Efficient protoplast regeneration for some homofermentative lactobacilli and pediococci

Conditions for protoplast regeneration were examined for several strains of homofermentative lactobacilli and pediococci isolated from silage. Attempts to regenerate protoplasts using previously published agar regeneration media for lactobacilli were unsuccessful for most of the strains. Replacing or increasing colloidal substances in a medium containing raffinose and MgCl(2) as osmotic stabilizers enabled efficient regeneration of the protoplasts at a frequency of 10-99%. A medium containing gelatin, polyvinylpyrrolidone (PVP) and no agar was effective for Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus rhamnosus protoplasts. An agar medium containing PVP (PVP medium) was effective for Pediococcus sp. protoplasts, and addition of agarose to the PVP medium enabled regeneration of Lactobacillus casei protoplasts. A medium containing calcium alginate gel and no agar was effective for Lactobacillus curvatus protoplasts. The type of colloidal substance required for protoplast regeneration varied from species to species. This result suggested that several kinds of media may be necessary to regenerate protoplasts for all the genera of lactobacilli and pediococci.     

米曲霉两株营养缺陷型原生质体的形成和再生的条件实验*

采用1%溶壁酶加1%玛瑙螺酶(褐云玛瑙螺消化液的冷冻干粉)的混合酶,自米曲霉(Aspergillus oryzae)的两株营养缺陷型中获得了大量的原生质体,并比较了渗透压稳定剂、温度、菌丝体的培养基成分等因素对原生质体形成和再生的作用。无机盐类稳定剂(NaCl、KCl)获得了高产量的原生质体,而有机类(蔗糖、甘露醇、山梨醇)做为稳定剂不甚理想。对120和720菌株的原生质体在高渗再生培养基上进行再生试验,再生率分别为52%和65%。     

Plant regeneration from mesophyll protoplasts of Solanum commersonii dun

Somatic fusion of Solanum commersonii, a frost tolerant wild potato species not crossable with Solanum tuberosum, relies on the possibility to isolate and culture protoplasts. This study was conducted to determine whether protoplasts could be isolated and plants regenerated in three S. commersonii accessions. Shoot cultures for protoplast isolation were maintained on Murashige and Skoog medium. Mesophyll protoplasts were isolated and cultured using a protocol originally described for S. tuberosum with some modifications. Differences were evident among the three accessions for protoplast yield, plating efficiency and regeneration frequency. Protoplast yield ranged from 3.0 to 8.5 × 10⁶ protoplasts per g of fresh tissue. At 1–2 × 10⁴ protoplasts ml⁻¹, which was the optimal plating density, 10–20% of plated protoplasts gave multicellular colonies. Regeneration of shoots was observed in two accessions only, the maximum regeneration frequency being 66%. In one of these accessions the reduction of sucrose concentration in regeneration media improved the regeneration frequency from 14 to 35%. About three hundred plants were rooted in vitro and successfully transferred to soil.     

木耳属种间杂交技术研究 Ⅰ.木耳菌丝原生质体的形成与再生

本文比较了不同酶液、渗透压稳定剂、酶解温室及菌丝培养基成份等因素对木耳属(Auricularia)中木耳(Auricularia auricula)和毛木耳(Auricularia polytricha)菌丝释放原生质体的作用及影响。用0.5%纤维素酶加0.5%蜗牛酶的混合酶液,以0.6M的MgSO_4为稳定剂,在34℃下可自两种菌丝体获得大量原生质体。对原生质体再生条件的研究表明,纤维二糖和菌丝体培养物浸提物对再生有明显促进作用,再生率达20%左右。本文还用VBL型荧光增白剂观察了菌丝脱壁以及原生质体细胞壁再生的过程。     

