水翁悬浮细胞系的建立及其悬浮培养的生长特性

建立了水翁悬浮细胞系,并对其悬浮培养的生长特性作了初步探讨。以水翁新生芽尖作为外植体,接种于添加有不同浓度和配比的生长调节物质及各种附加物的MS固体培养基中,诱导培养产生初代愈伤组织;挑选Ⅰ和Ⅱ型的愈伤组织进行继代培养改良,考察愈伤组织的生长状况和统计生长量来决定最佳继代培养基的配方和得到适合悬浮培养的愈伤组织;将以上得到的愈伤组织转接于最佳继代液体培养基中,于24±1℃,120r/min条件下振荡培养,筛选分散度好、较均匀、生长快、色浅透明的细胞作为种子传代,数次传代后得到性能良好的悬浮细胞系;以细胞生长量(鲜重)为指标,绘制了水翁悬浮细胞的生长曲线。研究表明:2.0mg/L的2,4-D的诱导率最高(92%,初代愈伤组织为Ⅰ型),Ⅱ型愈伤组织的最高诱导率为75%;最佳的继代培养基配方为MS 0.5mg/L 2,4-D 0.5mg/L 6-BA 1.0mg/L IAA 0.5mg/L IBA 0.5mg/L NAA 0.1mg/L KT 700mg/L LH,形成Ⅱ型愈伤组织的生长量可达3.28g/瓶(鲜重);液体继代培养3代后,可得到性能良好的悬浮细胞系;水翁悬浮细胞的生长曲线表明,最佳接种期为培养后的16~18d。     

白刺花胚性愈伤组织诱导及体细胞胚发生和萌发

探讨不同因素对白刺花下胚轴、子叶2种外植体胚性愈伤组织诱导及体细胞胚发生和萌发的影响。以B5和MS为基本培养基,研究2,4-D、6-BA和TDZ对白刺花下胚轴和子叶胚性愈伤组织的诱导;在MS培养基上添加不同浓度2,4-D,研究胚性愈伤组织增殖情况;采用ABA,探究对体细胞胚发生的影响。结果表明:下胚轴比子叶更易诱导胚性愈伤组织,筛选出2种外植最佳的胚性愈伤组织诱导培养基均为MS+2.0 mg/L 2,4-D+0.5 mg/L TDZ+0.5 mg/L 6-BA,胚性愈伤组织诱导率分别为77.3%和41.0%。15.0 mg/L ABA、0.2 mg/L 2,4-D和2.0 mg/L 6-BA有利于体细胞胚发生,1/3MS+0.2 mg/L NAA+0.1 mg/L 6-BA+2.0 g/L活性炭+25 g/L蔗糖+7 g/L琼脂的培养基可使体细胞胚萌发率达80%以上,再生植株移栽成活率高达90%。白刺花外植体种类及培养基类型均会影响胚性愈伤组织的诱导,其中下胚轴诱导效果优于子叶;MS培养基较适合启动细胞脱分化形成愈伤组织,2,4-D对胚性愈伤组织的增殖保持有调控作用,ABA有利于体细胞胚的发生。     

生姜离体叶片愈伤组织悬浮细胞系建立与植株再生

以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

水稻原生质体培养再生植株程序化的初步研究

从12个品种水稻成熟种子诱发愈伤组织并继代培养,通过MS培养基中2,4-D浓度的变换,研究了2,4-D对水稻愈伤组织生长的影响。用AA培养基建立适合原生质体培养的胚性细胞悬浮系仅需3个月。由悬浮细胞系游离的原生质体在改良的KPR培养基中进行液体浅层培养,有10个品种获得高植板率的细胞团。变换使用不同的分化培养基,从7个品种得到再生植株。实验重复性达到80%,初步实现了水稻原生质体培养的程序化。     

向日葵悬浮培养再生芽

从54份向日葵(Helianthus annuus L.)材料中筛选出三个再生能力较强的基因型,取其下胚轴诱导愈伤组织,再以愈伤组织制备悬浮细胞系。在 MS 添加 BA(0.5mg/l)和2,4-D(0.001mg/l)液体培养基上确定细胞生长量,悬浮培养细胞在去掉2,4-D 的 MS 培养基上能够诱导芽的再生。     

曼陀罗茎段愈伤组织诱导和再生植株的研究

本试验以曼陀罗茎段为外植体,在附加不同植物激素组合的培养基中对愈伤组织的诱导和植株再生进行研究。结果表明:采用修改的MS培养基(除去甘氨酸,维生素B1含量增加至0.5mg/L,pH5.5)附加2mg/L2,4-D可由曼陀罗茎段诱导大量胚性愈伤组织;愈伤组织继代选用0.5mg/L2,4-D为宜;不定芽的诱导采用MS培养基(20g蔗糖,8g琼脂,0.1g水解干酪素) 6-BA(0.5mg/L);幼苗进一步转接至1/2MS IBA(0.2mg/L)生根培养基中,可完成曼陀罗茎段愈伤组织诱导和再生植株的组织培养过程。     

苹果原生质体培养及芽分化的诱导

苹果原生质体培养难度较大。目前再生植株的仅有苹果砧木 M9、MM106及斯巴坦。许多研究均停留在愈伤组织阶段,可见诱导愈伤组织的分化,是苹果原生质体再生植株的关键。本文报道了从新红星苹果花蕾胚性愈伤组织建立的悬浮细胞系分离原生质体,经培养获得无根绿苗的结果。4月末,5月初采集大蕾期苹果花蕾,0.1%升汞消毒,接种于含激素的 MS 培养基上,诱导愈伤组织,再建立胚性悬浮细胞系。继代5天左右的悬浮细胞培养物可作为游离原生质体     

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.     

