EJBT-00378; No of Pages 7

Electronic Journal of Biotechnology xxx (2019) xxx–xxx

Contents lists available at ScienceDirect

Electronic Journal of Biotechnology

1 A comparison of callus induction in 4 Garcinia species

Q32Q2 Valerie Suwanseree a, Salak Phansiri b, Chinawat Yapwattanaphun c,⁎

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3 a
Graduate School, Kasetsart University, Chatuchak, Bangkok 10900, Thailand
4 b
Scientific Equipment and Research Division, KURDI, Kasetsart University, Chatuchak, Bangkok 10900, Thailand

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5 c
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Chatuchak, Bangkok 10900, Thailand
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7 a r t i c l e i n f o a b s t r a c t

8 17

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Article history: Background: This research is intended to determine suitable types and concentrations of plant growth regulators
9 Received 24 November 2018 (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana,18
10 Accepted 16 April 2019 Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base medium was MS medium containing 19
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Available online xxxx 30 gl-1 sucrose, 0.5 gl-1 polyvinylpyrrolidone (PVP), and 7 gl-1 agar, and for the different treatments, PGRs wereQ5
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16
15
14
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12 added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mgl-1; 6-(3- Q6
21
36
Q7 Keywords:
hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mgl-1; 4-amino-3,5,6-trichloro- 22
37 Biopharming
38 Callus
2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mgl-1; and 2,4-dichlorophenoxyacetic
D 23
39 Clusiaceae acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mgl-1. The occurrence of callus was observed after 4 weeks. 24
40 Garcinia Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mgl-1 and 1 mgl-1 TDZ 25
41 treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mgl-1 TDZ treatment26
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Genetic transformation
42 Leaf explants and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mgl-1 picloram treatment. The highest callus 27
43 Phytochemicals induction rate for G. cowa was 62% in the 1 mgl-1 TDZ treatment and for G. celebica was 56% in the 0.5 mgl-1·mT-1 28
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44 Plant growth regulators treatment. 29

45 Stem explants
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Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White,
46 Underutilized plants
31
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friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments
47 Woody plant tissue culture
resulted in minimal callus formation. 32
33
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© 2019 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 34
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 35
51
49
48
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52 1. Introduction properties [3]. For instance, recent research has illuminated the 69
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antibiotic and cytotoxic potential of garcinol found in Garcinia 70

53 Garcinia is a genus in the family Clusiaceae (formerly Guttiferae) cambogia, Garcinia indica, Garcinia atroviridis, and several other 71
54 comprising evergreen tropical trees and shrubs that originated mainly Garcinia species [4,5,6]. 72
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55 in Asia, as well as some species from Australia, Africa, and Polynesia. Garcinia species that are related to mangosteen were investigated 73
56 There are 397 species in the genus Garcinia that are accepted by The with a view to future breeding applications because mangosteen is an 74
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57 Plant List [1]. At least 22 Garcinia species are found in Thailand [2]. obligate apomict. Conventional hybridization has not yet been 75
58 The most well-known member of the genus is Garcinia mangostana L., achieved because mangosteen flowers do not produce viable pollen 76
59 commonly known as mangosteen, an important fruit crop in [7]. Gene transfer or protoplast fusion offers exciting alternatives for 77
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60 Southeast Asia. Thailand is the world's largest exporter of mangosteen, crop improvement, and callus induction in species genetically related 78
61 and the export value reached US$ 234,496,785 in 2017 (Office of to mangosteen is a step in this direction. 79
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62 Agricultural Economics). With many traditional uses in folk medicine, This study was conducted on 4 species, namely, G. mangostana L., or 80
63 Garcinia species have been found to be a rich source of secondary mangosteen, Garcinia celebica, or “seashore mangosteen,” Garcinia cowa 81
64 metabolites including xanthones (such as alpha-mangostin and (“chamuang” in Thai), and Garcinia schomburgkiana Pierre (“madan” in 82
65 gamma-mangostin (Fig. 1)), flavonoids, benzophenones, lactones, Thai). 83
66 triterpenes, and phenolic acids [3]. Many of these phytochemicals Mangosteen is valued for its delicious fruit, and the juice is promoted 84
67 have been shown to have antioxidant, antiprotozoal, antifungal, as a healthy beverage. Several studies have also investigated the 85
68 antibacterial, anti-inflammatory, and anti-immunosuppressive medicinal properties of crude extract of mangosteen rind and isolated 86
active compounds such as xanthones and alpha-, beta-, and gamma- 87
mangostin. For instance, alpha-mangostin exhibited an MIC value of 88
⁎ Corresponding author.
Q4 E-mail address: agrcwy@ku.ac.th (C. Yapwattanaphun). 1.95 μg/ml against methicillin-resistant Staphylococcus aureus [8,9]. A 89
Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. study conducted by Jang et al. [10] in mouse indicated that alpha- and 90

https://doi.org/10.1016/j.ejbt.2019.04.006
0717-3458/© 2019 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: V. Suwanseree, S. Phansiri and C. Yapwattanaphun, A comparison of callus induction in 4 Garcinia species, Electronic
Journal of Biotechnology, https://doi.org/10.1016/j.ejbt.2019.04.006
 2 V. Suwanseree et al. / Electronic Journal of Biotechnology xxx (2019) xxx–xxx

Fig. 1. (A) Alpha-mangostin; (B) gamma-mangostin; Credit: PubChem.

