草莓悬浮细胞系的建立及某些影响因素

草莓悬浮细胞系的生长受到激素浓度和细胞培养密度的调控,悬浮细胞系在继代5-9d时,pH值显著升高,细胞增殖明显加快,建立了草莓栽培品种宝交早生、丰香和野生种新疆3号的悬浮细胞系。     

朱砂根愈伤组织培养及悬浮细胞系建立

以朱砂根(Ardisia crenata Sims.)无菌苗的茎段、叶片、胚轴和胚根为外植体进行愈伤组织诱导研究。结果表明:胚根在含有2,4-D的培养基中的诱导率最高,在添加5 mg L-1 AgNO3的MS+2,4-D 0.5 mg L-1+KT 0.01 mg L-1培养基中继代培养的增殖系数高达8倍以上。培养中获得了5种类型的愈伤组织(I-白色湿软状、Ⅱ-白色冰砂状、Ⅲ-淡黄色颗粒状、Ⅳ-黄绿色块状和V-绿色块状),其中Ⅱ和Ⅲ型愈伤组织可以成功建立悬浮细胞系,用M S+2,4-D 0.5 mg L-1+KT 0.01 mg L-1培养基进行固-液轮回培养,可以较好地保持悬浮细胞系。     

超甜玉米单倍体悬浮细胞系的建立及其生长特性

从超甜玉米的花药培养物中,筛选出28个具有高度胚胎发生能力的类型Ⅱ愈伤组织系,这种愈伤组织质地松散,易于分散,可以长期进行继代培养。将3个月龄的类Ⅱ愈伤组织接种在液体培养基中进行振荡培养,建立起分散程度很好的8个悬浮细胞系。实验结果说明,培养基中加入酪蛋白水解物(CH)和2-(N-吗啉代)乙基磺酸(MES)对悬浮细胞的生长是有利的。培养基的 pH 迅速下降吋,细胞的增殖十分缓慢,当培养基的 pH 值开始升高时,细胞的增殖也显著加快。利用滋养培养法,可以使悬浮细胞的愈伤组织形成率大大提高(比对照高11.8—19.7倍),并在低密度下达到较高的(41.3%)植板率。染色体计数表明,继代培养6个月之后,88.0%的悬浮细胞和95.2%的再生植株的染色体数保持着2n=x=10的单倍性水平。     

麻疯树悬浮细胞系的建立与优化

以麻疯树无菌幼苗为外植体,研究疏松愈伤组织诱导方案及不同培养条件对麻疯树悬浮细胞生长的影响,旨在建立麻疯树悬浮细胞体系.结果表明,麻疯树疏松愈伤组织诱导的最适培养基及激素组合为:MS+2,4-D0.6mg/L+BA 1.0 mg/L+蔗糖30 g/L,此培养基上诱导出的愈伤组织湿润松散,颜色鲜艳.接种愈伤组织进行悬浮培养的液体培养基最适激素组合为:NAA 0.2mg/L+2,4-D 1.0 mg/L+BA 0.5 mg/L.初代愈伤组织适宜用于悬浮培养,摇床转速应低于120 r/min为宜,这样培养的悬浮细胞分散度最高.培养基中添加500 mg/L水解酪蛋白能有效地促进悬浮细胞的生长.悬浮细胞振荡培养过程中悬浮细胞生长的时间进程为起始培养的第5天前,细胞增殖十分缓慢;第5-11天期间生长迅速;第13天后基本停止生长.在上述优化培养条件下,麻疯树悬浮细胞系增长速率最快,细胞生长状态最佳.     

油松胚珠愈伤组织诱导和悬浮细胞系的建立

雌配子体处于游离核时期的油松胚珠以含不同浓度和配比生长调节剂的培养基诱导产生愈伤组织,并建立了悬浮细胞系.这一悬浮细胞系生长快,分散性好,稳定均一,适用于研究其生理、生化和细胞周期调控.     

