盐角草（Salicornia europaea L.）体外再生体系及floral-dip转化系统的初步研究

盐角草（Salicornia europaea L.）属藜科盐角草属（Salicornia），是迄今已报道过的最为耐盐的真盐生植物之一。跟与其同属的海蓬子（Salicornia bigelovvi Torr.）相比, 它的分布更为广泛。它的种子含油丰富，因此具有发展为油料作物的潜力。此外，它也可以作为蔬菜和饲料。盐角草的组织培养工作为未来的转化研究和胁迫相关基因功能分析提供了有力的工具。本工作中，通过器官发生途径，建立了盐角草的体外再生体系。以完整的成熟种子为起始培养材料，在添TDZ 0.1 mg/L与NAA 1 mg/L的MS培养基上，暗培养三周后在下胚轴处形成愈伤组织，形成愈伤的平均频率为99％。愈伤组织在含TDZ 0.1 mg/L与NAA 1 mg/L的培养基上培养3－4周后分化出芽，分化频率约26.7%。采用2,4-D短时处理法结合添加NaCl，经过6－8周的培养获得了丛生芽，提高了再生频率。分化芽3周后转入含IBA 0.5 mg/L、KN 0.1 mg/L与0.05％活性炭的1/2 MS培养基，3周后生根形成完整植株。同时，本研究也进行了从直接不定芽途径建立盐角草再生体系的试验，但未获成功。 此外，本工作借鉴拟南芥的floral-dip转化法，对建立盐角草floral-dip转化系统进行了尝试。

