Tissue culture studies of Fortunella

japonica Swingle (golden orange,

kumquat)
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: Sapindales
• Family: Rutaceae
• Subfamily: Aurantioideae
• Tribe: Citreae
• Genus: Citrus
• Species: C. japonica
• Binomial name
• Citrus japonica Thunb.
 Synonyms
Atalantia 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.
• × Citrofortunella 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 Severinia
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.
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Research International 44: 2190–2197.
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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.
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• 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.