The 12 cultivars of the Jeju native Citrus are considered to have originated from China. However, the origin of the cultivar ‘Byungkyool’ (Citrus platymamma Hort. ex Tanaka) is not clearly known. We performed PCR analysis by using three primer sets designed from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) to analyze the phylogenetic relationship between the traditional citrus cultivars and the Byungkyool cultivar. Sequence length of the nrDNA ITS1 region of JNCPCRI (Jeju Native Citrus platymamma Citrus Research Institute) cultivar was 247 bp, 8the ITS2 region was 228 bp and the total ITS region (ITS1-5.8S-ITS2) was 638 bp. Analysis of the genetic relationship based on the sequence analysis at the ITS region of the JNCPCRI cultivar revealed that the ITS1 region of the cultivar was genetically the same as that of the Byungkyool (JQ990189) cultivar, and the ITS2 region was genetically similar to the Binkyool (JQ990180), Hongkyool (JQ990178), Dangyooja (JQ990179), and Pyunkyool (JQ990181) cultivars. Moreover, the total ITS region in the 5.8S rDNA region was genetically similar to the Hongkyool (JQ990178) cultivar. In addition, the total ITS region of the JNCPCRI cultivar was the most closely related to the Cheongkyool (JQ990183) cultivar and has been reported to originate from the Binkyool (JQ990180) and Pyunkyool (JQ990181) cultivars. Although the JNCPCRI cultivar was morphologically the same as the Byungkyool (JQ990189) cultivar, the ITS region showed genetic heterogeneity. Taken together, we conclude that the genetic variation in the ITS region of JNCPCRI cultivar suggests that it was propagated through fertilization with the surrounding citrus cultivars.

Backgrounds : Pomelo (Citrus grandis Osbeck) is the kind of citrus fruit which is Dicotyledoneae belongs to Rutaceae and special product in Jeju island. According to the previous researches, coumarin, eliocitrin, naringin are identified and these kinds of constituents revealed to be effective as anticancer, antioxidant, and antidiabetic. Until recently, there are many investigations about its functional properties were reported, but investigation about biological activities depend on extraction conditions are not sufficient. Methods and Results : 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was performed by the method of Blois with minor modification. After adding DPPH radical to each sample solutions, the mixtures were incubated in 30 minutes at aphotic place. Then, the degree of scavenging activity was recorded by microplate reader at 490 nm. The scavenging activity was expressed by RC50, which is the concentration of sample solution necessitated to scavenge 50% DPPH radical against negative control. For α-glucosidase inhibitory activity, the method using p-nitrophenyl-α-D-glucopyranoside (pNPG) was applied. To each samples, α-glucosidase and pNPG were mixed and incubated, consequently, Na2CO3 was added to terminate the reaction. Finally, absorbance was read at 450 nm. The same as DPPH radical scavenging activity, the inhibition was explicitly expressed by the amount of sample solution to inhibit 50% α-glucosidase, IC50. The 80% MeOH extract demonstrated the highest radical scavenging activity with 74.32±8.45 μg/ml. α-Glucosidase inhibitory activity was carried out to evaluate the inhibitory activity of sugar digestion and absorption and 60, 80% MeOH extract exhibited 416.35±11.07 μg/ml and 336.57±2.03 μg/ml, respectively. Conclusion : The DPPH radical scavenging and α-glucosidase inhibitory activity were evaluated in this study. The highest DPPH radical scavenging activity was seen in 80% Citrus grandis Osbeck MeOH extract, following 60% MeOH extract exhibited the second highest scavenging activity. Also, 80% MeOH extract showed 336.57±2.03 μg/ml, which was the highest α-glucosidase inhibition among all extracts.