Box-Benhnken响应面法优化白及愈伤组织的增殖培养基配方

为了提高白及组培过程中愈伤的增殖系数,以白及种子为外植体,利用响应面法对诱导愈伤的增殖培养基中的激素水平进行优化。在添加不同浓度的6-BA（6-苄氨基嘌呤）和2,4-D（2,4-二氯苯氧乙酸）的正交试验诱导愈伤基础上,根据Box-Benhnken（BBD）试验设计原理,采用三因素三水平的方法对3个激素的不同浓度水平进行组合实验。结果显示,通过回归模型分析获得增殖诱导的最佳培养基为MS+0.801 mg/L 6-BA（6-苄氨基嘌呤）+1.192 mg/L 2,4-D（2,4-二氯苯氧乙酸）+0.724 mg/L NAA（萘乙酸）。以优化后培养基进行愈伤组织继代发现其增殖系数为6.154 2,为理论预测值的99.74%。利用响应面法对愈伤组织增殖培养基进行优化,为以愈伤组织为研究材料的细胞悬浮培养、遗传转化、种苗快繁等研究奠定了基础。     

Intra- and Interspecies Virus Transfer in Aspergilli via Protoplast Fusion

Intra- and interspecies transfer of dsRNA viruses between blackAspergilliandAspergillus nidulansstrains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of blackAspergillusandA. nidulansstrains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found amongA. nidulansstrains, transfer among blackAspergilliwas frequently unsuccessful. The lack of virus transfer between blackAspergillusisolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible blackAspergilli. The inefficient transfer of dsRNA viruses between blackAspergilliis not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of blackAspergilliare discussed.     

茶树菇原生质体再生及单核体菌株的特性

【背景】茶树菇遗传育种工作是茶树菇产业持续发展的保障和关键,原生质体的制备及单核体菌株的获得可为茶树菇遗传育种工作的开展提供技术支持。【目的】获得茶树菇原生质体的再生特性、单核化特性及其交配型,为开展茶树菇的杂交育种、融合育种、诱变育种、遗传转化和功能基因挖掘等奠定基础。【方法】以茶树菇保藏菌种Aa11的菌丝为材料,采用甘露醇溶液和溶壁酶溶液直接处理平板菌丝制备茶树菇原生质体,而后对原生质体进行分离和再生培养。通过原生质体单核菌丝体两两单单对峙培养,观察对峙培养过程中的菌落形态变化。【结果】当接种块数量为7、酶解温度为33-34℃、酶解时间为60-80 min时,原生质体数量为107个/mL。茶树菇原生质体在涂布平板7 d后肉眼才可见明显的再生菌落形成,在再生培养基上再生率为0.71%,单核化率为41.1%;再生异核体和再生单核体在形成再生菌落时有时间差,从第7天开始往后连续3 d的再生菌落均为异核体菌株,往后第4天开始陆续出现单核体菌落,之后时间内的菌落均为单核体菌株。试验共得到290个原生质体单核体,分为A1B1和A2B2两种亲本交配型,A1B1和A2B2二者的比例为138:152...     

黑果枸杞原生质体培养及植株再生

该研究以黑果枸杞(Lycium ruthenicum)无菌苗为材料,建立了愈伤组织来源的原生质体再生体系,采用ISSR和FCM技术对再生植株进行了遗传稳定性分析。结果表明：(1)黑果枸杞叶片愈伤组织是产生原生质体的最好材料,在含0.5 mg·mL^-1甘露醇的酶液中,继代1次的叶片愈伤组织中原生质体产量为7.77×10⁶个·g^-1,活力为92%。(2)改良MS培养基 固体液体双层培养(MS₂ 固液双层)是培养原生质体的最好方式,培养10 d的原生质体分裂频率为45.9%,培养20 d的细胞团形成频率为22.9%。(3)在1.5 mg·mL^-1 6 BA+0.1 mg·mL^-1 IBA+MS培养基中,叶片愈伤组织产生的原生质体可分化获得再生植株。(4)ISSR分析显示,再生植株的平均遗传相似系数为0.88;FCM显示再生植株为二倍体,与亲本植株一致。该研究结果为进一步研究枸杞体细胞杂交技术转移野生植物抗逆遗传性状提供科学依据,为枸杞优良品种的选育奠定了基础。     

Protoplast isolation and regeneration in the marine red alga Porphyra nereocystis

