山地杨MD-110原生质体的分离技术

山地杨MD-110(P.maximowiczii×P.deltoides)是黑杨派与青杨派的优良杂交品种.选择生长约1个月无菌苗的健壮叶片(长1.5～2 cm),切成长约0.5 mm的细条,在2%纤维素酶RS、0.05%果胶酶Y-23、0.6 M甘露醇(pH=5.6)组成的酶液进行分离,分离出大量的具有活力的原生质体,且大小均匀,细胞膜完整;酶解2 h后轻轻摇动培养皿利于酶解,酶解时间以6 h为宜.纯化后其原生质体的产量为3.6×107/gfr.wt,活力可达90%以上.纯化后的原生质体在B5(2倍浓度)培养基,附加2,4-D 10 nag·L-1、NAA 0.2 mg·L-1和BAP 1 mg·L-1中进行高密度液体浅层培养.     

银杏原生质体制备及其融合研究

以银杏品种湖南梅核成熟胚进行组织培养得到的无菌苗和愈伤组织为材料,用不同酶液处理进行原生质体制备,并用PEG法进行原生质体融合实验。结果显示,以2.0%纤维素酶+1.0%果胶酶+5.0mmol/LMES+6mmol/LCacl2+0.6mol/L甘露醇( 号酶液)酶解银杏组织、方法1提取原生质体效果最好,其最高原生质体产率为9.4×107个/g;以17%蔗糖溶液处理得到原生质体质量最好。用40%PEG6000+0.3mol/L钙离子+pH9.5液体处理原生质混合液,银杏原生质体融合率最高,为67%。     

地锦与五叶地锦原生质体分离及培养研究

用纤维素酶、果胶酶、甘露醇的混合酶液提取地锦和五叶地锦无菌苗幼叶、五叶地锦胚乳愈伤组织及地锦幼胚愈伤组织的原生质体,对影响原生质体提取得率及活力因素进行分析,对不同材料原生质体得率进行比较,对提取的原生质体进行液体浅层、固液结合及固体等方式培养,对原生质体形成的愈伤组织以MS和改良B5培养基附加不同浓度NAA、6-BA、2,4-D、PEG、KT、ZT等进行分化培养.试验结果表明纤维素酶对原生质体提取得率有极显著影响,适宜地锦幼叶原生质体提取的酶液组合为纤维素酶O.5%、果胶酶0.3%、甘露醇0.6 mol·L-1、酶解8 h;不同材料原生质体提取得率有显著差异;仅五叶地锦胚乳愈伤组织来源的原生质体在固体培养基中形成新的愈伤组织,其它方式培养的不同材料原生质体均无分裂或仅形成数十个细胞的细胞团后便解体;用30余组配方进行了五叶地锦胚乳愈伤组织原生质体形成的新愈伤组织的分化培养,继代6～10次后仍无分化迹象.     

不同酶解条件对金边瑞香幼叶原生质体分离的影响

以室内培养的金边瑞香浅黄绿色、质地幼嫩的叶片为材料,探讨了影响其原生质分离的因素。结果表明:对原生质分离效果影响最大的是纤维素酶浓度和酶解时间;采用纤维素酶质量分数为0.2%、甘露醇质量浓度为0.6 mol.L-1、酶解时间为10 h时原生质分离效果最好,原生质体的产量为2.2×105个.g-1。     

转基因741杨原生质体游离与纯化研究

以转双抗虫基因741杨无菌苗叶片为材料,对叶片原生质体游离、纯化方法及影响因素等进行了分析研究.结果表明,上部叶片产量和活力均高于其他叶位,在实验的浓度范围之内,Macerozyme R-10(离析酶)、Cellulase Onozuka R-10(纤维素酶)、Driselase (Sigma)(崩溃酶)对原生质体的产量和活力无显著影响.适宜转双抗虫基因741杨叶片原生质体游离与纯化的条件为无菌苗上部叶片为材料,0.7 mol/L甘露醇的CPW盐溶液预质壁分离1～1.5 h CPW+0.5％ Macerozyme R-10+0.25％ CellulaseOnozuka R-10+0.025％ Driselase (Sigma)+ 0.5 mol/L甘露醇(pH 5.7),酶解温度27℃,酶解时间10 h,用“过滤-离心-漂浮法”纯化原生质体.     

