Tissue culture studies of Fortunella japonica Swingle (golden , ) Hatice Demiray, Aylin Eşiz Dereboylu Ege University Science Faculty, Biology SectionDepartment of Botany 35100, Bornova- İZMİR

Scientific Classification

• Kingdom: Plantae • Divisio: Angiosperms • Classis: Eudicotiledones (Magnoliopyta) • Subclassis: Rosidae • Ordo: • Family: • Subfamily: • Tribe: Citreae • Genus: • Species: C. japonica • Binomial name • Citrus japonica Thunb.

Synonyms hindsii (Champ. ex Benth.) Oliv. • Citrus erythrocarpa Hayata • Citrus hindsii (Champ. ex Benth.) Govaerts • Citrus inermis Roxb. • Citrus kinokuni Yu.Tanaka • Citrus madurensis Lour. • Citrus margarita Lour. Citrus microcarpa Bunge • Fortunella bawangica C.C.Huang • Fortunella chintou (Swingle) C.C. Huang • Fortunella crassifolia Swingle • Fortunella hindsii (Champ. ex Benth.) Swingle • Fortunella japonica (Thunb.) Swingle • Fortunella margarita (Lour.) Swingle • Fortunella obovata Tanaka • Sclerostylis hindsii Champ. ex Benth. • Sclerostylis venosa Champ. ex Benth. • × madurensis (Lour.) D.Rivera & al.

Morphology and medicinal importance • Kumquat (Citrus japonica), or golden orange [Fortunella japonica (Thunb.) Swingle], is a fruit likes an orange (Citrus sinensis) with its similar size and unlikely Citrus species, it is the only species that can be eaten with its peel and is slightly larger than the olive and is separated from other Citrus species with cold-resistant. They are slow-growing evergreen shrubs or short trees naturally occurring in South Asia. Flowers are white and emerge individually or in the form of bundles from the leaf axis similarly to the other Citrus species. In recent years nutritional value of agricultural products containing significant amounts of biologically active compounds has attracted the attention of consumers (Lampila, van Lieshout, Gremmen, & Lähteenmäki, 2009). In numerous different nutraceuticals polyphenols and more specifically flavonoids have been the basic subject of numerous studies (Liu, Qiu, Ding, & Yao, 2008; Reddy, Sreeramulu, &Raghunath, 2010). Citrus fruits and juices in the plant kingdom are main food sources rich in flavonoids (Gattuso, Barreca, Gargiulli, Leuzzi, & Caristi, 2007; Tripoli, La Guardia, Giammanco, Di Majo, & Giammanco, 2007). Citrus flavonoids have been shown to inhibit angiogenesis and to slow the migration and proliferation of cancerous cells by their antioxidant effects. Flavonoids as they exhibit antiviral and antimicrobial properties, they play a role as protector of coronary heart disease by affecting the functionality of those biological membranes (Barreca et al., 2009; Benavente- García & Castillo, 2008; Patil, Jayaprakasha,Murthy, & Vikram, 2009). flavonoids in fruit peel and juice and its difference from Citrus • Kumquat, Fortunella genus, includes Rutaceae family, orange trees in the form of a small brush, particularly phenolic compounds isolated from the bark is used as relieving inflammatory respiratory diseases in traditional Chinese medicine (Choi et al., 2011; Zang, 2005). Despite Citrus ve Fortunella genus are taxonomically very close relatives , fingerprints of flavonoids are very different. While the main flavonoids of Citrus genus are: flavanone and flavone glycosides (Berhow, Tisserat, Kanes, & Vandercook, 1998; Kawaii, Tomono, Katase, Ogawa, & Yano, 1999; Nogata et al., 2006), in Fortunella genus floretin 3′,5′-di-C-glycoside is the mostly found compound as a dihydrochalcone (Ogawa et al., 2001). In the juice of immature and mature kumquat 13 C- ve O-glycosyl flavonoids: Acasetin 3,6-di-C- glukosid, vicenin-2, lucenin-2 4′-metil eter, narirutin 4′-O-glycoside and apigenin 8- C-neohesperidoside are determined as being more less than fruit peel by reverse- phase LC-DAD-ESI-ITMS analyses. Antioxidant feature of flavonoids of fruit juice and especially floretin 3′,5′-di-C-glycoside have been improved (Davide Barreca, Ersilia Bellocco, Corrado Caristi, Ugo Leuzzi, Giuseppe Gattuso ,2011. Kumquat (Fortunella japonica Swingle) juice: Flavonoid distribution and antioxidant properties. Food Research International 44: 2190–2197; Shyi-Neng Lou, Yi-Chun Lai, Ya-Siou Hsu, Chi-Tang Ho, 2016. Phenolic content, antioxidant activity and effective compounds of kumquat extracted by different solvents. Food Chemistry 197: 1–6). Nutritive value • Kumquat is very rich in vitamin C plant. While 50 mg of ascorbic acid per 100 grams of orange (Agriculture Handbook NO. 98, 1956 Chemistry and technology of Citrus, Citrus products; and byproducts. United States Department of Agriculture. Washington, D. C.), kumquat has 151 milligrams of vitamin C per 100 grams (Morton, J. 1987. Kumquat. p. 182–185. In: Fruits of warm climates. Julia F. Morton, Miami, FL.). This vitamin is necessary for muscle and tissue formation in helping to make better use of other minerals and vitamins. 30 milligrams of vitamin C daily requirement is clean. That daily 20 grams kumquat, meet our need for vitamin C daily. Fortunella japonica plant have a potential that can be used as food supplements and cosmetics, for this purpose proliferation of this plant tissue culture studies are performed in vitro standard conditions. Material and Methods • Seed pairs of the plant were sown in MS (Murashige&Scoog, 1962) nutrient medium after surface sterilization with Na hypochloride solution, and than rinsing with sterile distilled water and peeled in vitro for growing the golden orange plant in sterile conditions.

