Determinação de atividade antioxidante, compostos fenólicos totais e teor de resveratrol em cinco cultivares brasileiras de Arachis hypogaea L. com a utilização de duas metodologias extrativas

O amendoim A. hypogaea L. é a quarta oleaginosa mais consumida no mundo e suas sementes são altamente energéticas, com grandes quantidades de lipídios, proteínas, vitaminas e carboidratos. Diversas atividades farmacológicas já foram observadas em extratos de raízes, folhas e sementes, sendo a principal delas a atividade antioxidante. Neste trabalho, foi realizada a comparação entre duas metodologias (por maceração e assistida por micro-ondas) para a extração de compostos antioxidantes, incluindo o resveratrol. Também foi realizada a comparação entre extratos de diferentes órgãos de cultivares brasileiras (IAC 886, IAC Caiapó, IAC Tatu ST, IAC 8112 e IAC 99-1) quanto à atividade antioxidante, por DPPH, ao teor de compostos fenólicos, por Folin-Ciocalteu, e ao teor de resveratrol, por HPLC. Por fim, foram estabelecidos protocolos de cultura de tecidos para explantes de sementes, visando à produção de calos e plantas in vitro para posterior dosagem de compostos de interesse, tendo em vista a possibilidade de modulação das condições in vitro. As melhores condições determinadas para a extração por maceração de antioxidantes de A. hypogaea foram 80% de etanol em água como solvente, trituração com almofariz e pistilo, 50 mL solvente por grama de material vegetal seco, 120 minutos de incubação e dois estágios de extração. As melhores condições para a extração de resveratrol assistida por micro-ondas foram o uso de 37 mL de solvente/g material vegetal seco, com agitação de 1200 rpm por 15 minutos, a 37C. De uma maneira geral, os extratos de raízes e oriundos de micro-ondas apresentaram maior atividade antioxidante (até 92,36 2,71%), teor de compostos fenólicos (até 54,15 1,39 mg EAG/g extrato) e teor de resveratrol (até 1,614 0,356 mg/g extrato). Dentre as cultivares estudadas, IAC Tatu e IAC 99-1 foram as que apresentaram os teores mais elevados. Brotos e calos friáveis foram obtidos a partir de cotilédones, eixos embrionários e folíolos embrionários cultivados em meios suplementados com BAP e picloram, respectivamente.

Conservação in vitro de germoplasma de Petiveria alliacea L. utilizando cultura de tecidos e criopreservação

A implementação de estratégias visando à conservação de espécies medicinais apresenta grande importância, tanto do ponto de vista ecológico quanto econômico. Petiveria alliacea L., espécie da família Phytolacaceae, conhecida como guiné, erva-de alho, erva-tipi ou amansa-senhor, pode ser encontrada desde a América Central até a América do Sul. Esta espécie possui ampla utilização medicinal, devido à presença, em sua composição, de vários polissulfetos, como o DTS (dibenziltrissulfeto), com propriedades antifúngica, antineoplásica e imunomodulatória, entre outras. O objetivo deste trabalho foi a conservação in vitro de germoplasma de Petiveria alliacea obtido de diferentes populações ocorrentes no Rio de Janeiro, através de métodos baseados em cultura de tecidos e criopreservação. Plantas obtidas através da germinação in vitro foram utilizadas como matrizes para a micropropagação através da multiplicação de meristemas pré-existentes em meio MS sem reguladores de crescimento, e embriões somáticos foram obtidos de forma direta a partir da cultura de explantes foliares das linhagens estudadas (Magé MG; Marechal Hermes MH; Niterói NT; Vila Isabel VI e Planta envasada AL), em presença de PIC (20μM) e 2,4D (22,6 μM), após 60 dias de cultivo. Os ápices caulinares das plantas micropropagadas e os embriões somáticos obtidos foram submetidos à criopreservação através de técnicas de vitrificação, encapsulamento-vitrificação e encapsulamento-desidratação, com a avaliação da pré-cultura e de soluções crioprotetoras (PVS2 e PVS3), em diferentes concentrações e tempos de exposição. Os resultados mostraram uma taxa de 100% de germinação in vitro. As condições para a pré-cultura de ápices foram padronizadas, entretanto não foram obtidos resultados positivos após o descongelamento. Por outro lado, os embriões da linhagem AL (linhagem embriogênica em cultura há 24 meses) mantiveram sua capacidade multiplicativa (100%) (embriogênese secundária) quando submetidos à desidratação em sacarose (0,5M) e expostos às soluções de vitrificação PVS2 e PVS3 pelos menores tempos (15 e 30 minutos). Os diferentes meios de recuperação apresentaram respostas variáveis em relação à capacidade multiplicativa dos embriões. O encapsulamento/vitrificação foi a técnica que promoveu maior tolerância dos embriões ao congelamento. A recuperação dos embriões através de multiplicação após a criopreservação abre uma perspectiva de conservação de genótipos com alta produção de polissulfetos, de interesse para a indústria farmacêutica