Pectin은 식물 세포벽의 주요성분으로 과실이나 채소류의 세포막이나 세포막 사이의 엷은 층에 존재하 며, 고점도의 수용성 다당체로 염과 pH에 의한 점도 변화가 심하며, 알코올류와 만나 gelation 되는 특징을 가 지고 있다. 식품분야에서 펙틴은 점도의 증가 및 겔 형성제로 사용되어 왔으나, 화장품 분야에서는 그 사용이 극히 제한적이었다. 본 연구에서는 pectin 효소 분해물의 분해 정도 및 분자량 분포를 정확히 확인하기 위해, HPLC (GPC)를 이용한 분석 조건을 확립하였으며, 생물 전환 공정을 통해 저분자량 pectin oligomer가 형성 되는 것을 확인하였다. 그리고, 2종의 효소에 대한 저분자량 pectin oligomer 생산 최적 조건 실험을 진행하여 최적 생산 조건을 확립하였으며, 이로 부터 제조한 pectin 효소분해물에서 저분자량 pectin oligomer를 선별적 으로 분리하는 공정도 개발하였다. 이러한 공정을 통해 제조된 저분자량 pectin oligomer 소재 LMPH A 와 B는 약 200 ∼ 2,700 Da 정도의 분자량 분포를 가지는 것으로 확인되었다. LMPH A와 B의 생리활성을 확인 한 결과, 2종 모두 항산화 활성을 보였다. 게다가, 이들이 pectin 및 D-galacturonic acid 보다 상대적으로 우 수하며, 농도의존적으로 증가함을 보였다. 또한 자외선(UVB)에 의한 피부세포의 광손상 및 이로 인한 apoptosis를 방어하는 효과를 나타내었다. 세포 활성화 효과 측정결과는 LMPH A, B 모두 0.025% 이상의 농도에 서 세포 활성화 효과를 보였으며, 농도가 0.5%에 이를 때까지 농도 의존적으로 증가하는 것을 확인할 수 있었 다. 특히, LMPH B의 경우, 0.5% 농도에서 약 30%, LMPH A도 약 22%의 매우 우수한 세포 활성화 효과를 가지는 것으로 확인되었다. 결론적으로, 본 연구를 통해 개발된 2종의 LMPH가 우수한 생리활성과 동시에 우수 한 안전성을 보임으로써, 향후 화장품 소재로 응용 가능성이 매우 높을 것으로 기대된다.

Citrus canker caused by Xanthomonas citri is a notorious disease affecting a decrease in fruit productivity and quality. Citrus export to USA is also prohibited by the disease. Therefore, development of citrus canker resistant variety is essential and exploitation of markers for molecular breeding is urgent. To develop DNA molecular markers, we performed whole genome resequencing for 8 varieties: 4 citrus canker resistant varieties including C. hybrid ‘Kioymi’ and 4 citrus canker susceptible varieties including C. iyo ‘Miyauchiiyokan’. In total, 642 polymorphic SNPs were detected between resistant and susceptible varieties. Of the 642 SNPs, 50 SNPs were preferably selected based on integrative genomics viewer. To apply the markers in a broad range of citrus variety, we performed genotyping with 6 other varieties very well known as citrus canker resistant and susceptible varieties in addition to previous mentioned 8 varieties. Three of the 50 SNPs were identified as a marker to distinguish citrus canker resistant varieties from susceptible varieties. Secondly, we developed molecular markers to apply for F1 lines crossed by ‘Kiyomi’ and ‘Miyauchiiyokan’. Of the 50 SNPs, we identified 2 SNP markers to distinguish between F1 resistant and susceptible lines. One of them is a resistance gene that plays a role in plant defense mechanism. In this study, we developed 5 molecular marker candidates possible to apply for molecular breeding to develop citrus canker resistant variety. We are working on development of candidate markers related to citrus canker.

