Picloram enhanced the callus induction, growth

kinetics, antioxidant potentials, and secondary

metabolites production of Zingiber o cinale var.
rubrum callus cultures
Pavallekoodi Gnasekaran 
Universiti Sains Malaysia
Zuraida Abdul Rahman 
MARDI: Institut Penyelidikan dan Kemajuan Pertanian
Bee Lynn Chew 
Universiti Sains Malaysia
Jasim Uddain 
Sher-e-Bangla Agricultural University
Maheswaran Solayappan 
AIMST University
Nelson Yeou Chear Jeng 
Universiti Sains Malaysia
Suganthi Appalasamy 
Universiti Malaysia Kelantan
Vanitha Mariappan 
Universiti Kebangsaan Malaysia
Dwi Kusuma Wahyuni 
Universitas Airlangga
Sreeramanan Subramaniam  (  sreeramanan@gmail.com )
Universiti Sains Malaysia

Research Article

Keywords: Zingiber o cinale var. rubrum, Leaf-sheath, Callus, Secondary Metabolites, Gas
chromatography

Posted Date: April 7th, 2023

DOI: https://doi.org/10.21203/rs.3.rs-2698252/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  
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 1 Picloram enhanced the callus induction, growth kinetics, antioxidant potentials, and
2 secondary metabolites production of Zingiber officinale var. rubrum callus cultures.
3
4 Pavallekoodi Gnasekarana, Zuraida Abdul Rahmanb, Bee Lynn Chewa, Jasim Uddainc,
5 Maheswaran Solayappand, Nelson Jeng Yeou Cheare, Suganthi Appalasamyf, Vanitha
6 Mariappang, Dwi Kusuma Wahyunih,Sreeramanan Subramaniam a,h, i*
7
a
8 School of Biological Sciences, Universiti Sains Malaysia (USM), Georgetown, 11800 Penang,
9 Malaysia,
10
b
11 Biotechnology Research Centre, MARDI Headquarters, MARDI HQ, Persiaran MARDI-UPM,
12 43400 Serdang Selangor, Malaysia,
13
c
14 Department of Horticulture, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh
15
d
16 Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100, Semeling,
17 Kedah, Malaysia
18
e
19 Centre for Drug Research, University Sains Malaysia, 11800 Minden, Penang, Malaysia
20
f
21 Department of Natural Resource and Sustainability, Faculty of Earth Science, Universiti
22 Malaysia Kelantan (UMK), Jeli Campus, Locked Bag No. 100, 17600 Jeli, Kelantan, Malaysia
23
g
24 Centre of Toxicology and Health Risk Studies (CORE), Faculty of Health Sciences, Universiti
25 Kebangsaan Malaysia (UKM), 50300 Kuala Lumpur, Malaysia
26
h
27 Department of Biology, Faculty of Science and Technology Universitas Airlangga, Surabaya
28 60115, Indonesia
29
i
30 Chemical Centre Biology (CCB), Universiti Sains Malaysia (USM), Bayan Lepas, 11900, Penang,
31 Malaysia
32
33
34
35 ⁎Corresponding author. E-mail address: sreeramanan@gmail.com / sreeramanan@usm.my
36
37
38
39
40

1
41 ABSTRACT
42 Plant cells are driven by types and concentrations of plant growth regulators to produce callus

43 mass containing bioactive compounds. This study aimed to induce callus and to observe the

44 histological, phytochemicals, and antioxidant basis of the callus. An efficient callus induction

45 protocol was developed using picloram for Malaysian red ginger, Zingiber officinale var. rubrum.

46 The effect of auxinic picloram herbicide was studied using six different concentrations (0, 0.5, 1,

47 2, 4, and 8 mg/L) on various explants (leaf sheath, leaf, root) to optimise the callus induction. The

48 induced callus was studied for growth kinetics, anatomical features, antioxidant capacity, and

49 phytochemical content. The highest callogenesis frequency (93.75%) and biomass accumulation

50 (3.68 g) were observed on leaf sheath explant cultured on ½ strength Murashige and Skoog (MS)

51 medium supplemented with 8 mg/L which also requires earlier subculture duration (45 days post-

52 inoculation) in comparison to lower concentrations. Morphological investigation through

53 histological procedure demonstrated friable and non-embryogenic characteristics of the primary

54 and subcultured callus. Cultivated leaf sheath (CLS) methanolic extract showed the highest total

55 phenolic (191.26 mg GAE/g dry extract) and flavonoid (4.54 mg QE/g dry extract) contents

56 contributing to antioxidant activity with an estimated EC50 of 0.208 mg/mL. Although

57 comparatively lower than CLS extract, callus extracts showed higher antioxidant activity and

58 significantly lower EC50 values than in vitro leaf sheath extract. 4H-Pyran-4-one, 2,3-dihydro-3,5-

59 dihydroxy-6-methyl-, phenol, and phenolic glucoside were only present in callus cultures while

60 methyl esters, fatty acids, and phytosterols could be obtained from leaf sheath and callus extracts.