亚麻胚性细胞悬浮培养体系的建立和影响因素

亚麻品种‘双亚五号’的胚性愈伤组织诱导最佳培养基为MB（MS无机盐加B5维生素）＋1.0mg·L^-12,4-D;细胞初始悬浮培养的最佳培养基为MB＋0.2mg·L-16-BA;细胞继代悬浮培养的最佳培养基为改良的MB（NH4NO3减半,KNO3加倍）＋0.02mg·L^-12,4-D＋0.2mg·L^-16-BA;适宜的蔗糖量为50g·L^-1;适宜的继代接种量为1.0～1.5g·（25mL）^-1;继代间隔为5～7d。     

影响禾谷类作物胚性细胞悬浮系建立的一些因素

介绍了禾谷类作物通过愈伤组织继代培养建立胚性细胞悬浮系及其影响因素的研究进展,并对这一问题的研究前景作了展望。     

药用植物人参愈伤组织的诱导培养与悬浮细胞体系建立

人参(Panax ginseng C.A Meyer)是五加科人参属植物,其主要药效活性成分为人参皂苷,具有广泛的药理作用。为建立人参愈伤组织诱导培养体系,以探索实现工程化细胞大规模生产人参皂苷的有效途径。本研究以人参根切片作为外植体,成功诱导出质地松疏、生长迅速、易于分散、启动较早的淡黄色透明状的愈伤组织,建立了适合于长期继代培养的人参细胞系,为深入开展人参干细胞分化及人参皂苷生物合成分子调控研究提供了理想的模型与基础。     

高羊茅胚性悬浮细胞系的快速建立及其单细胞培养

本研究采用高羊茅(Millennium和Hundog Ⅴ)成熟种子为材料,以MS为基本培养基,通过添加2.5 mg/L CuSO4·5H2O,或提高NH4NO3浓度至2.5 g/L等措施均能明显提高愈伤组织的质量,经过3～5个月的筛选获得疏松干燥、颗粒状、生长旺盛、适合悬浮培养的Ⅱ型胚性愈伤组织.悬浮培养初期需用MSⅠ液体培养基进行一个月的启动培养,之后转用MSⅡ继代保持,约2个月左右即建立起来自高羊茅2个品种的3个悬浮细胞系.生长特性测定结果表明,悬浮培养的初始接种量以1.0～3.0 ml/40 ml为宜,生长周期内其pH值不断波动变化,最适范围在5.0～5.7之间.5～8 d为悬浮系的对数生长期,此时细胞分裂旺盛,增殖较快,是分离单细胞的最佳时期.单细胞培养方式以悬浮培养效果最好.     

Some Factors Affecting the Anthocyanin Formation by Populus Cells in Suspension Culture

In order to elucidate the mechanism of anthocyanin formation by Populus cells in suspension culture, the favourable conditions for anthocyanin formation were investigated. The influence of some factors affecting the anthocyanin formation, i.e. light, sucrose and riboflavin etc. were also examined. Light irradiation and high sucrose concentrations brought about a marked increase of PAL activity, which increased rapidly at the lag phase preceding the anthocyanin formation. The effect of blue light on anthocyanin formation was markedly superior to other kinds of monochromatic light (green and red) or white light. Riboflavin was effective only under light exposure. It was inferred that light, sucrose, riboflavin and PAL activity etc. were closely related with the anthocyanin formation. Especially, light and sucrose cooperated in the increase of PAL activity which was a key enzyme in the biosynthesis of anthocyanin.     

植物细胞悬浮培养影响因子研究

植物细胞悬浮培养已广泛应用于育种和生产次生代谢产物,在生物工程中发挥着重要作用。本文综述了愈伤组织继代改良的措施,影响胚性悬浮细胞系建立的几个因素,存在问题及前景展望。     

雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

为探索药用植物雷公藤(Tripterygium wilfordii)悬浮细胞原生质体提取的最优条件,并建立雷公藤原生质体瞬时转化体系,以雷公藤悬浮细胞为材料,对酶解液配比、酶解时间、甘露醇浓度及处理转速进行考察。用PEG介导的瞬时转化法将外源基因转化到雷公藤原生质体中。结果表明,以雷公藤悬浮细胞为材料提取原生质体的最佳条件是酶液配比为2.0%纤维素酶+0.5%果胶酶+0.5%离析酶,甘露醇浓度为0.6 mol·L–1,酶解10小时,处理转速为67×g;用PEG介导法将含有编码GFP的植物表达载体转化雷公藤悬浮细胞原生质体,激光共聚焦扫描显微镜下细胞显示绿色荧光。通过实验筛选得到雷公藤悬浮细胞原生质体的最佳提取条件,建立了雷公藤悬浮细胞原生质体的瞬时转化体系,为进一步开展雷公藤功能基因及合成生物学研究奠定了基础。     

Runner Formation in Everbearing Strawberry as Influenced by Growth-Promoting and Inhibiting Substances

The interactive effects of gibberellic acid, abscisic acid,and benzyladenine were studied in relation to runner formationof the everbearing strawberry. In two of three cultivars studied,Ozark Beauty and Superfection, exogenous gibberellic acid stimulatedrunner formation and inhibited flowering. In the Geneva cultivar,a shy runner producer, gibberellic acid application as a foliarspray failed to induce runners. When applied in combinationwith benzyladenine, gibberellic acid greatly stimulated runnerformation Benzyladenine alone had no effect on runnering. Exogenous abscisic acid inhibited petiole length and runnerformation and had no influence on the number of inflorescencesinitiated. Response to exogenous gibberellic acid or abscisicacid varied according to the reproductive or vegetative stagethe plant exhibited at the time of treatment. Activity of endogenous gibberellin-like substances was not substantiallydifferent between flowering and non-flowering field-grown Genevaplants when sampled in long day lengths of August. Endogenousinhibitors were highly active in flowering Geneva plants andrelatively inactive in non-flowering plants The implications of these findings on hormonal balance in thestrawberry are discussed.     

草莓高效离体叶片再生体系的建立

以草莓'明宝(Meiho)'和'红颊(Benihope)'的叶片为外植体,研究了不同基本培养基、暗培养时间、植物生长调节剂、叶龄、不同放置方式对其不定芽再生的影响.结果表明:各品种叶片不定芽离体再生的最佳条件不同.'明宝'叶片的最佳不定芽再生培养基为MS+2.5 mg/L TDZ+0.1 mg/L IBA+0.1 mg/L 2,4-D,叶片再生的最佳叶龄为30～40 d,再生率可达82.8%;'红颊'叶片的最佳不定芽再生培养基为MS+2.0 mg/L TDZ+0.1 mg/L IBA+0.1 mg/L 2,4-D,叶片再生的最佳叶龄在10～20 d,再生率可达79.8%.2个品种叶片暗培养14 d可以提高不定芽再生率;叶片正放比反放再生效果好;添加8 mg/L AgNO3和1 000 mg/L活性炭可有效提高再生率.     

毛白杨悬浮细胞系的建立及再生植株的获得

以毛白杨基因型TC152无菌苗为材料,研究毛白杨悬浮细胞系建立与植株再生,结果表明,通过悬浮培养和固体培养两种方法诱导毛白杨悬浮细胞分化不定芽,最终获得无菌生根苗。愈伤组织在MS＋1.5mg·L-12,4-D＋30g·L-1蔗糖的液体培养基中振荡培养,12d可建立悬浮细胞系;悬浮细胞系继代培养基为MS＋0.8mg·L-12,4-D＋30g·L-1蔗糖,继代周期为7d,悬浮细胞在MS＋1.0mg·L-16-BA＋0.1mg·L-1NAA＋0.5～1.0mg·L-1ZT＋30g·L-1蔗糖培养基中悬浮培养,可分化大量不定芽,每个培养瓶中可得到40～50个芽,个别不定芽玻璃化;不定芽在1/2MS＋0.6mg·L-1IBA＋20g·L-1蔗糖＋5.5g·L-1琼脂培养基上可分化不定根。悬浮细胞通过固体平板培养增殖为愈伤组织块后,在MS＋1.0mg·L-16-BA＋0.1mg·L-1NAA＋1.0mg·L-1ZT＋30g·L-1蔗糖＋5g·L-1琼脂的固体培养基上,不定芽分化率可达到70.00%。     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.