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91 gamma-mangostin may have therapeutic potential for the treatment of The focus of this research was to induce callus in vitro from leaf tissue 125
92 allergic asthma. Similarly, alpha- and gamma-mangostin were shown to of the selected Garcinia species. Aseptic callus tissue is useful for starting 126

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93 inhibit allergic inflammatory responses in the lab [11]. Xanthones were cell suspensions that can be scaled up for the efficient collection of 127
94 demonstrated to significantly suppress the degranulation in Ag- useful phytochemicals from these plants in a way that saves space 128
95 mediated activation of a high-affinity IgE receptor, a primary event in compared to growing them outdoors in plantations [21]. In addition, 129

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96 several allergic responses [12]. In an interesting product development the callus tissue has important applications for advanced plant 130
97 study, researchers at Silpakorn University demonstrated that chitosan- breeding techniques such as genetic manipulation through 131
98 132

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based nanofiber mats loaded with G. mangostana extracts exhibited Agrobacterium-mediated gene transfer, particle bombardment, or
99 antioxidant and antibacterial activities and could accelerate the rate of protoplast fusion. 133
100 wound healing when compared to the control in a rat study [13]. We tested varying concentrations of thidiazuron (TDZ), 4-amino- 134

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101 There is evidence that alpha-mangostin can be effective in killing 3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), 2,4- 135
102 cancer cells as well [4,14,15]. dichlorophenoxyacetic acid (2,4-D), and 6-(3-hydroxybenzylamino) 136
103 Less research has been carried out on other Garcinia species that are purine (meta-topolin) for inducing callus on young leaf sections of G.
D 137
104 not cultivated on a commercial scale; however, they also contain cowa and G. celebica and (due to greater availability of plant material) 138
105 phytochemicals with good potential for further development. on both leaf sections and immature stem sections from G. mangostana 139
106 Benzophenones and biflavonoids isolated from G. celebica were found and G. schomburgkiana Pierre in vitro seedlings. 140
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107 to have moderate to high antioxidant activity [16]. Garcihombronane Thidiazuron (TDZ) is a cytokinin-like urea derivative that has been 141
108 D (Fig. 2), a xanthone found in G. celebica, was found to inhibit the found to facilitate the efficient micropropagation of many recalcitrant 142
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109 growth of the malaria-causing protozoan parasite Plasmodium woody species. Huetteman and Preece [22] wrote in a review that at 143
110 falciparum [17]. Five xanthones extracted from the bark of G. cowa concentrations of higher than 1 μM, TDZ can stimulate the formation 144
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111 were also found to possess antimalarial activity against P. falciparum in of callus, adventitious shoots, or somatic embryos. In woody plant 145
112 laboratory tests [18]. Extract from the rind of G. cowa fruits was species, TDZ has successfully been utilized in micropropagation 146
113 reported to be effective in inhibiting the growth of 4 common forms systems for Semecarpus anacardium L. [23] and sandalwood (Santalum 147
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114 of gram-positive bacteria [19]. In addition, the strong antioxidant album L.) [24]. 148
115 properties of phytochemicals from rinds of the G. cowa fruit may have The auxin derivative 2,4-D has been used together with kinetin to 149
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116 a useful application for suppressing the synthesis of aflatoxin by induce callus in at least 3 species of woody plants grown in vitro: 150
117 Aspergillus sp. in peanuts and animal feed [20]. G. cowa is cultivated on Dalbergia sissoo [25], Ulex europaeus [26], and Prunus persica [27]. 151
118 a small scale in Thailand, where its leaves, rich in Vitamin A, are Picloram, an auxin-like synthetic PGR, was used to induce 152
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119 harvested for the preparation of soup or curry [2]. G. schomburgkiana embryogenic callus on cashew [28] and coffee [29]. Picloram was able 153
120 fruit, also rich in Vitamin A and calcium, is sold in local markets and to induce large amounts of callus on Rudgea jasminoides petioles as a 154
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121 may be used as pickle or candy or prepared as a dipping sauce [2]. All way to obtain phytoalexin, a defensive metabolite [30]. 155
122 these other Garcinia species have edible fruit, but it is not favored by Meta-topolin is a relatively newly discovered PGR; hence, not as 156
123 consumers as much as mangosteen. They may be considered much research has been carried out on its use in tissue culture. Meta- 157
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124 underutilized tropical fruit trees. topolin is a naturally occurring cytokinin first isolated from poplar 158
[31]. It was reported to induce callus on the herbaceous species 159
Spathiphyllum floribundum Schott cv. Petite [32] and the woody
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160
species Sclerocarya birrea [33]. 161
The objective of this study was to test varying concentrations of TDZ, 162
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picloram, 2,4-D, and mT to induce callus and/or shoots on leaf explants 163
of G. mangostana, G. schomburgkiana, G. celebica, and G. cowa to gain 164
more information that can be of use for biotechnological applications 165
in the future. 166

