음나무 배발생 캘러스의 증식 및 체세포배 발달을 위한 액체 현탁 배양조건 확립

문흥규,김선자 한국식물생명공학회 2009 식물생명공학회지 Vol.36 No.1

This study was conducted to establish the optimal suspension culture system for both the propagation of embryogenic cells (ECs) and the induction of somatic embryos (SEs) of Kalopanax septemlobus. The proliferation rate of ECs was reduced as the inoculum density was increased; the highest rate was obtained when 0.1 g/100 ml of cells was initially inoculated. According to the analysis of cell growth pattern and cell growth cycle (G1, S and G2/M), the cell growth started in 5 days culture initiation, grew rapidly until 15 days and then decreased gradually. Distinctive changes of the cell growth cycle by the culture periods was also observed; the growth cycle was doubled from initial 5.6% to 11.7% of S stage in 5 days culture and then reached in stable stages again. Therefore, the results indicated that a 15-day-cycle was the optimal culture period for the propagation of the ECs through the suspension culture. Furthermore, the cell inoculum density was also important for the induction of SE; more than 65% of SEs at the torpedo stage was induced by using the low level of cell inoculum (0.5 g/L), while the higher inoculum densities were rapidly reduced the proportion of SEs at that stage. Although the higher inoculum density delayed the development of SE, it did not affect the proportion of SEs at the globular and heart stage. In conclusion, this study showed that the suspension culture of the Kalopanax septemlobus ECs through the control of inoculum density was an efficient way for both the propagation of ECs and the induction of SEs, suggesting that the development of this system might help to reduce the culture period for the somatic embryo production. 본 실험은 음나무 배발생 세포의 증식 및 체세포배 발달을 위한 액체 현탁 배양조건의 확립을 위해 수행되었다. 배발생 세포의 생 장율은 접종 밀도가 증가할수록 감소하였다. 배발생 세포의 증식 에 가장 효과적인 접종 밀도는 0.1 g/100 ml 로서 이 농도에서 세포 의 생장율이 가장 높았다. 배양기간에 따른 배발생 세포의 생장 패 턴 및 세포 주기 (G1, S, G2/M) 분석 결과, 세포의 생장은 배양 5일 후부터 증가가 시작되어 15일 까지 급격히 생장하였으며 그 이후 에는 점차 감소하였다. 배양 기간 별 세포 주기 (cell cycle) 의 변화 가 명확하게 관찰되어 배양 5일째 S기는 초기의 5.6% 에서 11.7% 로 두 배 정도 증가하였으며, 배양 15일 이후부터는 다시 초기의 세포 주기로 되돌아 가면서 안정화되는 것으로 나타났다. 따라서 음나무 배발생 세포의 현탁배양은 15일의 주기로 배양하는 것이 증식에 가장 효과적인 것으로 생각되었다. 한편 배발생 세포에서 체세포배의 유도는 배양초기의 접종 밀도가 중요한 것으로 나타났 다. 0.5 g/L 의 낮은 밀도로 접종 시에는 65% 이상의 어뢰형 배가 유도된 반면 접종 밀도가 높아질수록 어뢰형으로의 배발달은 급격 히 감소하였다. 초기의 접종 밀도가 증가할수록 특히 어뢰형 배의 발달은 지연되었으나 구형 및 심장형 배의 유도는 초기 접종밀도 에 영향을 받지 않았다. 이상의 실험결과로 음나무 액체 배양 시 초기 접종 밀도를 조절함으로써 배발생 캘러스의 증식 및 체세포 배를 효과적으로 유도할 수 있었으며 이는 체세포배 생산을 위한 배양 기간의 단축이 가능함을 보여주는 결과이다.

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.

희귀 및 멸종위기 수종 개느삼의 액아배양을 통한 기내번식

문흥규,김용욱 한국식물생명공학회 2008 식물생명공학회지 Vol.35 No.3

An efficient micropropagation was established by using axillary bud explants from two-year-old tree (Echinosphorea koreensis Nakai), which has been known as a rare and endangered species. Among various basal media tested, DKW medium was shown to be the best for axillary shoot elongation. The addition of both BA and TDZ to the medium induced 6 to 10 shoots per explant during eight weeks of culture, without showing any abnormal morphology at the shoot proliferation stage. However, high concentration of TDZ (> 0.05 mg/L) appeared to cause hyperhydration on either leaf or shoot at the later developmental stage. Approximately 20 % of shoots produced roots by the addition of 1.0 mg/L NAA but not by IBA (0.2 ~ 1.0 mg/L). Ex vitro micro-cuttings were better source for root induction; up to 58.6% of the micro-cuttings rooted when 100 mg/L IBA was applied to the soil (vermiculite). More than 90% of plantlets with roots were successfully acclimatized and grew normally in the field. Therefore, we suggest that this endangered tree species can be effectively micropropagated by axillary bud culture system developed in this study.

