Produção de plantas e metabólitos de Cleome dendroides Schult. & Schult. f. (Cleomaceae) utilizando diferentes sistemas de cultivo in vitro

Cleome dendroides é uma espécie endêmica da Mata Atlântica dos estados do Rio de Janeiro e Espírito Santo, bioma alterado por intensa atividade antrópica, o que constitui uma ameaça à preservação de suas populações. Não existem estudos dos pontos de vista fisiológico, biotecnológico, fitoquímico ou farmacológico sobre a espécie. Considerando-se o perfil fitoquímico e o potencial medicinal do gênero, torna-se relevante definirem-se protocolos para a produção de plantas e metabólitos de C. dendroides utilizando diferentes sistemas de cultivo in vitro. No presente trabalho, foram realizados estudos sobre a germinação in vivo da espécie, avaliando-se a influência do substrato, temperatura e luz. Não se observou qualquer tipo de dormência, sendo as temperaturas de 20, 25 e 20-30C, em areia ou vermiculita, apropriadas para a germinação in vivo. Definiu-se também uma metodologia eficiente de germinação sob condições in vitro, e as plântulas obtidas foram utilizadas como fonte de explantes para os estudos de propagação in vitro. A resposta morfogênica foi avaliada considerando-se a origem e tipo do explante, tipos e concentrações de reguladores de crescimento e número de subculturas. A metodologia empregada mostrou-se eficiente para a produção de brotos e manutenção de estoques de plantas in vitro que serviram como fonte de explantes. A melhor condição para a propagação in vitro foi definida em meio solidificado contendo BA, independentemente do tipo de explante e da origem. Os brotos obtidos foram alongados, enraizados e aclimatizados. Também foi estabelecida a cultura de raízes e a regeneração de brotos a partir destas culturas. Avaliou-se o efeito da origem do explante, assim como dos tipos e concentrações de fitorreguladores sobre a proliferação de raízes e a regeneração de brotos. O fitorregulador AIB propiciou maior multiplicação das raízes, enquanto BA mostrou-se eficiente na regeneração de brotos a partir das raízes recém-formadas. Foi estabelecido ainda um protocolo de cultura de calos e de suspensões celulares. Avaliou-se o efeito da origem e do tipo de explante, dos tipos e das concentrações de fitorreguladores sobre a calogênese. A combinação de PIC com KIN foi a mais eficiente para a indução de calos em explantes de plântulas obtidas a partir de germinação in vitro, produzindo calos que foram mantidos por pelo menos dois anos. As suspensões celulares também foram estabelecidas em meio contendo PIC + KIN, mantendo uma produção de biomassa de cerca de cinco vezes o peso fresco inicial por três sucessivas subculturas. Análises histoquímicas e fitoquímicas revelaram a presença de alcaloides nos calos e nas suspensões celulares. Foram realizadas análises fitoquímicas de plantas de campo, plantas aclimatizadas, plantas mantidas em estoque in vitro e culturas de raízes, as quais indicaram a presença de derivados de glicosinolatos. Os resultados demonstraram a viabilidade de produção de material vegetal de C. dendroides por meio de métodos biotecnológicos e a produção in vitro de metabólitos de importância medicinal

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.

药蒲公英(Taraxacum officinale Weber)体外再生体系的建立、耐盐愈伤组织筛选和抗盐基因转化

药蒲公英(Taraxacum officinale Weber)是菊科蒲公英属的模式种，主要分布于欧洲和北美，在我国新疆也有少量分布。与Taraxacum mongolicum Hand-Mazz(我国中药市场的主流种和主要自然分布种)相比，药蒲公英的生物量更大，作为营养保健蔬菜具有更大的市场价值。药蒲公英的组织培养工作是开展基础研究的有力工具，本工作中，药蒲公英叶片外植体在含0.2mg/L IAA和1.0mg/L TDZ的MS培养基中培养2周后便产生大量的丛生芽，在含有0.5mg/L 2,4-D和2mg/L6-BA的MS培养基中培养30天后，形成明显的愈伤组织，愈伤组织块在含1.0mg/L 6-BA的MS培养基中成功再生。 体细胞无性系变异是植物愈伤组织培养中的普遍现象，我们将继代6次的愈伤组织接种于含盐培养基，得到了能够耐受1.0%NaCl的细胞系。耐盐细胞系在含盐培养基中的相对生长率和细胞活力明显高于对照（非耐盐细胞系接种于含盐培养基），由耐盐细胞系在含盐培养基中获得再生植株的工作正在进行。 直接不定芽再生途径对遗传物质具有高度保真性，是遗传转化的理想体系。我们利用此再生系统，将来源于耐盐植物山菠菜(Atriplex hortensis L.)BADH基因通过农杆菌介导的叶盘转化法导入药蒲公英，获得了PCR检测成阳性的转基因植株5株，从而建立了药蒲公英的转化体系。转基因植株的其他分子检测和耐盐性鉴定工作正在进行。

