大球盖菇原生质体再生及单核化特性的研究

大球盖菇原生质体再生条件及单核化特性结果。原生质体再生程度极快,涂布平板3d后肉眼即可见明显的再生菌落形成,在PGPM再生培养基上再生率为0．97－2．0％,渗稳剂种类对再生率无明显影响,但可影响再生菌落形态,液体预培养1－2d,再生率明显下降;大球盖菇原生质体单核化率高达77．6％,且再生双核体和再生单核体在形成再生菌落时无时间差,其生长速度亦无快慢之分,液体预培养可显著减少单核化率,再生单核体中存在亲本两种交配型,但二者的比率不为1。     

茯苓原生质体单核化及同核体杂交

茯苓Wolfiporia hoelen是一种食药兼用的大型真菌,在我国栽培历史悠久,但由于长期无性繁殖,导致菌种退化,栽培产量下降,严重影响产业发展。为解决茯苓育种的难题,我们前期建立了茯苓同核体鉴定方法,明确了其交配系统,建立了单孢同核体杂交育种体系,但是部分茯苓菌株子实体形成困难或子实体贴生,导致担孢子收集困难,因此原生质体单核化的研究具有重要意义。本研究以两株存在较大遗传差异且同核体类型不同的菌株776和775为材料,核荧光染色表明超过60%的原生质体具有细胞核,原生质体再生率分别为11.0%和7.6%,不易生长的再生菌株中既有同核也有异核菌株。原生质体单核化获得了两个菌株的同核体菌株,比例可达到10%以上,菌株776获得了两种交配型的同核体菌株(5:3),不存在偏分离(χ²=0.5),但菌株775原生质体单核化仅获得了一种交配型的同核体菌株,表现出严重的交配型偏分离。通过杂交菌株与两亲本对峙试验及基于rpb2的杂合位点验证,证实获得了原生质体同核体杂交菌株25株,原生质体同核体与单孢同核体杂交菌株50株,表明茯苓原生质体同核体杂交育种的可行性。     

大球盖菇原生质体再生及单核化特性的研究*

大球盖菇原生质体再生条件及单核化特性结果。原生质体再生速度极快,涂布平板３ｄ后肉眼即可见明显的再生菌落形成,在ＰＧＰＭ再生培养基上再生率为０．９７～２．０％,渗稳剂种类对再生率无明显影响,但可影响再生菌落形态,液体预培养１～２ｄ,再生率明显下降;大球盖菇原生质体单核化率高达７７．６％,且再生双核体和再生单核体在形成再生菌落时无时间差,其生长速度亦无快慢之分,液体预培养可显著减少单核化率,再生单核体中存在亲本两种交配型,但二者的比率不为１。     

斑玉蕈不同交配型的原生质体单核菌株差异分析

对2个斑玉蕈菌株进行了原生质体分离与交配型鉴定,并对不同交配型单核体的代谢特性进行了研究。结果表明,2个斑玉蕈菌株均出现2种交配型,2种交配型比例不一致,且2个菌株的两种单核体的分离比例均不符合1∶1分离比,交配型比例上出现明显的偏差现象。2个菌株中,不同交配型单核菌株的菌丝形态有明显差别,代谢过程中的菌丝生物量及发酵液的pH、还原糖含量和电导率均存在差异,表明在这2个菌株中不同交配型单核菌株的菌丝形态及代谢特性与交配型因子相关。     

毛栓菌原生质体制备和再生及单核菌株产漆酶特性

毛栓菌Trametes hirsuta能有效地降解木质素,在生物燃料、制浆和饲料工业等方面具有很高应用价值。为了获得遗传性能稳定的T. hirsuta单核菌株,研究了其菌丝生长培养基的类型、菌丝生长时间（菌龄）、酶解时间、原生质体纯化离心速度和原生质体再生培养基类型对T. hirsuta YJ-9-1原生质体制备与再生的影响;采用DAPI染色和锁状联合缺失的观察,从再生株中筛选单核菌株并考察其产酶特性。结果表明：采用YGM菌丝生长培养基、88h菌龄、1h酶解时间、4,000r/min原生质体纯化离心速度以及YGMS再生培养基,最终可获得密度大约为5.0×106个/mL的原生质体悬浮液和9.1%的再生率;从200株再生菌株中筛选出了3株单核菌株,其中一株单核菌株D-2-1的漆酶产量比原菌T. hirsuta YJ-9-1明显提高,在第12天其漆酶酶活为771.67U/L,是原菌的1.51倍。     