• Explants like as root, hypocotile, epycotile and cotyledones taken from the different parts of the seedlings grown in sterile conditions transferred to callus medium. MS medium modified with the addition of 30 gr/l sucrose, 100 mg/l myoinositol, 5 mg/l tiamine and 2 mg/l 2,4-D and 0.5 mg/l kinetin was used as callus medium. Explants taken to callus medium were subcultured fifteen days intervals in fresh medium and incubated in biotron at 16/6 h fotoperiod and 25°C temperature. Fresh weight differences occured in tissues were calculated throughout the eight weeks time. Root callus

Hypocotyl callus

Results

• It was observed that only hypocotyl and root explants propagated in tissue culture medium. • Weight gain was determined from the measurements of callus obtained (Table 1.) Table 1. Evaluation of root and hypocotyl embryonic calli weight measurements in two subcultures statistically (Steel&Torry, 1980).

First 1. Sub 1. Sub 2. Sub 2.Sub 3. Sub 3. Sub Applica Weight Culture Culture Culture Culture Culture Culture tion (g) (g) (increase (g) (increase (g) (increase %) %) % ) Root 0.046±0 0,463±0,2 906.5 0.082±0. 78.2 0.393±0. 754.3 Callus .033bd 37acd 029bd 208abc

Hypoco 0.023±0 0,366±0,2 0.063±0. 173 0.703±0. 2956 tyl .012bd 45acd 1491.3 113bd 174abc Callus • Differences between “a” and control group, “b” and 1. Sub culture, “c” and 2. Sub culture, “d” and 3. Sub culture are statistically important (p≤0,05). Discussion

• Embryogenesis is easily observed in callus obtained from premature or immature embryos of Citrus species. But this embryogenetic calli obtained from most of the Citrus genotypes can not differantiate into somatic embryos by loosing their somatic embryogenesis ability (Zhang et al. 2006) .

