냉동보존이 진주조개(Pincata fucata martensii) 유생의 형태 및 구조에 미치는 영향을 알아보기 위하여 냉동전후 D형 및 각정기 유생을 광학 및 전자현미경으로 조사하였다. 동해방지제는 0.2 M sucrose를 첨가한 2.0 M 를 사용하였다. 냉동후 유생은 일부 패각이 손상되긴 했지만 hinge와 prodissoconch가 뚜렷하게 나타났으며, 소포체, 지질 과립, 미토콘드리아, 핵 등을 포함한 세포내 소기관들이 고르게 분포되어 있었다

Somatic embryos do not survive at exposure to liquid nitrogen temperatures without cryoprotective treatments. A simplified technique which simultaneously induces and cryoprotects embryogenic calli using plant vitrification solution 2 (PVS2) followed by dehydration was developed for the cryopreservation of Soap berry genetic resources. Vitrification is a way of removing the moisture in vegetation through PVS2. The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% Dimethylsulfoxide (w/v) in B5 medium containing 0.4M sucrose. Two tests were done. The one was to eliminate moisture at 0℃ and the other at 25℃. In both cases the best results came out at a vitrification time of 10~20 minutes. It was also found that the survival rate was higher at 0℃ than at 25℃. In particular, the survival rate reached more than 80%. Water-damaged embryos turned brown and stoped growth, but energetic embryos took on a milky hue and show a very vigorous growth rate. Successful cryopreservation of somatic embryos of soapberry can be used to establish in vitro genebanks for long-term conservation of Soapberry genetic resources to complement field genebanks and other in vitro methods already being used.

In vitro-grown axillary buds of Melia aredarach were successfully cryopreserved by vitrification. On the MS medium supplemented with BA 1 mg/L, multiple shoots were developed within 4~5 weeks. Plantlets of Melia azedarach were cold-hardened at 10℃ for a 16-hr photo-period for 6 weeks. Excised axillary shoot-tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 25℃. Axillary shoot-tip meristems wert dehydrated using a highly concentrated vitrification solution (PVS2) for 60 min at 0℃ prior to a direct plunge into liquid nitrogen (LN). The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% DMSO (w/v) in MS medium containing 0.4M sucrose. After short-term warming in a water bath at 40℃, the meristems were transferred into 2 ml of MS medium containing 1.2M sucrose for 15 min and then planted on solidified MS culture medium. Successfully vitrified and warmed meristems resumed growth within 2 weeks and directly developed shoots without intermediary callus formation. The survival rate of cold-hardened plantlets for 3 and 4 weeks was 90%. We did not find any difference in PCR-band patterns between control and cryopreserved plants. This method appears to be a promising technique for cryopreserving axillary shoot-tips from in vitro-grown plantlets of Medicinal plants.

본 실험은 개갑된 인삼종자를 안전하게 장기보존하고, 또한 이를 효율적으로 발아시키기 위하여 인삼종자의 건조 저항성 및 동결보존을 위한 적정 수분함량을 분석하고, 배 발육과 발아에 관여하는 온도와 호르몬의 상호작용 효과에 대하여 조사하였으며, 이에 대한 결과는 다음과 같다. 1. 30일 후의 초기 발아율은 GA3 와 BA 등 생장조절제 처리 후 5℃ 와 10℃ 에서 발아시킬 경우에만 50~% 이상으로 높아졌다. 70일 후의 발아율은 생장조절제의 처리와 상관없이 5℃ 와 10℃ 에서 모두 90~% 이상으로 높게 나타났고, 15℃ 에서도 생장조절제 처리구에서는 일부 발아하였다. 2. 개갑 인삼 종자를 무균상 송풍구와 종자건조실에서 건조시킬 경우 각각 12시간(종실 수분함량 10.6~% ) 및 3일(6.1~% ) 까지는 내과피와 종실간에 수분함량에 차이를 보이면서 급속히 건조되다가 이후 서서히 건조되었다. 3. 무균상에서 0~~l8시간 동안 건조된 종자(수분함량 57.9~~8.7~% )는 95~% 이상이 발아하였으나, 30시간 건조된 종자(7.2~% )는 발아율이 85~% 로 감소하였다. 건조된 인삼종자를 액체질소에 저장 후 발아율을 조사한 바, 12~~30시간 건조(10.6~~7.2~% )된 종자는 90~% 이상이 발아하였다. 4. 건조실에서 건조된 인삼종자는 0~~2일 동안 건조(57.9~~9.0~% )하였을 때 90~% 이상이 발아하였으나, 6일 건조(3.8~% )된 종자는 발아율이 30~% 이하로 대폭 감소하였다. 동결보존 후의 발아율은 2일 건조 종자(9.0~% )만이 90.5~% 의 높은 발아율을 보였다. 5. 최적수분함량에서 두 건조방법간에 동결보존 전후의 발아율에 유의한 차이가 없었으며, 건조 장해를 받지 않으면서도 동시에 동결장해를 회피할 수 있는 적정 수분함량범위는 8~~l0~% 였다.

