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.

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 mm 2 ('Clementine') while the small seed measured 7.95±0.04 mm 2 ('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.

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 (χ 2 =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.

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 10 g․l -1 were added. The experimental layout followed a randomized block 2×6 factorial design (seed coat removal × fertilizer concentration) for each rootstock. Fertilizer concentrations were 0, 10, 20 and 30 g․l -1 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. (Fig. 1)

A new mandarin hybrid cultivar 'Shinyegam' was developed from a cross between 'Kiyomi' (Citrus unshiu × C. sinensis) and 'Wilking' (Citrus reticulata) at the Citrus Research Institute of the National Institute of Horticultural and Herbal Science in 2012. The 'Jegam na No. 33', a first selection seedling from the fruits obtained in 2008 was finally named 'Shinyegam' after field evaluation trials at five locations in Jeju island, Korea from 2010 to 2012. The tree vigor was intermediate and the tree was spreading in nature, similar to that of the 'Kiyomi' cultivar. The fruits produced were compressed to oblate and globose in shape, were a deep orange in color, and moderately weighed at 130 g. 'Shinyegam' produces fruit that mature in late December, and has few seeds, which are mono-embryonic. The average seed number of a fruit varied depending on the pollinator citrus tree. Cultivation of 'Shinyegam' along with the other cultivars possessing high pollen fertility resulted in seed numbers that was more than 20, while in the absence of a pollinator species the seed number was around 5. The fruit contained soluble solids at 11 to 12 ° Bx and the acidity was approximately 1% at maturity. The rind thickness of approximately 2.5 mm ensures easy peeling. The flesh is very tender and juicy, with a pleasant, aromatic flavor similar to that of 'Wilking' but is comparatively weak. 'Shinyegam' is moderately resistant to the citrus scab disease and melanose, but susceptible to citrus canker. Owing to the high quality of fruit, good aroma, and cold hardness characteristics, this cultivar is expected to satisfy consumer acceptability in terms of the diversity of the cultivar in open-field cultivation.