雪莲细胞培养物中黄酮类物质代谢调控及其生物活性成份分析

水母雪莲（Saussurea medusa Maxim.）和新疆雪莲（Saussurea involucrata Karel. et Kir.）是我国珍稀的药用植物资源，具有清热解毒、止痉镇痛、敛伤、消肿及治疗热病、风湿等多种功效。雪莲的主要药用成份为紫丁香甙（Syringin）、芦丁(Rutin)、高车前素（Hispidulin）和Jaceosidin等苯基丙酸类（phenylpropanoid）和黄酮类（flavonoids）物质。最新的药理研究表明，上述物质还具有抗菌消炎、保肝降压、延缓衰老和抑制癌细胞增殖等重要的研发价值。 雪莲生境恶劣，生长缓慢，人工引种困难，加上长期掠夺性采挖，已使雪莲处于灭绝的边缘。为了保存国家珍稀植物品种，保护生态环境，满足临床上对雪莲药物的需求，本研究在雪莲组织培养的基础上，应用诱导子添加技术和毛状根培养技术对雪莲中具有重要药用价值的次生代谢物质进行调控，并对雪莲MYB类转录因子的功能进行了初步探索，为保护珍稀植物资源、维护生态环境、开发野生雪莲替代产品、缩短雪莲药用成份的生产周期奠定了基础。另外，分析了野生雪莲和雪莲培养物中主要生物活性成份的种类及含量，为今后雪莲药理药效研究及品质评价奠定了基础。 为了提高雪莲黄酮的产量，满足工业化生产的需要，在细胞培养水平上，通过添加茉莉酸甲酯（MJ），对雪莲黄酮类物质的代谢进行调控。研究了诱导子的添加时间、添加浓度对水母雪莲红色系悬浮细胞的生物量和总黄酮产量的影响。发现在细胞培养的指数期（第9天）添加5.0 µmol/L的MJ，可以使总黄酮产量提高2.4倍（1134.5 ± 63.86 mg/L），而雪莲细胞干重（dw）仅比对照提高23.8 %（20.4 ±0.27 g/L）。另外，细胞中苯丙氨酸裂解酶（PAL）的活性分析表明，MJ添加后PAL活性的增加与雪莲总黄酮含量增长之间存在相关性。 在器官培养水平上，对雪莲毛状根的诱导频率及其培养条件进行了研究。结果表明，选择发根农杆菌R1601侵染预培养2天的新疆雪莲根段外植体，毛状根的诱导效率可达到83 %。毛状根的冠瘿碱检测、PCR和Southern分析表明，Ri质粒中的T-DNA已整合到植物基因组中并稳定表达。以新疆雪莲毛状根为外植体，能够容易地获得再生芽。在含有1.0 mg/L 6-BA的MS固体培养基上，其再生频率高达91 ± 5.9 %，是其正常根的2.4倍。而水母雪莲在该培养条件下，仅有少量的畸形芽出现。进而对毛状根的培养条件进行初步研究，结果表明在无激素附加的MS液体培养基中，新疆雪莲的HR1601根系在一个培养周期内（32 天），其生物量能够达到接种量的16倍，而紫丁香甙含量（43.5 ± 1.13 mg/g dw）能够达到野生雪莲的83倍。从而显示了雪莲毛状根培养体系的优良特性。 在基因水平上，对雪莲黄酮类物质代谢调控的研究已经展开。玉米P基因编码的Myb类转录因子能够调节黄酮类物质代谢途径关键酶基因的表达。根据P基因的保守序列设计引物，从雪莲细胞培养物中获得了SmP基因。核酸序列分析表明，SmP基因与烟草中涉及苯丙素类物质代谢途径的LBM 1、LBM 3和MybAS 1基因具有较高的一致性，分别为66 %、60 %和61 %。因此为了研究雪莲SmP基因的功能，构建了正义表达载体，并与先前构建好的反义表达载体分别导入烟草，分析了转基因植株的形态特征及黄酮类物质的含量变化。其中，约有30 %转反义SmP基因的株系表现叶片皱缩、叶脉紊乱、主侧脉角度缩小、叶片、花瓣失去对称性以及花粉败育等性状。 另外，通过正交试验设计优化了雪莲提取工艺的条件，并对雪莲细胞提取物进行了分离纯化。正交试验设计结果表明，温度对雪莲黄酮提取效率的影响极为显著，而分批多次提取比一次性浸提，能够收到较好的提取效果。考虑到工业生产中的实际问题，推荐在60 ℃水浴条件下，采用50 %乙醇对雪莲样品连续浸提2次的方案。对雪莲提取物的纯化研究表明，雪莲成份复杂，仅依靠单一的分离手段，往往难以奏效。另外，野生雪莲及雪莲培养物中生物活性成份的比色法、HPLC（High Performance Liquid Chromatography）、LC-ESI-MS（Liquid Chromotagraphy Electrospray Ionization Mass Spectrometry）分析表明，传统的NaNO2-AlCl3 法测定雪莲总黄酮的含量，结果偏高，不利于雪莲黄酮的实验室研究分析与今后工业化生产的质量监控。而AlCl3 法的显色反应较为特异，今后有望取代NaNO2-AlCl3 法，作为雪莲类药材品质评价的标准。而HPLC-DAD结合LC-ESI-MS可以对雪莲中的主要生物活性成份进行较为准确的定性分析，从而解决了由于缺乏相应的雪莲化合物标准品而难以对雪莲中的成份进行定性定量分析及比较的难题。最后综合利用上述分析方法，对雪莲细胞培养物中的花素类物质进行了分析。结果表明，雪莲细胞中至少含有7种花色素类物质，分别为矢车菊素-3-O-葡萄糖甙及其衍生物、天竺葵素糖甙衍生物和芍药色素糖甙衍生物。