A simplified technique which cryoprotects zygotic embryos by encapsulation-dehydration was developed for the germplasm conservation of herbaceous peony (Paeonia lactiflora Pall.). The highest survival rate (85~%) was obtained from embryos treated by encapsulation-dehydration. The zygotic embryos were precultured on MS medium containing 0.3mg/L GA3 for 1 day. The precultured embryos were encapsulated in 3~% (w/v) alginate beads and immersed for 1 h in MS medium containing 2 M glycerol and 0.5 M sucrose. The encapsulated embryos were dehydrated for 5h by air drying prior to direct immersion in liquid nitrogen. This encapsulation-dehydration method appears to be a promising technique for germplasm cryopreservation of a herbaceous peony.

본 연구는 산림 수목 종자를 대상으로 다양한 초저온 동결 조건을 적용하고, 초저온 저장 후에 나타나는 종자의 발아 특성 및 유묘의 생장 특성 조사하기 위해 수행되었다. 유리화 과정을 기초로 한 초저온 저장은 다릅나무 종자의 발아율을 크게 감소시켰으나, 초저온 저장 전 동결보호제 처리와 초저온 저장 후의 빠른 해빙은 종자의 발아율 감소를 완화시켰다. 초저온 저장 전 동결보호제 노출 시간은 종자의 발아율에 영향을 주었다. 즉 동결보호제 노출시간이 길어짐에 따라 종자의 발아율은 감소하였다. 그러나 초저온 저장 기간은 종자의 발아율에 영향을 주지 않았다. 또한 동결보호제의 노출 시간과 초저온 저장 기간은 유묘의 생장 특성에 영향을 주지 않았다. 따라서 수목 종자의 초저온 저장은 조직의 손상이나 변형이 없이 장기간 저장할 수 있는 현지 외 보존 기술로 적절하다고 판단된다. 그러나 산림 종자의 보다 효율적인 초저온 저장을 위해서는 각 수종의 종자 특성에 맞는 동결보호제 및 처리 기술이 개발되어야 할 것으로 판단된다.

Recently, human embryonic stem (hES) cells have become very important resources for ES cell basic research, cell replacement therapy, and other medical applications; thus, efficient cryopreservation methods for these cells are needed. This study examined whether a newly developed minimum volume cooling (MVC) vitrification method, which was tested through cryopreservation of sensitive bovine oocytes, can be used for freezing hES cells. Feeder-free cultured hES cell (MB03) colonies were mechanically dissected into several small clumps following enzymatic treatment. We compared the freezing efficiency of a slow-cooling method using a cryo-module (0.4-0.6C/min, 20-30 clumps/vial) and MVC vitrification using a modified 0.5-ml French mini-straw designated as a MVC straw (>/min, 10 clumps/straw) After thawing, in vitro survival of hES cell clumps was higher for MVC-vitrified cells (80.8%, 97/120) than for slow-cooled cells (38.2%, 39/102). Further, the proliferation rate of surviving MVC-vitrified cells was similar to that of control hES cells from 2 weeks after thawing. In addition, vitrified-thawed hES cells demonstrated a normal karyotype, were positively immunostained for surface marker antibodies (AP, SSEA-4 and TRA-1-60) and the Oct-4 antibody, and could differentiate into all three embryonic germ layer cells in vitro. This result demonstrates that hES cell clumps can be successfully cryopreserved by a newly developed MVC vitrification method without loss of human cell characteristics.

