Citrus Variety 'Jedae mandarin'

Abstract

The present disclosure relates to ‘Jedae mandarin’, a new citrus variety of early-ripening satsuma mandarin. The branch buds of C. unshiu Marc. cv. Miyagawa-wase, an early-maturing satsuma mandarin, underwent gamma irradiation and subsequent grafting for asexual propagation. As a result, the ‘Jedae mandarin’, a novel variety of citrus plant, showed uneven peels of fruits and changes in flavonoid content, compared to C. unshiu Marc. cv. Miyagawa-wase, although it exhibited characteristics very similar to those of C. unshiu Marc. cv. Miyagawa-wase in terms of blooming, fruit-bearing, sugar content, acidity, etc. Thus, ‘Jedae mandarin’ may be used as a new variety with high economic value.

Claims

1. A variant of citrus plant of a novel variety named ‘Jedae mandarin’, substantially as illustrated and described herein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 is a photograph showing the breeding process of named ‘Jedae mandarin’ bred in accordance with the Embodiment 1 of the present invention.

[0058] FIG. 2 is a photograph taken on Jul. 2, 2013, showing the fruit bearing of named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention (cultivated in Donghong-dong, Seogwipo-si, Jeju Special Self-Governing Province, Korea).

[0059] FIG. 3 is a photograph taken on Nov. 20, 2013, showing the fruit bearing of named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention (cultivated in Donghong-dong, Seogwipo-si, Jeju Special Self-Governing Province, Korea).

[0060] FIG. 4 is a photograph taken in early July 2013, showing the fruit of mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention.

[0061] FIG. 5 is a photograph taken in early November 2013, showing the fruit of mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention.

[0062] FIG. 6 is a photograph taken in early December 2013, showing the fruit of mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention.

[0063] FIG. 7 is a photograph taken in early December 2013, showing the fruit of C. unshiu Marc. cv. Miyagawa-wase cultivated under the conditions same as those for Jedae mandarin bred in accordance with Embodiment 1 of the present invention.

[0064] FIG. 8 is a side view illustration of mature fruit of mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention.

[0065] FIG. 9 is a photograph comparing the difference in external appearance of stalk(top) of citrus between the fruit of mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention(on the right side) and the fruit of C. unshiu Marc. cv. Miyagawa-wase(on the left side).

[0066] FIG. 10 is a photograph comparing the difference in external appearance of the rear(bottom) of citrus between the fruit of mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention(on the right side) and the fruit of C. unshiu Marc. cv. Miyagawa-wase(on the left side).

[0067] FIG. 11 is a photograph comparing the difference in citrus fruit cross-section of between mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention and C. unshiu Marc. cv. Miyagawa-wase(top and left side: C. unshiu Marc. cv. Miyagawa-wase/right side: Jedae mandarin).

[0068] FIG. 12 is a photograph comparing the difference in white area(albedo layer) inside fruit peel between mutant citrus named ‘Jedae mandarin’ bred in accordance with Embodiment 1 of the present invention and C. unshiu Marc. cv. Miyagawa-wase(bottom and left side: C. unshiu Marc. cv. Miyagawa-wase/right side: Jedae mandarin).

[0069] FIG. 13 is a photograph (20×) showing the results of microscopic observation of the peel of young fruit(harvested in June 2013) of C. unshiu Marc. cv. Miyagawa-wase at cellular level in accordance with Embodiment 3 of the present invention.

[0070] FIG. 14 is a photograph (20×) showing the results of microscopic observation of the peel of young fruit(harvested in June 2013) of Jedae mandarin at cellular level in accordance with Embodiment 3 of the present invention.

[0071] FIG. 15 is a photograph (40×) showing the results of microscopic observation of the peel of mature fruit(harvested in December 2013) of C. unshiu Marc. cv. Miyagawa-wase at cellular level in accordance with Embodiment 3 of the present invention(the arrow indicates the epidermal cell cytolysis).

[0072] FIG. 16 is a photograph (40×) showing the results of microscopic observation of the peel of mature fruit(harvested in December 2013) of Jedae mandarin at cellular level in accordance with Embodiment 3 of the present invention.