We have developed a method for isolating viable protoplasts from the blade phase of the epiphytic marine red alga Porphyra nereocystis Anderson, using a two-step enzymatic digestion with commercially available enzymes. The first step uses papain, the second step uses abalone acetone powder. The method is rapid and gives a high yield of viable protoplasts. In liquid culture in enriched seawater medium, the protoplasts can undergo regeneration along three pathways: they directly form filaments resembling the conchocelis phase of Porphyra; they form calli with relatively thick-walled, pigmented cells; and they indirectly form blades from the edges of these calli. Porphyra nereocystis protoplasts also may serve as an alternative propagation method in aquaculture and be useful for studies of cell-wall formation, cell division, and thallus differentiation. They may also be used in somatic selection, somatic hybridization and gene-transfection experiments.Abbreviations AAP abalone acetone powder - PAP papain - FDA fluorescein diacetate This paper is dedicated to the memory of the late Dr. Munenao Kurogi (1921–1988), Professor Emeritus of Hokkaido UniversityThis research was supported by the Washington Sea Grant Program (National Oceanic and Atmospheric Administration). We thank Professor Y. Fujita (Nagasaki University, Japan), Professor S.-J. Wang (Shanghai University of Fisheries, P.R. China) and Dr. H. Kito (Seikai Regional Fisheries Research Laboratory, Nagasaki, Japan) for sharing their experience with Porphyra protoplast production with us. We thank J.S. Charleston for expert technical assistance in preparation of the electron-microscopy specimens. We also thank Dr. S.K. Herbert and John Carrier (Friday Harbor Laboratories) and Dr. John Merrill and D. Gillingham (American Sea Vegetable Co. and Applied Algal Research, Seattle) for collections of P. nereocystis.     

蕨麻愈伤组织原生质体制备条件的优化

以青海‘蕨麻4号’诱导培养的愈伤组织为材料,采用4因素3水平L9(34)正交实验,研究酶类组合、酶解时间、甘露醇浓度及离心速度等主要因素对蕨麻原生质体分离的影响,建立高效、稳定的蕨麻原生质体分离体系,为进一步通过原生质体融合、基因工程等方法对蕨麻进行品种改良奠定基础。结果表明:各因素对蕨麻原生质体产量的影响顺序为:酶类组合酶解时间甘露醇浓度离心速度;青海‘蕨麻4号’愈伤组织原生质体的最适酶解条件为:2.0%纤维素酶+0.75%果胶酶,40r/min振荡酶解10h,甘露醇浓度为0.5mol/L,离心转速为1 000r/min时原生质体的产量达最大(8.96×10~5 cells/g),活力为92.77%。     

Protoplast fusion inStreptomyces avermitilis

Summary The power of protoplast fusion as a generally applicable method for obtaining genetic recombination is demonstrated by the recombination of genes involved in avermectin biosynthesis. A backcross ofStreptomyces avermitilis strain MA6202, an improved mutant that had lost the ability to carry out the methylation of the C-5 hydroxyl of the avermectin molecule, with the original soil isolate MA4680 resulted in the recovery of at least one unambiguous recombinant class despite the instability of rifampicin resistance, one of two markers initially used for recombinant selection. Such intrinsic instability is frequently encountered in streptomycete genetics, and this result delineates the utility of protoplast fusion as a genetic tool. Other difficulties addressed include recovery of complementary recombinant classes, differences in recombination frequency due to colony density on regeneration medium, and alteration in plating efficiency on diagnostic media following protoplasting and regeneration. The results of a cross between a nicotinamide auxotroph MRG1003 and a lysine auxotroph MRG 1004 are included to aid in the elucidation of these problems as well as to support the finding of homologous recombination inS. avermitilis.     

Protoplast responses in the epidermis of Allium cepa induced by penetration by Botrytis allii

Penetration of Allium cepa epidermal cells (white, yellow, and red varieties) by Botrytis allii induced a response by host protoplasts in normal tissue which was not observed when penetrations were made in protoplast-free host cell walls. Callose and auto-fluorescing substances (possibly phenolic compounds) were located at the penetration sites only in normal host cells containing protoplasts. Lignin tests were negative. Halos were clearly visible in both types of tissue. Autofluorescence was observed at penetration sites in normal cells of all cultivars but general wall background autofluorescence was not observed in white onions. Autofluorescence was generally yellow green and when treated with ammonium hydroxide became green. Treatment with sodium hydroxide abolished autofluorescence. No attempt was made to isolate the autofluorescing material.     

Protoplast isolation in the marine brown alga Dictyopteris prolifera (Dictyotales)

Protoplasts were isolated from thalli of Dictyopteris prolifera using a mixture of crude enzymes from vicera of live oysters (Crassostrea gigas) and the following commercial enzymes: an abalone enzyme, cellulase, polygalacturonase and hemicellulase. The enzyme mixtures produced up to 3.3 × 10⁷ cells per l g of tissue fresh weight. The conversion to protoplasts of the cells was about 100% using the oyster enzyme or the abalone enzyme alone. The optimum pH for protoplast isolation was 6.0 and 20 hours were required for conversion to protoplasts.     

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. The planting density was 2.5–3.0×10⁵ protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.