61 In conclusion, the callus culture of Z. officinale var. rubrum is a potential renewable source of

62 bioactive phytochemical compounds and can be employed for biotechnological practices such as

63 elicitor-induced accumulation of secondary metabolites and genetic modification.

2
64 Keywords: Zingiber officinale var. rubrum; Leaf-sheath; Callus; Secondary Metabolites; Gas

65 chromatography

66

67 Graphical abstract

68

69
70
71 1. Introduction
72
73 Manipulation of plant cells to produce unorganized masses of undifferentiated cells under

74 in vitro conditions is driven by types and concentrations of plant growth regulators as well as

75 abiotic factors (Mavrikou et al., 2020). Induced callus could be maintained for an extended period

76 by subculturing on a replenished growth media (Efferth, 2019). Picloram (4-amino-3,5,6-trichloro

77 picolinic acid) hormone with auxin-like activity selectively demonstrates callus induction ability.

78 Despite the ability of 2,4-D auxin for the induction of callus, several researchers have reported on

79 the callogenesis potentials of picloram (El-Mageid, 2019; Habibah et al., 2019; Salma et al., 2019).

80 Callus cultures are treated as in vitro biofactories for the production of bioactive

81 compounds. Biosynthetic cycles of cell cultures are higher due to enhanced metabolic rate in

82 comparison to differentiated plants (Saw et al., 2010). Therefore, experimental conditions are

83 studied to optimize secondary metabolite production on a large scale (Hundare et al., 2018). Thus,

3
 84 in vitro callus culture act as a means to facilitate indirect somatic embryogenesis or to produce

85 phytochemicals (de Oliveira et al., 2018). Callus growth kinetics is generally studied to identify

86 an appropriate timeline for subculture or harvest (da Luz Costa., 2015).

87 The callus growth curve generally follows a sigmoid pattern with distinct phases of

88 growth. The onset of the growth is initiated by a lag phase involving adaptation to the environment

89 before reaching an exponential phase marked by accelerated cell division and growth, whereby the

90 subsequent stationary phase involves the deceleration phase with zero growth rate and cell death

91 (Hussein et al., 2016).

92 Phenolics and flavonoids are natural antioxidative phytochemicals acting as reducing

93 agents by donating electrons to inactivate free radicals (Ullah et al., 2019). 2,2-diphenyl-1-picryl-

94 hydrazyl-hydrate (DPPH) assay has been perfected to determine the antioxidant activity while gas

95 chromatography-mass spectroscopy (GCMS) develops the chemical profiling of the plant extracts

96 and compounds (Choudhary et al., 2019; Gomathi et al., 2019). Preliminary analyses such as

97 antioxidant capabilities and chemical compound identification of plant extracts are explored to

98 assist biological activities.

99 The demand for Z. officinale var. rubrum metabolites, stimulated objective-driven tissue

100 culturing efforts of inducing ginger callus and withal, to conduct a comparative study between leaf

101 sheath explant and leaf sheath-induced callus cultures grown under differential picloram

102 concentrations. Hence, the present study aimed at determining the total phenolic and flavonoid

103 contents, antioxidant capacity, and secondary metabolites production of leaf sheath explant and

104 their callus culture.

