A simple and efficient protocol was developed for somatic embryogenesis from the cotyledon explant of Paeonia lactiflora Pall. Seeds of peony obtained from fieldgrown plants were disinfested and zygotic embryos were excised. For germination, excised embryos were cultured on the Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose, 0.8% (w/v) agar, and different concentrations of N6 benzyl-adenine (BA) and gibberellic acid (GA3). The greatest germination percentage (95%) was observed when embryos were cultured on the MS medium with 1.0 mg • L-1 BA and 0.5 mg • L-1 GA3, and maintained at 25 ± 2°C under a 16 h photoperiod. Thirty days old cotyledon explants were cultured on the MS medium supplemented with different concentrations and combinations of plant growth regulators viz., BA, GA3, 2,4-dichlorophenoxyacetic acid (2,4-D), and á-naphthalene acetic acid (NAA). After 90 days, the globular embryos were directly formed on the surface of explants. The highest frequency of somatic embryo induction (72.5) was obtained on the MS medium with 3.0 mg • L-1 BA, 1.0 mg • L-1 NAA, 1.0 mg • L-1 GA3, and 0.1% (w/v) activated charcoal (AC), with a mean number of 14 embryos per explant. Maturation of globular embryos into heart- and torpedo-shape was observed on the same medium. When the torpedo-shaped embryos were transferred onto the same MS medium supplemented with 3.0 mg • L-1 BA, 1.0 mg • L-1 NAA, 1.0 mg • L-1 GA3, and 0.1% (w/v) AC, secondary somatic embryos were observed on the surface of primary somatic embryos. When the embryos were transferred to the MS medium supplemented with 1.0mg • L-1 each of BA and GA3, all of them converted into plantlets, but their growth was very slow.

본 연구는 우리나라 주요 재배종인 Muscari armeniacum ‘Early Giant’ 품종을 사용하여 엽절편체로 부터 직접적으로 신초재생과 체세포배 발생에 미치는 생장조절제의 효과를 구명하였다. 무스카리의 엽조직으 로부터 캘러스 과정을 거치지 않은 직접 신초형성은 2,4-D 0.1 mg·L−1가 함유된 배지에서 가장 좋았다. 반면, 체세포배 발생은 생장조절제를 첨가하지 않는 대조구와 IPA 0.1~1.0 mg·L−1가 함유된 농도의 배지에서 비교적 양호하였다. 무스카리의 엽조직으로부터 재생된 자구를 기외로 이식했을 때 맹아율은 모든 처리구에서 80%이상 으로 높았으며 특히 NAA 0.1mg·L−1, IPA 1.0~3.0mg·L−1 배지에서 재생된 자구의 생장이 양호하였다.

알파파의 재분화된 식물체로부터 다량의 이차체세포배를 유도하였으며, 이들 체세포배로부터 재분화되는 식물체의 획득빈도를 향상시키기 위하여, 캘러스에서 유래된 배의 형태 및 형태별 유식물 분화 양상에 관한 실험을 수행하였다. 2,4-D 농도에 따라 체세포배의 형성에 차이를 나타내었는데, 생장조절제가 첨가되지 않았거나, 2,4-D가 0.1 m g / ℓ 첨가된 배지에서 형성된 배는 약 57% 이상이 2개의 자엽을 갖은 정상배였으며, 2,4-D 농도가 증가할수록 정상체세포배의 출현빈도는 감소하였고, 2,4-D 4 m g / ℓ 에서는 10%만이 정상배로 나타났다. 배의 형태에 따른 발아율 및 유식물 분화양상을 조사한 결과, 2개의 자엽을 갖는 정상배의 경우는 발아율이 85%로 가장 높았으며, 정상식물체로 발육되는 비율도 80%로 나타났다. 그러나 자엽이 1개 또는 3~4개인 배는 정상적으로 발육되는 식물체의 비율이 10% 이하로 매우 낮았으며, 자엽이 5개인 배와 나팔모양의 배는 정상적인 식물체로 발달하지 못하였다.