[0073] FIG. 17 is a photograph comparing the difference in leaf shape between Jedae mandarin citrus tree bred in accordance with this Embodiment of the present invention and C. unshiu Marc. cv. Miyagawa-wase citrus tree.

DETAILED DESCRIPTION

[0074] Hereinafter, exemplary embodiments of the present disclosure were described in detail by referring to the accompanying drawings to increase the ease of embodiment by those with general knowledge in concerned technical fields of the present invention. The present invention may, however, be embodied in many different forms and is not limited to the embodiments set forth herein.

Embodiment 1 Breeding of citrus mutated by radiation

[0075] FIG. 1 is a photograph showing the breeding process of named ‘Jedae mandarin’ bred in accordance with the Embodiment 1 of the present invention. The mutant citrus breeding process using radiation was described by referring to FIG. 1 hereinafter.

[0076] In the present invention, C. unshiu Marc. cv. Miyagawa-wase, cultivated the most widely in Jeju Province, was selected as a material for a breeding experiment. Thus, 5 kg of branches of C. unshiu Marc. cv. Miyagawa-wase of early-ripening satsuma mandarin was obtained from the Agricultural Research & Extension Services of Jeju Special Self-Governing Province for use in aforesaid experiment. At this time, the branches of C. unshiu Marc. cv. Miyagawa-wase used in the experiment had the scions collected for the period between early February and mid February for radiation-induced mutation of buds. The branches, collected in that way, were stored in 4 refrigerated conditions for 3 months from mid April to early May, the period of grafting, before being used.

[0077] The buds of branches of C. unshiu Marc. cv. Miyagawa-wase, obtained from the Agricultural Research & Extension Services of Jeju Special Self-Governing Province, were irradiated with gamma irradiation at the cobalt-60 (.sup.60Co) irradiation facility (source: C-188, Nordion International Ltd., Canada) in the Radiation & Applied Science Laboratory (currently, the Institute for Nuclear Science & Technology) of Jeju National University to induce mutation. Then, the irradiated buds mentioned above were joined with adult trees of C. unshiu Marc. cv. Miyagawa-wase through top-grafting. Cultivation and maintenance were carried out in accordance with customary methods.

[0078] Specifically, mutation was induced in the buds of branches of C. unshiu Marc. cv. Miyagawa-wase with gamma irradiation from the Cobalt-60 (.sup.60Co) irradiation facility at Radiation & Applied Science Laboratory of Jeju National University in April 2006. At this time, the buds of branches of C. unshiu Marc. cv. Miyagawa-wase were irradiated with gamma rays emitted at a distance of 0.68 m from the radiation source at a dose of 40 Gy for 22 hours. The buds of branches of C. unshiu Marc. cv. Miyagawa-wase, which were irradiated with gamma rays to induce mutation, were grated onto adult trees of C. unshiu Marc. cv. Miyagawa-wase which had been used as rootstock at Dongheung-dong packaging room of the Research & Practice Center affiliated with Jeju National University to induce branching. Selection of branches was performed after investigation of their traits. A total of 889 scions were grafted. Total germination rate stood at 80.3% (714 buds in all) as found in the germination rate survey after 2 months from the date of grafting.

[0079] The buds irradiated with gamma ray were grafted onto adult trees of C. unshiu Marc. cv. Miyagawa-wase (citrus packaging room of the Research & Practice Center affiliated with Jeju National University) to induce branching, and then the mutant branches were selected upon investigation of the traits based on whether the fruit shape was changed.

[0080] In addition, fruits generated from each mutant branch were stored in a low-temperature storage (5° C.) after they were harvested from late November to early December every year during the period spanning between 2009 and 2014.

[0081] Moreover, weight, diameter, peel thickness, sugar content, acidity, color, etc., of the fruits were measured.

[0082] The weight of fruit was measured up to 0.1 g unit by using an electronic indicator scale (CAS Co., Ltd, Korea). The longitudinal diameter and transverse diameter were measured up to 0.1 mm by using the digital caliper (Mitutoyo Corporation, Japan).