105

4
106 2. Materials and methods
107
108 2.1 Establishment of callus cultures
109

110 Leaf, leaf sheath, thin root, and thick root were dissected from 12-weeks-old in vitro Zingiber

111 officinale var. rubrum plants. Leaf-sheaths and thin roots were trimmed into 1 cm x 0.2 cm

112 measurements while leaves and thick roots were trimmed to 0.4 cm2 size. Explants were cultured

113 on full strength Murashige and Skoog (MS) media supplemented and enriched with picloram at

114 different concentrations (0, 1, 2, 4, and 8 mg/L) in addition to 20 g/L sucrose and solidified with

115 3g/L Gelrite. The pH of the media was adjusted to 5.8 with 0.1 M HCl or 0.1 M NaOH following

116 the sugar dissolved completely and sterilized by autoclave for 15 min at 121°C (Tomy High-

117 Pressure Steam Sterilizer ES-315, Japan). Cultures were maintained in complete darkness in the

118 plant tissue culture room maintained at 25±1°C. Each treatment consisted of nine replicates with

119 16 explants inoculated on each Petri plate. The callus induction was evaluated 90 days after

120 inoculation concerning callus fresh mass and callus induction frequency.

121

122 2.2 Establishment of the callus growth curve

123
124 Actively growing 14-day-old leaf sheath-derived callus (1g) was inoculated on callus induction

125 media supplemented with either 2, 4, or 8 mg/L picloram to allow proliferation and biomass

126 accumulation. Callus mass proliferated on hormone-supplemented medium were labelled as Callus

127 2, Callus 4, and Callus 8, respectively according to the concentration of the picloram to support

128 callus growth. The growth kinetics of callus proliferating in response to darkness was studied for

129 60 days at 5 days intervals with five replicates for each time point.

5
130 Callus was scooped out from the media and secured on a sterile filter paper (Whatman No.

131 1 England) to absorb excess moisture on the callus mass before measuring the fresh weight (FW)

132 of biomass on a weighing scale (Sortorious digital balance; Germany). The dry weight (DW) of

133 the callus biomass was obtained after achieving constant weight following the drying process at

134 40˚C in an incubator (Thermo Scientific; Germany). The growth index is defined as estimated

135 growth capacity by comparing the biomass accumulation at the sampling time to the pre-incubation

136 period (Godoy-Hernández and Vázquez-Flota, 2006). Hence, callus relative biomass growth

137 (CRBG) was determined according to the formula published by Huang et al. (2015).

𝑊𝑒𝑖𝑔ℎ𝑡 𝑔𝑎𝑖𝑛𝑒𝑑 𝑎𝑓𝑡𝑒𝑟 𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 𝑑𝑎𝑦𝑠 (𝑊2) − 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑊1)

138 𝐶𝑅𝐵𝐺 =
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡

139

140 2.3 Histological analysis of primary and subcultured callus

141 Histological steps established by Gnasekaran et al. (2016) were adapted to study

142 anatomical features of primary and sub-cultured callus. Induced and sub-cultured (4-weeks-old)

143 callus were fixed in formalin-acetic acid-ethanol (FAA) fixative for 4 days and rinsed with fresh

144 tap water at 1 hour interval for 6 hours on day 5. Fixation was followed by dehydration of the

145 samples in graded series of ethanol. On day 1, samples were dehydrated in 50% and 70% tert-

146 butyl alcohol (TBA) for 4 hours each and soaked in 85% TBA overnight. On day 2, the dehydration

147 step was continued with 90%, 95%, and absolute TBA for four hours each before leaving the

148 samples in the second change of absolute TBA overnight. On day 3, dehydrated samples were

149 submerged in xylene-substitute for an hour to remove pigments. Transparent samples were then

150 transferred to melted wax at 60oC by changing to a fresh wax solution at 1-hour intervals for three

151 times and blocked. During blocking, processed samples were immediately positioned on cubical

6
152 metal moulds half-filled with molten wax and coated with the extra wax solution. As precautionary

153 steps, metal moulds should be left on the cold platform for 30 min before and after blocking to

154 allow the wax blocks to detach easily and water droplets on the moulds are wiped off prior to

155 blocking. Wax blocks were fixed to a microtome cutter (Leica RM 2135) for slicing samples with

156 10 µm thickness. Thin strips of samples left floated on 40oC water for a few seconds and were

157 collected on a clean glass slide. Slides were dried on a slide warmer before leaving overnight at

158 60oC in the incubator (Memmert, Germany). The staining procedure was carried down the next

159 day in a transparent horizontal glass jar with a lining to hold the slides.