Produção in vitro, análise fitoquímica e avaliação da atividade antineoplásica de metabólitos de Hovenia dulcis Thunb. (Rhamnaceae) obtidos por diferentes estratégias biotecnológicas

Hovenia dulcis Thunberg, natural da Ásia Oriental, é cultivada no Brasil onde é conhecida como uva-do-japão. A espécie possui várias indicações na medicina popular e alguns estudos apontam o seu potencial antineoplásico, tripanocida e hepatoprotetor. Metabólitos secundários são substâncias não essenciais para a sobrevivência celular, mas que fornecem vantagens adaptativas aos vegetais, sendo atribuído, para algumas delas, atividades biológicas importantes. Substâncias de interesse medicinal têm sido obtidas por técnicas da cultura de tecidos vegetais, como a calogênese e a cultura de células em suspensão, que permitem a síntese de matéria-prima de forma contínua e homogênea, independentemente de fatores ambientais e sazonais. O presente estudo objetivou o estabelecimento de culturas in vitro de H. dulcis, visando à produção de metabólitos de interesse, com vistas à avaliação do seu potencial antineoplásico sobre células K562. Foram testados protocolos para o estabelecimento de diferentes sistemas, como culturas de calos, de células em suspensão (CCS) e compact callus clusters (CCC) e ainda a avaliação do uso de elicitores na otimização de metabólitos produzidos in vitro. Foi verificado que a adição dos fitorreguladores KIN e TDZ, substituindo o BAP, não foi capaz de induzir a formação de calos friáveis, bem como a manutenção das culturas em ausência de luz. O uso do nitrato de prata promoveu a friabilidade de calos em todas as concentrações testadas, considerando-se 2,0 mg.L-1 a melhor concentração. Foram alcançadas taxas de 100% de formação de CCS tanto na presença, quanto em ausência de AgNO3. O maior acúmulo de biomassa foi verificado na concentração mais baixa de PIC (0,625 mg.L-1). A análise dos espectros de RMN indicou a presença de (+)-dihidromiricetina, (+)-galocatequina, hovenitina II, hovenosideo G, hodulosideo III, hodulosideo IV, hodulosideo I e hovenidulciosideo B1 nas culturas de calos friáveis. No estabelecimento de culturas CCC, observou-se a formação de calos compactos verdes em todas as concentrações de ANA testadas. O aumento da velocidade de rotação para 135 rpm aumentou a dispersão das células com consequente formação dos agregados celulares desejados. A seleção de linhagens celulares demonstrou ser um método eficiente na uniformização do tamanho desses agregados e tal uniformidade se manteve estável por mais de cinco subcultivos em 100% das culturas. Uma fração rica em saponinas foi obtida a partir dos agregados celulares, correspondendo a 1,46% da massa seca. A análise por RMN sugeriu a presença das saponinas Hovenosideo G e dos hovenidulciosideos A2 e B2. O uso de elicitores em cultura de calos mostrou-se adequado à produção de metabólitos secundários, sem alterações morfológicas nos mesmos. A elicitação alterou o perfil cromatográfico analisado por HPLC. Na elicitação com 5,0 mg.L-1 de extrato de levedura foi verificado um aumento de quase três vezes (12,280 3,396 equivalentes de quercetina/mg de extrato) na síntese de flavonoides. Finalmente, os estudos de ação antitumoral in vitro demonstraram citotoxicidade dos extratos de calos não elicitados de H. dulcis sobre linhagem de leucemia mieloide crônica (IC50 de 74,05 μg.mL-1.) e inibição do crescimento de tais células (K562), sugerindo o potencial antineoplásico para um produto biotecnológio (calo) desta espécie.