2. Material and methods 167

For G. mangostana and G. schomburgkiana, seeds were removed from 168

the flesh of fresh fruit obtained from the local market. The seeds were 169
washed in dishwashing detergent for 2 min and then immersed in 170
1000 ppm antibacterial solution (Kanker-X) plus 1000 ppm fungicide 171
(Captan orthocide) for 2 h (shaken on an Innova 2300 orbital shaker 172
Fig. 2. 3b-Hydroxy-23-oxo-9,16-lanostadien-26-oic acid or garcihombronane D, a at 105 rpm). Subsequently, they were surface sterilized in 70% ethanol 173
triterpene found in G. celebica by Elfita et al. [17]. for 1 min, 20% Clorox solution for 15 min, and 10% Clorox solution for 174

Please cite this article as: V. Suwanseree, S. Phansiri and C. Yapwattanaphun, A comparison of callus induction in 4 Garcinia species, Electronic
Journal of Biotechnology, https://doi.org/10.1016/j.ejbt.2019.04.006
 V. Suwanseree et al. / Electronic Journal of Biotechnology xxx (2019) xxx–xxx 3

175 10 min and then rinsed with autoclaved distilled water 3 times. The 1000 ppm Captan orthocide for 2 h on an orbital shaker. Next, the 198
176 seeds were then placed on 1/2-macro nutrient MS medium leaves were immersed in 70% ethanol for 1 min, 10% Clorox solution 199
177 supplemented with 0.5 mgl-1 polyvinylpyrrolidone (PVP) to prevent for 15 min, and 5% Clorox solution for 10 min. Finally, they were 200
178 browning, 3% sucrose, and 0.7% agar in 8-ounce glass jars with 25– rinsed with autoclaved distilled water 3 times in a laminar flow hood. 201
179 35 ml of medium per jar. The jars were kept in the tissue culture lab Following surface sterilization, each leaf was cut into leaf sections 202
180 at 25 ± 2°C with an 8:16 h photoperiod. After the seedlings were measuring approximately 5–7 mm × 8–12 mm, each piece containing 203
181 large enough, they were taken out and sectioned under aseptic midrib. Each leaf section was randomly placed (adaxial side up) in a 204
182 conditions into leaf sections (5–7 mm × 10–15 mm pieces containing test tube containing 10 ml of one of the different treatment media 205
183 midrib) and stem sections (approximately 0.5–1 cm in length), and listed above or control (basal medium with no PGRs added), with a 206
184 placed in separate test tubes, each containing 10 ml of the different minimum of 12 leaf segments per treatment. 207
185 treatment media. The basal medium (control) was 1/2-macro nutrient Test tubes were kept in the tissue culture lab at 25 ± 2°C with an 208
186 MS medium supplemented with 0.5 gl-1 PVP, 3% sucrose, and 0.7% 8:16 h photoperiod. The percentage and appearance of callus or 209
187 agar. The experimental treatments were basal medium with the shoots were recorded after 4 weeks. The size of callus was visually 210
188 following PGRs added: 0, 0.5, 2.5, or 5.0 mgl-1 picloram; 0, 0.5, 1, 2.5, determined and recorded as “-” = no callus, “+” = minimal or slight 211

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189 or 5.0 mgl-1 meta-topolin; 0, 0.5, 1.0, 2.0, or 4.0 mgl-1 2,4-D; or 0, 0.1, callus, “++” = small callus, “+++” = medium callus, “++++” = 212
190 0.5, 1.0, or 2.0 mgl-1 TDZ. Twenty or more leaf sections and at least 10 large callus) and color and nature of callus (friable or compact) was 213

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191 stem sections were used for each treatment group. observed and photographed. 214
192 G. celebica seeds were obtained from Bogor Botanical Garden, Fig. 3 shows a graphical illustration of the process. 215
193 Indonesia. G. cowa seeds were obtained from The Park Adventure,

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194 Rayong Province, Thailand. Seeds of these 2 species were grown in 3. Results and discussion 216
195 potting soil in seedling trays outdoors. Young leaves were cut from the
seedlings and surface sterilized by first washing in dishwashing

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196 Callus was induced on explants of all 4 species tested, with all 4 PGRs 217
197 detergent for 2 min and then immersing in 1000 ppm Kanker-X plus tested, but the nature and amount of callus differed with species and 218

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Garcinia mangostana and Garcinia cowa and

G. schomburgkiana G. celebica
D
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Surface sterilize seeds Plant seeds in soil

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Grow sprouts in vitro

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Surface sterilize young leaves

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Aseptically excise and cut

leaf and stem sections
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Prepare medium:
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Control
Place on
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treatments TDZ 0.1, 0.5, 1, 2 mg -l

2,4-D 0.5, 1, 2, 4 mg -l
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Picloram 0.5, 2.5, 5 mg -l

Observe, record
results

Subculture for
subsequent

Fig. 3. Workflow diagram.

