액아배양에 의한 유묘 및 성숙 히어리나무의 기내변식

문흥규,노은운,하유미,심경구 한국식물생명공학회 2002 JOURNAL OF PLANT BIOTECHNOLOGY Vol.29 No.2

참나물(Pimpinella barchycarpa)의 체세포 경발생에 의한 식물체 대량증식

문흥규,윤양,이석구,Moon, Heung-Kyu,Yoon, Yang,Lee, Seok-Gu 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.2

Attempts were made to regenerate plants from petiole explane of Forest Pimpinella barchycarpa via repetitive somatic embryogenesis. Effective induction of somatic emb교ogenesis was achieved on both MS and modified $B_{5}\;(mB_{5})$ media containing BA + 2,4-D or BA + 2,4-D + NAA under light condition (16-h photoperiod/day) cultures. The explants exposed to the ligt produced numerous somatic embryos while those kept under the dark did not form any on the same medium. Somatic embryos at different developmental stages were observed to arise within a individual explants. Plantlets could be regenerated on $mB_{5}$ basal medium or $mB_{5}$ containing 0.1 mg/L NAA Secondary adventive embryos were formed on the surface of the somatic embryos. Therefore, repetitive somatic embryogenesis could be achieved by secondary embryogenesis. Although the treatment of 2,4-D or NAA alone was effective in callus formation and growth, but not in induction of somatic embryogenesis. Some explants, cultured on NAA-containing media in darkness, produced only adventive roots. The embryogenic potential was maintained for two years when subcultured to BA and 2,4-D containing media with 5 weeks inteval. Regenerated plantlets were maintained on $mB_{5}$ or MS basal media for 4 to 6 more weeks and transferred to soil of an artificial mixture for acclimation. Most plantlets (more than 97%) survived, and grew without any deformity. 기내증식 중인 참나물(Pimpinella brachycarpa)의 엽병을 절편으로하여 체세포배형성을 통한 식물체의 대량증식을 유도하였다. 체세포배 발생은 MS 및 $mB_5$ 기본배지에 BA와 2,4-D, 혹은 BA, 2,4-D 및 NAA를 혼용하여 명배양 하였을때 효과적이었으며, 농도는 BA 0.1 mg/L, 2,4-D 및 NAA는 0.5 mg/L 이었다. 광조건은 식물생창조절 물질의 반응에도 크게 영향을 미쳐, 명배양에서 효과적인 조건이 암배양에서는 전혀 다른 결과를 나타냈다. 즉 MS 및 $mB_5$ 배지에 0.5 mg/L 2,4-D를 각각 첨가하고 명배양한 조건에서는 100%의 체세포배 발생을 보인 반면, 암배양에서는 동일한 배지 조건에서 체세포배가 전혀 형성되지 않았다. 체세포배의 발육단계는 동일한 젤편에서도 많은 차이를 보였으며, 배발생한 캘러스를 $mB_5$ 기본배지 및 0.1 mg/L NAA가 첨가된 배지로 계대배양 하였을때 식물체의 재생에 주효하였다. 이미 형성된 체세포배는 배양기간이 길어지면 캘러스화 되면서 또다른 2차 체세포배가 형성되어 하나의 체세포배로 부터 연속적인 식물체의 재생이 가능하였다. 2,4-D와 NAA의 단독첨가는 캘러스의 형성에는 양호하나 체세포배로부터 식물체의 재생에는 비효과적이었다. NAA를 단독첨가하여 암배양한 절편에서는 2차 계대배양시 절편 표면으로부터 부정근이 형성되었다. 배발생한 캘러스는 BA와 2,4-D가 첨가된 배지에서 약 5주간 간격의 계대배양으로 2년 이상 유지가 가능하였다. 체세포배를 통하여 재생된 식물체는 $mB_5$ 및 MS 기본배지에서 4-6주간 생장시킨 다음, 인공 배양토에서 효과적으로 환경순화 되었다. 이 식물체는 엽병을 절편으로 체세포배 발생을 통한 기내 대량증식이 가능 하다고 사료되었다.