油桐叶肉细胞原生质体分离及瞬时转化体系的建立

【目的】探索油桐叶肉细胞原生质体分离的最适条件,建立油桐原生质体的遗传转化体系,使在油桐体内研究自身基因的功能成为可能。【方法】以油桐成熟叶片和组培苗幼叶为材料,通过酶解法成功分离得到油桐叶肉细胞的原生质体并确定最适分离条件。在此基础上,以获得的原生质体为受体系统,建立PEG介导的油桐原生质体基因转化方法。【结果】原生质体分离结果表明,酶解时间对原生质体产量和活性的影响最大,其次是纤维素酶浓度,而离析酶浓度和甘露醇浓度对原生质体产量和活性的影响较小。以成熟叶片为材料分离原生质体的最适条件为纤维素酶浓度1.5%、离析酶浓度1%、甘露醇浓度0.6 mol·L-1、酶解时间12 h,以组培苗幼叶为材料分离原生质体的最适条件为纤维素酶浓度2%、离析酶浓度1%、甘露醇浓度0.7 mol·L-1、酶解时间6 h。为了建立油桐原生质体的瞬时转化体系,通过PEG介导法将拟南芥MGT6基因导入到油桐原生质体中,结果发现MGT6蛋白定位于原生质体质膜,与之前报道的研究结果一致,这表明本研究建立的油桐原生质体转化方法可成功将外源基因导入油桐原生质体并使其表达。【结论】建立油桐成熟叶片和组培苗幼叶叶肉细胞原生质体的高效分离方法,综合考虑取材的便利性和对后续原生质体培养的影响,建议以组培苗幼叶为材料分离原生质体,分离条件为纤维素酶浓度2%、离析酶浓度1%、甘露醇浓度0.7 mol·L-1、酶解时间6 h。在分离得到油桐叶肉细胞原生质体的基础上,本研究建立的PEG介导的原生质体遗传转化方法能以油桐叶肉细胞原生质体为受体,高效地将外源基因导入其中并使外源基因表达。本研究结果不仅可促进油桐基础研究的发展,在通过细胞融合和基因工程手段进行油桐种质创新方面也具有重要意义。     

枣树原生质体分离条件的研究

枣树原生质体分离研究是其原生质体培养与融合、外源遗传物质转化等研究的基础工作．用胚性悬浮培养细胞、细粒状胚性愈伤组织、未细切愈伤组织和已细切愈伤组织等４种材料进行原生质体分离．结果表明,枣树胚性悬浮培养细胞是最佳起始材料,用浓度为１０ｇ／Ｌ纤维素酶＋１ｇ／Ｌ离析酶＋ＣＰＷ盐（１３２０ｍｇ／ＬＣａＣｌ２·２Ｈ２Ｏ和１００ｍｇ／ＬＫＨ２ＰＯ４·Ｈ２Ｏ组成的混合液）＋０．７ｍｏｌ／Ｌ甘露醇组成的混合酶液对起始材料进行１６ｈ的酶解,可获得较高的原生质体产量,且原生质体活力较高．     

朱红栓菌原生质体制备与再生的研究

将2016年7月在黑龙江省凉水国家级自然保护区采集的朱红栓菌Trametes cinnabarina子实体接种于PDA固体培养基平皿中,30℃下培养7 d,截取菌丝体接种于液体培养基中用于原生质体制备与再生实验。通过单因素试验和正交试验,研究酶解温度、酶解时间、菌丝质量分数、酶配比对原生质体制备的影响。结果表明,以0.6 mol·L-1甘露醇溶液作为高渗缓冲液,复合酶组成为1.5%溶壁酶﹢1.0%纤维素酶﹢1.0%蜗牛酶,菌丝质量分数为20%,30℃条件下酶解3.5 h为朱红栓菌原生质体制备的最适条件,产量达1.00×107个·m L-1;以0.6 mol·L-1蔗糖溶液作为再生高渗缓冲液,原生质体再生率最高达20.6%。     