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.

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.

두릅나무의 번식 기술

문흥규 한국수목보호연구회 2006 수목보호 Vol.11 No.-

희귀 및 멸종위기 식물 덩굴용담의 기내생장에 미치는 광질 및 환기효과

문흥규,박소영 한국식물생명공학회 2008 식물생명공학회지 Vol.35 No.3

Effects of light generated by LEDs on shoot growth and rooting of Tsuru-rindo (Tripterospermum japonicum) were evaluated. Apical shoots (one or two node with 3-4 leaves) were cultured on MS basal medium with 3% sucrose and maintained for four weeks under five different light qualities: fluorescent lamp (F), 100% red LED (R), 70% red LED+30% blue LED (R7B3), 50% red LED+50% blue (R5B5), or 100% blue LED (B). Rooting was promoted by both red light and fluorescent lamp, and the effect was further promoted under the ventilation. Red light enhanced shoot node elongation, whereas blue light appeared to suppress it. Growth of shoots and leaves were enhanced under the ventilation irrespective of the different light qualities. Under the ventilated condition, total fresh weight of plants was highest in R7B3 LED as 257.7 mg per plant. Dry matters, which are used for index of plant growth, were lowest under red light, whereas it was highest under blue light. The dry matter was inclined to getting higher by ascending the ratio of blue light and red light. Total chlorophyll content was highest in both R7B3 LED and R5B5 LED under ventilation as 29.5 and 31.2, respectively. Above results suggest that light quality optimization could be an important factor to foster in vitro growth of the species. Ventilation treatment appeared to be another important factor to induce normal shoot growth and rooting

참다래$\times$다래 교잡종의 액아배양 및 캘러스 배양에 의한 기내번식

문흥규,권영진,이병실 한국식물생명공학회 2001 식물생명공학회지 Vol.28 No.4

내한성이 강하여 수원지역에서 재배가 가능한 참다래$\times$다래 교잡종의 기내번식은 액아배양과 캘러스 배양으로 증식이 가능한 것으로 나타났다. 액아에서 줄기유도는 St 배지에 0.2 mg/L BA, 3.0 mg/L GA$_3$처리로 4주마다 약 3배의 증식이 가능했다. 줄기의 발근은 기내발근보다 기외삽목이 매우 효과적이었으며 환경순화 후 포지에서 95% 이상 활착되었다. 한편 캘러스 유도는 엽육을 절편으로 MS및 B$_{5}$배지에 2,4-D 및 NAA 처리로 100%가능하였으며 처리 농도에 따른 차이는 없었다. 캘러스에서 줄기의 재분화는 NAA 처리로 유도된 캘러스에서만 가능하였다. 줄기의 재분화는 MS 배지에 3.0 mg/L. zeatin 처리로 평균 64% 가능했으며 정상적인 생장이 가능했다. 이상의 결과로 볼 때 새로운 키위 교잡종은 기내배양으로 대량번식이 가능할 것으로 사료된다.다. Kiwi (Actinidia deliciosa) is exotic plant and thus susceptible to cold climate in the middle part of Korean peninsular. Several hybrids have recently been developed to enhance cold tolerance by crossing them with domestic species (A. arguta), We have developed an efficient micropropagation technique for the hybrids using both axillary bud and callus culture systems. Shoot proliferation from axillary buds was possible on St medium supplemented with 0.2 mg/L Bh and 3.0 mg/L GA$_3$. In vivo cuttings of the proliferated shoots were more effective for root induction and subsequent survival than in vitro rooting. More than 95% of the plantlets were successfully transferred to field. Effective callus induction was achieved on MS or B$_{5}$ medium with 2,4-D or NAA. Although callus induction could be made from any combinations of media and auxins, shoot regeneration was observed only from the callus induced on medium containing NAA.A.