed species extracts on germination, growth and protein in different varieties of Glycine max (L.) Merrill

Allelopathic effect of Ageratum conyzoides L., Cynodon dactylon (L.) Pers., Parthenium hysterophorus L., and Solanum nigrum L. were examined on seed germination, seedling growth, total protein content and protein profile on Ankur, Bhatt, Bragg, PK -416, PS-1042 and Shilajeet varieties of soybean (Glycine max (L) Merill). Aqueous extracts of weeds (10% w/v) showed both inhibitory and stimulatory influence on percent seed germination and seedling growth in different varieties of soybean. On the basis of per cent reduction in different parameters, the variety Bragg and PS-1042, and Shilajeet were resistant and susceptible respectively to different weed extracts, and among weed extracts, S. nigrum was most effective followed by P. hysterophorus compared to others. The total protein content (mg/g f wt.) in different varieties was increased with all the weed extracts except Bragg with C. dactylon and P. hysterophorus, PS-1042 with A. conyzoides and Shilajeet with C. dactylon, in which it was decreased. The protein banding pattern in different varieties not only differ between control and treatments but also among treatments. The order of susceptibility of different varieties with different weed extracts followed the order: Ankur > PK-416 > Bhatt > Shilajeet > Bragg and > PS-1042.     

Constitutive and induced defenses to herbivory in above- and belowground plant tissues

A recent surge in attention devoted to the ecology of soil biota has prompted interest in quantifying similarities and differences between interactions occurring in above- and belowground communities. Furthermore, linkages that interconnect the dynamics of these two spatially distinct ecosystems are increasingly documented. We use a similar approach in the context of understanding plant defenses to herbivory, including how they are allocated between leaves and roots (constitutive defenses), and potential cross-system linkages (induced defenses). To explore these issues we utilized three different empirical approaches. First, we manipulated foliar and root herbivory on tobacco (Nicotiana tabacum) and measured changes in the secondary chemistry of above- and belowground tissues. Second, we reviewed published studies that compared levels of secondary chemistry between leaves and roots to determine how plants distribute putative defense chemicals across the above- and belowground systems. Last, we used meta-analysis to quantify the impact of induced responses across plant tissue types. In the tobacco system, leaf-chewing insects strongly induced higher levels of secondary metabolites in leaves but had no impact on root chemistry. Nematode root herbivores, however, elicited changes in both leaves and roots. Virtually all secondary chemicals measured were elevated in nematode-induced galls, whereas the impact of root herbivory on foliar chemistry was highly variable and depended on where chemicals were produced within the plant. Importantly, nematodes interfered with aboveground metabolites that have biosynthetic sites located in roots (e.g., nicotine) but had the opposite effect (i.e., nematodes elevated foliar expression) on chemicals produced in shoots (e.g., phenolics and terpenoids). Results from our literature review suggest that, overall, constitutive defense levels are extremely similar when comparing leaves with roots, although certain chemical classes (e.g., alkaloids, glucosinolates) are differentially allocated between above- and belowground parts. Based on a meta-analysis of induced defense studies we conclude that: (1) foliar induction generates strong responses in leaves, but much weaker responses in roots, and (2) root induction elicits responses of equal magnitude in both leaves and roots. We discuss the importance of this asymmetry and the paradox of cross-system induction in relation to optimal defense theory and interactions between above- and belowground herbivory.     

施肥对落叶松人工林植物养分及生理特性的影响

以东北林业大学帽儿山实验林场的14年生落叶松人工幼龄林为研究对象，对白浆化的暗棕壤林地进行了施肥试验。采用完全随机排列的方法，在林分内设置了9个施肥处理和1个无肥对照。在生长旺盛期测定了叶片和根系的全N和全P质量分数以及光合速率、呼吸速率、叶绿素质量分数等指标。结果表明，单施520kg/hm^2尿素，在提高全N、全P质量分数的同时，还能促进根系对磷素的吸收和利用，增加光合速率和叶绿素质量分数；单施314kg／hm^2磷酸二铵，能加快根系对养分的转移速度，提高光合系数(光合速率／呼吸速率)，且是这些施肥处理中效果最好的施肥水平，这说明此林龄的落叶松林容易缺磷，应适量多施磷肥；处理436kg／hm^2尿素 236kg／hm^2磷酸二铵 133kg／hm^2氯化钾，由于K肥的混入，加快了根系对N、P的转移，使叶片中的质量分数增加，有利于光合作用的进行。因此，合理的施肥措施，不仅能提高落叶松人工林叶片与根系的养分质量分数，还能提高叶片的光合能力。对于此幼龄林，少N多P或N、P、K混合施肥效果最佳。