猕猴桃属两个种的原生质体培养与体细胞杂交

猕猴桃是重要的栽培果树，但目前栽培品种过于单一，不能满足生产和消费的需求。由于猕猴桃的雌雄异株特性、种间杂交亲合性差、遗传上高度杂合以及育种周期长等特点，常规杂交育种困难很大。现代生物技术，如原生质体培养和体细胞杂交等，为培育新品种提供了新途径。 毛花猕猴桃(Actinidia eriantha)和软枣猕猴桃(A.arguta)是猕猴桃属中具有重要利用价值的两个种。毛花猕猴桃果实大小在猕猴桃属中次于中华猕猴桃(A.chinensis)和美味猕猴桃(A.deliciosa)列第三位，果实维生素C含量达1014 mg/l00 g FW。软枣猕猴桃极耐寒，在-40℃下可安全越冬，其果实表面光滑无毛。这两个种是品种改良的重要种质资源。 作为生物技术基础的组织培养与植株再生系统，在毛花猕猴桃上尚未见报道。软枣猕猴桃的组织培养仅有一例报道，且芽分化率和分化系数都很低。这两个种的原生质体培养及与美味猕猴桃的原生质体融合也未见报道。针对这种情况，本试验对毛花猕猴桃和软枣猕猴桃的组织培养、原生质体培养及其与美味猕猴桃品种“Hayward”的原生质体融合进行研究，结果建立了较理想的毛花猕猴桃和软枣猕猴桃组织培养系统;首次从毛花猕猴桃原生质体得到再生植株和从软枣猕猴桃原生质体培养再生愈伤组织;通过改进融合方法，建立了毛花猕猴桃+美味猕猴桃和软枣猕猴桃+美味猕猴桃的原生质体融合体系，并将异核体培养分裂得到细胞团。这些结果有利于今后毛花猕猴桃和软枣猕猴桃资源的开发利用。主要试验结果如下： 以毛花猕猴桃试管实生苗叶片和茎段为外植体，培养在附加一定浓度Zea或CPPU的MS培养基上，产生的愈伤组织不经转代就可分化芽。试管苗茎段在附加0.0025 mg/L CPPU和0.1 mg/LIAA的MS培养基上愈伤组织产生、芽分化和苗生长都较理想;试管苗叶片则以附加0.025 mg/L CPPU和0.l mg/LIAA或0.5 mg/L Zea和0.1 mg/LIAA的MS培养基较好。当苗生长至1.0 cm时经诱导生根形成完整植株。 在软枣猕猴桃组织培养中，外植体种类、诱导培养基的激素种类和诱导分化时细胞分裂素种类都有重要影响。无菌苗茎段容易愈伤组织化，但分化困难;叶片外植体产生愈伤组织较难，但分化容易。在含Zea的MS培养基上，两种外植体产生的愈伤组织不经转代即能分化芽。分化培养基中添加Zea能有效地诱导芽分化，其中以2.0 mg/L Zea芽的分化最好，而Kin和BAP在0.5- 2.0 mg/L浓度范围内愈伤组织不分化。 以毛花猕猴桃或软枣猕猴桃试管苗叶片为分离原生质体的材料。试管苗的培养条件对原生质体分离效果及其培养反应有显著影响。弱光培养条件对两个种试管苗的原生质体分离及其培养都有好处，试管苗培养基也有重要影响。毛花猕猴桃和软枣猕猴桃试管苗合适培养基分别为MS基本培养基（大量元素减半）和MS+0.00025 mg/L CPPU+ 0.1 mg/LIAA。在此条件下培养的两个种的试管苗叶片，经酶解后原生质体产量分别为0.7-1.8×l06和3.0-3.5×l06/1 g FW，其原生质体在合适培养基上能够分裂。 毛花猕猴桃原生质体培养在MS培养基（去除NH4N03）附加l.0mg/L2，4-D液体培养基中，约10天时发生第一次分裂，分裂能持续下去并在培养3个月时形成约2mm大小愈伤组织。直接将其转入固体培养基中使其增殖和分化。在附加Zea 0.5 mg/L+ O.l mg/L IAA的MS培养基上继代2次，愈伤组织开始分化芽。芽伸长后切下诱导生根，形成完整植株。软枣猕猴桃原生质体培养基中，MS培养基附加2，4-D配合Zea或Kin对启动分裂是必须的，其中以MS＋2，4-D 0.5 mg/L+ Zea 0.5 mg/L最好，在此培养基上原生质体第一次分裂发生在4-6天时，培养12-14天时见到第三次分裂，培养三周的分裂频率为23%。培养45天后形成许多小愈伤组织块。软枣猕猴桃原生质体再生的愈伤组织从液体培养基转入固体培养基后未见进一步分裂。 对18株毛花猕猴桃原生质体再生植株的体细胞染色体数目作了观察，其中12株为整倍体，二倍体和四倍体各六株;另外六株为混倍体，其染色体数目变化在59-203之间。还发现原生质体再生植株有丝分裂间期细胞存在多核现象，有多核细胞的共10株，细胞内多核数目以双核和三核较常见，最多的有七个核。原生质体供体植株为2n=2x=58，未发现多核细胞。原生质体再生植株体细胞多核现象未见报道。 利用毛花猕猴桃或软枣猕猴桃叶片原生质体分别与愈伤组织来源的美味猕猴桃原生质体进行融合，融合方法为高Ca++高pH值PEG法。对Kao等(1975)报道的融合步骤作了修改。影响融合效率的因素主要有PEG种类、融合作用时间和融合液中DMSO浓度。最佳的融合条件为40%PEG (Sigma，MW3350)+10%DMSO，作用40 min。毛花猕猴桃+美味猕猴桃和软枣猕猴桃+美味猕猴桃的融合频率分别可达14.5%和13.6%。异核体经培养可分裂并形成细胞团。