药蒲公英（Taraxacum officinale weber）耐盐变异体的筛选及其耐盐性分析研究

以药蒲公英（Taraxacum officinale Weber）叶片外植体为材料诱导愈伤组织。以NaCl作为选择因子，从愈伤组织直接筛选。在选择培养基上，大部分愈伤组织褐化死亡，在一些褐化死亡的愈伤组织周围有少量新的细胞团生长，挑选生长存活状况好的细胞团转接到新鲜培养基上，每3周继代一次，经3个月继代筛选获得了耐1.5% NaCl的药蒲公英细胞团。以普通愈伤为对照，发现随着NaCl浓度的升高，耐盐愈伤的相对生长率下降但显著高于对照;且随着盐胁迫处理时间的延长持续升高，而普通愈伤对照几乎停止生长，说明耐盐愈伤具有相对稳定的耐盐性。在蛋白水平上，耐盐愈伤与对照愈伤差异明显，SDS-PAGE分析显示：耐盐愈伤比对照多出一条34 KD大小的蛋白带，且30 KD，18 KD左右的蛋白带明显上调。相同处理条件下耐盐愈伤脯氨酸的增加幅度高于对照。盐胁迫条件下，耐盐愈伤的超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性明显高于对照，且随着处理时间的延长和盐浓度的增加呈现升高的趋势，而对照则呈现先升高后下降的趋势。1.5% NaCl处理前后，耐盐愈伤的总黄酮含量显著高于对照。结果说明耐盐愈伤一方面通过积累蛋白和其他小分子有机溶质的方式调节其渗透平衡，另一方面还可通过提高抗氧化能力降低盐分造成的次级伤害。 将耐1.5% NaCl的药蒲公英愈伤组织接种在分化培养基上分化出芽，之后将再生芽转接到生根培养基中进行生根培养，经4个月得到了12株耐1.5% NaCl的药蒲公英再生植株。与野生型相比，耐盐植株叶片宽大、叶柄粗短、叶表面覆盖白色细毛，根粗壮较短，花茎中部具有2 cm左右的苞叶。RAPD和SDS-PAGE检测表明，耐盐植株与对照植株在DNA及蛋白水平上均存在明显差异。1.5% NaCl处理后，与普通再生植株相比，耐盐株系的抗氧化酶活性明显提高，脯氨酸含量上升幅度更为显著，而丙二醛含量降低，其主要药用成分黄酮的含量显著增加。这些结果说明耐盐植株的抗氧化防御能力明显增强。以上结果表明耐1.5% NaCl的药蒲公英再生植株为耐1.5% NaCl药蒲公英变异体，这些耐盐变异体有望成为抗盐耐海水蔬菜家族的新成员。同时，这些耐盐变异体植株比普通植株具有更高的医用商业价值。耐1.5% NaCl的药蒲公英再生变异体遗传稳定性的研究正在进行中。

玉簪品种的引种，栽培和组织培养

本研究通过对玉簪属植物的组织培养，发现以腋芽、花序、试管苗的叶片为外植体，均可达到迅速繁殖的目的，但对于不同的玉簪品种来说，所能采用的外植体的种类又不尽相同：对于非嵌合体玉簪品种，以腋芽、花序、试管苗的叶片作外植体都可以;而对于嵌合体玉簪品种，如果以花序或试管苗的叶片为外植体，通过诱导不定芽进行快繁，在所得的试管苗中，超过50%的个体失去斑叶特性，这在实际生产中是不可行的，如果以腋芽为外植体，所得试管苗中多于95%的可保持原有嵌合体特征，故为理想的外植体。 在以腋芽为外植体的离体培养中，取嵌合玉簪品种H. ‘Francee’和H. ‘Ground Master’的芽作材料，通过对试验结果的分析、比较，选出了合适的繁殖培养基：MS ＋ BA0.5mg/L + NAA0.5mg/L + KH2PO4150mg/L + 水解乳蛋白500mg/L + 蔗糖30g/L + 琼脂 5.6g/L，其可提高腋芽分化率，达到了快速繁殖的目的，此外，该培养基还可保持原来的生长势，降低斑叶特性分离比;同时选出了生根培养基：MS + IAA1mg/L + 蔗糖30g/L + 琼脂5.6g/L。 以花葶为外植体的试验，证实了此种方式仅适合非嵌合体玉簪的快速繁殖。 在以叶片为外植体的研究中，取了嵌合体品种H. ‘Francee’、H. ‘Ground Master’、H. ‘Gold Standard’、H. ‘Color Glory’和H. ‘Little Ming’的再生叶征作材料，通过对试验结果的分析、比较、观察到在培养基：MS + BA4mg/L + NAA0.1mg/L + 蔗糖 30g/L + 琼脂 5.6g/L上只有芽的形成，培养基：MS + BA0.4mg/L + NAA0.4mg/L + 蔗糖 30g/L + 琼脂 5.6g/L则利于根的发生，而培养基：MS + BA4mg/L + NAA0.4mg/L + 蔗糖 30g/L + 琼脂 5.6g/L促进根或（和）不定芽的产生，则因品种的不同而变化，另外还发现，经诱导不定芽途径所得到的试管苗，仅少于5%的个体保持斑叶特性，所以不可用于嵌合体玉簪的组织培养，但可快速繁殖非嵌合体玉簪。