알팔파의 하배축(hypocotyl)으로부터 캘러스 유도 및 식물체 재분화를 위하여 2.4-D 와 kinetin이 조합 처리된 MS 배지에 조직을 치상하였을 때 4주 후 캘러스가 유도되었으며, 2.4-D 4 m g / ℓ 와 kinetin 0.1 m g / ℓ 그리고 2.4-D 4 m g / ℓ 와 kinetin 0.5 m g / ℓ 조합에서 체세포배가 형성되었다. 성숙한 체세포배를 MS 기본배지로 계대배양하였을 때 정상적인 식물체로 재분화 하였다. 이차 체세포배 발생을 위하여 재분화된 기내식물의 자엽으로부 터 이차 캘러스를 유도하였다. 2.4-D의 농도에 따라 배발생 캘러스의 형성률에 차이를 보였으며, 2.4-D 4 m g / ℓ 의 MS 배지에서 배양하였을 때 배발생 캘러스의 유도가 가장 좋았다. 배발생 캘러스로부터 이차 체세포배의 발생률을 2.4-D 0.1 m g / ℓ 첨가한 MS 배지에서 가장 좋았으며, 캘러스당 배 발생률이 일차 캘러스 보다 평균 18배 증가하여, 이차 체세포배 배양에 의한 재분화 식물체의 대량증식이 가능하였다. 성숙한 이차 체세포배는 MS 기본배지에 계대배양 하였을 때 뿌리가 유도되었으며 정상적인 식물체로 발달하였다.

본 연구(硏究)는 식물(植物)의 발생(發生)과 분화(分化)에 관(關)한 생화학적(生化學的) 분자생물학적(分子生物學的) 구명(究明)을 위해 조직(組織) 발생(發生) 단계(段階)의 특이적인 유전자(遺傳子)의 발현조절 기작과 역할(役割)을 연구(硏究)하기 위한 기초(基礎) 지식을 얻고자 수행(遂行)되었다. 벼를 실험재료(實險材料)로 하여 현미로부터 callus를 유도(誘道)하고 이들 callus에서 여러가지 방법(方法)을 통(通)해 embryogenic callus와 nonembryogenic callus를 분리(分離)해 somatic embryogenesis에 관련된 특수한 단백질(蛋白質)을 찾고자 하였고 그 결과(結果)를 요약(要約)하면 다음과 같다. 지금까지의 보고(報告)에 의하면 재분화가 잘되는 callus와 그렇지 않은 callus간에는 여러가지 면에서 차이가 난다고 알려져 있다. 재분화가 잘되는 callus는 표면이 거칠고 흰색에 가까우며 조직(組織)이 단단하다. 반면에 재분화가 되지않는 callus는 표면(表面)에 윤기가 나며 단단하지 않다. 이러한 보고(報告)를 기초(基礎)로 하여 media에 1mg/1의 2,4-D가 첨가(添加)된 배지(培地)에서 2주 정도 callus를 유기시키고 유기된 callus는 다시 배양(培養)하였다. 7-8차 계대배양하면서 embryogenic callus와 nonembryogenic callus를 분리(分離)한 다음 이를 시료(試料)로 하여 protein을 추출(抽出)하여 SDS-PAGE 상에서 비교하여 보았다. 그 결과(結果) 두 callus간에 차이가 있음을 알 수 있었다. 7-8차 계대배양한 두 종류(種類)의 callus로 부터 protein을 분리하여 two-dimensional gel 전기영동에 의해 비교하여 본 결과(結果) 몇 개의 단백질(蛋白質)이 서로 다름을 알 수 있었다.