160 Slides were deparaffinized by rinsing in Histo-Clear solvent (National Diagnostics, USA)

161 for 10 minutes and rehydrated in a series of reducing ethanol concentrations (100%, 90%, 70%,

162 and 50%) for 2 min each. Rehydrated slides were stained with safranin for 3 hours before rinsing

163 in 70% and 80% ethanol for a minute each. Counterstaining with fast green was done for 2 mins

164 before dehydrating twice in 95% ethanol for a min, rinsing once in 100% ethanol for 2 mins, and

165 twice in Histo-Clear for a total of 6 mins subsequently. Stained slides were air-dried and mounted

166 with a coverslip using Shandon Histomount xylene substitute Mountant (Thermo Scientific). The

167 slides were observed via a light microscope (Olympus BX50, Olympus Optical Co. Ltd., Japan)

168 fitted to a JVC K-F55B colour video camera (JVC Victor Company of Japan, Limited, Japan) and

169 Docu Version 3.1 image analysis system (Soft Imaging System, GmbH, Münster, Germany) was

170 used to analyse the images.

171

172

173

7
174 2.4 Phytochemical analysis of Zingiber officinale var. rubrum extracts
175 2.4.1. Preparation of methanolic extracts

176 Callus and leaf sheath extraction was prepared using a modified protocol of Ghasemzadeh

177 and Jaafar (2011). Callus (4 weeks old), in vitro leaf sheath (IVLS) (3 months old), and leaf sheath

178 sourced from cultivated plants (6 months old) were oven-dried (Memmert, Germany) at 40oC to

179 constant weight to ensure complete moisture removal prior to grinding and extraction. Powdered

180 material (1 g) was extracted with 50 mL analytical grade methanol (MeOH) (Merck, Darmstadt,

181 Germany) for 15 mins via sonication in B-5510 ultrasonic cleaning bath powered by 42 kHz and

182 135 kW (Branson Ultrasonics Corporation, Danbury, CT, USA). Sonicated extracts were then

183 swirled at 100 rpm for an hour on an orbital shaker (Lab Companion SK-71) at room temperature.

184 Filtration of the crude extracts was done by a suction-filtering technique using filter paper

185 (Whatman® No. 1, 110 mm diameter) layered in a porcelain Büchner funnel connected to an

186 aspirator vacuum pump (EYELA A1000S, Tokyo Rikakikai Co. Ltd.) and evaporated to dryness

187 under reduced pressure at temperature < 40°C. Extracts were maintained in a -20ºC deep freezer

188 until further analysis. The percentage yield of each extract was computed according to the formula

189 of Moriasi et al. (2020) as below:

𝐷𝑟𝑖𝑒𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 (𝑔)

190 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑦𝑖𝑒𝑙𝑑 (%) = × 100%
𝐷𝑟𝑖𝑒𝑑 𝑝𝑙𝑎𝑛𝑡 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑢𝑠𝑒𝑑 𝑓𝑜𝑟 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 (𝑔)

191
192 2.4.2. DPPH free radical scavenging assay
193
194 DPPH free radical scavenging activity of the extracts was estimated by bleaching of purple

195 colouration (Chear et al., 2019). First, the extract was dissolved in MeOH to a concentration of

196 2 mg/mL and treated as a stock concentration to carry out serial dilution. In brief, 100 µL aliquot

197 of 0.2 mM methanolic DPPH was allowed to react with 100 µL of extract serially diluted to the

8
198 concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 mg/mL in a 96-well microtitre plate.

199 MeOH served as blank while MeOH and DPPH mixture was treated with negative control. After

200 30 min of dark incubation, the optical density (OD) of each well was measured via UV-visible

201 microplate spectrophotometer (Thermo Scientific™ Multiskan™ GO) at 517 nm. Percentage

202 inhibition and IC50 value of extracts at various concentrations were computed to infer the

203 scavenging activity which was constructed by plotting inhibition (%) versus extract concentration.

𝑂𝐷 𝐵𝑙𝑎𝑛𝑘 − 𝑂𝐷 𝑆𝑎𝑚𝑝𝑙𝑒
204 % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = × 100
𝑂𝐷 𝐵𝑙𝑎𝑛𝑘

205 Data were obtained from independent triplicate assays.

206

207 2.4.3 Total phenolics content (TPC)

208
209 Evaluation of total phenolic content (TPC) of callus and leaf sheath extracts based on Folin-

210 Ciocalteu reagent carried out according to the protocol of Basma et al. (2011). Solutions in the

211 order of standard or extracts (200 µL) at 1 mg/mL concentration, 500 µL 10% Folin-Ciocalteu’s

212 reagent, and 500 µL distilled water were aliquoted into 2.5 mL clean Eppendorf tubes. The mixture

213 was vortexed immediately and allowed to stand for 5 mins before being loaded with 800 µL 7.5%

214 saturated aqueous sodium carbonate (Na2CO3) and vortexed once again. Finally, 200 µL of the

215 mixture was aliquoted to 96 well-plate and incubated for 30 min in darkness at room temperature.

