Su Hyun Yun

Agrobacterium -mediated transformation using embryogenic calli in Satsuma mandarin ( Citrus unshiu Marc.) cv. Miyagawa wase

Agrobacterium-mediated transformation in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase was achieved with reasonable transformation efficiency of about 22%, which was the percentage of transgenic plantlets confirmed by genomic PCR (37 plantlets/168 hygromycin-resistant calli). Embryogenic calli of Miyagawa wase were infected with Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA1300 that contained miraculin gene (a taste-modifying protein) and hygromycin as a selection marker. After 5 days of co-culture in the medium containing 100 μM acetosyringone, calli were transferred to the liquid half embryogenic cell culture medium (half concentration of Murashige and Tucker’s (MT) medium modified with the addition of 500 mg·L− malt extract, 50 g·L− sucrose and 1.55 g·L− glutamine) with 15 mg·L− hygromycin and 250 mg·L− cefotaxime, and were cultured for two weeks. Then, the calli were grown on the solid selection medium with 20 mg·L− hygromycin for four weeks and 25 mg·L− hygromycin for another four weeks. The resistant embryos were selected and transferred to the embryo maturation medium. After 3 weeks of culture, the heart shaped embryos were transferred to the MT medium containing 1.0 mg ·L− GA3, 20.0 mL·L− coconut water, 0.02 mg·L− NAA and 0.0146 mg·L− coumarin for embryo germination. Finally the germinated embryos were cultured on MT medium containing 3.0% sucrose and 0.8% agar for growing to the normal plant. Stable integration of the transgene in the plant genome was confirmed by PCR and Southern blot analysis.

Development of a SCAR Marker Linked to Male Fertility Traits in 'Jinkyool' (Citrus sunki)

In Citrus, an F1 segregation population of 150 plants was constructed from a cross between ‘Kiyomi’ (C. unshiu × C. sinensis) carrying the male sterility trait and ‘Jinkyool’ (C. sunki). Sequence-related amplification polymorphism (SRAP) combined with bulked segregant analysis was used to develop markers linked to male fertility. In the F1 population, 66 out of 150 seedlings had aborted anthers and the ratio of male sterile plants to fertile plants in the progenies matched the expected Mendelian segregation ratio of 1:1 (χ² =2.16 at p=0.05). From the profiling of the 197 SRAP primer sets, three SRAP primer sets (F4/R27, F39/R60, and F15/R37) that were closely linked to the target trait were identified and successfully converted into a sequence characterized amplified region (SCAR) marker for selection of male fertility in citrus. The SCAR marker, using the pMS 33U/pMS 1462L primer set specifically, produced a single 1.4-Kb fragment that was linked to male fertility. Our results suggested that this SCAR marker can be useful for marker-assisted selection of male sterile individuals in breeding F₁ progenies in Citrus.

Antioxidative Activities of New Citrus Hybrid 'Hamilgam' Peel Extracts

*Citrus Research Station, NIHHS, RDA, Jeju 699-943, Korea.**Planning and Coordination Division, NIHHS, RDA, Suwon 440-706, Korea.ABSTRACT : The major objective of this study was to investigate the usability as cultivar for processing of new Citrushybrid ‘Hamilgam’. We investigated various antioxidant activities, such as DPPH radical, ABTS radical, Hydroxyl radicaland Superoxide anion radical effect, along with total polyphenol content and total flavonoid content of Citrus hybrid ‘Hamil-gam’, 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 narirutinand hesperidin, as determined by HPLC. Especially, the neohesperidin content of Hamilgam showed much higher (100times over) than that of Unshu. However, the aglycone form was not found. DPPH and ABTS radical scavenging activitieswere the highest in Hamilgam peels. Hydroxyl radical scavenging activity was high in the order of Unshu, Hamilgam andNatsudaidai. Superoxide anion radical scavenging of 3 cultivars peels displayed low activities compared to DPPH and ABTSradical scavenging. Based on these results, Hamilgam peel extracts possess antioxidant activities and may thus serve aspotential sources of functional food, cosmetic products, etc.Key Words : Citrus, Hamilgam, Polyphenol, Antioxidant Activities, Functional Food

Micrografting and Heat Treatment Combination for Eliminating Virus of CTV-infected Citrus

This study was conducted to eliminate viruses from citrus-infected plants using micrografting and thermotherapy. Six citrus cultivars including a ‘Setoka’ hybrid were used as plant sources. The TAS-ELISA technique demonstrated that several plants were CTV positive. However, no CTV symptoms were detected in plants obtained from shoots and treated at a high temperature of 40°C during the day and night and micrografted for two weeks with old trifoliate orange rootstock in vitro. Indexing of CTV, SDV, and CTLV for RT-PCR analysis of the eleven citrus seedlings, including ‘Setoka’, ‘Samdajosang’, ‘Pungkwang’, ‘Shiranuhi’, and ‘Ehimekashi dai28go’ was virus free following the micrografting and thermal therapy.