大百合属间杂交亲和性、离体培养及耐热生理研究

本实验以大百合和百合东方杂种系“索蚌”为材料，对大百合、百合杂交的亲和性、大百合离体培养及其耐热性进行了研究，以期为大百合与百合杂交育种及相应的耐热百合材料的筛选、种质保存、新品种快繁及栽培应用提供理论依据。 　　以大百合为母本，百合为父本，对属间杂交授粉后花粉管的行为进行观察，结果表明：大百合与百合属间杂交授粉后，百合的花粉在大百合的花柱内的伸长过程中，出现少部分花粉管末端分叉、膨胀或变细，胼胝质大量不规则沉淀，及部分花粉管在伸长过程中受阻等不亲和现象，但大部分花粉仍能够正常萌发，穿过花柱道，进入子房，到达胚珠，且能够观测到早期的胚。虽然杂交亲和性与花粉管的行为有关，但杂交的成功与否还受到受精后诸多因素的影响，还需要从胚胎学和遗传学方面进一步探讨。 　　以大百合的鳞片、叶柄和子房为外植体，进行离体培养，结果表明：大百合的鳞片和叶柄外植体均可成功地诱导小鳞茎，叶柄相对更容易。鳞茎诱导小鳞茎的最佳培养基为MS＋NAA0.5－1.0mg/ml +BA2.5mg/ml +KT2.5mg/ml +蔗糖3%＋琼脂0.7%，28周后，每个外植体平均可以分化4-11个小鳞茎;叶柄诱导小鳞茎的最佳培养基为MS＋NAA1.0－2.0mg/ +BA2.5-3.0mg/ml +KT2.5-3.0mg/ml +蔗糖3%＋琼脂0.7%，26周后，每个外植体平均可以分化3-9个小鳞茎。同时也发现，用鳞茎作为外植体，污染率较高。在大百合的子房离体培养实验中发现：BA和KT 是影响大百合子房分化途径的关键因素，其浓度分别为0.1-1.0mg/L、2.0-4.0 mg/L和高于4.0mg/L时，外植体分别分化为愈伤组织、芽和叶。外植体分化的基本培养基以N6、B5为佳。愈伤组织诱导小鳞茎的最佳培养基为MS+0.1-0.5mg/L NAA +2.5mg/L BA+2.5mg/L KT +10%蔗糖+0.7%琼脂。在1/2MS +3%的蔗糖+0.7%琼脂+1%活性炭的生根培养基上，生根率为100%。炼苗一周后移栽，长势良好。 　　对长至5－6片真叶的大百合植株在不同高温（30℃、35℃和40℃）下，分别进行4h、10h及24h(热胁迫10h，然后在22℃对照温度下缓苗14h)的热胁迫处理，测定了不同处理下，植株的净光合速率（Pn），实际光化学效率（φPS2），最大光化学效率（Fv/Fm）和叶片的相对电导率，游离脯氨酸含量，可溶性蛋白含量，以及叶片中超氧化物歧化酶（SOD）和过氧化氢酶(CAT)的活性。结果表明：大百合对30℃的高温胁迫有较好的适应能力，表现为可溶性蛋白、游离脯氨酸等渗透调节物质的积累，抗氧化酶活性的提高，以及缓苗后细胞膜的自我修复和光合能力的恢复;随着胁迫温度的升高（35℃、40℃）和胁迫时间的延长（4h、10h），大百合一方面对高温胁迫做出了积极的响应，另一方面，光系统的光合能力，细胞膜的稳定性，抗氧化酶的活性，也受到了一定程度的伤害，在缓苗后,细胞膜的稳定性、细胞的渗透势、抗氧化酶的活性等都在一定程度上得到恢复。 　　