Please cite this article as: V. Suwanseree, S. Phansiri and C. Yapwattanaphun, A comparison of callus induction in 4 Garcinia species, Electronic
Journal of Biotechnology, https://doi.org/10.1016/j.ejbt.2019.04.006
 4 V. Suwanseree et al. / Electronic Journal of Biotechnology xxx (2019) xxx–xxx

219 concentration of PGR. The highest callus induction rate at 4 weeks was Table 2 t2:1
220 100%, observed both in G. mangostana leaf sections cultured in Callus induction in Garcinia schomburgkiana stem sections at 4 weeks. t2:2

221 medium with 0.5 mgl-1 TDZ and in G. schomburgkiana stem sections Plant growth regulators Percentage of Callus Nature of callus t2:3
222 cultured in medium with 1 mgl-1 TDZ (Table 1 and Table 2). The added to callus induction size t2:4
223 second highest callus induction rate observed was 93%, from the MS media (mgl-1) t2:5

224 0.1 mgl-1 TDZ treatment on G. mangostana leaf sections and the Control t2:6
225 0.5 mgl-1·mT-1 treatment on G. mangostana stem sections (Table 1 0 (n = 54) 8.33 + Yellow, compact t2:7
t2:8
226 and Table 3). Other treatments that resulted in a high percentage of TDZ t2:9
227 callus formation were 2 mgl-1 TDZ on G. mangostana stem sections 0.1 (n = 10) 70 ++++ Green/yellow, embryogenic t2:10
228 (90.5%) and 0.5 mgl-1 picloram on G. schomburgkiana leaf sections 0.5 (n = 10) 70 +++ t2:11
1 (n = 8) 100 +++ t2:12
229 (89%) (Table 3 and Table 4).
2 (n = 10) 70 +++ t2:13
230 G mangostana young stem sections from in vitro seedlings (height t2:14
231 approximately 5–10 cm) appeared to readily form callus even on basal 2,4-D t2:15
0.5 (n = 24) 50 +++ Yellow, compact t2:16
232 medium with no PGRs added because the mean callus induction rate

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1 (n = 24) 33.33 ++ t2:17
233 for G. mangostana stem sections in the control group was 55.36% 2 (n = 24) 8.33 + t2:18
234 (Table 3). This may be because the cambium tissue is actively growing 4 (n = 24) 8.33 ++ t2:19

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235 in small seedlings. However, G. schomburgkiana green stem tissue, t2:20
Meta-topolin t2:21
236 although it appeared softer and less woody than stems of G. 0.5 (n = 21) 0 - Yellow, compact t2:22
237 mangostana seedlings, was less prone to forming callus, with a mean 2.5 (n = 21) 14.29 + t2:23

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238 callus induction rate of only 8.33% in the control group (Table 2). 5 (n = 21) 12.5 + t2:24
239 On the control leaf sections with no PGRs added to the medium, the t2:25
Picloram t2:26
240

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mean callus induction rate was 8.5% for G. mangostana, 0% for G. 0.5 (n = 24) 41.67 + White to brown, nodular t2:27
241 schomburgkiana, 7.6% for G. cowa, and 16% for G. celebica (Table 1 and 2.5 (n = 24) 0 - t2:28
242 Table 4, Table 5, Table 6). The formation of small amounts of callus on 5 (n = 24) 8.33 ++ t2:29

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243 the control leaf sections was likely a stress response to the wound Mean callus induction rate for G. schomburgkiana stem: 34.77%, S.D. 32.01; - = no callus; t2:30
244 caused when the leaves were cut into sections. The amount and size + minimal or slight; ++ small; +++ medium; ++++ large. t2:31