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated two full-length cDNA gene, CuCRTISO and CuCRTISO-like, from Citrus unshiu. To confirm whether these two genes have the function of the carotenoid isomerase, The full-length cDNA of CuCRTISO and CuCRTISO-like gene, respectively, were fused with 35S promoter and NOS terminal region and then transformed into tomato CRTISO mutant, Tangerine, which shows orange fruit due to lack of carotenoid isomerase activity. The mature fruit color of the transgenic line expressing CuCRTISO gene changed from orange to red, which was similar to the fruit color of the tomato “Money Maker”. We also carried out HPLC analysis to detect all-trans lycopene, which is produced from prolycopene by carotenoid isomerase. In the transgenic line expressing CuCRTISO the all trans lycopene was detected from mature fruit but in the tangerine mutant several prolycopenes were detected from it. On the other hand, the transgenic line expression of CuCRTISO gene retained the orange-color fruit at the mature stage as Tangerine mutant. These studies indicate that the CuCRTISO gene has a function of carotenoid isomerase and also plays a role of it in other plant species, and that the CuCRTISO-like gene might be not enough to produce the all trans lycopene or has a another unknown function(s).

Polyembryony in many citrus varieties is an impediment in breeding because it makes hard to identify hybrids after crossbreeding. So, it has become imperative for developing efficient methods to distinguish zygotic seedling generated from polyembryonic seed depending on citrus variety. Simple sequence repeat(SSR) marker is one of useful systems for such purpose. However, SSR markers to separate zygotic seedlings derived from the crossbreeding between ‘Marita unshiu’ (Citrus unshiu) ‘Seongjeon’ and ‘Shiranuhi mandarin’ [(C. unshiu x C. sinensis) x C. reticulata] ‘Hallabong’ have not been developed yet. In this study we tried to identify an effective SSR marker to screen zygotic seedling after crossbreeding between ‘Seongjeon’ and ‘Hallabong’. For this investigation, 387 seedlings were generated from 114 seeds produced from crossing those two varieties. A total of 116 SSR markers were tested to identify a special marker for distinguishing origin, zygotic or nucellar seedling. As a result, two markers, SSR012 and SSR093, were found to be more effective than other markers. These two SSR markers might be useful to select zygotic individuals in crossbreeing between ‘Seongjeon’ and ‘Hallabong’.

CMA banding patterns of chromosomes of eleven Jeju citrus landraces were characterized and compared by means of sequential staining using fluorochromes of chromomycin A3 (CMA) and 4’,6-diamidino-2-phenylindole (DAPI). The somatic metaphase chromosomes examined in this study were all diploids (2n = 18). Chromosomes were classified into five types based on the number and distribution of CMA positive bands; A: two telomeric and one proximal bands, B: one telomeric and one proximal bands, C: two telomeric bands, D: one telomeric band, E: no band. Four to five types of chromosomes and unique chromosome compositions were observed from each accession. The CMA banding patterns of Jeju citrus landraces were 1A+1B+1C+9D+6E in jinkyul, 1A+1B+1C+8D+7E in cheongkyul, 1B+1C+10D+6E in hongkyul, 2A+1B+3C+6D+6E in sadoogam, 1A+2B+1C+8D+6E in dangyooza, 1A+1B+3C+7D+6E in dong-geongkyul, 2B+2C+7D+7E in pyunkyul, 2A+2B+2C+6D+6E in gamza, 1A+2B+1C+7D+7E in byungkyul, 1A+1B+1C+9D+6E in jigak, 1A+1C+10D+6E in binkyul. Type D and E chromosomes were predominant in all Jeju citrus landraces. The chromosome composition with an even number distribution in gamza was observed, hence it could be recognized as a non-hybrid species. The results indicated all Jeju citrus landraces except gamza seemed to be hybrids, but might be diverged from species originated or cultivated in Jeju, Korea and other countries.

The peel of Citrus sunki exhibits multiple biological activities such as anti-oxidant, anti-inflammation and anti-obesity, but little is known about neurodegeneration-related activities. In this study, we investigated the protective effect of ethanolic extract from both immature and mature Citrus sunki peel on neuronal cell death. Treatment of the neuroblastoma cell line SH-SY5Y with MPP+, an inducer of Parkinson disease model, increased cell death in a dose dependent manner. Increased levels of active caspase-3 and cleaved PARP were detected. Treatment with immature Citrus sunki peel extract significantly reduced MPP+-induced neurotoxicity. Cytoprotection with immature Citrus sunki peel extract was associated with a decrease in caspase-3 activation and PARP cleavage. In contrast, mature Citrus sunki peel extract had no significant effects. These data suggest that immature Citrus sunki peel extract may exert anti-apoptotic effect through the inhibition of caspase-3 signaling pathway on MPP+-induced neuronal cell death.