두릅나무 15개체의 체세포배 유도 및 식물체 재분화에 미치는 유전자형의 효과

문흥규,홍용표,김용욱,이재순 한국식물생명공학회 2001 식물생명공학회지 Vol.28 No.3

Winter bud explants from 15 individual angelica tree (Aralia elata) were cultured in vitro to find out optimal conditions for somatic embryo induction as well as plant regeneration. Calli are induced and grown on MS medium supplemented with 1.0 mg/L 2,4-D for 4 weeks and subcultured on a half-strength MS medium without phytohormones to induce somatic embryos. Inter-simple sequence repeat (I-SSR) markers were analyzed with total DNAs extracted from the trees. Genotype effects on somatic embryo induction were examined by cluster analysis. Callus induction rate varied from 58.5 to 100% among the genotypes. Somatic embryo induction rate also greatly varied from 0 to 100% among the genotypes. There was a significant difference in somatic embryo induction rate even among the individual trees that showed close genetic relationships each other. This suggested that somatic embryo induction rate in Aralia elata be influenced by a few major specific genes rather than whole genomic similarity among individual trees. Four individuals of Ulneong-7, Cheju-1, Shingu and China, which are recalcitrant to somatic embryo induction, turned out to have a close genetic relationship, suggesting that both physiological and genetic factors affect somatic embryo induction. The results suggest that genotype selection be the most important factor to achieve an efficient propagation, although cultural optimization through medium and explant manipulation may also play crucial roles in somatic embryogensis as well as plant regeneration of these species.

두릅나무 체세포배 유래 소식물체의 순화에 미치는 배양토 및 공급액의 효과

문흥규,배찬호,김용욱,이재순,이재선 한국식물생명공학회 2001 식물생명공학회지 Vol.28 No.5

두릅나무의 체세포배 유래 소식물체의 효과적인 순화 방법을 개발하고자 플라스틱 용기를 사용 4가지 인공배양토 및 MS 액체배지 등 7가지 수용액 처리를 통해 활착률 및 생장을 조사하였다. 상토는 perlite가 vermiculite보다 활착 및 생장에 양호하였고, 입자가 클수록 활착률 및 생장이 좋았다. 액체배지는 2차 증류수 처리가 가장 양호하여 96%의 활착률을 나타냈으며, 염류농도를 1/4 및 1/8로 낮춘 MS배지는 각각 92%의 활착률을 보인 반면 기본배지 및 1/2 MS배지는 활착률이 저조하였다. 한편 0.1% hyponex와 2% sucrose 처리는 84% 및 76%의 활착률을 각각 나타냈다. 결론적으로 두릅나무의 체세포배 유래 소식물체의 순화는 입자가 큰 perlite를 사용하고 증류수 처리 혹은 염류 농도를 낮춘 MS배지 처리로 효과적인 순화가 가능함을 보여주었다. In order to develop effective acclimatization methods for Aralia elata plantlets regenerated from somatic embryos, various acclimatizing conditions were compared regarding both survival rate and growth of the plantlets. The plantlets were transplanted into plastic boxes containing artificial soil in the presence of either several levels of MS liquid media, distilled water, 2% sucrose or 0.1% hyponex solution. They were then cultured by spraying of distilled water twice a week and maintained in the normal tissue culture room. Perlite was proved to be better than vermiculite on survival rate and growth of the plantlets. As the size of perlite (larger than 0.2 cm in diameter) increased, both the survival rate and growth of the plantlets improved. Among the various MS liquid media and different aqueous solutions tested, distilled water appeared to result in the best survival rate and growth. MS media were also effective in increasing survival rate and supporting growth when diluted to 1/4 and/or 1/8. The acclimatized plantlets could be transplanted directly onto the nursery bed and grown normally. The above results suggest that plantlets regenerated from somatic embryos of Aralia elata be effectively acclimatized using a plastic box containing perlite with distilled water treatment.