• Embryogenic calli of C. sinensis ve Fortunella hindsii Swingle are obtained from MT medium by using 2% glycerol as carbon source (Murashige and Tucker 1969). Non-embryogenic callus (NEC) of ‘Valencia’ is obtained from the somatic embryos derived from leaves of seedlings in 1.5 mg/l 2,4-D added MT medium. The potential of occurring embryogenesis is related by the level of CsL1L expression. Acording to sub-cellular localization analyzes CsL1L is a nuclear protein of plant. Microsatellite in CsL1L is improved with polymorphism between Citrus species (Shi-ping Zhu, Jun Wang, Jun-li Ye, An-Dan Zhu, Wen-wu Guo, Xiu-xin Deng. Isolation and characterization of LEAFY COTYLEDON 1-LIKE gene related to embryogenic competence in Citrus sinensis. Plant Cell Tiss Organ Cult 2014, 119:1–13). In another study 11 rootstock of Citrus are grown in vitro and embryogenic callus is obtained from their root, cothyledone, epicothyl, leaf and embryo explants. • Explants are cultured in different nutrient media. The highest embryogenic callus occurance ratio is obtained in root stock of Troyer sitranjı (Citrus sinensis (l) Osb. X Poncirus trifoliata (L) Raf) by 80% in 2,4-D (2 mg/l)+BAP (0.5 mg/l) added nutrient medium (İlkay ŞEN, Bazı turunçgil anaçlarında eksplant kaynağı ve büyüme düzenleyicilerin embriyogenik kallus eldesi üzerine etkileri. Yüksek Lisans Tezi, Çukurova Üniversitesi Fen Bilimleri Enstitüsü Bahçe Bitkileri Anabilim Dalı, ADANA, 2010). • The best nutrient medium is 2,4-D (2 mg/l)+KİN (0.5 mg/l)+NAA (2 mg/l) for embryogenic callus (EK) obtainment. In epicotyl explant experiment, the best EKK ratio is shown in Rubidoux (Poncirus trifoliata (L) Raf.), trifoliat genotype, while in leaf explant buxifolia genotype have given the best EK ratio. Troyer sitranjı genotype gave the best EK ratio in cotyledon explant experiment. Gou Tou orange (Citrus aurantium L.) genotype gave the highest EK in root explant experiment with the best nutrient media of 2,4-D (2 mg/l) + KİN (0.5 mg/l). In this study; embryogenic callus obtainment is aimed by using root, cotyledone, epycotyl, leaf and hypocotyl explants from the seedlings of Fortunella japonica (Thunb.) Swingle grown in vitro. Long lived embryogenic callus is obtained from only root and hypocotyl explants. • Through embryo culture success can be achieved in less time while with conventional methods of breeding can not have effective results. In addition, embryo culture technique allows to examine the effects of nutrients, plant growth regulators and other chemical and physical factors on embryonic growth and differentiation. Because of embryogenic callus cultures of plant stem cells can be used in cosmetics as well as plant breeding programs, our work is of high value in terms of exhibiting antioxidant properties of kumquat future evaluation in different areas. References

• Lampila, P., van Lieshout, M., Gremmen, B., & Lähteenmäki, L. (2009). Consumer attitudes towards enhanced flavonoid content in fruit. Food Research International, 42(1), 122−129. • Liu, H., Qiu, N., Ding, H., & Yao, R. (2008). Polyphenols contents and antioxidant capacity of 68 Chinese herbals suitable for medical or food uses. Food Research International, 41(4), 363−370. • Reddy, C. V. K., Sreeramulu, D., & Raghunath, M. (2010). Antioxidant activity of fresh and dry fruits commonly consumed in India. Food Research International, 43(1), 285−288. • Gattuso, G., Barreca, D., Gargiulli, C., Leuzzi, U., & Caristi, C. (2007). Flavonoid composition of Citrus juices. Molecules, 12(8), 1641−1673. • Tripoli, E., La Guardia, M., Giammanco, S., Di Majo, D., & Giammanco, M. (2007). Citrus flavonoids: Molecular structure, biological activity and nutritional properties: a review. Food Chemistry, 104(2), 466−479. • Barreca, D., Laganà, G., Tellone, E., Ficarra, S., Leuzzi, U., Galtieri, A., et al. (2009). Influences of flavonoids on erythrocyte membrane and metabolic implication through anionic exchange modulation. The Journal of Membrane Biology, 230(3), 163−171. • Benavente-García, O., & Castillo, J. (2008). Update on uses and properties of Citrus flavonoids: New findings in anticancer, cardiovascular, and anti-inflammatory activity. Journal of Agricultural and Food Chemistry, 56(15), 6185−6205. • Patil, B. S., Jayaprakasha, G. K., Murthy, K. N. C., & Vikram, A. (2009). Bioactive compounds: Historical perspectives, opportunities, and challenges. Journal of Agricultural and Food Chemistry, 57(18), 8142−8160.

