Kee-Hwa Bae

Effects of NaOCl treatment on in vitro germination of seeds of a rare endemic plant, Oreorchis coreana Finet

Oreorchis coreana Finet is threatened globally by over-collection from its natural habitats for horticultural purposes. Its rarity in nature makes this plant one of the most endangered species in Korea. In this study, we investigated the effects of sodium hypochlorite (NaOCl) on orchid seed viability and seed germination. An in vitro bioassay swelling test using immature seeds was compared with a standard chemical procedure using triphenyl tetrazolium chloride (TTC) to test seed viability. In general, the bioassay was more appropriate for estimating embryo viability after a prolonged pre-treatment (more than 1 h) in 1% NaOCl, a surface sterilant often used to enhance germination of seeds of terrestrial plants. Therefore, an efficient method for investigating in vitro swelling of immature seeds is urgently needed. We established a method for determining the viability and swelling of O. coreana seeds via in vitro examination of immature seeds. Treatment of immature seeds with 1% NaOCl for 10 min greatly enhanced the extent of swelling of immature zygote embryos when compared to untreated seeds. These data obtained here appear to be comparable to viability and swelling that occurs in O. coreana seeds via asymbiotic germination.

In vitro germination of Gastrodia verrucosa Blume and Hetaeria sikokiana Tuyama treated by NaOCl

Abstract An optimization in vitro seed germination was established by using triphenol tetrazolium chloride (TTC) test, which has been known as two rare orchids ( Gastrodia verrucosa Blume and Hetaeria sikokiana Tuyama) in Jeju island. We have established proper NaOCl treatment for in vitro germination of G. verrucosa and H. sikokiana through TTC test. In the case of H. sikokiana, seed viability through TTC test was high with 95% in control. However, NaOCl 1% treatment for 30 minutes showed the highest embryo swelling rate to seed viability. Likewise, swelling formation of embryos, diameter of embryos, protocorm formation and diameter of protocorms of G. verrucosa was 87%, 59 μm, 91% and 138 μm through NaOCl 1% treatment for 30 minutes. This result will be applied on the basic information for improving in vitro seed germination rate of G. verrucosa and H. sikokiana. Keywords rare plants, Orchidaceae, seed viability, swelled embryo 서 론 제주도는 우리나라에서 식물다양성이 가장 풍부한 곳으로 알려져 있다. 제주도의 식물 수는 167과 770속 1,819종, 121변종, 50품종으로 총 1,990분류군으로 정리된바 있다(Kim et al. 2006). 이중 제주특산종은 77종으로 분류되는데, 이는 국내 472분류군의 약 16%를 차지하는 비중이다(Kim 2006). 이처럼 제주도에 특산종이 많이 분포하는 이유는 육지와 격리된 섬이라는 환경과 독특한 아열대성 기후 때문인 것으로 보여 진다. 한라천마(

In vitro propagation and multiple shoot induction of Rhodiola rosea L. by axillary bud culture

An efficient in vitro propagation was established by using axillary bud explants of roseroot (Rhodiola rosea L.), which has been known as a medicinal plant in East Asia. Among various media tested, MS medium supplemented with 1.0 mg/L BA and 1.0 mg/L GA3 was found to be the best for multiple shoot formation (15 axillary shoots per axillary bud). In addition 1/2MS medium containing 50 g/L sucrose was best for shoot elongation (7.8 cm) and increasing total chlorophyll contents (8.64 mg/g) best. Maximum number of roots (17.7 roots per explant) was observed on the medium without plant growth regulators. Propagated plants were successfully acclimatized to ex vitro conditions, with a sur- vival frequency of 97% after 12 weeks. Most rooted shoots grew well and produced viable seeds when grown in vitro culture conditions. Therefore, R. rosea can be effectively propagated in vitro by the system we developed in this study.

In vitro propagation of endangered species, Hylotelephium ussuriense (Kom.) H. Ohba

멸종위기수종인 둥근잎꿩의비름 기내 부정아유도에 미치는 식물생장조절물질의 종류 및 배양부위의 영향과 기내생육 및 엽록소함량에 미치는 배지, sucrose 농도 및 배양용기에 따른 환기효과를 연구하였다. 부정아 유도는 3.0 m g/L의 BA와 0.01 mg/L의 IBA가 첨가된 배지에 액아포함 줄기를 배양하였을 때 가장 효과적이었다. 기내 부정아의 줄기와 뿌리 신장에 MS배지의 농도와 sucrose의 농도는 영향을 주지 않았다. 환기처리가 기내 건전 식물체 생산에 미치는 영향을 조사한 결과, 기내 환기처리구는 줄기신장에 효과적이었고, 뿌리의 신장은 유의적 차이는 없이 모든 실험구에서 10 cm 이상 증식하였다. 엽록소의 함량은 환기처리 시 총엽록소 함량이 3.12 mg/g으로 높게 나타남을 확인하였다. 둥근잎꿩의비름의 부정아 유도에 농도와 배양부위가 중요한 요인으로 작용하고 기내식물체 신장에 미치는 배지 및 sucrose농도의 영향은 크지 않은 것으로 나타났다. 기내 건전식물체의 생산은 환기가 용이한 배양용기하에서 양호한 것으로 나타났다. 【To establish the system of in vitro plant regeneration, the different explants (stem with axillary bud and stem without axillary bud) of Hylotelephium ussuriense were cultured on the Murashige and Skoogs medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). The adventitious shoot induction was more effective in the stem with axillary bud explants than the stem without axillary bud explants, and was the best on MS medium containing 3.0 mg/L BA and 0.01 mg/L IBA. Frequency of plantlet growth was not significantly treated on MS and sucrose. Total chlorophyll contents under ventilation treatment were higher than those in control (non-ventilation). This in vitro propagation protocol will be useful for conservation and mass propagation of this endangered plant.】

In vitro culture of rare plant Bletilla striata using Jeju magma seawater

This experiment was conducted to investigate the effect of various type of Magma seawater (MSW) con- centrations on plant growth and useful mineral contents in Bletilla striata. In the RO (Reverse Osmosis) and ED (Electronic Distal) treatment, hardness of medium was poored in 3.0 g/L gelrite but increased in 8.0 g/L plant agar, 38,000 and 2,000 g/cm 2 respectably. We analyzed the morphological and physiological characteristics differences of B. striata treated various MSW. Survival frequency of plant and growth (shoot length, shoot diameter, root length, root diameter, shoot/root ratio) were significantly increased in RO and ED treatment at 50% and 10%, especially. Chlorophyll contents in ED treatments were higher than those in control and RO treatment. The content of strontium (Sr) in 20, 50, 75, 100% ED treatment, were higher than those in the control and RO, ED 1, 5, 10% treatment. These results showed that treatment of ED with the range of 20 ~ 100% could be used to supply the strontium enriched orchid plant. It is considered that MSW may be applied for use in Magma seawater to promotion of growth and produced functional plant.

Callus induction and plant regeneration of Iris dichotoma Pall. in endangered species

Iris dichotoma Pall. is an important endangered plant belonging to the family Iridaceae. A method was developed for the rapid micropropagation of I. dichotoma through plant regeneration from leaf, rhizome, and root explant-derived calli. Leaf, rhizome, and root segments were cultured on Murashige and Skoog (MS) medium supple- mented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0-3.0 mg·L -1 ) for callus induction. Callus production was highest at 1.0 mg·L -1 2,4-D, where 73.8% and 45.5% of cultured rhizome and root cuttings, respectively, produced calli. The viable calli were maintained at an induced concentration of 2,4-D (3.0 mg·L -1 ). They were then transferred to MS medium supplemented with various concentrations of 2,4-D (0-3.0 mg·L -1 ) in combination with 6-benzyladenine (BA: 0, 1.0 and 3.0 mg·L -1 ) for adventitious shoot regeneration. The addition of a low concentration of 2,4-D into BA-containing medium significantly increased the frequency of shoot regeneration in leaf, rhizome, and root-derived calli. The highest number of adventitious shoots (26.4 per callus) formed at 0.5 mg·L -1 2,4-D and 1.0 mg/l BA. For rooting of the shoots, half- strength MS medium supplemented with different concentrations of indole 3-butyric acid (IBA) 0-3.0 mg·L -1 was tested. The optimal results were observed using half-strength MS medium supplemented with 1.0 mg·L -1 IBA, on which 98% of the regenerated shoots developed roots with an average of 3.5 roots per shoot within 45 days. The plantlets raised in vitro were acclimatized and transferred to soil with 95% success. This in vitro propagation protocol will be useful for conservation and mass propagation of this endangered plant.

