• Applied Microscopy
• /
• 제13권1호
• /
• pp.49-61
• /
• 1983

Protoplast releasing mechanisms from Trichoderma koningii, fine structures of the released protoplsts, and their regeneration mode were studied by scanning and transmission electron microscopy. Two types of protoplast releasing mechanisms were observed. In one mechanism, cytoplasm emerged through a cell wall pore developed by cell lytic enzymes and formed a spherical protoplast. In the other mechanism, as the cell wall became progressively thinner, the inner cytoplasm partially rounded to form nonspherical bodies which became spherical protoplasts after being released into the enzyme solution. But, these two types of protoplast releasing mechanisms did not seem to be. mutually exclusive but could occur on the same mycelium simultaneously. And it appeared that cytoplasm which did not become a protoplast by the first mechanism could from a protoplast by the second mechanism. The preparations contained two types of protoplasts, released from different sites of the mycelia. Those released from younger mycelia had dense cytoplasm and small vesicles. Those released from the older mycelia had less dense cytoplasm and larger vacuoles. In the case of regeneration, before producing normal mycelia, most of the protoplasts assumed aberrant tube and yeast-like-forms. Normal mycelia were produced at the end of the yeastlike-forms and sometimes in the middle of the aberrant tube.

• Journal of Microbiology and Biotechnology
• /
• 제11권1호
• /
• pp.72-78
• /
• 2001

It is possible ot prepare protoplasts of the zygomycete fungus, Phycomyces blakesleeanus, by digesting the cell wall of spore germlings with commercially available chitinase and chitosanase. However, the cells without any cell walls immediately form large aggregates, and thus, it is difficult to isolate the individually separated protoplasts. Inherent problem with the formation of aggregates in preparing protoplasts could be solved by the use of bovine serum albumin (BSA). As a result, we were able to prepare a large number of single protoplsts quickly and easily. We took time-lapse photomicrographs of the formation of protoplasts, and found that there were certain regions of the cell wall of spore germlings that were sensitive to chitinase and chitosanase, although the cell wall of the original spores is known to be insensitive to these enzymes. There are two kinds of cell walls on a spore germling; one with a bound wheat germ agglutinin (WGA), and the other a bound concanavalin A (ConA). Furthermore, only cells with walls which had bound WGA were able to regenerate, while those with walls with bound ConA were not able to regenerate.

• KSBB Journal
• /
• 제5권1호
• /
• pp.43-47
• /
• 1990

약난초는 내장산 이남에서만 자생하는 상록성 난으로 관상용 뿐 아니라 점질이 높은 구경은 약용(점활제)으로써 이용가치가 높은 중요한 자원식물중의 하나이다. 본 연구에서는 이와같은 약난초의 다량증식을 위하여 몇 가지 실험을 하여 다음과 같은 결과를 었다. Green pod를 이용하여 미숙종자를 개체로 유도시키는 것은 그 유도율이 낮았고 생장점을 이용, 캘러스를 유도하여 PLB를 형성시켜 개체로 유도하는 것이 용이하였다. 그러나 미숙종자의 이용도 배지조건등의 개량으로 가능하리라 사료된다. 캘러스의 형성은 Hyponex배지에 2.4-D(2mg/l)와 Kinetin(1mg/l)을 첨가하였을 때 높았다. 한편 업육조직 및 배양세포로부터 분리시킨 원형질체는 70%이상의 생존력을 나타내었고 형광현미경 관찰을 통하여 세포벽의 재생을 확인하였다. 이와 같은 실험결과는 자생란의 다량증식 뿐 아니라 잡종난 식물체를 유도하는 제반 실험에 응용될 수 있는 기초 자료가 될 수 있으리라 사료된다.

• 한국자원식물학회:학술대회논문집
• /
• 한국자원식물학회 2003년도 심포지엄
• /
• pp.50-58
• /
• 2003

Isolation and culture of leaf protoplasts from two chilli cultivars (Capsicum annuum var. accumnatum and Bird chilli) were developed to enhance selection process in the somatic hybridization programmes. In order to isolate the protoplasts from leaves of these two chilli cultivars different incubation periods (3, 5 and 10 hours) were tested with combinations of enzyme mixtures containing cellulase and macerozyme. Leaves were incubated on three enzyme mixtures (2% cellulase + 0.4% macerozyme, 1% cellulase + 0.2% macerozyme and 0.5% cellulase + 0.1 % macerozyme in 13% mannitol) at 251oC in the dark. Three hours of incubation using 2% cellulase and 0.4% macerozyme was the best for the protoplast isolation of both chilli cultivars tested. The yield was 5 ${\times}$ 108protoplasts/ml/ g leaf tissue in both chilli varieties. It was found that in the mixed nurse method using Nagata and Takebe (NT) medium supplemented with 1.0mg/12,4-D, NAA and BAP with 0.5M mannitol and 1.2% Sea Plaque agarose is the best medium for protoplast culture. Protoplasts of Capsicum annum var. accumnatum were alive for 14 days forming cell walls and initiating cell division.