References

• Choi, M. Y., Chai, C., Park, J. H., Lim, J., Lee, J., & Kwon, S. W. (2011). Effects of storage period and heat treatment on phenolic compound composition in dried citrus peels (Chenpi) and discrimination of Chenpi with different storage periods through targeted metabolomic study using HPLC-DAD analysis. Journal of Pharmaceutical and Biomedical Analysis, 54, 638–645. • Zang, K. T. (2005). Small encyclopedia of Chinese herbal medicine. Heliopolis Culture Group, Taiwan: Neptune Culture Publishing Ltd. • Berhow, M., Tisserat, B., Kanes, K., & Vandercook, C. (1998). Survey of phenolic compounds produced in Citrus. USDA ARS Technical Bulletin, 1856, 1–154. • Kawail, S., Tomono, Y., Katase, E., Ogawa, K., & Yano, M. (1999). Quantitation of flavonoid constituents in citrus fruits. Journal of Agricultural and Food Chemistry, 47, 3565–3571. • Nogata, Y., Sakamoto, K., Shiratsuchi, H., Ishii, T., Yano, M., & Ohta, H. (2006). Flavonoid composition of fruit tissues of Citrus species. Bioscience Biotechnology and Biochemistry, 70(1), 178–192. • Ogawa, K., Kawasaki, A., Omura, M., & Yoshida, T. (2001). 30,50-Di-C-bglucopyranosylphloretin, • a flavonoid characteristic of the genus Fortunella. Phytochemistry, 57, 737–742. • Davide Barreca, Ersilia Bellocco, Corrado Caristi, Ugo Leuzzi, Giuseppe Gattuso (2011) Kumquat (Fortunella japonica Swingle) juice: Flavonoid distribution and antioxidant properties. Food Research International 44: 2190–2197. • Shyi-Neng Lou, Yi-Chun Lai, Ya-Siou Hsu, Chi-Tang Ho (2016) Phenolic content, antioxidant activity and effective compounds of kumquat extracted by different solvents. Food Chemistry 197: 1–6.

References

• Agriculture Handbook No. 98. (1956)Chemistry and technology of Citrus, Citrus products; and byproducts. United States Department of Agriculture. Washington, D. C. • Morton, J. 1987. Kumquat. p. 182–185. In: Fruits of warm climates. Julia F. Morton, Miami, FL. • Murashige T, Skoog F. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plant 15: 473-497, 1962.

References

• Steel RGD, Torrie JH. Principles and Procedures of Statistics. pp. 403-447. 2nd Ed. McGraw-Hill Inc., New York, 1980. • Zhang JE, Guo WW, Deng XX (2006) Relationship between ploidy variation of citrus calli and competence for somatic embryogenesis. Acta Genet Sinica 33(7):647–654. • Murashige T and Tucker DPH (1969). Growth factor requirements of Citrus tissue culture. Proc. First Intern. Citrus Symp., 3: 1155–1161. • Shi-ping Zhu, Jun Wang, Jun-li Ye, An-Dan Zhu, Wen-wu Guo, Xiu-xin Deng, 2014. Isolation and characterization of LEAFY COTYLEDON 1-LIKE gene related to embryogenic competence in Citrus sinensis. Plant Cell Tiss Organ Cult, 119:1–13.

References

• İlkay ŞEN, Bazı turunçgil anaçlarında eksplant kaynağı ve büyüme düzenleyicilerin embriyogenik kallus eldesi üzerine etkileri. Yüksek Lisans Tezi, Çukurova Üniversitesi Fen Bilimleri Enstitüsü Bahçe Bitkileri Anabilim Dalı, ADANA, 2010.