In present study, attempts were made to preserve abalone (Haliotis discus hannai) sperm in liquid form at low temperature, to evaluate the effect of various diluents in short-term storage on sperm, and cryopreservation procedures were optimized for the cryoprotectants as well as freezing rates, in terms of the motility and survival rate, and the ultrastructural changes of sperm after short-term storage and cryopreservation were observed. The abalone sperm reached maximum motility until about 4min after activation. The motility was constant for about 16min, after which it dropped gradually, and about 50min later all motility ceased. Threshold activation of sperm was found in 40% artificial seawater (ASW), and motility increased as the concentration of ASW increased. In Hanks balanced salt solution without calcium (Ca-Free HBSS, 300 and 400 mOsmol/kg) and 10%, 20%, and 30% ASW the sperm was immotile, and motility once again restored incompletely only in HBSS of 300 and 400 mOsmol/kg, 20% and 30% ASW after 100% ASW was added. Sperm motility was extended following 20 days of cold storage only in 70% and 100% ASW. A high motility index of 3.5-4.5 was observed for the first 8 days in 70% and 80% ASW. In other diluents sperm motility was constant less than 10 days, and the motility index was obviously lower than that of sperm in 70% and 100% ASW. After 20 days of cold storage survival rates of 10.2%-20.7% were obtained in ASW and 300 mOsmol/kg HBSS, and that in 400 HBSS (65.3%) was significantly higher than others. The constant period of sperm motility stored in 70% ASW was longer obviously than that in 100% ASW after 6 days of storage, and the time to maximum motility of sperm stored in 70% increased gradually, while the difference in which of sperm in 100% ASW was not significant. The sperm plunged into liquid nitrogen all died except that sperm using 15% glycerol as cryoprotectant restored 10.4% of motility. The highest motility index (3.4) was obtained with 5% glycerol and freezing procedure: 50℃/min from to .

The present study examined the possibility of long term storage, by cryopreservation in liquid nitrogen, of the sperm of Filefish (Thamnaconus septentrionalis), and the changes in motility, survival rate and ultrastructure of the sperm after freezing and thawing. The sperm was collected by stripping and stored on ice until experiments. For selection of the immobilizing solution, diluted artificial seawater (ASW) of 20, 30 and 40% were tested. The sperm motility was significantly inhibited in 30% ASW, and restored entirely after 100% ASW was added again. Two cryoprotectants, dimethyl sulfoxide (SO) and glycerol, were added to 30% ASW to formulate the extenders at the concentrations between 5 to 20% by volume for freezing. The sperm was diluted at the ratio of 1 :6 with the extenders, inserted into 0.5ml plastic straws and frozen at a freezing rate of /min to after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The straws were thawed in a water bath for 15 sec. The highest post-thawed sperm motility and survival rate were obtained with 5% glycerol Afterward, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of /min to showed the best result Some ultrastructural changes of sperm, such as the detachment of plasmatic and nuclear membranes, destruction of mitochondria, were observed after cryopreservation. Morphological normality of the sperm in 5% glycerol frozen at the ratio of 1/min to was better than that of others.