245 of callus induced on the control leaf sections were very small D
246 compared to most of the treatments with PGRs added.
247 There were some exceptions, however, because some PGR less than that of the control with the 1 mgl-1, 2 mgl-1, and 4 mgl-1 2,4- 258
248 treatments appeared to be toxic, as the leaf sections tended to turn D treatments and with the 0.5 mgl-1 and 5 mgl-1 picloram treatments 259
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249 brown and die more quickly than the control, especially when PGRs (Table 6). For G. cowa, the percent callus formation at 4 weeks was 260
250 were added at a high concentration. For example, in the case of G. less than that of the control with the 4 mgl-1 2,4-D treatment and the 261
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251 mangostana stem sections, picloram tested at all concentrations and 5 mgl-1·mT-1 treatment (Table 5). 2,4-D is used as a herbicide in 262
252 2,4-D tested at all concentrations failed to induce callus or induced agriculture; hence, it is not surprising that it would be harmful to 263
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253 callus at a lower rate than the control (Table 2). In G. mangostana leaf plants at high concentrations. 264
254 sections, picloram at the higher concentrations of 2.5 mgl-1 and 5 mgl- Looking at all treatments together, the range of callus induction rates 265
1
255 and 2,4-D at concentrations of 2 mgl-1 and 4 mgl-1 resulted in a were 0–100% for G. mangostana leaf sections, 0–93% for G. mangostana 266
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256 lower callus induction rate than the control (Table 1). Additionally, for stem sections, 0–89% for G. schomburgkiana leaf sections, 0–100% for 267
257 G. celebica leaf sections, the percent callus formation at 4 weeks was G. schomburgkiana stem sections, 0–86% for G. cowa leaf sections, and 268
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t1:1 Table 1 Table 3 t3:1

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t1:2 Callus induction in Garcinia mangostana leaf sections at 4 weeks. Callus induction in Garcinia mangostana stem sections at 4 weeks. t3:2
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t1:3 Plant growth regulators Percentage of Callus Nature of callus Plant growth Percentage of Callus Nature of callus t3:3
t1:4 added to callus induction size regulators added callus induction size t3:4
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t1:5 MS media (mgl-1) to MS media (mgl-1) t3:5

t1:6 Control Control t3:6

t1:7 0 (n = 62) 8.5 + White/yellow 0 (n = 46) 55.36 ++ Yellow, embryogenic t3:7
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t1:8 t3:8
t1:9 TDZ TDZ t3:9
t1:10 0.1 (n = 24) 74.5 ++ Green/yellow, embryogenic 0.1 (n = 20) 80 ++++ Green/yellow, embryogenic t3:10
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t1:11 0.5 (n = 26) 100 +++ 0.5 (n = 23) 72 ++++ t3:11

t1:12 1 (n = 26) 93 ++++ 1 (n = 20) 85 ++++ t3:12
t1:13 2 (n = 24) 78 +++ 2 (n = 21) 90.5 ++++ t3:13
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t1:14 t3:14
t1:15 2,4-D 2,4-D t3:15
t1:16 0.5 (n = 71) 59 ++ White, friable 0.5 (n = 33) 36.36 ++ White to yellow-brown, t3:16
t1:17 1 (n = 25) 12 ++ 1 (n = 22) 27.27 +++ compact t3:17
t1:18 2 (n = 25) 0 - 2 (n = 22) 18.18 ++ t3:18
t1:19 4 (n = 25) 0 - 4 (n = 23) 28.57 ++ t3:19
t1:20 t3:20
t1:21 Meta-topolin Meta-topolin t3:21
t1:22 0.5 (n = 28) 29 + Yellow, compact 0.5 (n = 14) 93 + Yellow, compact t3:22
t1:23 2.5 (n = 28) 46 + 2.5 (n = 14) 64 +++ t3:23
t1:24 5 (n = 28) 18 + 5 (n = 14) 64 ++ t3:24
t1:25 t3:25
t1:26 Picloram Picloram t3:26
t1:27 0.5 (n = 22) 18.5 + White to brown, nodular 0.5 (n = 21) 0 - n/a t3:27
t1:28 2.5 (n = 22) 0 - 2.5 (n = 21) 0 - t3:28
t1:29 5 (n = 22) 0 - 5 (n = 21) 0 - t3:29

t1:30 Mean callus induction rate for G. mangostana leaf: 35.8%, S.D. 36.27. - = no callus; Mean callus induction rate for G. mangostana stem: 47.06%, S.D. 33.92. - = no callus; t3:30
t1:31 + minimal or slight; ++ small; +++ medium; ++++ large. + minimal or slight; ++ small; +++ medium; ++++ large. t3:31

Please cite this article as: V. Suwanseree, S. Phansiri and C. Yapwattanaphun, A comparison of callus induction in 4 Garcinia species, Electronic
Journal of Biotechnology, https://doi.org/10.1016/j.ejbt.2019.04.006
 V. Suwanseree et al. / Electronic Journal of Biotechnology xxx (2019) xxx–xxx 5

t4:1 Table 4 Table 6 t6:1

t4:2 Callus induction in Garcinia schomburgkiana leaf sections at 4 weeks. Callus induction in Garcinia celebica leaf sections at 4 weeks. t6:2

t4:3 Plant growth regulators Percentage of Callus Nature of callus Plant growth regulators Percentage of Callus Nature of callus t6:3
t4:4 added to callus induction size added to callus induction size t6:4
t4:5 MS media (mgl-1) MS media (mgl-1) t6:5

t4:6 Control Control t6:6

t4:7 0 (n = 69) 0 - na 0 (n = 47) 16.45 + Yellow, compact t6:7
t4:8 t6:8
t4:9 TDZ TDZ t6:9
t4:10 0.1 (n = 24) 0 - Green/yellow, 0.1 (n = 12) 41.67 +++ Green/yellow, t6:10
t4:11 0.5 (n = 24) 12.5 ++ embryogenic 0.5 (n = 20) 18.18 ++ embryogenic t6:11
t4:12 1 (n = 24) 20.83 ++ 1 (n = 12) 0 - t6:12
t4:13 2 (n = 24) 37.5 ++ 2 (n = 12) 16.67 ++ t6:13
t4:14 t6:14
t4:15 2,4-D 2,4-D t6:15
t4:16 0.5 (n = 24) 79.17 +++ Yellow, compact 0.5 (n = 19) 41.18 +++ Yellow, compact t6:16