두릅(Aralia elata)의 체세포배 유도, 발아 및 식물체 재분화에 미치는 요인

문흥규,오경은,손성호 한국식물생명공학회 1999 식물생명공학회지 Vol.26 No.4

두릅나무 (Aralia elata Seem.) 2년 생 11개체의 동아를 1.0 mg/L 2,4-D, 3% sucrose, 0.3% gelrite를 포함한 MS 고체 배지에서 체세포배 형성, 발아 및 식물체 재분화에 미치는 개체목의 효과를 조사하였다. 배발생 캘러스의 유도는 개체목에 따른 차이가 있었으나 발아 및 식물체 재분화는 차이가 뚜렷하지 않았다. 배발생 캘러스는 대체로 노란색을 띠었고, 조성은 비배발생 캘러스보다 부드럽고 생장은 다소 느렸다. 체세포배의 발아 및 식물체 재분화는 MS배지의 염류 농도가 크게 영향하였고, 기본배지보다는 염류 농도를 반감시킨 1/2 MS배지에서 BA및 ABA의 처리에 관계없이 양호한 배발아 및 식물체 재분화를 나타냈다. BA처리는 0.1mg/L 농도에서 배축과 자엽 발달에 다소 촉진효과를 나타냈으나 발아와 재분화율은 기본배지보다 우수하지 못했다. 발아되는 배는 자엽수 및 형태에 있어 다양한 변이를 나타냈다. ABA처리는 두릅 체세포배의 발아 및 재분화에 비효과적이었다. 재분화된 식물체는 인공배양토에서 95%이상 환경순화되어 포지에 이식 후 형태적 변이 없이 정상생장이 가능하였다. In order to find optimum conditions for somatic embryogenesis from different individual (2-year-old) in Aralia elata were cultured on MS medium supplemented with 1.0 mg/L 2,4-D, 3% sucrose, and 0.3% gelrite. We also investigated the effect of MS medium salt concentration, BA and ABA on the embryo germination and plant regeneration. While noticeable difference was observed on somatic embryo induction among different individual tree, no apparent difference was seen in both germination and regeneration frequencies. Compared with nonembryogenic calli, embryogenic calli tended to look yellow and/or pale brown in color, slowly growing and soft in their texture. Regardless of BA or ABA treatment, half-strength MS salt medium proved to be better than full strength MS medium in both embryo germination and plant regeneration. Both hypocotyl and cotyledon developments were slightly promoted by adding 0.1 mg/L BA. However, its effect on germination and regeneration seemed inferior to control. ABA treatment on somatic embryos at their torpedo and early cotyledonary stages resulted in poor response in germination and regeneration. Although most regenerated plantlets varied greatly in cotyledon number and shape, they could be developed into normal plants after 4 weeks in culture. More than 95% plantlets were acclimatized in an artificial soil mixture, successfully transplanted to nursery bed and grew normally without any phenotypic abnormalty.

상수리나무 유목과 성숙목의 기내번식

문흥규,윤양,이재선 ( Heung Kyu Moon,Yang Youn,Jae Seon Yi ) 한국산림과학회 1997 한국산림과학회지 Vol.86 No.3

Present study describes a method on the application of efficient tissue culture systems for the micro-propagation of juvenile and mature sawtooth oak(Quercus acutissima). Nodal segments with axillary buds were used as initial explant sources. WPM(Woody Plant Medium) was the best in growth and proliferation of shoot among the media tested. Although the single effect of zeatin revealed on two dorminant shoot elongation with normal growth until the elevation of levels up to 3.0㎎/ℓ, BAP(N^6-benzyl amino purine) usually showed better response than zeatin on shoot multiplication and/or elongation. In addition, the incorporation of BAP and zeatin onto the culture media represents more effectiveness in shoot proliferation and its growth. Optimum concentrations of BAP and zeatin were 0.5 and 0.05-1.0㎎/ℓ, respectively. Ninety percent of the proliferated shoots was rooted on half-strength GD (Gresshoff and Doy, 1972) medium containing 0.5㎎/ℓ IBA(indole butyric acid) in 4 weeks after culture. Mores than 70% of the rooted plantlets survived after 5 months of transplanting into artificial soil mix containing equal amount of peatmoss and perlite. Among 27 plus tree clones which were grafted twice onto the juvenile rootstocks, only 4 clones revealed the possibility for shoot multiplication through tissue culture system. The capacity for the micropropagation using mature explant sources was highly depended on clonal differences compared with those of octet age. More than 90% of rooting ratio was obtained from the best responding clone. Among the 7 rooting media tested, GD medium was the best far rooting. The most effective rooting was obtained on half-strength GD medium containing 0.2 to 2.0㎎/ℓ IBA. More than 60% of rooted plantlets survived after 5 months of transplanting into the artificial soil mix.