利用原生质体融合研究凤尾菇中性菌株的交配反应

凤尾菇营养缺陷型突变型中性菌株与另一株营养缺陷型的正常交配型菌株进行了原生质体融合反应融合子之间以及与亲本之间在菌落形态和菌丝生长速度上表现出了较大差异。融合子菌落角变的菌丝体多核体转变为双核体,并且在菌丝体上产生了圆桶状的特殊结构而不是通常的锁状联合。初步观察表明这些结构似乎与细胞核的迁移有关,对融合子子实体的担孢子进行了遗传分析,结果确实了实体是由两个亲本菌株的融合子产生,同时也表明要对中性菌株交配反应中基因重组进入深入分析还需要进行更多的研究。     

紫芝不亲和性因子分析

为开展紫芝的杂交育种,对6个紫芝菌株的不亲和性因子进行分析。通过原生质体单核化,6个紫芝获得9个不同亲本型的单核化菌株,共测得7个不同的A因子和7个不同的B因子,A、B因子的多态性各占单核体的77.8%,表明紫芝有较丰富的交配型因子,为紫芝的杂交育种奠定基础。     

蛹虫草单孢菌株的生物学特性及稳定性

蛹虫草菌株在继代培养过程中极易退化。本研究选取5株蛹虫草菌株作为出发菌株,基于单孢分离和显微技术从每株出发菌株中获得50株单孢菌株。通过PCR方法对单孢菌株进行交配型类型鉴定,全部为单一交配型单孢菌株,且不同出发菌株分离得到的MAT1-1和MAT1-2交配型单孢菌株比例差异较大,分别为27:23、34:16、42:8、28:22和7:43。从不同出发菌株获得的单孢菌株中随机选择MAT1-1和MAT1-2交配型单孢菌株各5株(共计50株),进行菌落直径、产孢量和虫草素含量测定。与出发菌株相比较,14株单孢菌株菌落直径具有显著差异(其中10株显著减小),24株产孢量具有显著差异且全部下降,29株单孢菌株虫草素含量具有显著差异(其中21株显著下降)。进一步,将50株单孢菌株连续继代培养,测定菌株菌落直径、产孢量和虫草素含量,计算第七代与第一代比值评估菌株性状稳定性。结果表明,与出发菌株相比较,14株单孢菌株菌落直径比值具有显著差异且全部增加,41株单孢菌株产孢量比值具有显著差异(其中40株显著下降),44株虫草素含量比值具有显著差异且全部下降。本研究表明同一菌株中的不同单孢菌株个体之间,在生...     

植物微原生质体的融合

文章就植物微原生质体融合技术的基本原理和技术的应用作了介绍和展望。     

香菇单核菌丝9101（12）原生质体再生条件的研究

本文对香菇（Ｌｅｎｔｉｎｕｓｅｄｏｄｅｓ）单核菌丝９１０１（１２）原生质体制备后的再生条件,进行了探索。结果表明,原生质体经过合０．６Ｍ．甘露醇的麦芽糖酵母粉培养基（ＭＹＧ）液体培养５天,用双层培养法,在含０．６ＭＭｇＳＯ４的高渗ＭＹＧ平板上经再生,再生率达５×１０－５。本文将Ｋｎｏｗｌｔｏｎ等于１９８４年创造的一种分离高频再生衍生株的方法首次运用至食用菌中,所得９１０１（１２）再生株Ｒ３,再生率提高２～３倍,为香菇原生质体融合操作打下基础。     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

Protoplast culture and plant regeneration in Glycine canescens F.J. Herm

Protoplasts were isolated from seedling hypocotyls of Glycine canescens F.J. Herm. and cultured in agarose to form multicellular colonies. Colonies transferred to agar medium enlarged to form calli which were tested for morphogenic potential on a range of media. Protoplast-derived calli produced shoots with a low but reproducible frequency; these shoots were rooted and transferred to the greenhouse.     