Early Identification of Putative Zygotic Seedlings in Citrus Crosses between ‘Morita unshiu’ (Citrus. unshiu Marc.) and ‘Ponkan’ (C. reticulata Blanco) Using RAPD and SRAP

The purpose of this study was to evaluate the methods of identifying zygotic seedlings of crosses between ‘Morita unshiu’ (Citrus. unshiuMarc.) and ‘Ponkan’ (C. reticulataBlanco). In order to investigate the frequency and position of zygotic seedlings and to determine the efficiency of zygotic seedling identification, random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) were performed using UBC (9, 27, 229, 230, and 254) primers and F4/R27, F7/R14, F12/R10, and F44/R62 primer sets, respectively. A total of 37 putative zygotic seedlings out of 55 individuals were selected by RAPD and SRAP. The F7/R14 primer pair showed a screening ability of 45.5% (25/55), whereas the primer UBC27 revealed the highest efficiency of zygotic seedling identification (50.9%, 28/55). When both UBC27 and F7/R14 were properly used for selection of hybridized seedlings of ‘Morita unshiu’ (C. unshiuMarc.) and ‘Ponkan’ (C. reticulataBlanco), screening efficiency was increased to 60% (33/55) for putative zygotic seedlings. Thus, it is possible to select putative hybrid zygotic seedlings in an accurate and effective manner by RAPD and SRAP.

Molecular characterization of an S-adenosylmethionine decarboxylase gene from floral organ differentiating axillary bud in calamondin (× Citrofortunella mitis)

Summary Calamondin (× Citrofortunella mitis J. Ingram & H. E. Moore) is known for its ability to develop floral organs to flower in the adult phase during the four seasons. To provide information on molecular events during floral differentiation from axillary buds, we constructed a cDNA library from floral differentiating axillary buds of adult phase calamondin. Ninety-six cDNA clones were randomly selected from this library and sequenced. Ninety-six nonredundant ESTs were identified. Two clones encoded S-adenosylmethione decarboxylase, a key enzymes in polyamine biosynthesis. CmSAMDC 1 (Citrofortunella mitis s-adenosylmethionine decarboxylase 1) contained an 1083 bp ORF that encoded a putative SAMDC precursor of 361 amino acids. Northern blot analysis revealed that CmSAMDC 1 was expressed in axillary buds prior to floral differentiation and in axillary buds immediately after floral differentiation, in immature flower (5 mm), in fully developed flowers (120 mm long), in floral parts (petals, pistils, and stamens) of trees in the adult phase of development, but not in leaves or axillary buds of juvenile phase nucellar seedlings or leaf tissue from trees in adult phase of development. In situ hybridization revealed expression of the CmSAMDC 1 gene in the floral apex of axillary buds after differentiation, and in the phloem of axillary buds and petioles. These results suggest that SAMDC could play a role in actively differentiating and developing reproductive and vegetative organs.

Frequency of Spontaneous Polyploids in Monoembryonic Jeju Native Citrus Species and Some Mandarin Cultivars

Polyploids are a potentially important germplasm source in seedless citrus breeding program. Seedlessness is one of the most promising traits of commercial mandarin breeds that mandarin triploid hybrids possess permanently. The formation of new constant triploid hybrids can be recovered through diploid species hybridization from the fusion of divalent gametes at low frequencyor intra-and inter-ploidy crosses. However, extensive breeding work based on small F1 hybrid seeds developed is impossible without a very effective aseptic methodology and ploidy event. In this study, in vitro embryo culture was employed to recover natural hybrids from monoembryonic diploid, open-pollinated mandarin. Flow cytometry was used to determine ploidy level. A total of 10,289 seeds were extracted from 792 fruits having approximately 13 seeds per fruit. Average frequency of small seeds developed was 7.1%, while the average frequency of small seeds per fruit were: 8.9% for ‘Clementine’ 10.2% for ‘Harehime’ 2.6% for ‘Kamja’ 3.1% for ‘Pyunkyool’ 2.8% for ‘Sadookam’ and 7.0% for ‘Wilking’ mandarin. Average size of a perfect seed was 49.52±0.07 mm2 (‘Clementine’) while the small seed measured 7.95±0.04 mm2 (‘Clementine’), which was about 1/6 smaller than the perfect seed. In total, 731 small seeds were obtained and all of them contained only one embryo per seed. The efficiency of ‘Clementine’ was 14 times higher than ‘Wilking’ and more than 109 times higher than ‘Pyunkyool’. The basic information on spontaneous polyploidy provides for the hybridization of constant triploids and increases the efficiency of conventional cross.

Improvement of Seed Germination in a Spontaneous Autotetraploid of Poncirus and Chlorophyll Fluorescence of Seedlings in Salt Stress

Speed germination success and robust vegetative growth of citrus rootstock through improved sowing methods and fertilizer inputs offer the usage of root system for the citrus. The current study evaluated the influence of seed coat removal and different fertilizer concentrations on plant germination and plant growth of spontaneous rootstock siblings. Decoated and coated seeds of diploid and tetraploid plants were sown in tubes. Commercial fertilizer concentrations of 0, 2, 4, 6, 8 and were added. The experimental layout followed a randomized block factorial design (seed coat removal fertilizer concentration) for each rootstock. Fertilizer concentrations were 0, 10, 20 and of the fertilizer for the resistance of the strength on the salt level. The germination rate of seeds without testa sown in vitro was improved (67-80%) compared to that of nontreated seeds. The eventual tree height of the seeds without testa in the diploid group was increased due to higher fertilization compared to that in the nontreated group. The removal of seed testa promoted the seed germination of both diploid and tetraploid trifoliate orange and resulted in greater height. Their vegetative development was also increased due to the increased fertilization of the rootstock. The Fv/Fm value for the diploid plants was 0.4 and 0.8 for the tetraploid ones under salt stress after 11 days of treatment. The removal of seed testa may improve the seed germination of trifoliate orange. Tetraploid trifoliate orange appears to possess resistance to salt stress compared to the diploid variety.