• KSBB Journal
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• v.25 no.2
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• pp.142-154
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• 2010

The protein-bound innerpolysaccharides (IPS) produced by suspended mycelial cultures of Inonotus obliquus have promising potentials as an effective antidiabetic as well as an immunostimulating agents. To enhance IPS production, intensive strain improvement process should be carried out using large amount of UV-mutated protoplasts. During the whole strain-screening process, the stage of solid growth-culture was found to be the most time-requiring step, thus preventing rapid screening of high-yielding producers. In order to reduce the cell growth period in the solid growth-stage, therefore, solid growth-medium was optimized using the statistical methods such as (i) Plackett-Burman and fractional factorial designs (FFD) for selecting positive medium components, and (ii) steepest ascent (SAM) and response surface (RSM) methods for determining optimum concentrations of the selected components. By adopting the medium composition recommended by the SAM experiment, significantly higher growth rate was obtained in the solid growth-cultures, as represented by about 41% larger diameter of the cell growth circle and higher mycelial density. Sequential optimization process performed using the RSM experiments finally recommended the medium composition as follows: glucose 25.61g/L, brown rice 12.53 g/L, soytone peptone 12.53 g/L, $MgSO_4$ 5.53 g/L, and agar 20 g/L. It should be noted that this composition was almost similar to the medium combinations determined by the SAM experiment, demonstrating that the SAM was very helpful in finding out the final optimum concentrations. Through the use of this optimized medium, the period for the solid growth-culture could be successfully reduced to about 8 days from the previous 15~20 days, thus enabling large and mass screening of high producers in a relatively short period.

• Korean Journal of Food Science and Technology
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• v.21 no.1
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• pp.154-163
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• 1989

This studies were designed to improve the productivity of L-lysine by protoplast fusion and immobilized system of fusants using strains of Brevibacterium flavum ATCC 21528, Brevibacterium lactofermentum ATCC 21086 and Corynebacterium glutamicum 820. Mutants were isolated with concentration method of $300{\mu}g/ml$ penicillin-G after treatment of $250{\mu}g/ml$ N-methyl-N-nitro-N-nitrosoguanidine. B. flavum $37-2(Hos^-,\;Kan^r,\;AEC^r)$, B. lactofermentum $6-2(Ile^-,\;Val^-,\;Str^r,\;AEC^r)$ and C. glutamicum 57-5$(Met^-,\;Thr^-,\;Rif^r,\;AEC^r)$ were isolated from mutants. Protoplasts were induced by being incubated with $500{\mu}g/ml$ lysozyme of lysis solution for 6 hr and the ratio of protoplast formation and regeneration were ranging from 97-99% and 33-37%, respectively. Fusion frequencies of fusants of BBFL 21, BCFG 37 and BCLG 59 were shown in the range from $1.25{\times}10^{-6}\;to\;5.83{\times}10^{-7}$ under the optimum conditions. The fusant BBFL 21 showed the highest productivity of $411.1\;ng/ml{\cdot}hr$ L-lysine in the lysine productivity broth at $30^{\circ}C$ for 72hr. In the immobilization systems, fusant BBFL 21 was employed in various polymer matrices such as sodium alginate, polyacrylamide, agar and ${\alpha}-carrageena$. The immobilization of sodium alginate showed the highest productivity of $413\;ng/ml{\cdot}hr$ L-lysine in the batch system. Continuous fermentation of immobilization system by using tube fermentor was produced the highest productivity $416.7\;ng/ml{\cdot}hr $ L-lysine under optimum condition.

• The Korean Journal of Mycology
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• v.21 no.3
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• pp.200-211
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• 1993

Somatic hybrids of Pleurotus florida ASI 2016 and Pleurotus ostreatus ASI 2018 were obtained by protoplast fusion. The 40 fusants($P1{\sim}P40$) was examined for the yield on fermented and pasteurized rice straw in a tray. The carpophore yield of them were showed as the range of $27.0{\sim}155.2$, based on parental values of 100(ASI 2018), The pilei of fusants between orange white colored P. florida and dark grey colored P. ostreatus had mixed colors in the young stage. Other breeding programmes were performed to improve new varieties with high yield and good quality. A new oyster mushroom variety, Wonhyeongneutaribeosus(P72), was developed at the Agricultural Sciences Institute, Rural Development Administration in 1990. This P.florida-ostreatus-ostreatus hybrid P72 was selected from 38 protoplast fusion products($P41{\sim}P78$) between P.florida-ostreatus recombinant P5-M 43-arg rib and P. ostreatus ASI 2-13-0 2001-19-pro orn. The yield indexes of 38 hybrids ranged $40.5{\sim}152.7$ compared with the parental values of 100(ASI 2001). Hybrid P72 was characterized by the large fruiting bundle of semispherical shape with long stipe and by the small and circular pileus, resulting in lower harvesting cost. A significant increase in carpophore production was observed in somatic hybrids of protoplasts due to heterosis. A comparision of hybrid with parents P72 was made using isozyme analysis. The esterase banding patterns could be characterized by new bands in the hybrids. Seven fusion products of four crosses between P.florida ASI 2016 and P. ostreatus ASI 2018 were analysed with respect to the distribution of progenies and segregation of gene markers by random basidiospore analysis. Segregation of alleles should yield progeny of four genotypes in a Mendelian ratio of 1 : 1 : 1 : 1 for prototrophs, auxotrophs of one parental type, auxotrophs of the other parental type, and auxotrophic recombinants, respectively. However, five fusants of them did not detect one parental, P.ostreatus, type. Basidiospores could yield progeny of 16 genotypes in the cross of one of the recombinant P5-M43-arg $rib{\times}P. ostreatus$ ASI 2-13-pro orn but the segregants of three fusants were not detected clearly. The allele ratio of loci could be expected 1 : 1 : 1 : 1 for arg, rib, pro and orn. The ratio, however, would be changed to 4 : 1 : 1 : 1 with increasing proportion of argo In almost all the fusants, prototrophic recombinants were recovered in large numbers against auxotrophic markers. Parental genotypes were recovered with the recombinant progeny amounting to $38.68{\sim}99.56%$. The analysis provides proof of heterokaryosis and strong evidence for haploidy of vegetative nuclei, a sexual cycle consisting of nuclear fusion and meiosis.

• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.