陆地棉抗虫基因工程研究

以陆地棉（Gossypium hirsutum L.）栽培品种新陆早4号、系550、冀资492、衡无89-30、邯93-2、冀资123等为材料，进行了组织培养及植株再生研究，建立了一套陆地棉体细胞植株再生速成体系。通过调整激素种类与比例以及改善培养条件，降低了畸形胚发生频率（从80%降为41%），并可将畸形苗转化为正常苗（转化率约为78%）;通过水培和嫁接，结合试管扦插、扩繁技术，解决了棉花生根及移栽难题，为农杆菌介导法转化棉花奠定了基础。 用绿色荧光蛋白基因（gfp）作为报告基因，构建了pBGb1m（含Bt和gfp二价基因）、pBGbf（含Bt-gfp融合基因）和pBGbfg（含Bt-gfp融合基因和gna基因）等三种植物表达载体。通过农杆菌介导法转化烟草，转基因再生植株经过荧光、虫试、PCR、Southern blot和Western blot等检测，表明三种植物表达载体能够在转基因植物中有效表达，同时，绿色荧光蛋白（GFP）的检测表现出了简便、经济、快速、可靠等优点，为大量棉花转基因苗的检测提供了一种有效方法。 采用花粉管通道法将携带细胞间隙定位信号肽的Bt基因的pBin438-S1m质粒导入棉花品种冀资492，经过田间卡那霉素筛选、虫试、PCR、PCR-Southern blot和Southern blot检测，证明Bt基因已整合至棉花基因组中，而且可能是以单拷贝形式插入。 同时，通过农杆菌介导法将三种植物表达载体（pBGb1m、pBGbf和pBGbfg）转化陆地棉栽培品种新陆早4号、冀资492、衡无89-30和邯93-2等材料，获得了大量转化再生棉株。经过PCR和PCR-Southern blot检测，转基因阳性植株为转为再生植株总数的89.45%。目前，虫试、Southern blot及Western blot正在进行之中。

