白Pian体细胞胚悬浮培养的动力学研究

白(ＰｉｃｅａｍｅｙｅｒｉＲｅｈｄ.ｅｔＷｉｌｓ.)是我国特有的云杉属树种,在林业生产和环境绿化中均具有重要地位。其体细胞胚胎发生的研究,一方面可用于优良种质的大规模快速繁殖,为植树造林和园林绿化提供优质苗木;另一方面可作为遗传转化的再生系统,进行树种遗传...     

重组人干扰素ω在对数生长期毕赤酵母中的高效表达特性研究

用不同比生长速率μ的毕赤酵母探讨其表达外源重组蛋白的差异性,通过起始pH值、甲醇诱导浓度和周期、菌体浓度、装液量等实验,优化具有较高μ的对数生长期毕赤酵母表达rhIFNω的摇瓶条件。结果表明,μ对毕赤酵母表达rhIFNω有显著影响。μ为0.1612h-1的毕赤酵母表达rhIFNω最高为558mg/L,较μ为0.1321、0.0505和0.0052h-1的毕赤酵母分别提高50%、68%和99%。对数生长期的毕赤酵母表达rhIFNω的最适摇瓶表达条件为:250mL摇瓶装入30mL BMMY,控制菌体浓度达到200~300g/L(WCW),起始pH值自然,每24h添加甲醇15g/L一次,诱导表达周期为4d。通过表达条件的优化,rhIFNω的表达量达到1070mg/L,较优化前提高149%。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

海石竹的离体快繁及核型分析

以海石竹 (Armeriamaritima)的叶片为外植体 ,经离体培养诱导产生愈伤组织 ,再分化形成不定芽 ,并经过继代增殖和壮苗生根 ,获得完整的再生植株 ,最后对其再生植株进行核型分析。结果表明 ,海石竹叶片的愈伤组织诱导和分化的适宜培养基为MS +BA 1 .0mg/L +NAA 0 .2mg/L ,诱导初期进行 7d暗培养 ,最佳增殖培养基为MS+BA 1 .0mg/L +NAA 0 .1mg/L ,生根培养基为MS+NAA 0 .2mg/L。以上培养基均含蔗糖3 0 g/L ,琼脂 5g/L ,pH 5 .8。海石竹的核型公式为 2n=2x=1 8=1 0m +8sm ,存在染色体数目变异的现象。     

Somatic Embryogenesis and Plant Regeneration from Protoplasts of Cowpea (Vigna sinensis)

Immature cotyledons of cowpea (Vigna sinensis Endlo) were used for protoplast isolation. Enzyme solution for protoplast isolation contained 40% cellulase Onozuka R-10,0.30% Macerozyme R-10 and 2% hemicellulase. The purified protoplasts were cultured in Bs,MS or KM8p liquid medium in dark (25℃) at a density of 1 × 105–5 × 105/ml. The protoplasts started cell division in 3–5 days . Sustained cell divisions resulted ill formation of cell clusters and small calli,with cell division frequency reaching 23%–28% in MS medium . Calli of 2 mm in size were transferred onto MSB (MS salts+B5 vitamins) medium with 2 mg/L 2,4-D, 0. 5mg /L BA forfurther growth. Embryogenic calli appeared on this medium. After passage to fresh medium with the same composition, the embryogenic calli were transferred into MSB liquid medium to establish suspension culture. When the suspended calli were transferred back onto MSB agar medium with 0. 1 mg /L IAA, 0.5mg/L KT, 5% mannitol (cultured in light,2000 lx,12h/d), a lot of adventitious roots formed in 7–10 days, and then somatic embryos formed from the protoplast derived calli. But only a few embryoids developed further into the cotyledonary stage ,and the others died at globular, heart-shaped, or torpeto stage . Finally, some cotyledonary embryoids germinated and developed into plantlets or shoots with leaves.     

Plant Regeneration from Protoplasts of Coriandrum sativum

Calli produced from stem segments of seedling of Coriandrum satwum which were cultured on MS agar medium containing NAA 1.0mg/L. The embryogenic cell colony suspension was estabilished on MS liquid medium containing NAA 1.0mg/L%2,4-D 0.2mg/L+BA 0.5 mg/L. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained in the enzyme mixture containing 2.0% Onozuka R-10, 1.0% pectinase, 0.5% snailase, 0.5% dextran sulfate potassium Salt, 0.6mol/L mannital CPW solution at pH 5.8 and 25℃. Cultured in a KM8P liquid medium containing NAA 1.0mg/L+2,4-D 0.2mg/L+6-BA 0.5 mg/L, glucose 0.4mol/L and CM 20mi/L; the protoplasts entered the stage of derision after three days, cell clusters formed in 10 days and calli formed after about 50 days. When the calli were transferred to MS agar medium containing many growth substances, they differentiated into embryoids, and then developed into plantlet with many green leaves and roots on the 1/2 MS agar medium.     