Protoplast isolation and regeneration in Streptomyces clavuligerus

The regeneration of streptomycete protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. Reports of studies on the regeneration of protoplasts from Streptomyces clavuligerus are limited and for this reason the experiments described in this paper were carried out. An investigation of protoplast formation and cytology was made to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency were isolated from mycelium, grown in a two-stage culture system (without glycine), using lysozyme dissolved in a sucrose osmoticum containing 1% bovine serum albumin. The latter promoted improved protoplast viability. A systematic survey was made of the components of regeneration medium R5, previously used for S. clavuligerus, and other potentially advantageous components and conditions, in an attempt to raise the regeneration frequency of the protoplasts. An improved regeneration medium (R6) and protocol which supported higher and more consistent levels of regeneration of S. clavuligerus protoplasts resulted from these experiments. These improved procedures for protoplast isolation and regeneration proved to be suitable for other streptomycete species.     

Optimal Cultural and Physiological Conditions for Handling Streptomyces rimosus Protoplasts

A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. After cell wall degradation with lysozyme, protoplasts about 1,000 to 1,500 nm in diameter appeared. The reversion process exhibited normal and aberrant regeneration of protoplasts to hyphae and to spherical cells, respectively. Spherical cells contained no alpha, epsilon-ll-diaminopimelic acid and were colorless or red after Gram staining. They showed consistent stability during at least five subsequent subcultivations. However, the omission of glycine from the precultivation medium reduced the unusual process of regeneration almost completely. After normal protoplast regeneration, the production of oxytetracycline by single isolates was not affected.     

Studies of protoplast fusion in Streptomyces chrysomallus

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 degrees C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1(-1) to the medium and incubation at 23 degrees C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.     

A procedure for regenerating Japonica and Indica varieties of rice from protoplasts

Fertile rice plants have been regenerated from protoplasts of two japonica rice varieties (Radon and Baldo) using a protocol initially developed for plant regeneration from protoplasts of an indica rice. Embryogenic calli were developed from immature embryos of Radon and Baldo rice on a callus induction medium, and then used to establish cell suspensions. Protoplasts were isolated from the cell suspensions, and cultured on a Millipore filter placed on a Kao/agarose medium that contained cell clusters from suspensions of IR52 or IR45. The protoplasts grew vigorously on Kao medium and developed into embryogenic calli within two to three weeks. Somatic embryo development occurred during a subsequent transfer of the calli to an LS medium for two to three weeks. The calli were then transferred to MS or N6 plant regeneration medium, and within one to three weeks, plants regenerated from 21 to 32% of the Radon calli, and 33 to 35% of the Baldo calli. Based upon these results and the previous success in regenerating an indica variety from protoplasts, this procedure has great promise for regenerating a range of rice varieties, and probably for regeneration of other monocotyledonous plants from protoplasts     

黄曲霉菌原生质体的制备和再生技术优化

黄曲霉菌的遗传转化是研究黄曲霉菌致病相关功能基因的前提和基础,而原生质体是研究和建立真菌遗传转化系统的重要工具。本文分别以黄曲霉孢子和菌丝为材料,研究不同条件下黄曲霉原生质体的形成和再生,结果表明,黄曲霉孢子在酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3 h,原生质体制备率高达97.3%,再生率达89.2%;黄曲霉菌丝在菌龄为42 h,酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解1 h,可获得最高原生质体产量为2.0×10^6个/m L,再生培养基中以1 mol/L蔗糖作为渗透压稳定剂时,原生质体再生率达5.5%。故本实验条件下,黄曲霉孢子原生质体的形成和再生优于菌丝。     

γ-亚麻酸生产菌深黄被孢霉原生质作的形成和再生

采用2％的溶壁酶加4％的蜗牛酶,从深黄被孢霉的菌丝中获得了大量的原生质体。同时对菌丝的培养时间、渗透压稳定剂、酶解系统和酶解温度等因素进行了系统的观察,从而获得了制备深黄被孢霉原生质体的最适条件。并对该原生质体在高渗培养基上进行了再生实验,其再生率为32％。     