The present study examined the possibility of cryopreservation of the D-shaped and umbo larvae of arkshell (Scapharca broughtonii), in terms of the survival rates after freezing and thawing. D-shaped and umbo larvae of arkshells were obtained from a shellfish farming on Yosu city. The average shell lengths were m and , respectively. Five cryoprotectants (CPAs), dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol, were tested at the concentrations of 1.5, 2.0 and 2.5 M. After larvae suspended in CPAs, cryoprotectants were loaded in 0.5 ml straws at a larval density of 50-100 larvae per straw, and epuilibrated for 10 and 20 minute at room temperature (), repectively. Straws were cooled at a rate of /min from to , held for 5 min at , and then cooled at /min to and equilibrated for 5 min followed by plunging in liquid nitrogen. After storage in liquid nitrogen for 1 day, straws were thawed in a water. As soon as straws were observed to melt, larvae were diluted with an equal volume of ASW and then washed twice with a large volume of ASW at an interval of 2 min to unload the CPAs. The results showed that after equilibration for 10 and 20 minute at room temperature, no larvae survived using methanol as CPAs, and it was observed that larval shells all open slightly, and larval flesh broke down and slopped over the shells. The highest survival rates (D-shaped larvae: 77.6%, umbo larvae: 59.3%) were obtained with 2M DMSO, and 1.5M glycerol yielded survival rates of 53.8% for D-shaped larvae and 37.5% for umbo larvae. The surviving D-shaped larvae showed active rotary motion and perfect membrane integrity and cytoplasmic normality, and the vigorous movement of veliger cilia was observed inside the closed shells. The breakdown of tissue occurred in the abnormal larvae, and the isolated cell often run out of shells.

The rol e of osm otol erance and dehydr ation, in terms of both medium com posi tion and exposur e duration, on sur vi val ofem bryos of Citrus m adurensi s after cryopr eser vation usi ng the encapsul ation-dehydr ation and modified encapsul ation-dehydr

자작나무 식물유전자원의 효과적인 장기보존 방안을 구명하기 위해 동결전처리 된 겨울철 동아를 건조처리에 의해 초저온보존 시험하여 다음과 같은 결과를 얻었다. 자작나무 동아는 내동성이 최고조에 달한 1월 20일경의 함수율이 42.43%로 가장 낮게 나타났다. 1월 20일에 채취한 동아를 10분간의 건조처리 와 5~-20℃까지 1분당 1℃ 하강조건으로 완속동결 전처리하여 -196℃의 액체질소에 초저온 보존한 경우의 세포생존율은 83.33%로 가장 이상적인 처리조건으로 조사되었다. 자작나무 동아의 시료조제는 동아에 약간의 목질부 조직을 붙여 초저온 보존하는 경우에 86.7%로 세포생존율이 높게 나타났다. 24시간 이상 액 체질소에 초저온 보존한 자작나무 동아는 40±5℃의 온수 중에서 급속 해동하는 경우가 86.6%의 세포생존율을 비롯하여 60%의 식물체 재생율을 나타내어 본 실험에서 가장 효과가 좋은 것으로 조사되었다.

컴퓨터 세포동결기를 이용하여 생쥐 배아를 동결할 때 액체질소 (L)의 분사속도가 해빙 후 배아의 미세구조, 기능 및 발달에 미치는 영향을 알아보고자 하였다. 이를 위해 배아는 동결을 하지 않은 대조군 (control) 및 동결군에서 L의 분사속도에 따라 고속분사군 (120 infusion/min group 1), 저속분사군 (50 infusion/min; group 2)으로 나누었다. ICR 계열의 생쥐의 2 세포기 배아를 사용하였으며, 동결 및 해빙은

우리나라에서 진주양식의 대상패류인 진주조개 (Pinctada fucata martensii) 담륜자를 냉동보존하기 위하여 4가지 동해방지제를 사용하여 냉동실험을 실시하고, 적합한 동해방지제를 찾고자 하였다. 동해방지제인 dimethyl sulfoxide (DMSO), ethylene glycol, glycerol과 1, 2-propanediol을 희석액인 0.2 M sucrose에 각각 0.01, 0.1, 1.0과 2.0 M이 되도록 혼합한 다음, 10