骆驼蓬的组织培养及植株再生

以骆驼蓬(Peganum harmala L)无菌苗下胚轴切段为材料,在不同的培养基上进行愈伤组织的诱导,发现在MS基本培养基附加2．0mg/L 2,4—D、0．5mg／L 6—BA和3％蔗糖时,可100％的诱导出愈伤组织。愈伤组织在附加2．0mg／L 6—BA、0．5mg／L NAA、500mg／L CH和3％蔗糖的MS培养基上诱导出丛生芽,进而发育成苗,苗的分化频率在30％左右。分化苗或其茎切断在附加0．2mg／L IBA、0．2mg/L NAA和3％蔗糖的l／2MS培养基上出现根的分化,分化频率在90％以上。再生植株经炼苗后移栽成活,成活率在80％以上。     

Plant Regeneration from Protoplasts of Suspension Cells of Saposhnilkovia divaricata (Turcz.) Schischk

Embryogenic calli were produced from the segments of the young roots, hypocotyls or petioles of test-tube seedlings on MS agar medium containing 1 mg/1 2,4-D. When shaken in the MS liquid medium, the calli formed cell suspension with many embryogenic cell clumps.Using the enzyme mixture: Onozuka R-10 1.5%+MacerozymeR-10 0.3%+Snailase 0.5%+CaCl2 5 mmol/l + Mannitol 0.6 mol/1 (pH=5.8), protoplasts were obtained from the cell clumps which had been subcultured for three to' seven days. When cultivated, the protoplasts grew and began to divide after four days, and formed cell clumps about l—2 mm within fifty days. Protoplast-derived calli were formed from the cell clumps on the MS agar medium with 0.5 mg/l 2,4-D. When transferred onto the MS agar medium containing 0.1 mg/1 6-BA or 0.1 mg/1 2,4-D and 0.5 mg/1 6-BA, the calli differentiated into embryoids. On the MS agar medium without phytohormone, the embryoids grew into plantlets.     

太白米组织培养的研究

太白米地下鳞茎在MS附加不同浓度KT、BA、IAA、NAA、2,4－D的培养基上,可诱导产生愈伤组织,其中在MS附加NAA 0.5mg/L,KT0.1mg/L的培养基上愈伤组织诱导率最高,可达65％,且生长快;培养在MS附加2,4－D 1.0mg/L,KT 0.1mg/L培养基上的鳞茎可经不定根直接发育成新的鳞茎,由此建立了太白米的鳞茎再生体系,鳞茎再生率达2．17倍。     

Plant Regeneration from Cell Supension Derived Protoplasts of Heracleum moellendorffii

Heracleum moellendorffiz Hance is a herb belonging to Umbelliferae used in traditional medicine in China. The young stem-nodes were induced for callus formation on MS medium containing 1 mg/L 2,4-D. After subcultured for about five months, the embryogenic calli were used for cell suspension culture. The protoplasts were prepared from this suspension by digestion with enzyme mixture containing 1. 5% cellulase Onozuka R-10 +0. 3% macerozyme R-10 + 0. 5% snailase + 5 mmol CaCl2 + 0. 6 mol/L mannitol, at pH 5.8, and cultured in modified MS and modified N6 media with 0.3 % agarose. They divided after 3 days and developed into small cell colonies after about 2 weeks. From this time on, the glucose concentration in the culture media was decreased to 0. 2 mol/L,which led to futher growth of the colonies to small calf . After a period of proliferation on solid medium with 0. 5 mg/L 2,4-D, the calli were transferred to a medium with 0. 1 mg/L zeatin on which somatic embryos differentiated and developed to plantlets     

枸杞髓组织离体培养及高频率植株再生的研究

枸杞髓组织在４种ＭＳ培养基上都能诱导出愈伤组织,诱导率５３．７％～１００％。在培养基ＭＳ＋６－ＢＡ０．１ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ获得的愈伤组织,呈颗粒状,分散性能好,胚性细胞多．将其转移到ＭＳ＋６－ＢＡ０．５ｍｍ／Ｌ＋ＮＡＡ０．０１ｍｇ／Ｌ的分化培养基上获得大量绿色小芽,小芽在ＭＳ＋６－ＢＡ０．２ｍｇ／Ｌ的培养基上得到快速繁殖,繁殖系数５０～１５０株／芽·月。丛生芽在ＭＳ＋ＮＡＡ０．２ｔｍｇ／Ｌ的培养基上形成完整植株     