水稻OsGF14-c基因功能的初步分析

GF14是一类在高等植物的基本代谢﹑信号转导﹑抗逆以及转录调节等多种生理生化途径中发挥重要作用的基因。这类基因是14-3-3基因家族的成员，由于启动子结构中具有一个保守的G-Box元件而得名。我们根据该类基因的保守序列合成特异性探针，从水稻中花10号花的cDNA文库中筛选到一个基因，通过全长测序以及同源比较，发现它的核酸序列与NCBI数据库中注册的水稻GF14-c基因有高达99%的同源性，并在其上游启动子区也发现了一个保守的G-Box元件，从而确定该基因是水稻的GF14-c基因，我们把它命名为OsGF14-c。OsGF14-c的cDNA全长1154bp，编码256个氨基酸。序列分析表明该基因与酵母同源基因BMH2有71%的同源性，构建真核表达载体pdYES2，借助LiAC转化法将pdYES2导入BMH2缺陷型酵母菌株GG3000，筛选获得了补偿BMH2功能的重组酵母菌株PYGG。OsGF14-c基因的启动子区域具有一个典型的﹑高等植物特有的顺式作用元件G-Box元件，提示该基因可能与转录因子相互作用，洋葱表皮瞬时表达实验验证了OsGF14-c是核定位的。构建原核表达载体pGF-c2转化大肠杆菌XL1-Blue，筛选获得了表达GF14c-MBP融合蛋白的重组菌株，利用直链淀粉亲和层析纯化了OsGF14c-MBP融合蛋白。为了研究该基因在水稻的生长发育过程中所起的作用，我们以OsGF14-c基因的全长cDNA序列构建反义表达载体载体p35sCAMVF，并分别以OsGF14-c cDNA序列的特异区段以及保守区段构建了RNAi表达载体US和UT。将p35sCAMVF﹑US和UT借助农杆菌介导的转化分别导入水稻愈伤组织,培养成苗并进行遗传学分析。我们的工作对今后深入研究OsGF14-c基因的功能奠定了一定的基础。

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。

砷超富集植物蜈蚣草(Pteris vittata L.)中砷解毒机制的研究

砷是一种具有致癌、致畸、致突变的有毒元素，在地表的含量本来很低。然而，随着现代社会的发展和工业活动的增加导致砷污染日趋严重。土壤和水体中的砷污染可以通过食物链进入人体，对人类的健康造成极大的危害。植物修复是一种利用植物对污染物的超富集能力来清除或减低污染的新型环境生物技术。植物修复的实际应用依赖于超富集植物的发现和超富集机制的阐明，特别是砷解毒过程(砷的吸收、还原和区域化)的研究及相关基因的克隆。砷超富集植物蜈蚣草(Pteris vittata L.)中砷解毒机制的阐明将为砷污染的植物修复及新型工程植物的研发提供理论基础。 本论文以蜈蚣草为试材，针对蜈蚣草的砷解毒机制取得了如下研究进展： 1.以砷超富集植物蜈蚣草为材料，建立了一个适于研究蜈蚣草砷吸收和解毒机制的新系统—愈伤组织悬浮培养体系。首次证明蜈蚣草愈伤组织与其孢子体及配子体一样具有对砷的抗性和砷超富集的能力。 2.以蜈蚣草愈伤组织为材料，通过比较亚砷酸盐、砷酸盐和二甲基胂酸盐对蜈蚣草和拟南芥植物毒性的差异，表明砷的还原可能是蜈蚣草对砷解毒的重要机制之一而砷的甲基化对蜈蚣草的砷解毒作用甚微。 3.以蜈蚣草愈伤组织为材料，通过对砷在蜈蚣草愈伤组织细胞中的亚细胞定位，首次直接证明植物液泡对砷具有非常明显的区隔化作用。暗示区隔化作用在蜈蚣草对砷的解毒过程中发挥着重要的作用。 4.通过测定蜈蚣草愈伤组织对不同化学态的砷处理下抗氧化物质的变化发现酸溶性巯基在蜈蚣草砷解毒中也发挥着重要作用。 5.以蜈蚣草愈伤组织为材料，发现磷和砷的吸收在高浓度范围下（﹥0.2 mM）存在明显的协同效应。对蜈蚣草高亲和磷酸盐转运蛋白基因－PvPHT基因功能的初步分析则表明PvPHT参与了蜈蚣草对磷和砷的吸收过程。