[0083] After measuring the diameter and thickness of each fruit, juice was collected from each fruit. Sugar content and acidity were measured by the acidity and sugar content analyzer NH-2000 (HORIBA, Japan), and 4-5 mL of juice was inserted into the syringe as specified in the operation manual of the device.

[0084] Additionally, chroma meter (CR-400, Minolta) was used to measure the chromaticity of mutant fruits that exhibited changes in fruit color.

[0085] Changes in fruit shape were observed through naked eyes, while changes in the fruit peels were observed additionally at the cellular level with a microscope (Nikon microphoto type 114).

[0086] Mutant buds were collected again from the branches selected as mentioned above, and then, were grafted again onto rootstocks of adult trees and trifoliate orange trees(purchased from seedling suppliers) to induce fruit-bearing of citrus (see FIG. 1).

Embodiment 2 Analyses on Characteristics of Mutant Citrus, an Early-Maturing Satsuma Mandarin

[0087] 1) Comparison of general traits

[0088] The citrus fruits produced in accordance with aforesaid Embodiment 1 were characterized by comparing general traits of citruses and fruit trees, which bore fruits in 2013, with those of C. unshiu Marc. cv. Miyagawa-wase (see FIG. 2 and FIG. 3).

[0089] Specifically, the mutant citrus bred in accordance with aforesaid Embodiment 1 showed characteristics similar to those of C. unshiu Marc. cv. Miyagawa-wase, cultivated commonly in Jeju Province, in terms of general traits such as leaf shape, leaf color, blooming period, vitality of tree, etc., as shown in Table 1 below.

TABLE-US-00001 TABLE 1 Vitality Blooming Coloring of Period of Variety of Tree Period Peel Harvesting C. unshiu Moderate From May Late From late Marc. cv. 10 to 20 November November to Miyagawa-wase mid December Jedae mandarin Moderate From May Late From late 10 to 20 November November to mid December

[0090] 2) Comparison of characteristics of fruits

[0091] FIG. 4 to FIG. 7 present the photographs of fruits of mutant citrus named ‘Jedae mandarin’ bred in accordance with the Embodiment 1 of the present invention, which were taken in early July, early November, and early December, respectively, and the photographs of fruits of C. unshiu Marc. cv. Miyagawa-wase, the mother plant, which were taken in early December. Moreover, FIG. 8 shows side view photograph of mature fruit of Jedae mandarin and FIG. 9 shows the outer appearance of citrus leaf stalk which is compared with that of C. unshiu Marc. cv. Miyagawa-wase. FIG. 10 shows the photograph that compares the outer appearance of rear part of the citrus with that of C. unshiu Marc. cv. Miyagawa-wase.

[0092] Besides, the mutant citruses, which had been bred in accordance with aforesaid Embodiment 1 of the present invention and thereafter bloomed between May 10 and May 20(cultivated on open field in Donghong-dong, Seogwipo-si, Jeju Special Self-Governing Province), were harvested in early December to mid December for the period of 4 years. Harvested mutant citruses were investigated to determine their traits, and the results were summarized in Table 2.

TABLE-US-00002 TABLE 2 Longitudinal Transverse Hunter Color Value Diameter Diameter Fruit Peel Sugar L a b of Fruit of Fruit Weight Thickness Content Acidity (Light- (Red- (Yellow- Harvesting Year (mm) (mm) (g) (mm) (°Brix) (wt %) ness) ness) ness) Period Jedae mandarin 2009 62.3  78.1  156.73  4.2 10.2   0.97 64.41 33.95 64.66 Mid December 2010 54.8  66.5  131.6  2.1 7.8  0.78 67.78 29.56 72.44 Early December 2011 52.8  62.6  110.3  2.6 10.8   0.69 69.5  26.5  72.4  Early December 2013 34.9  46.3  41.1  2.5 11.8   0.85 66.48 30.69 68.77 Early December 2014 65.35 64.64 126.3   4.02 7.4  1.25 59.55 30.51 34.84 Early December C. unshiu Marc. cv. Miyagawa-wase 2009 43.7  50.1  67.4  2.3 8.2  0.83 66.88 26.96 73.37 Early December 2010 51.7  56.6  94.0  2.0 8.8  1.00 67.56 31.13 70.31 Early December 2011 50.1  60.0  94.6  2.4 9.6 0.9 39.36 28.77 72.18 Early December 2013 45.5  60.4  88.2  2.4 10.2   0.82 67.58 32.36 69.57 Early December 2014 57.6  61.38 102     3.44 7.4  1.25 58.33 30.74 33.32 Early December