海甘蓝愈伤组织再生植株的研究

海甘蓝种子在附加有２～５ｍｇ／Ｌ６－ＢＡ＋０１ｍｇ／ＬＮＡＡ的ＭＳ培养基上,幼苗生长健壮。幼苗的下胚轴和子叶柄在ＭＳ＋１ｍｇ／Ｌ２,４－Ｄ＋０５ｍｇ／Ｌ６－ＢＡ的培养基上可以获得较好的愈伤组织。将来源于下胚轴的愈伤组织培养于含有０５ｍｇ／ＬＮＡＡ,２ｍｇ／Ｌ６－ＢＡ的ＭＳ培养基上分化出的丛生芽状态最好。最佳生根培养基为１／２ＭＳ＋０５ｍｇ／ＬＢＡ。     

海洋中海藻糖产生菌的筛选及发酵条件优化

从180余份海水、海泥样品中筛选得到60株产海藻糖较高的菌株,编号为2-14的菌株海藻糖产量最高,为127.9mg/g cell。对2-14菌株进行形态特征、培养特征及生理生化试验,鉴定该菌株为红酵母属(Rhodotorula sp.)。研究摇瓶发酵条件对红酵母海藻糖产量的影响,结果为:初始pH5.5,发酵温度28℃,装液量75mL(250mL三角瓶中)。采用优化后发酵条件红酵母海藻糖产量为193.3mg/g cell,优化前对照值为132.1mg/g cell,优化后的结果是优化前的1.46倍。在5L发酵罐中培养得到最佳发酵时间为54h,发酵罐培养发酵液中海藻糖含量最高达2.5g/L,为摇瓶培养的1.6倍。     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度,在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明,以下条件：初始甘油浓度40g／L、初始甲醇浓度3.1g甲醇／gDCW、每24h添加0.51g甲醇／gDCW、诱导表达周期72h、250mL三角瓶诱导培养基装液量30mL、初始pH6,0,最适于菌体生长与产物表达。在此基础上,7L罐上通过恒速流加甘油进一步提高细胞密度,诱导阶段甲醇采取前期恒速流加和后期DO-stat,发酵结束菌体干重达80g／L,酶活为217U／mL,比摇瓶结果提高了66.2％。     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度, 在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明, 以下条件：初始甘油浓度40 g/L、初始甲醇浓度3.1 g甲醇/g DCW、每24 h添加0.51 g甲醇/g DCW、诱导表达周期72 h、250 mL三角瓶诱导培养基装液量30 mL、初始pH 6.0, 最适于菌体生长与产物表达。在此基础上, 7 L罐上通过恒速流加甘油进一步提高细胞密度, 诱导阶段甲醇采取前期恒速流加和后期DO-stat, 发酵结束菌体干重达80 g/L, 酶活为217 U/mL, 比摇瓶结果提高了66.2%。     

毕赤氏酵母工程菌高密度发酵表达恶性疟原虫融合抗原的研究

疟疾目前仍是危害人类健康的主要传染病,现全球每年新增疟疾病例3～5亿人,其中约300万人死于该病.特别是病原虫抗药性的产生和扩散已给疟疾防治工作带来极大困难,因此研制新的预防措施已成为当务之急.研制有效的疟疾疫苗被认为是人类控制乃至消灭疟疾的重要途径,已越来越受到重视,并已构建和鉴定多个疫苗候选抗原[1,2].本研究进行高密度优化表达的融合抗原就是一个疫苗候选抗原.     

水母雪莲体细胞胚胎发生及其植株再生

水母雪莲（Saussurea medusa Maxim.)茎和叶片的切段接种于MS＋2mg/L NAA 0.5mmg/L 6-BA的培养基上,20d后产生黄褐色的愈伤组织,经过几个月的继代培养,愈伤组织仍保持旺盛的增殖能力,但部分由黄褐色逐渐变为红色,将红色愈伤组织转到MS＋0．1mg/L NAA＋0．2mg/L 6-BA 5mg/L GA3的培养基上,30d后可分化出大量的体细胞胚,体细胞胚成熟后转到1／2MS＋0．2mg/L IAA 0.5%活性炭的培养基上,30d后可长出2－4cm的根,带根的小苗经锻炼后移栽到土壤中,成活率达76％,细胞组织学观察表明,发育成熟的体细胞胚具有胚根,胚轴和胚芽的完整结构,具有独立的维管系统。