Protoplast culture and plant regeneration in Lycium barbarum L.

Protoplasts were isolated from leaves of the woody plant Lycium barbarum L. and cultured in liquid nutrient medium TM-2 at a density of 10⁴–10⁵ cells ml^-1. After ten days of culture, regenerated colonies were transferred to the agar-solidified medium TM-3, and 5–7 days later to regeneration media PRM or TM-4. Formation of shoots was observed after 30–40 days. Completely formed and rooted plants were transferred to the soil. Cytological and morphological analysis of the regenerated plants revealed relative genetic stability of this species in the process protoplast — plant. The results obtained allow us to conclude that L. barbarum can be used in the experiments on somatic hybridization or direct gene transfer.     

Optimisation of Protoplast Production in White Lupin

The influence, was investigated, of abiotic parameters on the isolation of protoplasts from in vitro seedling cotyledons of white lupin. The protoplasts were found to be competent in withstanding a wide range of osmotic potentials of the enzyme medium, however, −2.25 MPa (0.5 M mannitol), resulted in the highest yield of protoplasts. The pH of the isolation medium also had a profound effect on protoplast production. Vacuum infiltration of the enzyme solution into the cotyledon tissue resulted in a progressive drop in the yield of protoplasts. The speed and duration of orbital agitation of the cotyledon tissue played a significant role in the release of protoplasts and a two step (stationary-gyratory) regime was found to be better than the gyratory-only system. This revised version was published online in July 2006 with corrections to the Cover Date.     

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。     

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%。     

Genetic variability of the wild incompatibility alleles of the tetrapolar basidiomycete Agrocybe aegerita

Summary The variability of the sexual incompatibility genes of Agrocybe aegerita was investigated in the homokaryotic progeny of 13 wild dikaryotic strains from five distinct European geographic origins. Results of mating tests allowed identification of 18 A alleles and 16 B alleles out of a possible 26 different alleles for each in the sample. The determination and the comparison by a contingency ² test of the frequencies of allele replications between intra- and interregional matings showed no departure from a random distribution of incompatibility alleles. The allelic series estimated for the incompatibility genes of the entire population of A. aegerita, 30 A and 25 B aleles, are significantly less extensive than those already hypothesized for other tetrapolar hymenomycetes. However, the low variability of incompatibility genes has little effect on the outbreeding efficiency (92.6%) of this mushroom. The low variability of the incompatibility alleles and the apparent absence of intrafactorial recombination could relate to a single-locus structure of the incompatibility genes in A. aegerita.     

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.     

Protein mapping and genome expression variations in the basidiomycete Agrocybe aegerita

Summary A procedure suitable for the extraction and mapping of total proteins from the basidiomycete, Agrocybe aegerita, was developed. A. aegerita mycelia were fragmented either with a Dangoumeau grinder, an X-press bomb or a sonicator and the efficiency of these three disruption methods were compared. The extraction buffer composition was optimized to avoid proteolytic activities. 2D-SDS-PAGE analysis of protein extracts showed that the rate of reproducibility depending on extractions and electrophoretic separations was always greater than 96% for all strains. The differences in efficiency observed between the breaking procedures indicate that the A. aegerita cell wall is more mechanically resistant than that of other basidiomycetes. The efficient action of protease inhibitors (PMSF and SDS) showed that A. aegerita mycelia contains numerous and/or highly active proteases. Reproducibility of protein extraction and separation methods allowed the establishment and the comparison of standard maps. Qualitative and quantitative variations in gene products between a wild dikaryotic strain and 11 homokaryotic strains from its progeny were examined. The genetic diversity, determined by comparing the distribution of proteic variations in 11 homokaryons from the same progeny, was comparable to that observed between co-isogenic homokaryons of another basidiomycete.     

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.