Somatic embryogenic calli were obtained from different native citrus species in Jeju island, South Korea. Undeveloped ovules were cultured on 5 different media, respectively; MSI (MS, modified, with the addition of malt extract 500 mg･L-1 and sucrose 50 g･L-1 and agar 2 g･L-1), MSII (MS, modified, with the addition of malt extract 500 mg･L-1 , kinetin 1 mg･L-1 and sucrose 50 g･L-1 and agar 2 g･L-1), MSIII (MS, modified, with the addition of malt extract 500 mg･L-1, 6-benzyladenine, 3 mg･L-1 and sucrose 50 g･L-1 and agar 2 g･L-1), EME-S (MT, modified, with the addition of malt extract 500 mg･L-1 and sucrose 50 g･L-1 and agar 2 g･L-1), 1/2 EME-S (half concentration of MT marcronutrients, half concentratrion of BH3 marcronutrients, malt extract 500 mg･L-1, glutamine 1.55 g･L-1 and sucrose 50 g･L-1 and agar 2 g･ L-1). Embryogenic calli were induced in the surface of undeveloped ovules in different manners, depending on citrus species and culture conditions. Somatic embryos developed into plantlets with a high frequency. Citrus embryogenic calli can be applied widely to somatic hybridization, genetic transformation, and in vitro germplasm conservation

Buckwheat sprout is used as vegetable, and also flour for making noodles, and so on. Currently, information about tissue culture in buckwheat is limited and restricted to micro-propagation. We carried out somatic embryogenesis and plant regeneration using hypocotyl segments as explant of the cultivated buckwheat species, Fagopyrum esculentum which differs from existing studies in the growth regulator combinations used. Maximum callus regeneration was induced on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg · L-1, benzyladenine (BA) 1.0 mg · L-1 and 3% sucrose. Friable callus was transferred to solidified MS media containing BA (1.0 mg · L-1) with various concentrations of 2,4-dichlorophenoxyacetic acid for the induction of embryogenesis. The optimum concentrations of growth regulators (for regeneration of plantlet) were indole-3-acetic acid (2.0 mg · L-1), Kinetin (1.0 mg · L-1), BA (1.0 mg · L-1). Only 2,4-D did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 5% to 20%. Whole plants were obtained at high frequencies when the embryogenic calli with somatic embryos and organized shoot primordia were transferred to MS media with 3% sucrose. The main objective of this research was to develop an efficient protocol for plant regeneration for common buckwheat, and to apply in future for genetic transformation.

Somatic embryogenesis is a process where a plant or embryo is derived from a single somatic cell or group of somatic cells. Application of this process include: clonal propagation of genetically uniform plant material; elimination of viruses; provision if source tissue for genetic transformation; generation of whole plants from single cells called protoplasts; development of synthetic seed technology. In this study tissue culture was carried out for mass propagation of cassava using somatic embryogenesis. For tissue culture set up, we used cassava variety (“Rayong5”, “Rayong7”, “Rayong9”, “Rayong11”) developed in Rayong Field Crop Research Center(RYFCRC). In induction of callus step, the callus formed from each cassava variety. “Rayong 7“ showed the highest induction rate of 95%, while induction rate of other varies were ranged from 50% to 85%. In the case of weight of callus ”Rayong5“ has the highest weight. Results in the present study would be useful in mass propagation of cassava by somatic embryogenesis.

Buckwheat sprout is used as vegetable, and also flour for making noodles, and so on. Currently, information about tissue culture in buckwheat is limited and restricted to micropropagation. We carried out somatic embryogenesis and plant regeneration using hypocotyl segments as explant of the cultivated buckwheat species Fagopyrum esculentum, differs from existing studies in the growth regulator combinations used. Maximum callus regeneration was induced on MS medium containing 2,4-D(2.0 mg/L) and benzylaminopurine BAP (1.0 mg/L) and 3% sucrose. Friable callus was transferred to solidified MS media containing BAP (1.0 mg/L) and at various concentrations for the induction of embryogensis. The optimum concentrations of additives were IAA (2 mg/L), KIN(1.0 mg/L), BAP (1.0 mg/L), and 3% (w/v) sucrose. Only 2,4-D did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 5 % to 20%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to MS media with 3% sucrose. Regenerated plants after acclimation will transfer to green house. The main objective of this research was to develop a efficient protocol for plant regeneration for common buckwheat, and to apply in future for genetic transformation.

