生防真菌寡雄腐霉原生质体的制备及再生

寡雄腐霉Pythium oligandrum是一种对动、植物和环境无害,兼具杀菌和增产效果的生防真菌。本研究以0.8mol/L甘露醇与25mmol/L CaCl_2和10mmol/L Tris-HCl作为渗透压稳定剂,使寡雄腐霉菌丝体在含有裂解酶、纤维素酶、破壁酶的复合酶系中酶解得到其原生质体。此原生质体通过液体振荡培养的途径恢复再生1d后涂于平板上可生长为成熟菌体。本研究所述方法制备得到的寡雄腐霉原生质体数量超过1×10~9CFU/m L,活性超过80%;通过液体培养再生途径使得寡雄腐霉原生质体再生率达32.9%。此方法操作简便,稳定性好,获得的寡雄腐霉原生质体品质高、数量多,完全能够满足后期基因遗传转化、细胞融合培养等研究应用。     

Preparation of protoplasts and regeneration of intact cells ofStreptomyces cinnamonensis

Protoplasts were prepared and intact cells were regenerated inStreptomyces cinnamonensis— a monensin producer— to make genetic manipulations with this strain possible. 70–80% of protoplasts were formed and up to 90% of them could regenerate into intact cells.     

Reversion of Bacillus megaterium protoplasts to the bacillary form.

Photomicrographic evidence of reversion of Bacillus megaterium protoplasts to the bacillary form on soft agar plates hypertonic medium is demonstrated.     

Preparation and regeneration of protoplasts of the strain Streptomyces hygroscopicus 155]

Protoplasts of Streptomyces hygroscopicus 155 producing nigericin, a polyether antibiotic, were prepared. Conditions for protoplasting and optimal concentrations of lysozyme and glycine, and the age of mycelium were studied. With the method of the experiment design, concentrations of four components: CaCl2, MgCl2, KH2PO4 and TES buffer in the regeneration medium were determined. The levels of CaCl2 and TES buffer proved to be the most important parameters.     

Polyethylene glycol-induced fusion of heat-inactivated and living protoplasts of Bacillus megaterium.

Protoplasts of Bacillus megaterium, incubated at 50 degrees C for 120 min, lost the ability to revert to bacillary form. Such heat-inactivated protoplasts, however, produced recombinants when fused by polyethylene glycol treatment with normal protoplasts. Although this differential inactivation effect is not yet fully reproducible, reciprocal inactivations of the parental protoplasts in genetic crosses have clearly shown that for protoplast fusion (i) either of the parents may serve as the viable recipient for markers coming from the heated parental protoplasts, and (ii) either of the parents may be rendered nonviable and yet, when fused with a viable partner, contribute to formation of a recombinant. Heat inactivation seems to provide a way to counterselect when few markers are available and one of the parents is prototrophic.     

Experiments with Bacillus thuringiensis protoplasts. II. Study of the potential interspecies fusion of bacterial protoplasts: Bacillus thuringiensis and Bacillus megaterium

In the present study the possibility of obtaining interspecific bacterial hybrids by polyethylene glycol-assisted fusion of protoplasts from Bacillus thuringiensis and Bac. megaterium has been examined. Electron microscopic and genetic data allow to confirm with great probability that cytological fusion takes place. However, genetic analysis revealed that neither of methods applied for protoplast fusion gave stable recombinants. Apparently, it is due to the lack of recombination or the death of recombinants that follows the functioning of the cell correction system. The mechanism of protoplast fusion and its most important steps are also studied in the present work.     

Adventures with Bacillus megaterium – fusion of bacterial protoplasts

This essay describes the author's studies with bacteria in post-war Hungary; the difficulties encountered, with funding, collaboration and publication; and how the Szeged Institute consolidated itseif as the one outstanding scientific Institute in Eastern Europe, with the author at the helm.