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t4:17 1 (n = 20) 79.17 ++ 1 (n = 23) 13.64 ++ t6:17
t4:18 2 (n = 24) 79.17 + 2 (n = 24) 9.52 + t6:18
t4:19 4 (n = 24) 37.5 ++ 4 (n = 24) 4.17 ++ t6:19

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t4:20 t6:20
t4:21 Meta-topolin Meta-topolin t6:21
t4:22 0.5 (n = 19) 0 - Yellow, compact 0.5 (n = 25) 55.56 ++ Yellow, compact t6:22
t4:23 2.5 (n = 19) 0 - 2.5 (n = 23) 29.41 + t6:23

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t4:24 5 (n = 19) 8.33 + 5 (n = 28) 20 + t6:24
t4:25 t6:25
t4:26 Picloram Picloram t6:26

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t4:27 0.5 (n = 24) 88.89 + White to brown, 0.5 (n = 18) 11.11 + White, nodular t6:27
t4:28 2.5 (n = 24) 75 + nodular 2.5 (n = 18) 17.65 + t6:28
t4:29 5 (n = 24) 37.5 ++ 5 (n = 17) 5.89 ++ t6:29

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t4:30 Mean callus induction rate for G. schomburgkiana leaf: 39.68%, S.D. 34.47; - = no Mean callus induction rate for G. celebica leaf: 34.5%, S.D. 15.54; - = no callus; + minimal t6:30
t4:31 callus; + minimal or slight; ++ small; +++ medium; ++++ large. or slight; ++ small; +++ medium; ++++ large. t6:31

269 6–82% for G. celebica leaf sections. The mean callus induction rate across
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270 all PGRs at all concentrations and control was 35.8% for G. mangostana in the greatest amount of large-sized callus growth was TDZ, followed 280
271 leaf sections (S.D. 36.27), 47.06% for G. mangostana stem sections (S.D. by 2,4-D. The nature of the callus differed noticeably between 281
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272 33.92), 39.68% for G. schomburgkiana leaf sections (S.D. 34.47), 34.77% different PGRs. Callus induced by TDZ was generally compact, nodular, 282
273 for G. schomburgkiana stem sections (S.D. 32.01), 42.71% for G. cowa yellow to green, and, especially in the case of G. celebica, embryogenic 283
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274 leaf sections (S.D. 16.73), and 34.5% for G. celebica leaf sections (S.D. (Fig. 4 and Fig. 5). Callus induced by 2,4-D was mostly white, with a 284
275 15.54). fluffy appearance on G. cowa leaf sections but yellower and more 285
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276 Looking at each PGR separately, the mean callus induction rate compact on the other species (Fig. 4). Large amounts of white callus 286
277 across species and across concentrations was 52.53% for TDZ (S.D. with a fuzzy appearance were induced by picloram on G. cowa leaf 287
278 23.69), 28.16% for 2,4-D (S.D. 24.79), 27.31% for mT (S.D. 25.84) and sections but only smaller amounts on the other species. 288
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279 20.23% for picloram (S.D. 25.27). Qualitatively, the PGR that resulted Some research has been carried out on tissue culture of other species 289
in the genus Garcinia. For example, Te-chato [34] reported the induction 290
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of callus from leaf explants of G. speciosa on MS medium supplemented 291

t5:1 Table 5
t5:2 Callus induction in Garcinia cowa leaf sections at 4 weeks. with BA at 3 different concentrations (0.1, 0.5, and 2.5 mgl-1) in 292
combination with 0.5 or 1.0 mgl-1 TDZ or 0.1, 0.5, or 0.25 mgl-1 NAA. 293
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t5:3 Plant growth regulators Percentage of callus Callus Nature of callus