[0093] FIG. 4 to FIG. 10, and Table 2 above showed that fruits of Jedae mandarin bred in accordance with the Embodiment 1 had the shape different from that of C. unshiu Marc. cv. Miyagawa-wase, the mother plant. In particular, the photographs of Jedae mandarin fruits in the drawings indicated that the fruit shape of Jedae mandarin was different significantly from that of C. unshiu Marc. cv. Miyagawa-wase, the mother plant, from which the mutation had occurred, depending on variations of peel traits.

[0094] Specifically, fruits of Jedae mandarin exhibited characteristics almost similar to those of fruits of C. unshiu Marc. cv. Miyagawa-wase in terms of sugar content and acidity. Fruits of Jedae mandarin were slightly larger than those of C. unshiu Marc. cv. Miyagawa-wase. In addition, fruits of Jedae mandarin had the peels that were less smooth and more rugged on outer surface in terms of fruit shape, compared to fruits of C. unshiu Marc. cv. Miyagawa-wase.

[0095] Fruits of Jedae mandarin had groove of radial shape from the fruit stalk and were found to have vertical corrugation in overall way when they were viewed from the side. Thus, fruits of Jedae mandarin were considered to have excellent fruit shape and have the potential to be developed into decorative citrus fruits.

[0096] In addition, fruits generated from Jedae mandarin maintain their altered shapes described above from young fruit bearing stage to maturation stage in early December, and can be harvested with those of C. unshiu Marc. cv. Miyagawa-wase almost around the same time. However, fruits of Jedae mandarin showed distinctive difference from those of C. unshiu Marc. cv. Miyagawa-wase in terms of fruit shape arising from changes in peel traits.

[0097] FIG. 11 shows the photograph that compares the citrus section of mutant citrus named ‘Jedae mandarin’ bred in accordance with the Embodiment 1 of the present invention with the fruit section of C. unshiu Marc. cv. Miyagawa-wase (top and left side: C. unshiu Marc. cv. Miyagawa-wase; right side: Jedae mandarin). FIG. 12 shows the photograph comparing the white portion (albedo layer) inside the peel with that of C. unshiu Marc. cv. Miyagawa-wase(bottom and left side: C. unshiu Marc. cv. Miyagawa-wase; right side: Jedae mandarin).

[0098] FIG. 11 and FIG. 12 above show that the mutant citrus named ‘Jedae mandarin’ is also characterized by smooth fiber not protruding roughly, regarding the surface that comes into contact with fruit flesh in albedo layer, unlike C. unshiu Marc. cv. Miyagawa-wase. Therefore, fruits of Jedae mandarin showed good adhesion between fruit and fruit flesh and good storability, but could not be easily peeled.

[0099] As shown in Table 2 and FIG. 4 through FIG. 12, the fruits of Jedae mandarin, a mutant citrus bred in accordance with aforesaid Embodiment 1, exhibited difference in traits, depending on climate (amount of precipitation, amount of sunshine, etc.) and fruit size of the year where irradiation occurred. However, the changes in fruit shape were found to be maintained stably.

[0100] 3) Comparison of content of functional component(flavonoid)

[0101] The harvested citrus fruits were washed twice with water to remove pesticides and other impurities. Fruit flesh and peel were separated and sliced to the thickness of 0.3 cm and then frozen for 3 days in a −70° C. ultra-low temperature refrigerator, followed by lyophilization(freeze-drying) for 72 hours. 100% methanol was selected as the extraction solvent based on the studies, such as the study by Senevirathne (2010, Journal of Food Engineering, 97, 168-176), et al., which involved the use of citrus, among existing literatures on analyses of flavonoid.