苦苣苔科植物的组织培养和离体保存

苦苣苔科(Gesneriaceae)植物种类繁多, 全世界约150属3700余种,我国有58属470余种，大部分具有极高的观赏价值，许多是传统的民间草药。虽然我国苦苣苔科植物资源丰富，然而很多种类分布区域狭窄，种群数量稀少，加之生境受到破坏，许多已经面临灭绝的危险。本研究拟通过组织培养、玻璃化超低温保存以及快速繁殖，达到保护和扩繁珍稀濒危苦苣苔科植物的目的。 以药用唇柱苣苔（Chirita medica D. Fang ex W. T. Wang）和粉绿异裂苣苔（Pseudochirita guangxiensis W.T.Wang var. glauca Y. G. Wei et Y. Liu）为材料，取幼嫩叶片为外植体，通过组织培养实验得到最佳诱导不定芽培养基：MS培养基附加30 g l-1蔗糖，7.5 g l-1琼脂，药用唇柱苣苔附加0.10 mg l-1 BA ，0.10 mg l-1 NAA，粉绿异裂苣苔附加0.05 mg l-1IAA，1.00 mg l-1BA。最高不定芽诱导率分别为：90.3%和85.0%。最佳生根培养基：1/2MS培养基附加30 g l-1蔗糖，5 g l-1活性炭，7 g l-1琼脂，生根率为100%，诱导产生6.11条根，根长为18.8mm（药用唇柱苣苔）；1/2MS培养基附加10-20g l-1蔗糖，1 g l-1活性炭，7 g l-1琼脂，诱导生成6.8-7.4条根，均长17.7-22.0mm（粉绿异裂苣苔）。 在组织培养的基础上进行了苦苣苔科植物的玻璃化超低温冷冻保存研究。以烟叶唇柱苣苔（C. heterotricha Merr.）和濒危植物药用唇柱苣苔叶片外植体为材料，经过自然干燥、装载液处理、玻璃化溶液处理、液氮冷冻保存，成功实现了玻璃化超低温冷冻保存，经过液氮冷冻保存后的材料可以继续分化、生长。适当时间的玻璃化试剂处理对于材料无致死作用，不经液氮冷冻，可以达到100%存活。-20 oC 、-40 oC、液氮保存后，存活率随温度下降而下降，表明冷冻致死的原因在于冰晶形成；提高冷冻后成活率的关键是控制干燥脱水，经过适当的自然干燥，材料存活率分别达到50.0%和27.8%。 以叶片为外植体材料，通过组织培养和快速繁殖可以大规模扩繁苦苣苔科植物。主要步骤为：外植体叶片消毒→不定芽诱导培养→生根诱导培养→继代保存或炼苗移栽，经过3-4个月时间可获得大量栽培植株。已成功保存并培养了40余种苦苣苔科植物，包括濒危苦苣苔及高观赏价值苦苣苔。

Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.

The role of NH4+ toxicity in the decline of the submersed macrophyte Vallisneria natans in lakes of the Yangtze River basin, China

Experimental and field studies were conducted to evaluate the effects of NH4+ enrichment on growth and distribution of the submersed macrophyte, Vallisneria natans L, in lakes of the Yangtze River in China, based on the balance between free amino acids (FAA) and soluble carbohydrates (SC) in the plant tissue. Increase of NH4+ rather than NO3- concentrations in the water column caused FAA accumulation and SC depletion of the plant. The plant showed a unimodal pattern of biomass distribution along both FAA/SC ratios and external NH4+ concentrations, indicating that a moderate NH4-N concentration (< 0.3 mg L-1) benefited the plant, whereas the high NH4-N concentration (> 0.56 mg L-1) eliminated the plant completely. Therefore, 0.56 mg NH4-N mg L-1 in the water column was taken as the upper limit for V. natans in lakes of the Yangtze River basin. The mesocosm experiment showed that at a high external NH4-N (0.81 mg L-1), V. natans failed to propagate with a loss of half SC content (5 mg g(-1) DW) in the rhizomes, indicating that the consumption of carbohydrates for detoxification of excess NH4+ into non-toxic FAA significantly diminished carbohydrate supply to the rhizomes. This might consequently inhibit the vegetative reproduction of the plant, and also might be an important cause for the decline and disappearance of the plant with eutrophication. The present study for the first time reports substantial ecophysiological evidences for NH4+ stress to submersed macrophytes, and indicates that NH4+ toxicity arising from eutrophication probably plays a key role in the deterioration of submersed macrophytes like V. natans.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