银耳原生质体分离与再生条件优化研究*

应用正交设计,研究不同菌株(Tr01、Tr21)、材料(芽孢、菌丝体、子实体)、溶壁酶浓度和酶解温度对原生质体产量的影响。实验结果表明,实验材料对原生质体产量影响最大,以芽孢为材料原生质体产量可达到2.75×107个/ml,而菌丝体和子实体的原生质体产量仅为2.5×106个/ml 和1.0×106个/ml;在35℃下酶解,原生质体产量高;溶壁酶浓度在1%~3%范围内对原生质体产量影响不大;不同菌株原生质体产量差异不显著。本实验还研究了稳渗剂浓度对原生质体再生率的影响,结果表明,0.5 mol/L~0.7 mol/L的KCl 对原生质体再生没有显著差异,再生率最高可达32.3%。     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus.

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

小麦白粉病生防菌G-1菌株原生质体诱变育种

以链霉菌G-1(Streptomyces sp.G-1)为出发菌株,通过研究菌株G-1原生质体形成与再生的条件,发现该菌株在含0.5%甘氨酸的菌丝体培养基中经过二次培养后,所得菌丝体用2 mg/mL溶菌酶在30℃下处理90 min,可获得大量原生质体,其再生率可达8.2%。菌株G-1的原生质体经紫外诱变和宁南霉素抗性筛选后,得到一高产突变株G-1-125,其有效组分A的产量达到794mg/L,较出发菌株提高了180%。     

链霉菌11371原生质体的制备与再生

为选育链霉菌11371的高产Tetramycin菌株,摸索该菌株的原生质体的制备与再生。结果表明,链霉菌11371原生质体制备和再生的最佳条件为菌丝生长培养液中甘氨酸浓度为0．7％,培养温度为28℃,培养时间为42h,溶菌酶浓度为3mg／mL,酶解温度为37℃,酶解时间为90min。最佳再生培养基为R2YE培养基。     

Formation and regeneration of protoplasts of Streptomyces hygroscopicus

Techniques are described for the production and regeneration of protoplasts of Streptomyces hygroscopicus and related strains. No single technique was successful in producing protoplasts from all strains. Regeneration of protoplasts to the mycelial growth form was greatly improved by modifying the physical regeneration environment. Protoplast formation and regeneration was achieved in six out of the seven strains studied.     

Application of protoplast fusion technique to genetic recombination of Micromonospora rosaria

The optimum conditions for efficient formation and regeneration of Micromonospora rosaria protoplasts have been determined. The state of inoculum culture and stage of growth in a medium containing partially growth-inhibiting concentrations of glycine had significant effects on protoplasting. A high frequency of regeneration was accomplished with a hypertonic regeneration agar medium. A slight difference was found in the optimum culture age for formation and regeneration of protoplasts. Protoplast fusion was carried out using these optimum conditions. The recombinant frequency varied from 0.7 to 5.9% in the intraspecific crosses employing single and multiple auxotrophic markers. Electron microscopy showed stable and intact protoplasts when they were prepared with a hypertonic buffer. However, many protoplasts were shown to be damaged and many membraneous vesicles were observed when prepared in buffer without sucrose. The fusion process of protoplasts of Micromonospora was observed with the aid of electron microscopy.     

林肯链霉菌双亲灭活原生质体融合的研究

分别以紫外线、热灭活林肯链霉菌 94 7和 95 0 2原生质体 ,然后进行灭活双亲的原生质体融合 ,从 1 6株融合子筛选到林肯霉素高产株。用双亲的互补营养缺陷型对林肯链霉菌原生质体的制备、融合、再生的部分条件进行了研究。发现含 0 .4 %Gly和 34 %蔗糖的SM培养基最适于实验菌株原生质体的制备、再生。聚乙二醇 (PEG)分子量对原生质体融合影响不大 ,其在P缓冲液中的浓度却很重要。含 5 0 %PEG的P缓冲液最有利于原生质体融合     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

Release and regeneration of protoplasts from the fungus Trichothecium roseum

A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described. Protoplasts from T. roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer. A maximum number of 28 x 10(4) protoplasts/mL were obtained at pH 5.5. Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose--yeast extract agar) amended with 0.6 M KCl. The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.