• Journal of Plant Biology
• /
• 제30권1호
• /
• pp.1-9
• /
• 1987

Leaf mesophyll protoplasts of Nicotiana tabacum (nitrate reductase deficient mutant) were fused with cell suspension protoplasts of albino Petunia inflata in an electric field. Hybrid cell colonies were selected for nitrate reductase proficiency and chlorophyll synthesis. Five hybrid plant lines, regenerated from the selected calli lines, were analysed by electrophoresis, number of chromosomes and morphological characters. Somtic hybrid plants showed both parent patterns in the isozymesof isoleucine aminopeptidase and esterase. The hybrids had the expected chromosome number of 62 and exhibited an intermediate floral morphology when compared with the parents, but plant height and leaf arrangement were similar to N. tabacum.

• 한국환경과학회지
• /
• 제17권2호
• /
• pp.163-170
• /
• 2008

Protoplasts of Panax ginseng C. A. Meyer and Aralia continentalis K. (Araliaceae) were isolated from callus cells and mesophyll cells, respectively. The maximum yield of protoplasts isolated from callus cells of P. ginseng were obtained by incubation for 3 hrs in the enzyme mixture of 0.5% macerozyme, 1.5% cellulase, and 0.5 M mannitol as an osmoticum. In the case of mesophyll cells of A. continentalis, the highest yield of protoplasts were obtained by incubation for 5 hrs in the enzyme mixture of 1% macerozyme, 2% cellulase, and 0.6 M mannitol. A polyethylene glycol (PEG) treatment induced an intergeneric fusion of the protoplasts. The fusion products, that is, heterokaryocytes were obtained by treatment of 50% PEG containing 0.05 M Ca salts.

• 미생물학회지
• /
• 제23권3호
• /
• pp.209-214
• /
• 1985

The optimum conditions of protoplasts formation from Pyricularia oryzae were investigated with lytic enzymes and osmotic stabilizers. The mycelia were begun to refease the protoplasts in response to the complex enzyme solution after 30-60 minutes and reached to maximum after 2-3hrs. Among the lytic enzymes tested, the mixture solution containing ${\beta}-Glucuronidase$(0.01 ml/ml), Cellulase ONOZUKA-RS(20mg/ml), Driselase (10mg/ml), and Macerozyme R-10 (10mg/ml) resulted in the highest rate of protoplasts releasing of Pyricularia oryzae. The best stabilizer was 0.6M KCl at pH 7.0. Shen the mycelia were digested with enzyme mixture, the stationary culture was better than shaking culture for higher protoplast formation.

• 대한의용생체공학회:의공학회지
• /
• 제15권1호
• /
• pp.1-8
• /
• 1994

Electrofusion of cabbage protoplasts was performed with a developed equipment which consists of a parallel wire electrode, a AC field generator, and a pulse generator. Exposure of protoplasts to an alternating current field of 450 KHz causes attraction to each other which leads to chains of protoplasts extending from the electrode. Intra-specific protoplast fusion was effectively induced within the pearl chains by the additional application of a single high-intensity DC square wave pulse of 1 KV/cm for 1-3 msec. To improve the performance, micro fusion electrode fabricated from the semiconductor technology and microcontroller based field an, d pulse generator was proposed. The viability of protoplasts after an application of electric field at optimum condition was reduced by only 5 % compared with that of pre-application. A prototype of fusion electrode, which consists of insulator-structure, was successfully fabricated by using photosensitive polyimide.

• Journal of Microbiology and Biotechnology
• /
• 제1권4호
• /
• pp.246-250
• /
• 1991

Conditions for the production and regeneration in Lactobacillus bulgaricus protoplasts were investigated. Protoplasts of L bulgaricus strains were obtained by treatment with mutanolysin and lysozyme together in a protoplast forming buffer containing 0.02 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.0) and 0.5 M sucrose. High protoplast yield was obtained from cells cultured in the de Man, Rogosa and Sharpe(MRS) medium at the middle to late logarithmic growth phase. Regeneration was efficiently accomplished with a complex medium containing 1% sucrose, 20 mM $MgCl_2$, 5% gelatin, and 0.5% bovine serum albumin. The frequency of regeneration of protoplasts was 10~20% after 5 days of incubation at $30^{\circ}C$.

• Journal of Plant Biology
• /
• 제33권4호
• /
• pp.253-257
• /
• 1990

Callus-inducing ability of Alnus hirsuta was examined by culturing various tissues (leaf, hypocotyl, cotyledon and seed) on NT (Nagata & Takebe) medium, supplemented with 2.5$\mu$M 2,4-D. Leaf-originated callus was cultured on media varying in auxin (IBA and NAA) and cytokinin (BAP) concentrations to examine the effects of auxin and cytokinin on callus growth. Maximum growth was obtained at 10 $\mu$M IBA+10$\mu$M BAP and 10$\mu$M NAA without cytokinin. Cell suspensions established from cotyledon-originated callus yielded viable protoplasts after incubation for 16-18 hours in an enzyme mixture (1% (w/v) Onozuka R-10 0.5% (w/v) Macerozyme, CPW salts and 13% (w/v) mannitol, pH 5.8). Protoplasts were cultured on NT medium, supplemented with glucose, hormones and coconut milk. After 6 weeks of culture, protoplasts sustained cell divisions to form microcallus, which showed various colors from red to white.