음나무 (Kalopanax septemlobus) 체세포배를 이용한 인공종자 조제 및 발아

문흥규,최용의,이재선,김용욱 한국식물생명공학회 2007 JOURNAL OF PLANT BIOTECHNOLOGY Vol.34 No.3

Artificial seeds were produced by encapsulation of somatic embryos of Kalopanax septemlobus and investigated the effects of alginic acid concentration, size of somatic embryos, additives in capsules and nursery seedbeds for germination. The most suitable concentration of alginic acid was 3% for germination of encapsulated seeds. Germination was suppressed at higher concentration more than 3% alginic acid. For germination of artificial seeds, 1/2 MS medium with 0.02% activated charcoal was effective. There was no significant differences on the germination among the different size of somatic embryos. Additives in hydrated capsule was very important for germination and post-germinative growth of artificial seeds. Germination was severly inhibited in hydrated capsule containing only distilled water. Both sucrose and MS medium addition in hydrate capsule was effective for germination of artificial seeds. When artificial seeds were transferred to soilbed, germination rate was high in perlite containing 3% sucrose but very low in perlite with only water. These results indicate that nursery additives in both hydrate capsules and soilbeds was important for germination of artificial seeds in Kalopanax septemlobus.

액아유도에 의한 Eucalyptus pellita의 기내번식

문흥규,김지아,강호덕,이현신 한국식물생명공학회 2003 JOURNAL OF PLANT BIOTECHNOLOGY Vol.30 No.3

두릅나무의 번식 기술

문흥규 한국수목보호연구회 2006 수목보호 Vol.11 No.-

액아배양에 의한 유묘 및 성숙 히어리나무의 기내번식

문흥규,노은운,하유미,심경구,Moon, Heung-Kyu,Noh, Eun-Woon,Ha, Yoo-Mi,Shim, Kyung-Ku 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.2

히어리나무 1년생 및 10년생을 재료로 액아배양을 통한 기내번식을 시험하였다. 줄기의 증식은 zeatin과 BA의 공조처리가 효과적이었으며 MS배지에 zeatin 0.5∼3.0 mg/L, 0.2 mg/L BA 처리시 주효하였다. 1년생이 10년생보다 전반적으로 증식 및 생장이 양호하였으며, 배양 6개월 후에는 10년생에서도 매월 절편 당 3개의 줄기유도가 가능하였다. 기내 줄기의 발근은 1년생은 97%, 10년생은 62%를 나타내었고, 토양이식시 1 년생 유래 배양묘는 67%, 10년생은 48%생존되어 모수령에 따른 차이를 나타냈다. 본 실험결과 히어리나무의 액아배양으로 유시, 성숙목의 대량번식이 가능함을 보여주었으나 선발개체의 효율적인 기내번식을 위해서는 재유령화의 기술개발과 토양순화율을 증진시켜야 하는 것으로 나타났다. We have developed an in vitro micropropagation system via shoot formation from axillary buds using nodal segments of Corylopsis coreana. Explants from both juvenile tree (one-year-old greenhouse stock seedlings) and mature tree (ten-years-old tree in nursery) were compared with regard to propagation efficiency. Combined treatment of both BA and zeatin were effective on shoot proliferation since the best result was obtained on MS medium supplemented with 0.5∼3.0 mg/L zeatin and 0.2 mg/L BA. Generally, juvenile explants were better in both shoot proliferation and growth than mature explants. However, as the duration of in vitro culture was proceed to 6 months, explants from mature tree also produced three shoots per explant. Distinctive differences in rooting and adaptability to soil of shoots obtained from mother trees. Whereas shoots originated from juvenile explants rooted as high as 97%, those from adult explants showed 62% rooting. Similar result was also observed in soil acclimatization. The plantlets derived from juvenile plants survived 67%, while only 48% of those from adult trees survived. The results showed a possibility of the micropropagation of Corylopsis coreana through shoot formation from axillary buds. In addition, the advance of the research still remain to enhance the frequency of acclimatization of plantlets from mature trees for practical application.