216 OD was measured at 765 nm using a microplate spectrophotometer (Multiskan Go, Thermo

217 Scientific). MeOH was treated as a blank and TPC was estimated from the calibration curve of a

218 gallic acid standard prepared in the range of (0-250 mg/L) (Figure 1). TPC was expressed as gallic

219 acid equivalent per gram of DW (mg GAE/g) of extracts according to the below formula:

9
 𝑉
220 𝐶 = 𝐶1 ×
𝑀

221 C = Total phenolic content mg/g, in GAE

222 C1= Gallic acid equivalence (mg/mL) or concentration of gallic acid established from the

223 calibration curve (Y = 0.0044x + 0.1148; R2 = 0.985).

224 V = Volume of extract (mL)

225 M = Weight of the extract (g)

226
227
228 2.4.5. Total flavonoid content
229
230 The aluminium chloride (AlCl₃) method by Al‐Mansoub et al. (2014) was adapted to

231 estimate the total flavonoid content (TFC) of the extracts. In brief, solutions in the order of 100 µL

232 of standard or extract, 20 µL of 10% w/v AlCl₃, 20 µL of 1 mol L−1 sodium acetate, 300 µL MeOH,

233 and 560 µL distilled water were added to clean Eppendorf tubes and 200 µL of the mixture was

234 transferred to 96 well-plate. The well-plate was incubated for 30 mins in darkness at room

235 temperature before proceeding with OD measurement at 415 nm using a microplate

236 spectrophotometer (Multiskan Go, Thermo Scientific). TFC of each sample was estimated from

237 the quercetin calibration curve established in the range of 0-100 mg/L (Figure 2). TFC was

238 expressed in mg of quercetin equivalents per gram of dry extract (mg Quercetin/g) by computing

239 according to the below formula:

𝑉
240 𝐶 = 𝐶1 ×
𝑀

241 C = Total flavonoid content mg/g, in quercetin

10
242 C1= Quercetin equivalence (mg/mL) or concentration of quercetin established from the calibration
243 curve (Y = 0.0105x + 0.0493; R2 = 0.9995).
244 V = Volume of extract (mL)
245 M = Weight of the extract (g)
246
247
248 2.4.6. Gas chromatography-mass spectrophotometry
249
250 GCMS analysis of the crude methanolic extracts of Zingiber officinale var. rubrum was

251 conducted according to the protocol established by Chear et al. (2016). GCMS analysis of extracts

252 at 1 mg/mL concentration of cultivated leaf sheath (CLS) and IVLS, as well as callus maintained

253 on MS, supplemented with 2, 4, and 8 mg/L picloram were carried out on an Agilent 6890N

254 Network GC system coupled to an Agilent 5973i Mass Selective Detector (Agilent Technologies,

255 Waldbronn, Germany) fitted to HP-5MS column (30 m 0.25 mm, 0.25 mm; Agilent Technologies)

256 with inert helium gas flowing at 1.2 mL/min. Injected 1mL extract carried via splitless mode. The

257 initial temperature of the oven was maintained at 70oC for 2 mins and increased at the rate of

258 20oC/min to attain 280oC. Similarly, the column was maintained at 280oC for 20 mins while the

259 injector and detector were maintained at 280oC and 250oC, respectively. Mass acquisition by

260 electron-impact ionization was achieved at 70 eV over the range of m/z 40e550 (atomic mass unit,

261 AMU). The spectral match against Wiley Registry (John Wiley and Sons, Hoboken, NJ, USA) and

262 the National Institute of Standards and Technology database (Gaithersburg, MD, USA) libraries

263 identified separated chemical components. Mass spectrum similarity between compounds between

264 Z. officinale var. rubrum extracts and library platforms was confirmed by comparing molecular

265 ion mass, base ions, fragment ions, and peak intensities to ensure spectral matching of more than

266 90%. The correlation percentage maximum (CPM) of the identified compounds was calculated as

267 per the stated formula which was based on peak area as the unit for comparison.