Meristem nodular callus developed under all these treatments after 4– 294
t5:4 added to induction size
t5:5 MS media (mgl-1) 6 weeks, but the highest percentage of callus (90%) was formed in the 295
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treatment of 2.5 mgl-1 BA and 0.25 mgl-1 NAA, followed by the 296
t5:6 Control
t5:7 0 (n = 109) 7.61 + Yellow, dry treatment with 0.1 mgl-1 BA and 0.1 mgl-1 NAA (60% callus). In the 297
t5:8 same report, Te-chato also tested 3 different PGR treatments on G. 298
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t5:9 TDZ
atroviridis leaf explants and observed that no callus emerged in the 299
t5:10 0.1 (n = 29) 48.27 +++ Yellow, compact
t5:11 0.5 (n = 29) 24.14 ++++ treatment with 0.5 mgl-1 BA and 0.5 mgl-1 TDZ, but friable callus 300
developed on 15% of the explants in the treatment with 0.5 mgl-1 BA
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t5:12 1 (n = 29) 62.07 +++ 301

t5:13 2 (n = 29) 31.03 ++ and 0.5 mgl-1 thiourea (TU) and 35% of the explants in the treatment 302
t5:14 with 1.0 mgl-1 BA and 0.5 mgl-1 NAA. This is comparable to a 303
t5:15 2,4-D
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t5:16 0.5 (n = 22) 22.72 +++ White, fuzzy meristem nodular callus formation rate of 69% reported for G. 304
t5:17 1 (n = 24) 20.83 +++ mangostana on Te-chato's callus induction medium containing 305
t5:18 2 (n = 26) 7.69 ++ 0.5 mgl-1 BA and 0.5 mgl-1 TDZ. [34]. Together with our results from 306
t5:19 4 (n = 24) 0 -
the present study, this indicates that TDZ may be the most effective 307
t5:20
t5:21 Meta-topolin PGR for inducing callus in Garcinia species. 308
t5:22 0.5 (n = 29) 8.33 + Yellow, compact Kuo et al. [21] reported that callus culture can be an important 309
t5:23 1 (n = 29) 8.33 +
method for harvesting metabolites from plants such as the medicinal 310
t5:24 2.5 (n = 29) 20.83 +
t5:25 5 (n = 29) 0 - alkaloids Fangchinoline and Tetrandrine synthesized in the tree 311
t5:26 Stephania tetrandra S. Moore. Their experimental results showed that 312
t5:27 Picloram
t5:28 0.5 (n = 25) 24 ++++ White, fuzzy
MS medium supplemented with 1.0 mgl-1 BA and 0.5 mgl-1 TDZ was 313
t5:29 2.5 (n = 26) 24 ++++ the best medium for inducing callus growth and its proliferation in 314
t5:30 5 (n = 26) 11.54 ++ that species. Similarly, our results indicate that TDZ could be used to 315
t5:31 Mean callus induction rate for G. cowa leaf: 42.71%, S.D. 16.73; = no callus; + minimal or induce callus from Garcinia species for the extraction of beneficial 316
t5:32 slight; ++ small; +++ medium; ++++ large. phytochemicals. 317

Please cite this article as: V. Suwanseree, S. Phansiri and C. Yapwattanaphun, A comparison of callus induction in 4 Garcinia species, Electronic
Journal of Biotechnology, https://doi.org/10.1016/j.ejbt.2019.04.006
 6 V. Suwanseree et al. / Electronic Journal of Biotechnology xxx (2019) xxx–xxx

Fig. 4. Examples of medium- to large-sized callus on leaf explants of 5 species in the genus Garcinia. (a) Garcinia mangostana leaf section from in vitro seedling 4 weeks after culture on MS
medium with 1 mgl-1 TDZ; (b) G. schomburgkiana leaf section from in vitro seedling 4 weeks after culture on MS medium with 0.5 mgl-1 picloram; (c) G. celebica leaf section from outdoor
grown seedling 4 weeks after culture on MS medium with 1 mgl-1 2,4-D; (d) G. cowa leaf section from outdoor grown seedling 4 weeks after culture on MS medium with 0.1 mgl-1 TDZ;

318 Singh et al. [24] reported in vitro plant regeneration by indirect done using mT on woody plants. Moyo et al. [33] induced shoots from 357

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319 organogenesis from callus cultures of sandalwood (Santalum album L.). shoots, hypocotyls, and epicotyls excised from 30- to 60-day-old 358
320 They tested culturing leaf explants on Woody Plant Medium (WPM) seedlings of the drought-tolerant multipurpose African tree with 359

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321 supplemented with either TDZ or 2,4-D. The highest callus frequency edible fruit, marula (Sclerocarya birrea), a member of the family 360
322 (100%) was obtained when leaf tissue was cultured in the medium with Anacardiaceae. They reported a shoot induction rate of 63% in the 361
323 0.4 mgl-1 TDZ and the highest amounts of fresh weight and dry weight treatment with 8 μM mT [33]. In the present study, mT did not have as 362

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324 of leaf-derived callus were observed in the medium supplemented with strong an effect on callus induction in Garcinia species as the other 363
325 0.8 mgl-1 TDZ [24]. Our results were similar because the largest amount PGRs studied. Although the callus induction rate was as high as 56% 364
326 on G. celebica leaf sections at the concentration of 0.5 mgl-1, the size of 365

