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Bo-Kook Jang(장보국),Cheol Hee Lee 한국원예학회 2021 한국원예학회 학술발표요지 Vol.2021 No.10
Cryopreservation is the preferred method for gene banks because it allows safe and long-term preservation of various plant species and germplasm using only a small space. Moreover, it can be applied to a wide range of flora and is thus an ideal preservation method. Therefore, the present study have proposed the cryopreservation method for long-term conservation of eastern bracken gametophytes. Encapsulation-dehydration of gametophytes was performed, and conditions for exogenous sucrose and abscisic acid (ABA) preculture were investigated. Gametophytes are sensitive to dehydration and drying, and the following treatment conditions were required to survive from cryopreservation: encapsulation by alginate containing 0.75 M sucrose, 18 h loading treatment containing 0.75 M sucrose, and 6 h drying treatment. Encapsulation is essential for gametophyte survival. After dehydration and drying, the moisture content of alginate beads was less than 27.5%, which was appropriate for maintaining the survivability. In addition, sucrose, and ABA preculture were essential before encapsulation to achieve a survival of 90% or more. Various stress and preculture during the cryopreservation process regulated gene expression of PaSuSy, PaLEA14, and PaABI1b, as well as endogenous ABA content. These results suggest that the encapsulation-dehydration method is effective for long-term conservation of eastern bracken gametophyte, and that preculture can alleviate abiotic stress and increase its survival.
참지네고사리의 포자발아, 전엽체 및 포자체 번식에 영향을 미치는 배양 조건
장보국(Bo Kook Jang),조주성(Ju Sung Cho),이기철(Ki Cheol Lee),이철희(Cheol Hee Lee) 한국원예학회 2017 원예과학기술지 Vol.35 No.4
본 연구는 참지네고사리의 기내 포자발아, 전엽체 증식 및 포자체 형성에 적합한 배지 및 배양토를 구명하고 균일묘를 생산하고자 수행되었다. 포자는 배지의 종류와 농도에 관계없이 모든 처리구에서 발아율이 우수하였다. 그 중 Knop배지에서는 MS 계열 배지에 비해 전엽체로의 발달이 빠르게 진행되었다. 포자를 발아시켜 획득한 전엽체를 재료로 배지의 구성물질을 달리하여 배양하였다. 전엽체의 증식율은 Knop배지에 비해 낮았으나, 장정기 등 생식기관의 형성이 활발한 1MS배지가 전엽체 증식용 배지로 적합한 것으로 생각되었다. 또한 MS배지의 sucrose 농도는 2%, 총 질소급원의 농도는 60mM, NH4<SUP>+</SUP>와 NO3<SUP>-</SUP>의 비율을 20:40mM로 조절하는 것이 전엽체의 생육 및 형태형성에 효과적이었다. 포자체 형성에 적합한 배양토를 선발하기 위하여 믹서기로 분쇄한 전엽체를 인공토양(원예상토, 피트모스, 펄라이트, 마사토)을 단용 또는 혼용한 토양에 분주하여 14주간 재배하였다. 그 결과, 원예상토를 사용한 처리구에서 포자체의 형성 및 생육이 우수하였으며, 특히 원예상토와 마사토를 2:1(v:v)로 혼용하였을 때 포자체의 형성(0.83개/cm<SUP>2</SUP>)이 가장 왕성하였다. We investigated a suitable method for in vitro germination of spores, propagation of prothalli, and the formation of sporophytes in the fern Dryopteris nipponensis Koidz. Spore germination rate was relatively high regardless of culture medium. Prothallus development was faster in Knop medium than in Murashige and Skoog (MS) media. Prothalli used in all experiments were obtained from germinated spores, and were cultivated in different concentrations of media components. The active formation of sexual organs such as antheridium made 1MS medium suitable for prothallus propagation, although there was a lower propagation ratio compared to Knop medium. Growth and morphogenesis of prothalli were most effective on 1MS medium containing 2% sucrose, and 60 mM of total nitrogen source with 20:40 mM ratio of NH4<SUP>+</SUP>:NO3<SUP>-</SUP>.To select a suitable soil composition for sporophyte formation, ground prothalli were cultivated on single and mixed soils using bed soil, peat moss, perlite, and decomposed granite for 14 weeks. Bed soil promoted sporophyte formation and growth regardless of single or mixed use. In particular, a mixture of bed soil and decomposed granite in a 2:1 ratio (v:v) led to accelerated sporophyte formation (0.83/cm<SUP>2</SUP>).
조직배양을 이용한 가지고비고사리의 전엽체와 포자체 대량번식을 위한 배양조건
박경태,장보국,이철희,Park, Kyungtae,Jang, Bo Kook,Lee, Cheol Hee 한국식물생명공학회 2019 식물생명공학회지 Vol.46 No.2
본 연구는 다양한 분야에서 활용이 가능한 봉의꼬리과 가지고비고사리[Coniogramme japonica (Thunb.) Diels]의 전엽체와 포자체의 대량번식을 위한 적정 배양방법을 구명하고자 수행되었다. 전엽체 증식을 위해 기내에서 발아하여 계대배양한 전엽체를 재료로 사용하여 1/4-1배로 조절한 MS 배지와 Knop 배지에 10주간 배양한 결과, 1MS 배지에서 전엽체의 생체중이 14.5 g으로 가장 많이 증가하였다. 이후 1MS 배지에 sucrose와 활성탄, 질소급원의 농도를 다르게 조절하여 8주간 배양한 결과, 3%의 sucrose 농도에서 전엽체의 생체중이 10.8 g으로 증식률이 가장 좋았다. 활성탄의 경우 네 처리구가 8.8 ~ 10.8 g 범위로 농도에 따른 유의적인 차이를 보이지 않았다. 질소급원은 ${NH_4}^+:{NO_3}^-$의 비를 1:2로 하여 30, 60, 120 mM의 농도로 첨가한 결과, 세 처리구 모두 생체중에 유의한 차이가 없었으며 비슷한 수준으로 증가하였다. 토양종류에 따른 포자체의 형성을 실험한 결과, 원예상토와 마사토를 2:1(v:v)로 혼합한 토양에서 228.0개로 가장 많은 포자체가 형성되었다. 반면에 피트모스와 마사토를 2:1(v:v)로 혼합한 토양을 제외하고 피트모스가 첨가된 토양에서는 포자체가 형성되지 않았다. This study was conducted to investigate the optimal culture method for gametophyte and sporophyte propagation in Coniogramme japonica (Thunb.) Diels, which can be used in various fields. The propagation of prothallus were cultured in 1/4 - 1 Murashige and Skoog medium and Knop medium for 10 weeks. The results indicated that the fresh weight of prothallus was the highest (14.5 g) in 1MS medium. Subsequently, various concentrations of sucrose, activated charcoal and nitrogen source were also added to 1MS medium and cultured for 8 weeks. The results provided that the sucrose concentration was 3% and the fresh weight of prothallus was the highest 10.8 g. According to the concentration in the range of 8.8 ~ 10.8 g, in the case of activated charcoal, the four treatments showed no significant difference. The nitrogen source was added at a concentration of 30, 60 and 120 mM with the ratio of ${NH_4}^+:{NO_3}^-$ being 1 : 2. As a result, the fresh weight of all treatments increased to similar level but there was no significant difference. We investigated sporophyte formation according to soil type and the highest number of sporophytes at 228.0 was formed in soils mixed with horticultural substrate and decomposed granite at 2 : 1 (v : v). On the other hand, sporophyte was not formed in soils containing peatmoss except for the one with peatmoss and decomposed granite at 2 : 1 (v : v).