水稻同源四倍体三系法杂种优势利用研究

水稻是重要的粮食作物，其产量的增加和品质的改良都是关系国计民生的大事。就我国现阶段的国情而言，水稻产量在现有水平上稳步提升仍是未来十几年甚至几十年农业生产最重要的目标之一。尽管根据“超级杂交水稻育种”的战略设想和水稻育种实践，通过不断地改进育种技术可望在更高的产量水平上进行水稻杂种优势利用，在稻属植物内还具有很大的产量潜力可以挖掘。然而，仅仅从现有的种质基础出发，要更大幅度提高水稻单产，实现“超级杂交稻”的目标也存在一些困难：现有的推广品种是二倍体，尽管种类众多，但是其基因组的来源相对单一；同时，水稻基因组DNA含量也是作物中最少的，基因组内寻求开发潜力有一定困难；水稻作为C3植物，光合利用效率不高也是制约水稻产量提高的因素之一。因此，寻求常规手段以外的技术突破或者方法创新，是实现“超级杂交稻”的目标的迫切需求。本研究利用秋水仙素能抑制细胞分裂中纺锤丝的收缩、使细胞染色体加倍的作用，对水稻幼穗诱导的愈伤组织细胞进行加倍，并分化出再生植株；创制出水稻同源四倍体新的种质材料，在此基础上选育水稻同源四倍体雄性不育三系材料，并实现水稻同源四倍体的三系配套，开展水稻同源四倍体杂种优势利用和四倍体杂交水稻选育研究，建立水稻同源四倍体杂种优势利用的新技术体系。这不仅有助于倍性水平杂种优势的开拓和利用，同时也将为我国新世纪“超级稻”育种研究开辟一条新的技术途径。 水稻幼穗诱导愈伤组织并分化成苗是一项成熟、简单的组织培养技术。本研究以普通二倍体水稻亲本为材料，用秋水仙素进行水稻的多倍体化诱导，创制同源四倍体水稻三系亲本材料并对其进行鉴定。多倍体化以秋水仙素诱导的愈伤组织培养为基础，研究不同秋水仙素浓度梯度和愈伤组织诱导培养基组合对诱导四倍体植株的影响。结果表明在MS+2,4 D 1.0mg/L+ KT0.2mg/L+ IAA0.2mg/L 和500mg/L的秋水仙素处理下，水稻愈伤组织染色体加倍（有最高的效率）效果较好，平均加倍频率可达25.26%，其中，材料CDR22和IR26诱导较易成功，加倍频率分别达到75%和26.5%；相对材料94109 1.3%加倍频率和冈46B 10.8%加倍频率，诱导率差异极显著。 对水稻四倍体材料进行了形态学鉴定结果表明，与二倍体水稻对照相比其株高、穗长、花粉育性等主要农艺性状，确定四倍体材料在穗长和千粒重两方面极显著提高，种子的长度和宽度也显著增长。对花粉育性鉴定，确认水稻四倍体不育系材料仍为不育，保持系材料自交和杂交可育，恢复系材料自交和杂交可育。对四倍体材料进行细胞形态、染色体数目等方面进行细胞学鉴定，经核型分析表明水稻四倍体材料具有48条染色体，是二倍体水稻的两倍。水稻四倍体材料根尖分生组织细胞与二倍体的根尖分生组织细胞相比，细胞体积、细胞核和核仁显著增大。四倍体三系材料在细胞有丝分裂中期均可规则排列在赤道板，并能均等地移向两极；后期观察中没有发现染色体分离滞后现象，分裂末期细胞能够形成大小相对均一的子细胞。水稻同源四倍体三系材料细胞分裂未见异常，植株生长发育正常。 从1996年至2006年，针对结实率、有效分蘖、着粒数和穗长等主要农艺性状，通过系谱选育的方法，对培育的同源四倍体水稻亲本材料进行了连续选择和改良，取得较好成效。表现为结实率的改良效果极佳，所有改良材料的平均结实率均呈上升趋势，如D237（29.70％→72.70％）、DTB（19.55％→53.21％）等。有效分蘖总体呈现上升趋势，但在不同的年份，如1998和2002存在较大的负向波动。部分材料改良效果明显，如D19B（5.87→13.50）、D什香 (7.00→12.00)等；同时一些材料如DTB和D明恢63虽然总体略有提高，但在不同的年份波动很大，因此存在较大改良阻力，原因还有待进一步研究。着粒数的改良上升趋势比较显著，除保持系的DTB之外，其余材料的平均着粒数有显著提高。穗长的改良阻力较大，虽然不同材料总体上有所提高，但效果并不显著，并且不同年份有较大负向波动（2001）。此外还对株高、剑叶长等性状也进行了选择，但效果不显著，原因有待进一步提高。同源四倍体材料产量相关性状遗传改良幅度不一致，保持系和恢复系间的遗传改良效果也存在差异。这为同源四倍体水稻的进一步利用打下了良好的基础。 籼稻和粳稻亚种间杂交及杂种优势利用的主要障碍就是其低的结实率。而同源四倍体杂交水稻的研究为提高杂交水稻的杂种优势利用创造了新的途径。本研究通过随机区组设计方案，挑选性状优良的二倍体水稻材料，包括雄性不育系，保持系和恢复系进行秋水仙素诱导加倍，从而获得同源四倍体水稻对应的三系材料。利用选育的优良水稻同源四倍体三系材料，配制7个杂交组合，杂交F1代与其恢复系亲本进行比较，用于计算超亲优势（HB）；而杂交F1代与生产上大面积推广的二倍体杂交品种汕优63进行比较，用于计算杂种优势。结果显示，同源四倍体杂交水稻的超亲优势表现为：每株有效穗变化幅度为1.4%至105.9%，总粒数为0.5%至74.3%，每穗实粒数为17.6%至255.7%，结实率为9.6%至130.4%。这些农艺性状的改良使得这7个杂种F1的理论产量的超亲优势高达64.8%至672.7%。小区试验中四倍体杂交水稻组合T461A/T4002和T461A/T4193分别比二倍体对照汕优63提高46.3%和38.3%以上，除一个品种以外所有品种产量均接近或高于汕优63的产量。同源四倍体水稻强大的杂种优势表明，亚种间杂交育性低的问题可通过四倍体化及强化选择来解决。此外，同源四倍体杂交水稻器官的巨大性也是其产量提高的有利因素，水稻同源四倍体三系杂种优势利用研究具有一定的理论价值和商业生产潜力。 Rice is one of the major food crops, the improvement of the production and quality of it is an important thing related to the people's livelihood. On China's current national conditions, steadily increase of the rice yield based on the current level is still one of the most important goals in the next decade or even decades of agricultural production. According to the "super hybrid rice breeding" the strategic and rice breeding practice, improvement of the use of hybrid rice heterosis through continuous improvements in breeding technology is expected to get a higher level of rice yield, there are also a great yield potential can be exploited. However, there are also some difficulties to increase rice yield obviously and implement the goal of "super hybrid