[0102] For extraction, the lyophilized portion of citrus(peel and fruit flesh) were pulverized with a pulverizer and powdered by using 0.5 mm mesh. 250 mL of 100% methanol was added to 2.5 g of the powdered citrus samples(10 mg/mL each). Then, flavonoid was extracted using a shaking incubator at 25° C. for 24 hours and the solid contents were removed. Then, the flavonoid content was used for analyses with HPLC system mentioned. below.

[0103] The HPLC system used a 600 pump from Waters (Milford, Mass.). Flavonoid was detected at a wavelength of 280 nm using the W2998 UV-VIS detector.

[0104] Shim-pack VP-ODS (C18) column (4.6×150 mm, 5 μm) of Shimadzu Corporation was used for analyses. Chromatograms were prepared with HPLC by using the 7 standard substances (rutin, naringin, neohesperidin, hesperidin, narirutin, naringenin, and hesperetin) purchased from Sigma (St. Louis, Mo., US), and then the best detection wavelengths were selected. That was followed by establishment of mobile phase conditions[Eluent A, acetonitrile/acetic acid (1000/5); Eluent B, DW/acetic acid (1000/5); flow rate, 1 mL/min] suitable for analyses of flavonoid for isolation of peaks. Based on the established mobile phase conditions, 10 μL of each extract was injected, followed by analyses using the standard curve.

[0105] Table 3 shows respective contents of hesperidin (HD) and narirutin (NRT) in citrus peel, which were extracted as described above. Table 4 shows respective contents of hesperidin (HD) and narirutin (NRT) in citrus fruit flesh. All experiments were repeated 3 times. Experimental values were expressed as mean value and standard deviation value. ANOVA(Analysis of Variance) was performed using the SPSS program (12.0), and concerned significant values were verified by Duncan's multiple range test (p<0.05).

TABLE-US-00003 TABLE 3 Content (mg/100 g dry 2010 2014 weight of peel) HD NRT 1-1D NRT Control 463.1 ± 1.11  60.8 ± 1.51 305.3 ± 0.85  22.2 ± 1.34 (C. unshiu Marc. cv. Miyagawa-wase) Jedae mandarin 546.6 ± 3.38*  78.5 ± 0.61* 431.6 ± 2.37*  43.7 ± 0.82* *shows the value with significant difference in comparison to the control (p <0.05).

TABLE-US-00004 TABLE 4 Content (mg/100 g dry 2010 2014 weight of fruit flesh) HD NRT HD NRT Control 178.8 ± 0.43  92.4 ± 0.59 75.3 ± 0.85 12.2 ± 1.34 (C. unshiu Marc. cv. Miyagawa-wase) Jedae mandarin 276.3 ± 2.07* 132.2 ± 1.53* 125.6 ± 1.01*  22.4 ± 0.29* *shows the value with significant difference in comparison to the control (p <0.05).

[0106] As shown in Tables 3 and Table 4, the contents of hesperidin and narirutin among flavonoid components varied, depending on the harvest year in which the amount of precipitation and sunshine differed. However, their contents in mutant citrus which had been bred were significantly higher than those in C. unshiu Marc. cv. Miyagawa-wase which was the control.

Embodiment 3 Cell-level microscopic Characterization of the Peel of Mutant Citrus, An Early-Maturing Satsuma Mandarin

[0107] Observation of delayed decay of fruit peel

[0108] The observation materials were the young fruits and maturated fruits of Jedae mandarin and C. unshiu Marc. cv. Miyagawa-wase harvested altogether in 2013. Outer peel of citrus fruit was cut into the size of 5 mm×5 mm and fixed in FAA solution. Then, the cross-section of 10 μm in thickness was prepared by Paraffin method, followed by double staining with Safranine and Fast Green and microscopic examination.