影响菌根真菌黑核菌原生质体分离的因子研究

从菌根菌黑核菌菌丝出原生质体，并获得再生菌落。研究了预处理方法及不同水解酶组合，酶解温度和菌丝密度对原生质体产率的影响。在用胰蛋白酶预处理后，以０．８Ｍ山梨醇为渗透压稳定剂，温度为２０Ｃ，ｐＨ５．５及纤维素酶，溶解酶和浸解酶处理，可获得较高的原生质体产量。     

胡杨悬浮细胞原生质体游离及质膜单离子通道测定

该文以胡杨 (Populuseuphratica)悬浮细胞为材料 ,利用 1 0 %纤维素酶“onozuka”R 10、0 0 1%果胶酶Y 2 3、0 15 %离析酶R 10和 0 1%半纤维素酶的混合酶液消化细胞壁 ,得到原生质体 .并利用膜片钳细胞贴附技术分别测到质膜内向和外向单通道电流 .通道电流在不同水平上变化 ,表明并非只有一种类型的通道开放 .树木细胞原生质体的分离以及利用膜片钳测定细胞质膜离子通道的成功实践为深入研究木本植物抗盐的细胞学机制奠定了基础     

酶法提取侧柏叶总黄酮的研究

对酶法提取侧柏叶总黄酮类的工艺条件进行研究。以总黄酮得率为考察指标,探讨了影响提取的主要因素,并用正交实验确定了最佳提取工艺。结果表明,各因素对提取效果影响的顺序依次为酶解时间〉酶用量〉酶解温度〉酶解pH值,最佳工艺条件为：料液比1∶14,酶解温度45℃,介质pH3.5,酶用量0.3%,酶解时间2.0h,总黄酮得率可达1.026%。该提取工艺简便易行,重复性好。     

Plantlet regeneration from leaf protoplasts ofPopulus euphratica

Protoplasts were isolated from the leaves of sterile plants ofPopulus euphratica Oliv. by using 1% Cellulase “Onozuka” RS and 0.25% Pectolyase Y-23 in 0.6m of mannitol solution. Protoplasts were cultured in modified Murashige and Skoog's (MS) medium which contained no ammonium ions but was supplemented with BAP (6-benzylaminopurine), 2,4-D (2,4- dichlorophenoxy-acetic acid), and 1% sucrose at the cell density of 9×10⁴/ml. Cell divisions occurred in every culture medium, especially in the medium containing 0.5 mg/l of BAP and 0.1 mg/l of 2,4-D, in which callus was successfully induced by successive culture through cell cluster formation. Shoots were regenerated from the callus, and their growth was enhanced on 1/2 MS medium containing 0.8 mg/l of BAP. Finally, shoots were rooted and plantlets were regenerated on 1/2 MS medium without a hormone. A part of this paper was presented at the 106th Annual Meeting of the Jpn. For. Soc. (1995).     

Regeneration of protoplasts from hyphal strands ofVolvariella volvacea

A series of experiments on the preparation and regeneration of protoplasts from hyphal strands ofVolvariella volvacea (Bull. ex. Fr.) Singer were conducted with the aim of optimizing the conditions for its efficient regeneration. One commercial (Vvcl) and two wild (EAAC-0001 and EAAC-0002) strains ofV. volvacea from the Philippines were used and subjected to varying conditions to determine the most efficient means for regeneration of their protoplasts. The effects of age and type of strain, pH, type and concentration of osmotic stabilizer, enzymatic composition, treatment time, temperature, reciprocal frequency during enzymatic lysis of the cell wall, and centrifugation conditions were investigated. Results showed that the three strains ofV. volvacea had varying responses in terms of yield, size, and ability of their protoplasts to regenerate into the protoplast regeneration medium. Among the three strains, EAAC-0002 had the highest rate of regeneration. The 5-day-old culture ofV. volvacea, when subjected to a combination of 2% Novozyme 234 and 0.2% chitinase in 0.6M mannitol (pH 6.0) for 3 h at 30°C, 90 strokes/min and centrifuged at 1100 g for 10 min; produced an efficient yield of protoplasts with a relatively high regeneration rate.Part of this paper was presented at the 47th annual meeting of the Japan Wood Research Society, Kochi, Japan, April 2–5, 1997     

