The protoplast two-way fusions and fusant characteristics of Antrodia cinnamomea and Cordyceps militaris

This study generated two fused protoplasts of Antrodia cinnamomea and Cordyceps militaris in two ways.The protoplasts of A.cinnamomea were inactivated by heat to inactivate biochemical processes and enzymatic activities in the cytoplasm,and the protoplasts of C.militaris were inactivated by UV radiation to invalidate their genome function,then they were fused under optimal conditions to get a fusion rate as(7.42±0.8)×10^(-6) fusants/mL;the new fusants were abbreviated as Ac-Cm.On the other hand,when A.cinnamomea and C.militaris were treated with heat and UV oppositely using similar experiments,the fusion rate was(9.70±0.68)×10^(-5) fusants/mL,and the new fusants were abbreviated as Cm-Ac.We selected each of two best-growing fused colonies Ac-Cm-1,Ac-Cm-2,Cm-Ac-1,and Cm-Ac-2,together with parental A.cinnamomea and C.militaris,and studied their morphology,growth antagonism tests,and genetic relationships by 18 S rRNA sequencing.In comparison with the initial cultures of 4 fusants,the yields of adenosine,biomass,cordycepic acid,cordycepin,total polysaccharide,and total triterpenoids were increased up 1.305-50.1563 times in the optimal medium conditions.For gene stability tests,those of the four fusants and their outputs were stabilized within 10 generations.

Protoplast Fusion of Cosmos bipinnatus and Cosmos sulphureus

[ Objective] The aim was to study appropriate fusion conditions of protoplasts from 2 species Cosmos bipinnatus and Cosmos sulphureus. [ Method ] The protoplasts were separated from the blossom petals of Cosmos bipinnatus and Cosmos sulphureus availed with the function of pectinase and cellulase, and the fusion of two pretoplasts was performed with PEG. [ Results] A great deal of free pretoplasts were obtained from Cosmos bipinnatus by using 6% mannitol ＋ 1.3% cellu- lase ＋ 1.2% pectinase, and from Cosmos sulphureus by using 10% mannitol ＋ 1.3% cellulase ＋ 1.2% pectinase. Five hours after of enzymolysis treatment, large amount of dissociated protoplasts were observed in the solution. Pretoplast fusion was be obtained by using 30% PEG fusion liquid and 0. lmol/1 CaC12 solution （pH value = 9.5）. [ Conclusion] Our study established an optimal separation and fusion system of protoplasts from different coreopsis species, laying foundation for the breeding of novel coreopsis varieties.

Research Progress of Algae Protoplast Fusion Technique

This paper discussed the technique of algal protoplast fusion,including the method of protoplast-induced fusion,the screening and identification of hybrid cells and so on.In this paper,the research progress of protoplast-induced fusion of microalgae was reviewed,and the problems existing in the process of protoplast-induced fusion were discussed in order to provide a theoretical basis for the development of algae genetics and breeding.

Establishment and Identification of Cytoplasmic Male Sterility in Brassica napus by Intergeneric Somatic Hybridization

Exploitation of novel cytoplasmic male sterility (CMS) is a main approach for widening the cytoplasmic genetic background of hybrid oilseed rape and avoiding epidemic risk in oilseed rape production. In this study, symmetric somatic hybrids between Brassica napus var. Zhongshuang4 and Sinapis arvensis (Ye- you18) were produced by protoplast fusion. Two of the six established hybrids were male sterile showing trace or no pollen release upon flowering with non- or slightly extended stamens. Using Zhongshuang4 as a recurrent parent to pollinate the male sterile plants, the ratio of male sterile plants increased with the number of backcrosses. As early as in BC 3 generation, most of the sterile families had nearly 100% sterile plants. Up to BC 4 generation, the male sterility became stable and no fertility segregation was observed. All F 1 progenies from tested crosses using restorer and maintainer lines of Polima CMS were 100% sterile, indicating that the established CMS by somatic hybridization is different from Polima CMS. The origin of the cytoplasm and potential use of this novel CMS in oilseed rape breeding were discussed.

Construction of hybrid cell with Phanerochaete chrysosporium to degrade terephthalic acid wastewater——(I) phenotype evidence

The fungi Phanerochaete chrysosporium (PC) and Saccharomyces cerevisiae Y99 and the native bacterium YZ1 were the three parental strains for construction of hybrid cells through protoplast fusion to degrade terephthalic acid (TPA) wastewater. The results showed that the native bacterium YZ1 protoplasm could integrate with that of PC to form the hybrid cell Fhh and the fungus Y99 protoplasm also could integrate with that of Fhh to form the hybrid cell Fhhh. The protoplasts of YZ1 and Y99 could change the morphology of PC spore and mycelium for two times. The hybrid cell Fhhh got the best growth and degradation abilities in the wastewater. It suggested that the hybrid strains obtained from the inter\|kingdom protoplast fusion of the three parental strains could create potential for the purification of TPA wastewater.

Analysis of parental strain DNA fragments existing in GEMs-Fhhh

There were 6 target DNA fragments of the three parental strains existing in the cell of GEMs(genetically engineered microorganism strain) Fhhh measured in this research by PCR(polymerase chain reaction). The determination showed that GEMs Fhhh contained all the 6 target DNA fragments, mnp 1, mnp 2、 lip 1、 lip 2, FLO 1 and 16S rDNA, and had the molecular genetic stability. Meanwhile the PCR production of each parental strain could only had its target DNA fragments and was different from each other. It may illustrate that the technique of the inter kingdom protoplast fusion for the construction of GEMs Fhhh through the process of intercellular gene recombination could be used as a reliable bioengineering technique to create the specific functional stain for the pollution control.