An efficient somatic embryogenesis and plant regeneration protocol was developed for Schisandra chinensis Baill, using embryogenic cell suspensions and optimized media conditions. Friable embryogenic callus was induced from cotyledonary leaf and hypocotyl explants of 7 days old seedlings on MS agar medium supplemented with 1.0 to 4.0 mg l-1 of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast growing and well dispersed embryogenic cell suspensions were developed within two months when embryogenic calli were transferred to MS liquid medium containing 1.0 mg l-1 2,4-D. One third strength of MS medium was the best for both overall growth and development of somatic embryos in liquid culture. Over 3400 viable somatic embryos were produced from each 150 ml flask with an initial cell density of 30 mg in 30 ml medium. Germinated somatic embryos developed in liquid medium converted into plantlets after transferred to half-strength MS semi-solid medium. Approximately 90% of the converted plantlets were successfully transplanted to soil and grew into fertile plants.

본 연구를 통해 헛개나무의 기내유묘의 조직절편으로부터 배발생캘러스의 유기 및 2차 배형성, 그리고 배발생캘러스의 현탁배양계로부터 체세포배를 대량생산하여 식물체로 재분화시키는 시스템을 확립하였다. 현탁배양을 통해 배발생세포의 유도 및 증식에는 모두 30℃의 고온이 효과적이었는데 이와 같은 온도조건에서 배발생캘러스 유도율과 배발생캘러스의 생장량은 각각 100%와 894.6 mg로 25℃에 비해 1.53배와 9.19배로 높게 나타났다. 배발생세포를 18℃에서 배양할 경우 체세포배로 전환되었고 배양 5주후 체세포배로 발달하였으며 25℃ 이상의 배양온도에서는 배발생세포는 증식만 할뿐 체세포배는 형성되지 않았다. 체세포배로부터 식물체의 형성은 18℃ 저온에서만 가능하였다. 배지에 0.1과 0.5 mg/l BA를 첨가할 경우 식물체 재분화율은 37%와 28%로 생장조절물질을 처리하지 않은 배지에 비해 2.2배와 1.7배로 높게 나타났다 재분화된 식물체의 성장에 주는 무기염의 영향을 조사하기 위하여 유식물체를 MS와 1/3MS 고체배지에 옮겨 배양한 결과 1/3MS배지가 줄기신장과 뿌리의 유도에 적합하였다.

In vitro high-frequency plant regeneration of Muscari comosum var. plumosum through somatic embryogenesis was obtained via two developmental pathways: direct embryos and multiple shoots regenerated from embryogenic callus. Flower bud with pedicel, receptacle, petal and ovary wall, floral stalk and leaf as explants were cultured in MS medium supplemented with various plant growth regulators. Embryos formed directly from pedicel, receptacle and floral stalk. Depending on explant sources, the optimal medium was MS medium supplemented with 0.2 mg/L IBA and 0.3 mg/L BA, 3.0 mg/L IBA and 3.0 mg/L BA, and MS-free medium for pedicel, receptacle, and floral stalk, respectively. Multiple shoots regenerated from embryogenic cal]i which was initiated from petal, ovary and leaf were observed in MS medium with different concentrations and combinations of hormone. The most suitable medium for each type of explant was 3.0 mg/L IBA and 3.0 mg/L BA(petal and ovary) and 5.0 mg/L IBA and 5.0 mg/L BA (leaf) Furthermore, the combination of 0.1 mg/L 2,4-D and 1.0 mg/L BA was also good for all sources of explants not only for direct embryo formation, but also, for embryogenic callus induction.

An efficient plant regeneration system of Gentiana scabra through somatic embryogenesis was established. Leaves and roots of seedlings of Gentiana scabra excised after germination were cultured on MS basal medium with 2,4-D, NAA or BA. Embryogenic callus was obtained on MS medium with 0.5 mg/L 2,4-D alone or 0.1 mg/L 2,4-D combimation with 1.0 mg/L BA after 45 days of culture. These embryogenic calli gave rise to somatic embryos, which subsequently developed into plantlets on MS medium without PGRs. Also, shoots were effectively differentiated from embryogenic callus when root segments were cultured on MS medium supplement with 0.1 mg/L 2,4-D and 1.0 mg/L BA. Shoots were effectively rooted on MS medium without PGRs. In vitro flowers were formed from plantlets cultured on MS medium with 5% sucrose after 60 days of culture.