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of compact callus obtained (measured by visual observation) was from
327 the 0.5 mgl-1 TDZ treatment (Fig. 4 and Fig. 5). callus formed with mT was minimal compared to most TDZ treatments. 366
328 Collado et al. [35] reported that 2,4-D at the concentration of The interactions between endogenous PGRs in different plant 367

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329 4.0 mgl-1 combined with 1.0 mgl-1 kinetin was more effective for organs at different developmental stages are very complex, and the 368
330 inducing callus on immature cotyledons of Swietenia macrophylla King manner in which a given explant will react to different 369
331 (mahogany) than 2,4-D at the concentrations of 2.0 or 6.0 mgl-1 [35]. concentrations of different exogenous PGRs in vitro can vary greatly
D 370
332 This was in contrast to the results of the present study, in which we depending on the species, variety, age, and source of the explant 371
333 found 2,4-D at the concentrations of 2 and 4 mgl-1 to have a negative [38]. There were some important limitations to this study. The 372
334 effect on callus formation on leaf sections of G. mangostana, G. cowa, exact age and size of the leaves that were used as explants were 373
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335 and G. celebica. For Garcinia species, it appears that only lower not completely uniform. Variation in the extent of callus formation 374
336 concentrations of 2,4-D are useful for callus induction. may have been partly due to the age of the leaf from which each 375
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337 Kiong et al. [36] obtained a callus induction rate of 100% on leaf section was taken. In addition, individual leaves were not all the 376
338 explants of Melaleuca alternifolia (tea tree) with 2,4-D added to MS same size; hence, some of the leaf sections that were cut were 377
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339 medium at the rates of 1, 3, and 5 mgl-1. They described the callus as slightly larger and thicker than the other sections. This may have 378
340 greenish yellow, both friable and nodular. In the same report, Kiong et had an influence on the amount of callus tissue that developed. 379
341 al. [36] also reported callus induction rates of 93.3%, 96.7%, and 100% Finally, for this preliminary study, we tested each PGR separately in 380
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342 when picloram was added to the medium at the concentrations of 1, different concentrations but did not test combinations of PGRs. In 381
343 3, and 5 mgl-1, respectively. The callus from the picloram treatments future research, it would be interesting to test combinations of 382
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344 was noted to be yellowish brown and more friable [36]. We observed auxins and cytokinins for callus formation. 383
345 large amounts of white to green, fuzzy-looking callus on G. cowa in all
346 three picloram treatments (0.5, 2.5, and 5 mgl-1). In an experiment on
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347 another woody species, Anacardium occidentale (cashew), Cardoza and 4. Conclusions 384
348 Souza [28] found picloram at concentrations of 0.5 and 1 mgl-1 to be
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349 effective in inducing callus. In this research, we induced callus in vitro from young leaf and stem 385
350 Most of the research on mT has been done on herbaceous plants. For tissue from G. mangostana and G. schomburgkiana, and leaf tissue of two 386
351 instance, it was found to be effective for shoot induction in the underutilized woody plant species, G. celebica and G. cowa, using TDZ, 387
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352 endangered African plant Aloe polyphylla [37] and the ornamental 2,4-D, picloram, and mT. The largest amount of compact, nodular 388
353 plant Spathiphyllum floribundum Schott cv. Petite [32]. We were callus was obtained from the 0.5 mgl-1 TDZ treatment for G. 389
mangostana, G. schomburgkiana, and G. cowa and from the 0.1 mgl-1
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354 interested in testing the effect of mT on callus formation in Garcinia 390

355 species to contribute to the body of knowledge for culturing woody TDZ treatment in the case of G. celebica. The results may be useful for 391
356 species. Thus far, to our knowledge, only one other study has been biopharming and plant breeding efforts in the future. 392
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Fig. 5. Examples of medium- to large-sized callus stem explants of 2 species in genus Garcinia. (a) Garcinia mangostana stem section from in vitro seedling 4 weeks after culture on MS
medium with 0.5 mgl-1 TDZ; (b) Garcinia mangostana stem section from in vitro seedling 4 weeks after culture on MS medium with 0.1 mgl-1 TDZ; (c) Garcinia schomburgkiana stem
section from in vitro seedling 4 weeks after culture on MS medium with 0.1 mgl-1 TDZ; (d) G. schomburgkiana stem section from in vitro seedling 4 weeks after culture on MS medium
with 0.5 mgl-1 TDZ;

Please cite this article as: V. Suwanseree, S. Phansiri and C. Yapwattanaphun, A comparison of callus induction in 4 Garcinia species, Electronic
Journal of Biotechnology, https://doi.org/10.1016/j.ejbt.2019.04.006
 V. Suwanseree et al. / Electronic Journal of Biotechnology xxx (2019) xxx–xxx 7

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Please cite this article as: V. Suwanseree, S. Phansiri and C. Yapwattanaphun, A comparison of callus induction in 4 Garcinia species, Electronic
Journal of Biotechnology, https://doi.org/10.1016/j.ejbt.2019.04.006