[0109] Fixation was carried out by applying the FAA method. Each plant slice was cut into the size of 5 mm×5 mm and placed in a FAA fixative solution containing 90 ml of 50% alcohol, 5 ml of formalin, and 5 ml of acetic acid, and then, were fixed for 24-72 hours through vacuum processing which aimed to remove the air from the inside of plant tissues.

[0110] The fixed tissue was treated for 5 hours in 50% alcohol and then dehydrated for the following hours: for 5 hour in solution I (10 ml of tert-butyl alcohol, 40 ml of 95% alcohol, 50 ml of distilled water); for 37 hours in solution II (20 ml of tert-butyl alcohol, 50 ml of 95% alcohol, 30 ml of distilled water); for 5 hours in solution III (35 ml of tert-butyl alcohol, 50 ml of 95% alcohol, 25 ml of distilled water); for 18 hours in solution IV (10 ml of tert-butyl alcohol, 45 ml of 55% alcohol, crystol violet 1%); for 5 hours in solution V (75 ml of tert-butyl alcohol, 25 ml of 100% alcohol); and for 5 hours and 72 hours in solution VI (100 ml of tert-butyl alcohol).

[0111] The paraffin solution, which had already been dissolved in the dehydrated tissue, was slowly poured, allowing the paraffin to slowly penetrate into the tissue. For the tissue sectioning, the transverse slices, each with a cross-section of 10 μm in thickness, were prepared by using the microtome.

[0112] Double staining with Safranine and Fast Green was carried out twice for 60 minutes and 10 minutes, respectively, in xylene. The slices were treated for 10 minutes in Xylene:Alcohol and for 10 minutes, respectively, in 100%, 95%, 85%, and 75% alcohol. Then, they were treated with 1% Safranine for 2 hours, 75% alcohol for 5 minutes, 0.5% Fast Green for 30 seconds, 95%, 100% and 100% alcohol for 5 minutes each.

[0113] In addition, they were treated twice, i.e., with Xylene:Alcohol for 5 minutes and then with Xylene for 10 minutes. Following that, they were observed with a microscope (Nikon microphoto type 114).

[0114] By using the method described above, cell-level microscopic observation was performed of the peel shape of young fruits of C. unshiu Marc. cv. Miyagawa-wase (harvested in June 2013), young fruits of Jedae mandarin (harvested in June 2013), mature fruits of C. unshiu Marc. cv. Miyagawa-wase (harvested in December, 2013), and mature fruits of Jedae mandarin(harvested in December, 2013). The results are presented in FIG. 13 through FIG. 16.

[0115] When the peels of C. unshiu Marc. cv. Miyagawa-wase(control group) and those of Jedae mandarin(experiment group) were compared by referring to the FIG. 13 through FIG. 16, the cytolysis(indicated by arrows) of epidermal cells was not observed in the peels of Jedae mandarin, unlike the peels of the mature fruits of C. unshiu Marc. cv. Miyagawa-wase, which suggests that the cytolysis of peel epidermal cells was delayed in Jedae mandarin.

[0116] The longitudinally raised portion of Jedae mandarin fruit peels were distributed more in vascular bundle, compared to the flat portion of the peels. Meanwhile, the flat portion of the peels was found to contain more intercellular spaces with cell destruction, compared to the raised portion of the peel.

[0117] Overall, fruits of mutant citrus bred in accordance with the Embodiment 1 and those of C. unshiu Marc. cv. Miyagawa-wase can be harvested almost around the same time. However, the mutant citrus has altered fruit shape(uneven peel) and inherits genetic traits same as those of mother plant in connection with the characteristics of changes in flavonoid component and therefore can be mass-produced, unlike C. unshiu Marc. cv. Miyagawa-wase. In addition, the mutant citrus can be asexually propagated with grafting. In that sense, the mutant citrus, a new variety having all characteristics described above, was named “Jedae mandarin’.

[0118] While the present invention was described above in detail in connection with desirable exemplary embodiments, it is to be understood that the present invention is not limited to the disclosed embodiments, but on the contrary, is intended to encompass various modifications and improvements to be made by those skilled in relevant field of technology, which are defined within the spirit and scope of the appended claims as follows: