A survey of plant-parasitic nematodes (PPNs) was carried out in medicinal crop cultivated fields from July to August in 2023. Three-leaf ladybell, Adenophora triphylla var. japonica is a highly valued medicinal plant that is used to treat or prevent bronchitis, cough, cancer, and obesity in Korea. A. triphylla plants with small root-galls were observed in a field of Yeongju Agricultural Technology Center, which were identified as a root-knot nematode. Additional morphological and molecular analyses studies were performed and identified as Meloidogyne hapla, Northern root-knot nematode. Population densities of M. hapla ranged from 20~30 nematodes per 100 cm3 of soil. M. hapla was detected at lower densities in soil compared to other infected host crops, but there are concerns about damage to M. hapla since A. triphylla is cultivated for more than two years once planted. Our results indicate that A. triphylla roots damage by M. hapla were identifed, it is necessary to prepare control methods such as registration of applicable nematicides and crop rotation.

본 연구는 고부가가치 약용작물인 잔대(Adenophora triphylla var. japonica Hara)의 종자 형태특성과 휴면 유형을 조사하고, 발아율 향상을 위한 소독제와 광질을 선발하기 위해서 수행되었다. 종자 소독은 증류수(대조구), NaClO 4%, H2O2 4%와 benomyl 500mg·L-1를 이용하여 실시하였으며, 광질처리는 암조건(control I), 형광등 (control II), LEDs[red, blue, green, RB LEDs(red:blue = 8:2, 6:4, 4:6, 2:8)]를 광주기 12/12(light/dark), 광도 150μmol·m-2·s-1 photosynthetic photon flux density로 설정 하였다. 잔대 종자는 emryo (E):seed (S) ratio가 0.4로 미숙배 종자지만 30일 이내에 발아가 되며, 침지 6시간 만에 포화상태에 도달하였다. 종자 소독 후 benomyl 처리에서 곰팡이 발생이 유의적으로 억제되었으며, 최종 발아 율이 87%로 가장 높았다. 적색광에서 최종 발아율이 92%로 가장 높았으며, 일일평균발아수는 R2B8에서 가장 적었다. 따라서 잔대 종자는 휴면이 거의 없으며, benomyl 소독제와 적색광이 발아율 향상에 효과적이여, 향후 약용작물 잔대 재배에 이용가치가 높을 것이라 판 단된다.

Background : Adenophora triphylla var japonica is a perennial herb that belongs to Campanulaceae. Radix Adenophorae is a dried rhizome of A. triphylla and same genus plant. It has contains chemicals such as cycloartenyl acetate, lupenone, β-sitosterol, taraxerone, octacosanoic acid, and praeruptorin A. Radix Adenophorae considered to be effective regulating humoral and cellular immunity, antimutation, restraining adenocarcinoma cell, strengthening cardiac function, allaying a fever, and easing pain and cough. In this study, we tried to establish a mass production system of A. triphylla which has high economic value as a medicinal herb by plant tissue culture in order to cultivate standard varieties. Methods and Results : In this study, A. triphylla internode was used as a explant and it was surface sterilized by 1% sodium hypochlorite for 5 minutes, consequently several times washed with ddH2O. Further it was placed in to MS medium including with axillary bud. The 1/2MS, B5, SH was used in this research. And the plant growth regulator of 0.1 - 2 ㎎/ℓ auxins (NAA, IBA) and cytokines (BA) were used respectively to achieve multiple shoots. The whole study was carried out in the department of Herbal crop research, Eumseong, RDA. Conclusion : In this study we obtained, 6.2 multi-shoots per an explant, and the shoot growth was also favorable in the presence of 1.0 ㎎/ℓ BA and 1.0 ㎎/ℓ IBA.

Background : Seed germination for Adenophora triphylla var. japonica Hara. is not good when grown in direct seeding. This experiment was conducted to identify seedling box suitable for seedling transplantation and cultivation to improve farm income and public health. Methods and Result : To investigate the effect on growth, sizes of seedling box used were as follows (width × length × height, in ㎝); 2.5 × 2.5 × 4 (custom), 4 × 4 × 4, 4.5 × 4.5 × 6, 5.0 × 5 × 9.6, 5 × 5 × 11.2, 5 × 5 × 17, and for sowing in narrow strips; 33.5 × 55 × 13, 33.5 × 49 × 23, 33.5 × 49 × 30. The weight of the ground part decreased as box height became lower, highest at 17 ㎝ tall (0.65 g) and lowest at 4 ㎝ (0.23 g). In terms of root length of the seedlings, the longest was 9.0 - 9.3 ㎝ at box height of 17 ㎝ and decreased as height decreased. Root weight increased as box height decreased, the highest at 4 ㎝. After the seedlings were planted, more than 90% survived in seedling box compared to the 50% rooting rate on sown in narrow strips where root net formation did not occur. During harvesting period, plant height was 15.8 - 17.5 ㎝ with a shoot diameter of 13.3 - 20.7 ㎜, leaf length of 5.6 - 7.9 ㎝ and leaf numbers 15.6 - 22.3. Moreover, leaf width was the widest at 8.6 ㎝ at 6 ㎝ box height while dry weight was the most at 6.7 g at 11.2 ㎝. Survival rate and growth characteristics of the ground part were lower in seedling box for sowing in narrow strips. For the underground characteristics, root number increased as box height decreased while dry weight increased as box height increased, with 1.7 - 3.5 and 6.2 - 6.8 less in seedling box for sowing in narrow strips, respectively. The yield in a 17 ㎝ high seedling box was 152 ㎏ per 10 a, 16% higher than conventional level, and decreased as box height decreased. Conclusion : These results suggest that the optimum seedling box height is 11.2 - 17 ㎝ to have lower root numbers and higher dry weight and yield.

Background: Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth with radical leaves during the first year and shows reproductive growth with cauline leaves and bolting during the second year. In addition, the shape of the plant varies within the same species. For this reason, there are limitations to classifying the species by visual examination. However, there is not sufficient genetic information or molecular tools to analyze the genetic diversity of the plant. Methods and Results: Approximately 34.59 Gbp of raw data containing 342,487,502 reads was obtained from next generation sequencing (NGS) and these reads were assembled into 357,211 scaffolds. A total of 84,106 simple sequence repeat (SSR) regions were identified and 14,133 primer sets were designed. From the designed primer sets, 95 were randomly selected and were applied to the genomic DNA which was extracted from five plants and pooled. Thirty-nine primer sets showing more than two bands were finally selected as SSR markers, and were used for the genetic relationship analysis. Conclusions: The 39 novel SSR markers developed in this study could be used for the genetic diversity analysis, variety identification, new variety development and molecular breeding of A. triphylla.

To investigate skin-whitening effect of Adenophora triphylla var. japonica sprout extract, antioxidant activity, inhibitory effect on tyrosinase and melanin synthesis in B16/F10 melanoma cell were examined. Total phenolic content (246.25 ㎎ GAE/g) and total flavonoid content (303.94 ㎎ RE/g) of ethyl acetate fraction from Adenophora triphylla sprout (EFAT) showed the highest contents than other fractions (n-hexane, chloroform and distilled water). Antioxidant activities of EFAT has been evaluated using ABTS, DPPH radical scavenging activities, FRAP and inhibitory effect of lipid peroxidation. EFAT showed excellent radical scavenging activity and inhibitory effect on MDA production. Inhibitory effect of tyrosinase as a major enzyme of melanin synthesis was also measured. In these results, EFAT showed higher inhibitory effect against L-DOPA (51.27%) than L-tyrosine. IC50 value on α-glucosidase was 41.93 ㎍/㎖. In B16/F10 melanoma cells, EFAT inhibited melanin synthesis at 200 ㎍/㎖ concentration (about 42% decrease). Finally, main physiological compounds of EFAT were identified as a rutin and a chlorogenic acid using high performance liquid chromatography.

Background : This study aimed to determine the use of proper organic fertilizer through side dressing application in the environmental cultivation of Adenophora triphylla var. japonica Hara. to improve farming income. Methods and Results : In the cultivation of eco-friendly and environmental medicinal plant like Adenophora triphylla var. japonica Hara., it is necessary to use organic instead of chemical fertilizer. Organic fertilizers such as HL, CK, OK were applied three (3) times during the last ten days of July, August, and September. There was no significant difference between CF and NT, in terms of the number of rooting per 1㎡ which was 12.1 -12.9. There were also no significant differences in plant length, leaf length, leaf width and dry weight. The root length was 19.9 ㎝, which was the longest in CK, and 16.8 ㎝, the shortest in ST, but described as no significant difference. The root width was 14.8 – 5.5 ㎜, and the number of rootlet was 5.7 - 7.1, but was considered not a significant difference. The dry weight was 16.6 g in HL, 16.3 g in CK and ST, and 15.6 g in OK, but still there were no significant differences observed. However, it was 14.6 g in NT, which was considered the lowest, thus there was a significant difference. The number of dry root per 10 a was 149 ㎏ which is considered the most in HL, 141 ㎏ in CK, 140 ㎏ in ST, and 138kg in OK yet there were no significant differences observed. While NT only had 123 ㎏ hence, there was a significant difference on the number of dry root per 10a as compared with the group applied with the three organic fertilizers. Conclusion : In conclusion, it is proper to use Organic Fertilizer HL and CK through side dressing application in growing Adenophora triphylla var. japonica Hara.

Background : This project was conducted to determine proper seed harvest time and seed storage methods of Adenophora triphylla var. japonica Hara. Methods and Results : When Adenophora triphylla var. japonica Hara. transplanted this year, seeding can only be done the following year. Therefore, the seeds harvested in the interval of ten (10) days were investigated for seed harvest amount, germination rate, seed storage temperature and storing on container, between the period late August and mid-October. It is when the seed fructification started and ended. The seeds harvested from the first ten (10) days of October to mid October were 87.9% in whole seeds. The proper seed harvesting time was mid October which has a germination rate of 75% in whole seed harvest time. The lowest germination rate of 27.3% was at the germination temperature of 15℃. As the temperature rises, the germination rate increases. In 29℃, the germination rate was at 79% which is the highest; and from 30℃, the germination rate decreased to 71%. When the storage temperature was at 0℃ after storage of 5 months, the germination rate was at 83%. However, after storage of 12 months, it decreased by 7% that was 76%. When in 4℃, after storage of 5 months the germination rate was at 85%. However, after storage of 12 months it decreased by 8% which was 77%. In room temperature after storage of 5 months, germination rate was at 77%, but after storage of 12 months it decreased by 22% which was 55%. When seeds were stored in vinyl container, it was observed that the germination rate was at 86% after 5 months of storage, which was 9% higher than seed stored in paper which was 77%. After 12 months storage, seed in vinyl container had a germination rate of 71%, which was 3% higher than seed in paper at 68%. Conclusion : It is concluded that the proper time of seed gathering of Adenophora triphylla var. japonica Hara. is during the first ten (10) days of October to mid-October. When the long time storage is needed, below zero temperature with vinyl container is more appropriate.

Background : Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth by radical leaf until 1 year after sowing and shows reproductive growth during the second year and there is a characteristic of bolting by turning into cauline leaf. In addition, the phenotypes of plants varies even though they are belonging to the same species. For this reason, there is a limit for the classification of the species by the method of visual examination. Methods and Results : Simple sequence repeat (SSR) markers were developed based on the genomic sequence of A. triphylla using next generation sequencing to prepare the basis of molecular breeding and analyze the genetic diversity. Ninety-five primer sets including tri-, tetra- and penta-nucleotide motif types were randomly selected and they were applied to mixed genomic DNA and finally 39 primer sets showing from two to four bands were selected and used for genetic relationship analysis. Conclusions : Using the next generation sequencing, 39 polymorphic SSR markers were developed.

Background : Adenophora triphylla var. japonica Hara. is one of the important oriental medicinal herbs being used to cure lung/respiratory diseases. It belongs to the five Sam (ginseng) including Panax ginseng, Salvia miltiorrhiza, Sophora flavescens, and Scrophularia buergeriana. In 2015, Korea produced 72 tons of medicinal herbs in a 14-hectare area and imported 24 tons valued at US$95,000. This study was carried out to investigate the effect of several biopesticides to control leaf spot of A. triphylla var. japonica Hara. Methods and Results : One year plants at the Department of Herbal Crop Research, NIHHS, RDA were used. The treatments used biopesticides such as plant extracts (BP), microbial agent (DM), microbial agent (PJ), sulfur powder (HS), and controlled chemicals (ST), while non treatment (NT) was used as control. After the early part of disease outbreak, the plants were treated four times with 7 days interval. The incidence of leaf spot, efficacy, phytotoxicity and control value were investigated. Based on the efficacy test, incidence of leaf spot is lowest at 15.6% in plant extracts (BP). Other biopesticides showed higher incidence - 16.7% for microbial agent (DM), 17.8% for microbial agent (PJ), 18.9% for sulfur powder (HS) and 71.1% for non treatment (NT), compared with 11.1% in controlled chemicals (ST). The microbial agent (PJ), sulfur powder (HS), plant extracts (BP) and microbial agent (DM) were not damaged in the phytotoxicity test. In the test of application dose, time and methods, the control value was highest in plant extracts (BP) at 78.1%, followed by microbial agent (DM) at 76.6%, microbial agent (PJ) at 75.0%, and sulfur powder (HS) at 73.4% , compared with controlled chemicals (ST) at 84.4%. Conclusion : It is possible to use biopesticides at the proper time to prevent leaf spot in Adenophora triphylla var. japonica Hara.

Background : Adenophora triphylla var. japonica Hara. is used to cure respiratory/ lung diseases, which makes it an important oriental medicinal herb. It is one of the five Sam (ginseng), which includes Panax ginseng, Salvia miltiorrhiza, Sophora flavescens, and Scrophularia buergeriana. In 2015, Korea imported 24 tons of medicinal herbs worth US$95,000. This study was carried out to investigate the effect of several mulching materials on the growth and root yield of A. triphylla var. japonica using organic fertilizer. Methods and Result : Seeds of A. triphylla var. japonica were collected at the nursery of the Department of Herbal Crop Research, NIHHS, RDA in 2015. Mulching materials used are black plastic, black woven fabric, and biodegradable plastic. Hand weeding was done to serve as control. The soil was fertilized two months before planting. Seeds of A. triphylla var. japonica were sowed in tray in early March and were allowed to grow for two months. It was planted in early May with planting density of 30 ㎝ x 15 ㎝ and was covered with various mulching materials. Parameters investigated are survival rate, aerial part and root growth characteristics, and yield at harvest time. Results showed that biodegradable plastic is most favorable for plant growth with height of 17.2cm, leaf length of 13.4cm and leaf width of 5.6 ㎝. Dry plant weight was heaviest in biodegradable plastic treatment at 2.8 g, and lightest at 2.5 g when planted without mulch. Biodegradable plastic has the highest dry root weight of 19.3 g, followed by black vinyl (18.4 g), woven fabric (18.3 g) and without mulch (17.9 g). The yield obtained per 10 a was highest in biodegradable plastic treatment at 174 ㎏, while black vinyl and woven fabric treatments produced 169 ㎏ and 160 ㎏, respectively. Plants without mulch had the lowest yield of 157 ㎏. Conclusion : In the above results, biodegradable plastic and woven fabric are considered suitable mulching materials for the cultivation of A. triphylla var. japonica.

Background : Adenophora triphylla var. japonica Hara. is used to cure internal organ, which is called to important one of the five “Sam (ginseng)” oriental medicinal herbs included Panax ginseng, Salvia miltiorrhiza, Sophora flavescens, Scrophularia buergeriana. Korea imported 24 ton 129 thousand dollar for medicinal herbs and 23 ton 29 thousand dollar for food at 2013. This study was carried out to investigate the effect of several organic compost on the growth and root yield of A. triphylla var japonica with organic fertilizer. Methods and Results : Seeds were collected from Adenophora triphylla var. japonica Hara. at last year in nursery of Department of Herbal Crop Research, NIHHS, RDA. Four types organic fertilizer (germiculture (GG), granular mix oil cake (CJ), mixed organic (DJ), plant oil cake (TG)), chemical fertilizer (CF) and non-fertilizer (NF) were treated. It was fertilized on soil before 2 month of planting. Seeds A. triphylla var. japonica were sowed in tray at early March and then were grown during 2 month. It were planted in furrowed nursery at early with 30 × 10 cm planting distance and black vinyl covering condition. Soil chemical property, survival rate, aerial part and root growth characteristics and yield were investigated at harvest time. For main composition analysis, 10 mg of standard (b-sitosterol, lupenone) was taken, 80% MeOH was added to dissolve in 10 ㎖ flask. 0.5, 1, 2, 4 ㎖ was taken from dissolved solution and then was added to 10 ㎖ of 80% MeOH. It was filtered through 0.45 ㎛ filter and then 5 μℓ was taken to make standard solution. Analytical calibration curve was measured to diluted solution within sample concentration. 2 g of ground sample was taken, 40㎖ of 80% MeOH was added to extract by ultrasound sonication during 60 minutes and then it was filtered through 0.2 ㎛ filter to analyze component content. Conclusion : GG and DJ were the most suitable for organic cultivation of denophora triphylla var. japonica. Content of beta-sitosterol showed the highest value in DJ treatment and the lowest value in CF. Content of lupenone showed the highest value in DJ and the lowest value in CJ.

Adenophora triphylla var. japonica HARA is a herbaceous plant belongs to Campanulaceae. Adenophora root is mainly used for medicinal purpose. It is effective for lung cleaning, sputum remove, viscera strengthening, cough stopping and cancer treatments. Adenophora has about 70 species in the world and 17 of the species are distributed in Korea. Genetic resources of A. triphylla var. japonica HARA are valuable as the habitat is concentrated in East Asia. The intraspecies variation is very high according to the environmental conditions. A new A. triphylla var. japonica HARA variety, ‘Harang’, was developed through polyploid breeding in 2011. But, low domestic production and passive studies caused our country to rely on imports for almost all amount of the A. triphylla var. japonica HARA demands. In this experiment, genetic diversity between the collections were analyzed using 32 RAPD primers. Through this study, limit of morphologic classification could be solved and genetic diversity of this plant could be assured.