rice" based on the existing germplasm: Rice varieties in promotion are diploid, although there are many varieties, but their genome are from a comparatively single source; Meanwhile, the rice genome DNA are the least among the crops, it is difficult to exploit the development potential within the genome; Rice as C3 plants, photosynthetic efficiency is not high, it is one of the factors constraint rice yield. Therefore, seeking technological breakthroughs or innovative methods different from conventional means is the urgent needs to reach the target of "super hybrid rice". Using colchicine inhibit spindle contraction during cell division, double the cell chromosome, we induced callus cells from rice panicle to be doubled, and differentiated regeneration; we created a new autotetraploid rice germplasm material, and on that basis we bred male sterility three line autotetraploid rice materials, and the achieved the three line rice autotetraploid matchmaking, researched in autotetraploid rice heterosis usage and tetraploid hybrid rice breeding, constituted a new technology system of autotetraploid hybrid rice heterosis utilization. This not only helps the tetraploid rice heterosis exploration and use, but also inaugurates a new technical means for China in the new century "super rice" breeding research. We chose ordinary diploid rice as materials, using colchicine to induce the polyploidization, created the autotetraploid rice three-line materials and identified them. The polyploidization was based on the colchicine-induced callus tissue culture, and we experimented different colchicine concentrations and culture mediums to induce tetraploid plants, confirmed that the optimal concentration for inducement was 500 mg/L, the average induce rate was 25.26 %. Among all the materials, CDR22 and IR26 had higher induced rate; in contrary, 94109 and GANG46B had lower induced rate, the difference was significant. Autotetraploid materials was identified of both morphological and cytological, compared plant height, length of pollen sterility, and other major agronomic traits with a diploid rice as the control plant, identified that the autotetraploid materials had very significant advantages in ear length and thousand-grain weight, as well as the size of the seeds. Cytology identification included observation of the cell morphology, the number of chromosomes, and karyotype analysis on the autotetraploid materials confirmed that their chromosome number was 48, twice of the diploid rice. Mitoses in the three lines were common: chromosomes arrayed normally in metaphase and separated balanced into the two poles, chromosome moved without lagging in anaphase and daughter cells normally formed in telophase except one. It has been proved that tetraploid rice has normal meiosis as their diploid relatives, which usually including series of sub-phases as interphase, prophase I (five sub-phases), prophase II, metaphase I, II, anaphase I, II and telophase I, II. However, abnormal phenomena, such as formation of tetravalent, trivalent and univalent, chromosome lagging and so on, which would finally block