新疆紫草细胞培养产生紫草宁衍生物的代谢调控

本文对新疆紫草培养细胞的生长及紫革宁衍生物形成的调节与控制进行了研究．结果表朗：在不同时期照射白光、蓝光，对紫草培养细胞生长及紫草宁衍生物的积累均有明显的抑制作用;而第三周照红光一天、第二周以及整个培养过程均照远红光、第19天照紫外光等，都对生产培养基中紫草宁衍生物的积累有促进作用;其中，远红光与紫外光的最佳照射时间长度都为30分钟，且细胞内苯丙氨酸解氨酶的活性与紫草宁衍生物的含量有明显的正相关性．研究了不同照光条件下，紫草培养细胞生长，紫草宁衍生物积累以及苯丙氨酸解氨酶活性变化的动态，基本上找出了它们的变化规律以及它们之间的相互关系． 通过试验发现，紫草细胞培养的适宜温度为25℃，适宜pH值为5.3—5.8：在培养基中加入O,l%-0.30%的活性炭、用磁水器处理培养基等，对紫草宁衍生物的积累有良好的促进作用。 提高培养基中的Cu2+，Mg2+，Zn2+．K+浓度，有利于紫草宁衍生物的积累，而在生产培养基中加入NH：，则显著抑制了紫草宁衍生物的产生;培养基中蔗糖浓度为50g／L时，对紫草细胞生长及紫草宁衍生物的积累最有利，而生产培养基中不加IAA，KT对紫草宁衍生物的积累有利．以密环菌为诱导子，促进了紫草细脆生长及紫草宁衍生物的积累。 此外，对Cu2+、密环菌发酵液处理条件下，紫革宁衍生物积累与苯丙氨 酸解氨酶活性的关系以及不同蔗糖浓度条件下，生长培养基中紫草细胞生长、 紫草宁衍生物的积累，细胞内可溶性糖含量变化的动态进行了研究．

高山植物唐古特红景天（Rhodiola algida Var.tangutica）抗冻蛋白的研究

抗冻蛋白（AFP）或热滞蛋白（THP）最早是从极区鱼和昆虫中发现的一类特殊蛋白质，其共同特性是能阻止体液内冰核的形成和生长，降低体液的冰点，使其冰点低于熔点（热滞效应）。近几年的研究表明，AFP在某些植物、细菌、真菌中也存在。生长在极端低温环境中的高山植物体内是否也存在AFP，据我们掌握的资料，还未见这方面的报道。为此，我们选择了青海高原海拔4000米高山生长的唐古特红景天为实验材料，并在这种植物中发现了抗冻蛋白。主要实验结果概括如下： 1. 唐古特红景天叶片有很强的抗冻性，可耐受-26.5 ℃的冰冻温胁迫。在北京地区夏季（7月）驯化20天后，半致死温度（LT_(50)）升高为-15.5 ℃。叶片质外体蛋白抽提液有明显的热滞效应（0.2 ℃），在AFP中存在糖基。SDS-PAGE分析表明，质外体AFP为分子量在43-85KD范围内的五条多肽。 2. 光镜组织化学切片显示，在红景天叶片中存在蛋白质量丰富的细胞;电镜细胞化学研究揭示，在细胞壁外层及细胞间隙中分布着明显的经钌红特异染色的糖蛋白，这种糖蛋白因环境温度升高而减少。结合前述的实验结果，确认这种细胞壁外层及细胞间隙中的糖蛋白，即抗冻蛋白。 3. 以唐古特红景天叶片为外植体，在MS+BA_2+NAA_(0.2)固体培养基上可诱导出黄绿色，松脆愈伤组织。愈伤组织细胞在同样成分的液体培养基中培养获得成功。在悬浮培养液中可检测到分泌蛋白的存在。经SDS-PAGE分析表明，经低温锻炼或ABA诱导后，细胞分泌蛋白的多肽谱带数增加。与此相对应的是，细胞的抗冻能力也明显提高。PAS染色揭示，多肽中均含有糖基。 4. 通过测定热滞值，确信细胞分泌蛋白是具有抗冻活性的糖蛋白。

Agrobacterium-mediated transformation of Kentucky bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

长俊木瓜体细胞胚胎诱导的初步研究

以长俊木瓜为材料,研究了其体细胞胚胎诱导过程中各个环节的影响因素。结果表明:愈伤组织诱导的适宜外植体是叶片,培养基为MS+6-BA1.0 mg.L-1+2,4-D0.2 mg.L-1,黑暗培养;非胚性愈伤组织向胚性愈伤组织转化的适宜培养基是MS+6-BA1.0 mg.L-1+NAA1.0 mg.L-1;胚性愈伤组织的保持与增殖应在黑暗条件下进行,培养基为MS+6-BA1.0 mg.L-1+2,4-D0.2 mg.L-1;长俊木瓜叶片体胚的发生以加入ABA2.0 mg.L-1的MS培养基最为有利。

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.

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.

An efficient in vitro method for mass propagation of Potentilla potaninii wolf

This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and alpha-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mg l(-1) BA and 1.0 mg l(-1) NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mg l(-1) NAA and 0.5 mg l(-1) indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.