鞑靼荞麦（Fagopyrum tataricum Gaertn.）离体再生与遗传转化研究

鞑靼荞麦是我国特有的农业产品，具有抗寒耐旱特性和较高的营养保健功能。荞麦的开花习性及遗传特点导致其人工杂交授粉难以成功，这成为荞麦杂交育种难以获得突破的重要原因。因此利用转基因技术导入有益基因有可能成为荞麦遗传改良的新途径，而再生及转化体系的建立是开展转基因研究的基础。 本文研究了苗龄、外植体、几种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6-8d；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，下胚轴的可高达86.62％；愈伤组织分化的最适培养基为MS 0.1mg/L IAA＋2.0mg/L 6-BA＋1.0 mg/L KT＋0.5mg/L TDZ，下胚轴的分化率可达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。培养基中加入AgNO3后，能有效降低褐化率。生根最适培养基为含有0.5mg/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%。 在上述研究基础上，本文还对鞑靼荞麦的遗传转化体系进行了探索性研究。分别利用根癌农杆菌（Agrobacterium tumefaciens）介导法和微粒轰击法（基因枪法）对黑水苦荞下胚轴进行遗传转化。 在农杆菌介导的方法中，携带有质粒pCAMBIA2301的农杆菌菌株EHA105用于转化。载体质粒pCAMBIA2301包含有gus和npt-II 基因, 并受35s启动子驱动。研究结果表明，在侵染方式选择上，浸泡方式比吸打方式更有效，根癌农杆菌侵染的较适浓度为OD600＝0.5，共培养3天，恢复培养7天，能检测到gus基因的表达。 基因枪法使用质粒pBI121，同样包含有gus和npt-II基因, 并受CaMV35s 启动子驱动。轰击距离为9cm较合适，甘露醇前处理在本研究中未表现出明显优势。 两种转化方法比较，基因枪法比农杆菌介导法更快速有效。 本研究为进一步的遗传操作研究打下基础。 Tartary buckwheat (Fagopyrum tataricum Gaertn.), the traditional and unique agricultural product of China, is a kind of crop with strong drought and cold tolerance, abundant nutrition and high medical value. Artificial hybridization is hard in buckwheat because of its flowering habits and genetic characteristics, which leads to no breakthrough in tartary buckwheat breeding. However, biotechnological approaches, especially genetic transformation for the direct introduction of good genes into tartary buckwheat for quality improvement, hold great promise. In this study, we established tartary buckwheat regeneration system in vitro. It is the foundation for genetic manipulation of this crop. The effects of seedling age, hypocotyl and cotyledon as explants, and proportions of several growth regulators were tested in tissue culture of tartary buckwheat for establishing its in vitro regeneration system. The results showed that the best seedling age for callus induction was 6 to 8 days. On the MS medium containing 2.0mg/L 2, 4-D and 1.5mg/L 6-BA, the induction rate of callus from hypocotyls was up to 86.62%, while from cotyledons was about 75％. The suitable shooting medium was the MS medium+0.1mg/L IAA+2.0mg/L 6-BA+1.0 mg/L KT+0.5mg/L TDZ, and the shooting rate from hypocotyls was 9.52%. The callus induction and shooting rates were higher from hypocotyls than from cotyledons. Browning reduced when the medium mixed with AgNO3. Half strength MS supplemented with 0.5mg/L NAA was the best for rooting, the rate was around 50% after 30 days culture. TDZ can accelerate the shoot differentiation distinctively, and it could improve the shooting rate nearly 20%. On the base of above, the explorative research of the genetic transformation in tartary buckwheat was done. In the study, hypocotyls from Heishui tartary buckwheat were transformed by Agrobacterium-mediated method and microprojectile bombardment method (gene-gun), comparatively. In Agrobacterium-mediated method, a disarmed Agrobacterium tumefaciens strain EHA105 harboring plasmid pCAMBIA2301 was used. The vector pCAMBIA2301 contains gus and npt-II genes, driven by CaMV35s promoter. The results showed that the appropriate concentration of Agrobacterium tumefaciens for infecting was OD600＝0.5, and co-culture time was 3d. Seven days later after coculture, GUS expression could be tested. In particle bombardment transformation, plasmid pBI121 was used. pBI121 also contains gus and npt-II genes, driven by 35s promoter. Hypocotyls pretreated with mannitol, no effect was observed, and the suitable distance of bombardment is 9cm. Comparing with Agrobacterium-mediated method, gene-gun method is more convenient and effective. All above results could be a basic work for further study in tartary buckwheat transformation.