The major objective of this study was to investigate the usability as cultivar for processing of new Citrus hybrid 'Hamilgam'. We investigated various antioxidant activities, such as DPPH radical, ABTS radical, Hydroxyl radical and Superoxide anion radical effect, along with total polyphenol content and total flavonoid content of Citrus hybrid 'Hamilgam', C. unshiu, C. natsudaidai. The total polyphenol content and total flavonoid content were the highest in the Hamilgam. The major flavonoids of Hamilgam and Natsudaidai were naringin and neohesperidin, and those of Unshu were narirutin and hesperidin, as determined by HPLC. Especially, the neohesperidin content of Hamilgam showed much higher (100 times over) than that of Unshu. However, the aglycone form was not found. DPPH and ABTS radical scavenging activities were the highest in Hamilgam peels. Hydroxyl radical scavenging activity was high in the order of Unshu, Hamilgam and Natsudaidai. Superoxide anion radical scavenging of 3 cultivars peels displayed low activities compared to DPPH and ABTS radical scavenging. Based on these results, Hamilgam peel extracts possess antioxidant activities and may thus serve as potential sources of functional food, cosmetic products, etc.

Citrus canker caused by Xanthomonas citri pv. citri is one of economically important diseases in the citrus industry. The devastating bacterial disease results in unattractive quality and a significant reduction in fruit production. Citrus growers and industry in Korea has been struggling with the serious disease causing the prohibition of export market. Korea also became the top import market for oranges. The development of markers linked to citrus canker resistance is strongly needed. In this study, we investigated molecular markers between ‘Kiyomi’ (Citrus unshiu ｘ C. sinensis), a resistant cultivar, and Natsudaidai (C. natsudaidai), a susceptible cultivar. To develop markers, we focused on structural variation (copy number variation, CNV, and presence/absence variation, PAV). It has been well documented that CNV and PAV of defense-related genes are associated with resistant cultivars. Using a read depth approach following next-generation sequencing, we performed genome-wide analysis of CNV and PAV in two varieties. As a result, 633 genes showing at least two times difference between the mapping reads from two varieties and 61 genes showing presence of the mapping reads in only either one of them were screened. Visual inspection using the Integrative Genomics Viewer (IGV) was performed and experimental validation is being investigated. Interestingly, one of PAV candidates showed polymorphism in ‘Kiyomi’ and ‘Natsudaidai’ as well as other resistant and susceptible cultivars. Our results suggest a necessity for the detection of structural variation and indicate that the candidates may be useful for molecular breeding for citrus canker resistance and understanding disease resistance mechanism.

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

Miraculin can modify a sour taste into a sweet taste and is an alternative to traditional sweeteners. Mirculin gene was introduced into the Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.) callus by Agrobacterum-mediated transformation to produce citrus transgenic plant. Transgenic plant candidates were selected via hygromycin resistance. Transformation was confirmed by Polymerase Chain Reaction, Southern blotting, and Western blot analysis. Expression of this gene in transgenic citrus resulted in the accumulation of miraculin protein in the leaves. Multiple Shoots of transgenic citrus planets were micrografted onto trifoliate rootstocks in the sterile soil. Plants were established in the greenhouse 2 years after planting.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes cinnamyl aldehydes into cinnamyl alcohols, the final step in lignin biosynthesis. In this studied the purification, identification and characterization of a new cinnamyl alcohol dehydrogenase gene isolated from Citrus platymamma hort. Ex Tanaka. We expressed CAD potential gene in E.coli and then characterized its features in variety of specificity aldehydes substrates. The recombinant CAD protein was shown highest efficiently catalytic toward cinnamyl and coniferyl aldehydes and the shown lowest efficiently catalytic toward sinapyl aldehydes. We used a new improved analytical HPLC method in CAD enzymatic assay for fast and accurately measurement in various aldehydes substrates. In conclusion, our studies indicated the enzymatic activity of cDNA cloned CAD protein from Citrus platymamma hort. Ex Tanaka.