酶法降解黑木耳多糖工艺研究

采用酶法对黑木耳多糖进行降解试验研究。用β-葡萄糖苷酶以温度、pH值、酶用量、反应时间为影响因素,响应面法优化设计,DNS法测定其还原糖释放量。研究结果表明:最佳工艺为当每3mL多糖加入0.02%葡萄糖苷酶0.4mL时多糖浓度为0.3%、温度55℃、pH值5.5、反应时间20min。响应面优化后得出最佳工艺条件为:降解温度55.9℃、pH值5.58、酶用量0.475mL。     

酶法提取荆芥总黄酮的研究

研究了酶法提取荆芥总黄酮的工艺。通过单因素实验,探讨了粉碎度、料液比、酶解温度、酶解pH值、酶解时间、酶的用量对总黄酮提取率的影响,筛选出影响提取的主要因素,再通过正交实验,优化得到酶法提取荆芥总黄酮的最佳工艺条件。实验结果表明,各因素对提取率的影响顺序依次为粉碎度〉酶解时间〉酶的用量〉料液比。通过正交实验优选得到的最佳提取工艺为：药材粉碎度为50目,料液比为1：16,酶的用量为0．8％,酶解温度为40℃,酶解pH值为5,酶解时间为20min后,煎煮1．5h。该法操作简单、提取率高,可用于荆芥总黄酮的提取。     

<Emphasis Type="Italic">Nitraria sibirica</Emphasis> cell suspension culture: establishment,characterization and application

Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L^?1 6-benzylaminopurine (6-BA) and 1.0 mg L^?1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 × 10⁶ cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L^?1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).     

树脂吸附法分离高纯茶多酚新工艺研究

研究了树脂吸附法分离纯化茶多酚的绿色工艺。浸提低品位茶叶得到的浸提液，乘热粗沙沙滤，调pH值至1．5，使色素、咖啡因和大分子物质得到预分离，然后细沙沙滤、上AB-8树脂进行吸附，依次用蒸馏水和pH值为2-4的5％的乙醇溶液洗脱处理，洗脱剂为质量分数60％的乙醇，洗脱液喷雾干燥得到茶多酚产品，纯度可达90．22％。     

Nitraria sibirica cell suspension culture:establishment,characterization and application

Nitraria sibirica Pall.is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value.The objectives of this study include induction and multiplication of callus,establishment of a suspension cell line,and isolation of protoplasts from cell suspensions.Murashige and Skoog(MS) medium was used for callus induction from mature seeds of N.sibirica.Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mgL~(-1) 6-benzylaminopurine(6-BA) and 1.0 mgL~(-1) 2,4-dichlorophenoxy(2,4-D) acetic acid.Suspension cultures of N.sibirica were initiated by transferring friable calli to the same liquid multiplication medium.Characterization of the suspension culture was assessed based on fresh mass,dry mass,cell viability and p H value of the culture.A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium,including a lag phase,an exponential growth phase,a stationary phase,and a negative acceleration phase.The effect of factors such as pre-plasmolysis,enzyme combination,enzymolysis time and mannitol concentration,on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures.The maximum yield(9.79 9 106 cells/g) and highest viability(79.97%) of protoplast were reached when approximately 1 g of cell suspension(cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 molL~(-1) mannitol mixture solution,cellulose onozuka R-10 2%(w/v),hemicellulose 0.2%,macerozyme R-10 1%,and pectolyase Y-23 0.5%.Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10(P0.05).