Effect of Seawater Concentration on Seed Germination and Seedling Growth of Artemisia fukudo

Abstract Halophytes are plants of saline habitats that grow under conditions that may vary in extremes of temperatures (freezing to very hot), water availability (drought to water logging) and salinity (mild to almost saturation). Halophytes may also face sudden micro-environmental variations within their habitats. In this study, we examine some of the factors that determine the ability of seeds of Artemisia fukudo to germinate when conditions are optimal for seedling growth and survival. Germination percentage was the highest at 77.5% when treated in 0% seawater and seedling growth was best in 0% seawater plug cell tray. Physiological quality (chlorophyll contents) and mean germination time were best in the condition in which seeds were treated with 0% seawater (control). Germination performance index in the control group showed the highest value to 3.8. Seedling growth, seedling vigor index in the length of seedling growth andthe relative growth rate were the highest values of 0.76 cm, 1.43, 0.0099, respectively.Key Words: Artemisia fukudo, Chlorophyll, germination, seawater

Plant regeneration through the callus culture induced from bulb scales of an endangered species Lilium cernum Komarvo.

Lilium cernum Komarvo. is an important endangered plant belonging to the family Liliaceae. A method was developed for the rapid micropropagation of L. cernum through plant regeneration from bulb scales explant-derived calli. The bulb scales segments were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.5, 1.0, 3.0 mg·L -1 kinetin and 0, 0.1, 0.5, 1.0 mg·L -1 NAA or 2,4-D for callus induction. In media with 0.5∼3.0 mg·L -1 kinetin and 0.1∼1.0 mg·L -1 NAA and 2,4-D, 95∼100% of explants produced callus. They were then transferred to MS medium supplemented with various concentrations of NAA (0, 0.01, 0.05 and 1.0 mg·L -1 ) in combination with BA (0, 1.0 and 2.0 mg·L -1 ) for bulbet formation. Bulbet induction (78%), weight (468 mg) and size (15.5 mm) were obtained the highest on MS medium containing 2.0 mg·L -1 BA and 1.0 m g·L -1 NAA. In vitro frequency of plant regeneration was not significantly treated in strength of MS and sucrose concentration. Chlorophyll contents in 1/2MS with 50 g·L -1 sucrose treatments

Plant regeneration from callus of Iris odaesanensis Y. N. Lee native to Korea via organogenesis

Abstract Iris odaesanensis Y. N. Lee. is an important endangered and native plant belonging to the family Iridaceae in Korea. This study describes a method for rapid micropropagation of this species via from leaf, rhizome and root explants derived calli. Leaf, rhizome and root explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) for callus induction. Rhizome explants yielded calli at a frequency of 72% when cultured at 1.0 mg/l 2,4-D. Calli were maintained at 1.0 mg/l 2,4-D. These calli were transferred to MS medium supplemented with 0, 0.5, 1.0, and 2.0 mg/l 2,4-D in combination with 0, 0.5, 1.0, and 3.0 mg/l BA for adventitious shoot induction. The highest number of adventitious shoot (228.9 per petri-dish) were formed at 1.0 mg/l 2,4-D and 1.0 mg/l BA. WPM medium was the best to convert calli into plantlets, where up to 98.2% of calli were regenerated into plantlets. This in vitro propagation protocol should be useful for conservation of this endangered plant.