meiosis. Configurations of chromosome in metaphaseⅠwere versatile in structure and form accept the bivalent. That condition varied in different strain, suggesting more complex paring configurations and more versatile genetic characters in tetraploid rice. All these abnormalities in meiosis contributed to low fertility of gamete and might consequently resulted in low seed setting. Successive selection and improvement on seed set, productive tiller per plant, total grains per panicle, panicle length and so on had been carried out from 1996 to 2006. The raise of seed sets was significant in both restorers and maintainers. Seed sets of some strains were improved more significantly than others, for example D237（29.70％→72.70％）、DTB（19.55％→53.21％）and et al.. Productive tiller per plant was improved to some extant. The tendency of improvement was rising on the whole but changed in some years such as 1998 and 2002. Part of the stains increased greatly, such as D19B（5.87→13.50）、Dshixiang (7.00→12.00) and so on, but some strains including DTB and Dminghui63 only increased little and decreased in some years by unknown reason. Total grains per panicle increased significantly and all strains except DTB increased. Improvement of panicle length termed to be hard. Different strains showed different capacities for improvement and floating existed in different years for example 2001. It has been proved that other agronomical traits including plant length, flag leaf length and so on could be improved but not significantly by selection also. In a word, agronomical traits could be raised by successive selection that is prerequisite for further utility of autotetraploid rice. Poor fertility is the main barrier for utilizing heterosis between the two rice (Oryza stiva L.) sub-species, indica and japonica. Recently, the development of autotetraploid hybrids (2n=4x=48) has been suggested as a new method for increasing heterosis in hybrid rice. Using standard experimental protocols, the elite diploid rice male sterile, maintainer, and restorer lines were colchine-doubled and autotetraploid counterparts were obtained. Seven resulting hybrids were analyzed for heterobeltiosis (HB), where the F1 was compared to the male parent, and the degree of heterosis, where the F1 was compared to the diploid commercial hybrid, Shanyou 63. The HB among the autotetraploid hybrids ranged from 1.4 to 105.9% for the productive panicles per plant, 0.5 to 74.3% for total kernels per panicle, 17.6 to 255.7% for filled kernels per panicle, and 9.6 to 130.4% for seed set. Improvements in these yield components resulted in the HB for kernel yield ranging from 64.8 to 672.7% among the seven hybrids. Hybrids T461A/T4002 and T461A/T4193 yielded 46.3 and 38.3% more, respectively than Shanyou 63, and all other hybrids but one yielded the same or more than Shanyou 63. The high heterosis for yield suggests that hybrid sterility between two rice sub-species may be overcome by using tetraploid lines followed by intensive selection. Also, the gigantic features of the autotetraploid hybrids may establish a plant structure able to support the higher yield.

