CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing has recently been applied to Ganoderma lucidum as a promising tool for functional genomics and strain improvement. However, the multinuclear nature of this basidiomycete can result in genetic mosaicism, raising concerns about the long-term stability of edited strains. In this study, we report the occurrence of revertant phenotypes in CRISPR/Cas9-edited transformants of G. lucidum. Although the edited colonies initially exhibited the expected phenotype, repeated subculturing led to the reappearance of wild-type phenotypes. PCR and sequencing analyses revealed the coexistence of edited and non-edited nuclei, and the progressive loss of edited genotypes over successive generations. These findings demonstrate that multinuclearity is a key factor contributing to the instability of CRISPR/Cas9-based edits in G. lucidum. This brief report provides the first direct documentation of revertant occurrence in edited G. lucidum strains and underscores the need for rigorous selection strategies and novel approaches to secure stable homokaryotic transformants in mushroom genetic engineering.

This study reports the development of a new pure white Flammulina velutipes variety, ‘Seoldan’. The breeding process involved a primary cross between a brown strain (KMCC02267) collected by the Rural Development Administration and a white cultivar ‘Baekwoon’ (KMCC05472). From this cross, a white line ‘Fv-23-553’ was selected and subsequently crossed with a superior pure white resource (KMCC05474), resulting in the novel cultivar Seoldan. Mycelial growth tests showed that both varieties grew best at 25°C; however, Seoldan exhibited faster mycelial growth than the control variety under four temperature conditions except at 20°C. In media tests, Seoldan also demonstrated superior growth on three media types, with the exception of YM medium. In sawdust bottle cultivation, The time from scratching to the first fruiting was 2 days shorter in Seoldan than in the control variety, contributing to an overall reduction in cultivation time. The most distinct morphological trait of Seoldan is its pure white cap color. The average yield was 218.8 ± 10.6 g per bottle, comparable to that of the control. Morphologically, Seoldan produced smaller pileus and thinner stipes than the control. Furthermore, somatic incompatibility tests confirmed that Seoldan is genetically distinct from the control variety. These results indicate that Seoldan is a promising pure white variety with stable productivity, improved cultivation efficiency, and clear genetic differentiation from existing varieties.

Despite their historical use, studies on the genetic functions of mushrooms and varietal improvement via biomolecular techniques are limited compared to other organisms. Recent advancements in CRISPR/Cas9 have enabled precise genetic modifications in mushrooms, with RNP-based systems offering high editing efficiency without foreign gene insertion. In this study, we optimized gene-editing conditions for Ganoderma lucidum (Yongji 2) by utilizing RNP/nanoparticle complexes to enhance efficiency. The optimal conditions included a 0.2 M sorbitol buffer (pH 7.0) and a protoplast-to-complex ratio of 10:1. Among eight gRNAs designed for the catA gene, three were identified with high activity, and PEG-mediated transformation resulted in successful gene edits, primarily involving 1 bp deletions. The editing efficiency reached 7–8%, demonstrating that nanoparticle-supported RNP systems are effective for marker-free gene editing in mushrooms. These findings highlight a promising approach for advancing genetic research and varietal improvement in G. lucidum and other mushroom species.

Oyster mushroom is one of the most widely cultivated and consumed mushrooms in Korea, and mechanization and automation of cultivation systems have enabled mass production. Many cultivars have been developed to replace the old ones such as ‘Suhan‘ and ‘Chunchuneutari 2 ho,‘ which have been cultivated for over 20 years. Among these, ‘Soltari‘ was developed in 2015. Although it has excellent quality, its cultivation is challenging and the productivity is somewhat lower. To address these issues, the Mushroom Division at the National Institute of Horticultural and Herbal Science selected the genetic resource KMCC05165 and attempted hybridization between monokaryons from KMCC05165 and ‘Soltari(KMCC04940)’. Through repeated cultivation tests and evaluation of fruiting body characteristics, the superior strain ‘Po-2019-smj22’ was selected and finally named ‘Otari‘. The optimal mycelial growth temperature of ‘Otari’ was between 25 and 30°C and optimal fruiting body growth temperature was between 13 and 18°C. Mycelial growth on PDA medium was best at 25°C, and at the same temperature, mycelial growth was similar across four media: PDA, MEA, MCM, and YM. In 1,100 mL bottle cultivation, the yield was approximately 174 g, which is about 5% higher than the control cultivar ‘Soltari‘, and the number of valid individuals was also higher at about 25. The diameter and height of the pileus were 29.8 mm and 17.6 mm, respectively, slightly smaller than ‘Soltari‘, and the stipe was thin and long with a thickness of 12.2 mm. Additionally, the pileus’ lightness index (L index) was 30.7, indicating a darker brown color compared to 'Soltari.' With excellent mycelial growth, ease of cultivation, and high yield, the new cultivar ‘Otari‘ is expected to be widely adopted by domestic oyster mushroom farms.

Recently, active research in Korea and worldwide has begun to focus on gene function and cultivar development using gene editing tools. This research, in addition to studies on edible mushroom, aims to enhance the physical and biochemical characteristics of mushrooms for applications in materials and substance production. For these studies, efficient isolation of protoplasts from the target mushroom is critical. However, several commercial cell wall-lysing enzyme cocktails, including Novozyme234, Glucanex, and Lysing enzymes, have recently been discontinued. In this study, we aimed to identify alternative enzyme systems to replace the discontinued cell wall-lysing enzymes for stable isolation of protoplasts from Ganoderma lucidum. To select an optimal osmotic buffer, enzyme function in 0.6 and 1.2 M Sorbitol, Sucrose, Mannitol, and KCl was assessed. The effect of reaction time was also evaluated. Protoplast isolation efficiency of each alternative enzyme was tested using lysing enzymes from Trichoderma harzianum, Chimax-N, and Yatalase, either individually or in combination. This matrix of studies identified enzymes and optimal conditions that could replace the discontinued lysing enzymes.

배관 세정 공정은 조선소 선박 건조과정에서 배관 설치 후 장비를 시운전하는 단계로 넘어가기 전에 최종적으로 배관 내부의 이물질을 제거하는 매우 중요한 공정이다. 만약에 배관 내에 이물질이 있는 상태에서 장비를 시운전하는 단계로 넘어갈 경우 이물질이 고가의 장비에 유입되어 펌프 및 기어, 베어링 등이 파손되는 요인이 된다. 특히 펌프나 유압 밸브 같은 경우는 아주 작은 이물질이라도 장비 속으로 유입이 되면 대형 사고로 이어지며, 이런 장비 사고는 주변에 장비 운전을 하는 작업자의 인명사고까지 연계되어 중대 재해 의 잠재 원인이 되므로 조선소 고객인 선주들도 매우 집중적으로 확인하고 관리하는 공정이다. 이러한 문제점을 해결하기 위해 본 연구 에서 기존의 배관 세정 공법에서 유세정 효과를 증가시킬 수 있도록 배관 내 세정 유체의 흐름을 증가할 수 있는 시스템을 제안한다.

We aimed to develop outstanding domestic varieties suitable for both columnar and cylindrical-shape substrates, aiming to replace foreign varieties. and bred a high-quality new strain named ‘Charmjon’, using genetic resources collected from Japan and China. The optimal cultivation temperature for Charmjon’s mycelial growth was found to be 25°C, and its mycelial growth at 15°C and 25°C was superior to the control variety. In terms of mycelial growth characteristics based on the substrate, Charmjon exhibited excellent mycelial strength on PDA medium compared to the control variety. Through columnar and cylindrical-shape substrates cultivation, we assessed the quantity and morphological characteristics of the fruiting bodies. The results confirmed that Charmjon can be produced stably using both cultivation methods, and it showed higher yields and individual weights than the control variety. In addition, the color of the pileus was notably darker, and the shape of the pileus varied depending on the cultivation method. The test of genetic diversity revealed that Charmjon has distinct genetic characteristics compared to the control varieties.

Flammulina velutipesis a popular edible mushroom in South Korea, accounting for approximately 30% of the total mushroom export in the country. Despite its significant position in the domestic mushroom industry, approximately 65% of the cultivated F. velutipes are foreign varieties. To secure variety rights and enhance competitiveness in the export market, there is a need to develop domestic varieties with stable production and excellent shelf life. We focused on breeding a new variety called 'Baekwoon' through mon–mon crossing, using 'Baekseung' and an international collection strain (KMCC02260) as parents. Baekwoon exhibited faster mycelial growth rates at 15 °C and 25 °C and higher mycelial growth across four tested media compared to that of the control variety. Under bottle cultivation, Baekwoon had a mycelial cultivation period that was shorter by approximately 2 days compared to that of the control. The yield of Baekwoon was 228.0±10.9 g, 8.3% more than that of the control. Furthermore, Baekwoon displayed distinct morphological features characterized by a larger pileus and thicker stipe compared to that of the control variety. Genetic tests confirmed that Baekwoon is genetically different from both parental strains and the control variety. It is expected that the newly developed F. velutipes variety, Baekwoon, will be evaluated for its adaptation in different regions and actively promoted in the field, ultimately increasing the market share of domestic varieties.

Mushrooms have a unique taste and aroma, so in the processing of mushroom products with other ingredients, a separate pre-processing step is often taken to eliminate the mushroom aroma. In this study, we analyzed the changes in the concentration of volatile compounds according to drying conditions to promote the activation of processing using the fruiting bodies of yellow oyster mushrooms(Pleurotus citrinopileatus) and pink oyster mushrooms(P. djamor). The caps and stipes of yellow oyster and pink oyster mushrooms were separated and freeze-dried at -70oC for 120 hours. Subsequently, they were hot air-dried at temperatures of 40, 50, 60, and 70oC for 24, 24, 16, and 12 hours, respectively. The dried samples were pulverized and quantitatively analyzed by SPME-GC-MS. In the case of yellow oyster mushrooms, the concentration of t-2-nonenal in caps and stipes during freeze-drying was 164.43 g/g d.w. and 174.80 g/g d.w., respectively, whereas during hot air-drying, it significantly decreased to 0.35~3.41 g/g d.w. and 0.98~59.88 g/g d.w. In a similar manner, for pink oyster mushrooms, the concentration of 1-octen-3-ol during freeze-drying in caps and stipes was 31.05 g/g d.w. and 176.17 g/g d.w., respectively, whereas during hot air-drying, it significantly decreased to 1.59~9.66 g/g d.w. and 1.96~15.77 g/g d.w. Furthermore, most volatile compounds showed a tendency to decrease in concentration as the temperature during hot air-drying increased.