Linear energy transfer dependence of the effects of carbon ion beams on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

Purpose: To determine the effects of carbon ion beams with five different linear energy transfer (LET) values on adventitious shoots from in vitro leaf explants of Saintpaulia ionahta Mauve cultivar with regard to tissue increase, shoots differentiation and morphology changes in the shoots. Materials and methods: In vitro leaf explant samples were irradiated with carbon ion beams with LET values in the range of 31 similar to 151 keV/mu m or 8 MeV of X-rays (LET 0.2 keV/mu m) at different doses. Fresh weight increase, surviving fraction and percentage of the explants with regenerated malformed shoots in all the irradiated leaf explants were statistically analysed. Results: The fresh weight increase (FWI) and surviving fraction (SF) decreased dramatically with increasing LET at the same doses. In addition, malformed shoots, including curliness, carnification, nicks and chlorophyll deficiency, occurred in both carbon ion beam and X-ray irradiations. The induction frequency with the former, however, was far more than that with the X-rays. Conclusions: This work demonstrated the LET dependence of the relative biological effectiveness (RBE) of tissue culture of Saintpaulia ionahta according to 50% FWI and 50% SF. After irradiating leaf explants with 5 Gy of a 221 MeV carbon ion beam having a LET value of 96 keV/mu m throughout the sample, a chlorophyll-deficient (CD) mutant, which could transmit the character of chlorophyll deficiency to its progeny through three continuous tissue culture cycles, and plantlets with other malformations were obtained.

Effects of ion beam irradiation on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiation on adventitious shoots from in vitro leaf explants of two different Saintpaulia ionahta (Mauve and Indikon) cultivars were studied with regard to tissue increase, shoots differentiation and morphology changes in the shoots. The experimental results showed that the survival fraction of shoot formation for the Mauve and Indikon irradiated with the carbon ion beam at 20 Gy were 0.715 and 0.600, respectively, while those for both the cultivars exposed to the Xray irradiation at the same dose were 1.000. Relative biological effectiveness (RBE) of Mauve with respect to X-ray was about two. Secondly, the percentage of regenerating explants with malformed shoots in all Mauve regenerating explants irradiated with carbon ion beam at 20 Gy accounted for 49.6%, while that irradiated with the same dose of X-ray irradiation was only 4.7%; as for Saintpatdia ionahta Indikon irradiated with 20 Gy carbon ion beam, the percentage was 43.3%, which was higher than that of X-ray irradiation. Last, many chlorophyll deficient and other varieties of mutants were obtained in this study. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the leaf explants of Saintpaulia ionahta is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy to 25 Gy for carbon ion beam irradiation.

Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Freaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinitera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73 +/- 0.15, 1.45 +/- 0.04, 0.77 +/- 0.024 and 0.27 +/- 0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% for V vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line, showed greater potential in enhanced the anthocyanin production.

Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

A switch in the control of growth of the wing imaginal disks of Manduca sexta.

BACKGROUND: Insulin and ecdysone are the key extrinsic regulators of growth for the wing imaginal disks of insects. In vitro tissue culture studies have shown that these two growth regulators act synergistically: either factor alone stimulates only limited growth, but together they stimulate disks to grow at a rate identical to that observed in situ. It is generally thought that insulin signaling links growth to nutrition, and that starvation stops growth because it inhibits insulin secretion. At the end of larval life feeding stops but the disks continue to grow, so at that time disk growth has become uncoupled from nutrition. We sought to determine at exactly what point in development this uncoupling occurs. METHODOLOGY: Growth and cell proliferation in the wing imaginal disks and hemolymph carbohydrate concentrations were measured at various stages in the last larval instar under experimental conditions of starvation, ligation, rescue, and hormone treatment. PRINCIPAL FINDINGS: Here we show that in the last larval instar of M. sexta, the uncoupling of nutrition and growth occurs as the larva passes the critical weight. Before this time, starvation causes a decline in hemolymph glucose and trehalose and a cessation of wing imaginal disks growth, which can be rescued by injections of trehalose. After the critical weight the trehalose response to starvation disappears, and the expression of insulin becomes decoupled from nutrition. After the critical weight the wing disks loose their sensitivity to repression by juvenile hormone, and factors from the abdomen, but not the brain, are required to drive continued growth. CONCLUSIONS: During the last larval instar imaginal disk growth becomes decoupled from somatic growth at the time that the endocrine events of metamorphosis are initiated. These regulatory changes ensure that disk growth continues uninterrupted when the nutritive and endocrine signals undergo the drastic changes associated with metamorphosis.