绞股蓝离体培养中影响皂甙产量的几个因素研究

绞股蓝(Gynostemma pentaphyllum)系葫芦科绞股蓝属植物，药用价值广泛，但其野生资源日趋减少，绞股蓝主要药效成分为绞股 蓝皂甙。利用组织和细胞培养生产绞股蓝皂甙是合理开发利用和保护绞股蓝资源的可能途径之一。本文对绞股蓝组织培养中培养基 的蔗糖和激素的组成以及各种胁迫条件：渗透压、重金属离子、真菌诱导物等对皂苷产量的影响进行了初步研究。其中，渗透压、 重金属离子、真菌诱导物对绞股蓝愈伤组织皂甙产量的影响尚未见报道。1. 蔗糖对绞股蓝愈伤组织之生长影响显著，2,4-D对绞股 蓝愈伤组织皂甙含量、产量影响显著。增加蔗糖用量，减少2，4-D的用量可提高皂甙产量。2. Mn++ 用量的提高抑制绞股蓝愈伤组 织的生长，但可促进皂甙含量、产量的提高。Mn++用量提高至MS培养基的20-30倍时可使皂甙产量增加近一倍，而提高Cu++浓度的 作用不明显。3. 甘露醇用量增加抑制绞股蓝愈伤组织的生长，但可使皂甙含量、产量提高。0.680mol·l-1甘露醇可使皂甙产量提 高83%，而Nacl较大抑制愈伤组织的生长并使皂甙产量降低。4. 米曲霉粗提物对绞股蓝愈伤组织生长先略微促进，然后抑制，而根 霉粗提物则使愈伤组织生长受抑制；两者对皂甙含量、产量的作用相似：在较低浓度范围内升高，然后下降。米曲霉粗提物可提高 产量一倍，根霉粗提物可提高42%。这些结果为高产细胞系的筛选和生长、生产培养条件的优化积累了资料。在综述部分，对植物 细胞培养中组织和器官分化、细胞结构变化、生化水平的变化与次生物合成和积累的关系作了讨论。Gynostemma pentaphyllum blongs to Gynostemma, Cucurbitaccae. It has a wide medical use, but its wild resource is threatened by people's excessive use. Its effective medical components are gypenosides. For reasonable use and protect its resource, it is a possible way to product gypenosides by plant tissue and cell culture. This paper has a primary study on the components of sucrose and hormones and a variety of stress conditions: osmostic pressure, heavy metal ion, fungal elicitors in the medium for the calli culture. The effects of osmostic pressure, heavy metal ion and fungal elicitors on the calli of Gynostemma pentaphyllum have not been reported. 1. Sucrose had a significant effect on the growth of the calli, 2,4 D had notable effects on the gypenosides content and production of the calli. Increased the concentration of sucrose and decreased the concentration of 2,4 D improved the production of gypenosides. 2. Increased the concentration of Mn++ inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 20-30 times as MS medium which improved the production 100%. Increased the concentration of Cu++ had not a notable effect. 3. Increased the concentration of mannitol inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 0.680mol·l-1 which improved the production 83%. Nacl apparently inhibited the growth of the calli and decreased the production of gypenosides. 4. The crude preparation of Aspergillus oryzae inhibited the growth of the calli that in low concentration. The crude praparation of Rhizopus formosensis inhibited the growth of the calli throughout. Their effects on the content and production of gypenosides are alike, but the former is higher than the latter. On the optimum concentration, each crude preparation improved the production 100% (Aspergillus oryzae), 42%(Rhizopus formosensis). These results has accumulated some informantion on the select of high yield cell strains and choose the best culture conditons for the growth and gypenosides product of the calli. In the review, it is discussed that the differentiation on tissue-organal, cellular and biochemical levels related to the synthesis and accumulation of secondary metabolites in plant culuture.

Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.

Electroactive oligoaniline-containing self-assembled monolayers for tissue engineering applications

A novel electroactive silsesquioxane precursor, N-(4-aminophenyl)-M-(4'-(3-triethoxysilyl-propyl-ureido) phenyl-1,4-quinonenediimine) (ATQD), was successfully synthesized from the emeraldine form of amino-capped aniline trimers via a one-step coupling reaction and subsequent purification by column chromatography. The physicochemical properties of ATQD were characterized using mass spectrometry as well as by nuclear magnetic resonance and UV-vis spectroscopy. Analysis by cyclic voltammetry confirmed that the intrinsic electroactivity of ATQD was maintained upon protonic acid doping, exhibiting two distinct reversible oxidative states, similar to polyaniline. The aromatic amine terminals of self-assembled monolayers (SAMs) of ATQD on glass substrates were covalently modified with an adhesive oligopeptide, cyclic Arg-Gly-Asp (RGD) (ATQD-RGD). The mean height of the monolayer coating on the surfaces was similar to 3 nm, as measured by atomic force microscopy. The biocompatibility of the novel electroactive substrates was evaluated using PC12 pheochromocytoma cells, an established cell line of neural origin. The bioactive, derivatized electroactive scaffold material, ATQD-RGD, supported PC12 cell adhesion and proliferation, similar to control tissue-culture-treated polystyrene surfaces.

Co-electrospun poly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds

In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic acid), PLGA, 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and (x-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting PLGA-gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 80 mn, which was considerably smaller than fibers made under identical conditions from the starting materials (PLGA, 780 +/- 200 nm; gelatin, 447 +/- 1.23 nm; elastin, 1060 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking, and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds.

Formation and growth of Bryopsis hypnoides lamouroux regenerated from its protoplasts

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to Delta pH-based translocation (Delta pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.

Controle do crescimento e identificação de microrganismos contaminantes visando à micropropagação de gemas laterais de mangueira.

2008

A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Effect of oxygen carriers in microbial and plant cell cultures.

Dissolved oxygen concentration is one of the most limiting factors in aerobic cultures, due to the poor solubility of oxygen in aqueous media. In many processes, the microorganisms growth and production can be affected as a result of insufficient oxygen supply to the broths [1, 2]. To increase oxygen solubility, some methods can be used, such as the increment of aeration or agitation rates or decrease of the solution temperature.

Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.