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated a full-length promoter region of CuCRTISO from Citrus unshiu. We determined if the promoter encoded organ-specific or developmental-specific expression, and identified possible cis-acting promoter elements. The full-length promoter and two truncated versions were fused to the β-glucuronidase (GUS) gene and transformed into Arabidopsis thaliana. Transgenic lines expressing the full-length promoter (pCiso-Prom1) and truncated promoters (pCiso-Prom2 and pCiso-Prom3) showed the same developmental and organ-specific activity. GUS expression was detected in the cotyledon and root at 5 and 10 days after germination, mature leaf, and anther. The CuCRTISO promoter contained several cis-acting elements involved in hormonal and environmental stress. Drought stress or abscisic acid treatment did not induce GUS expression in any transgenic lines. Heat stress induced GUS expression in the pCiso-Prom1 line; this promoter construct contains the heat-stress responsive element (HSE). Ethylene and cold-stress treatments induced GUS expression only in the pCiso-Prom3 line, although all transgenic lines contained the same cis-acting ethylene and low-temperature response elements. which could indicate the existence of unknown repressor element(s) in the CuCRTISO promoter. These studies indicate that CuCRTISO promoter activity is regulated in a developmental and organ-specific manner that responds to heat, cold, and ethylene. These results provide new insights into the role of cis-acting element(s) in CuCRTISO promoter activity. (This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2010-0007627 and 2009-0094059), and by Golden Seed Project, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS), Republic of Korea)

we analyzed the survival rate through the measurement of germinability of the gamma-ray irradiated buds of 4 Citrus species, C. hybrid ‘Kiomi’, C. hybrid ‘Setoka’, C. hybrid ‘Kanpei’, and C. junos, in greenhouse. To induce mutation, spring buds of Citrus with 0, 10, 20, 40, 60, 80 and 100 Gy of gamma radiation were irradiated and then were vgrafted onto trifoliate orange rootstock. The rootstock number per gamma ray dosage was two hundreds. After after 6 months growth, the shoot germination rate from gamma ray-irradiated buds presented lower viability than those of non-irradiated buds. Higher dossage of irradiation more inhibited bud germination at all experimental groups of citrus. The viability of unirradiated citrus plants was C. junos > ‘Setoka’ > ‘Kiomi’ > ‘Kanpei’. We have determined LD50 of gamma ray-irradiation. This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2010-0007627 and 2009-0094059), and by Golden Seed Project, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS), Republic of Korea

본 연구는 유자나무에 발생하는 주요 병을 방제하기 위한 친환경 유기농업자재를 선발하기 위하여 수행되었다. Phomopsi scitri에 의한 유자 검은점무늬병은(Meloanose)과 Elsinoe fawcettii에 의한 유자 더뎅이병(Scab)은 유자나무에서 가장 일반적인 병으로 알려져 있다. 병 발생은 6월 하순경에 피해가 큰 것은 6월 동안의 강우량과 상대습도와 밀접한 연관관계를 가지고 있다. 또한 이 병들은 수령이 낮은 유자보다는 수령이 높은 유자에서 심하게 발생하는 경향이 있다. 유자 검은점무늬병에 대한 방제효과는 농가의 관행재배구가 78.3%였으며, 친환경유기농업자재는 49.1~72.6% 방제효과를 보였다. 반면, 유자 더뎅이병 시험에서 농가의 관행재배구에 대한 방제효과는 82.6%였으며, 공시자재인 친환경유기농업자재는 41.1~71.8%의 방제효과를 보였다. 각각의 시험에서 통계적 유의성을 검정한 결과 통계적으로 유의성이 있었다. 본 시험결과에서 친환경유기농업자재는 유자의 검은점무늬병 및 더뎅이병에 대한 방제방법으로 제시할 수 있었다.