11
 𝐴𝑟𝑒𝑎 𝑜𝑓 𝑎 𝑠𝑒𝑙𝑒𝑐𝑡𝑒𝑑 𝑝𝑒𝑎𝑘
268 𝐶𝑜𝑟𝑟𝑒𝑙𝑎𝑡𝑖𝑜𝑛 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑚𝑎𝑥𝑖𝑚𝑢𝑚 = × 100
𝐴𝑟𝑒𝑎 𝑜𝑓 𝑡ℎ𝑒 𝑙𝑎𝑟𝑔𝑒𝑠𝑡 𝑝𝑒𝑎𝑘

269 CPM was calculated by considering the largest peak defined by ethyl alpha-d-glucopyranoside

270 which scored 100%.

271

272 2.5. Statistical analysis

273
274 IBM Statistical Package for Social Sciences (SPSS) version 24 software (SPSS Inc.,

275 Chicago, IL, USA) was used for data analysis. One-way ANOVA and Duncan's multiple range

276 tests at p≤0.05 level of significance were applied to identify the significance of the differences

277 between the mean values of different treatments.

278

279

280 3. Results and Discussion

281
282 3.1. Establishment of callus cultures in Zingiber officinale var. rubrum

283
284 Callus induction capabilities of Zingiber officinale var. rubrum explants comprising of the

285 leaf sheath, thin root, thick root, and the leaf were studied on callus induction media enriched with

286 picloram at 0, 0.5, 1, 2, 4, and 8 mg/L. The control leaf sheath incubated in medium lack of

287 picloram showed no morphological changes and turned brown by the end of the incubation period

288 (Figure 3A). Leaf-sheath exhibited callus initiation by 9th-week post-inoculation and notably most

289 responsive explant for callus induction with the highest biomass FW (Table 1) and high callusing

290 frequency (Table 1). Leaf-sheath exposure to a longer duration for callogenesis up to 40 days was

291 observed on Curcuma longa leaf sheath cultured on MS medium supplemented with 3 mg/mL 2,4-

12
292 D and 0.8 mg/mL kinetin (Gurav et al., 2020). Callus initiation was initiated at the cut end of the

293 explants which was in contact with the medium. Excision of the explant caused epidermal rupture

294 which exposed the underlying tissue to the exogenous auxin supplemented with the callus

295 induction medium. Nutrients and growth hormones from the induction medium are absorbed

296 efficiently via the cut edges (Lee and Pijut, 2017). Subsequent rapid cell division at the epidermal

297 and sub-epidermal regions covered the explants with callus mass.

298 Higher concentrations induced massive callus formation on all explants except the leaf. For

299 example, in comparison to 2 mg/L picloram (1.74 g) (Figure 3B), 8 mg/L picloram (3.68 g) (Figure

300 3C) produced twice the callus mass on the leaf sheath (Table 1). Lower picloram concentration

301 caused the cut end of the leaf sheath to expand and swell which remained stagnant and turned

302 brown by the end of the experiment similar to the control treatment. Significant callogenesis on

303 the leaf sheath was observed from 2 mg/L picloram onwards (Figure 3C). Although leaf sheath

304 explants changed in colour from light green to brownish by the end of 12 weeks of dark incubation,

305 the proliferation of whitish to pale yellowish friable callus was continued (Figure 3C).

306 Increasing the concentration of picloram from 2 to 8 mg/L picloram proportionally

307 amplified the callus induction frequency of leaf sheath from 77% to 93% (Table 1). In general,

308 2,4-D is applied to induce callogenesis in ginger (El-Nabarawy et al., 2015; Ibrahim et al., 2015;

309 Musfir Mehaboob et al., 2019) because of DNA replication and mitosis potentials (Sen et al.,

310 2014). However, the present study on Z. officinale var rubrum is the first to prove the callogenesis

311 potentials of picloram on Zingiber species. Previously, Stanly et al. (2011) observed that picloram

312 did not induce callogenesis on a leaf, roots, and rhizome explants of Zingiber zerumbet Smith at

313 the end of 6 weeks of the incubation period. The differential callogenesis responses by the explants

13
314 are also driven by stimulating the callogenesis capacity of picloram in the dark (Habibah et al.,

315 2018) and the endogenous hormone content of the explant itself.

316 The competence of leaf sheath cells to induce callus was reported previously by Awan et

317 al. (2019) and Kaewthip et al. (2021). The highest callus initiation frequency by Z. officinale var.

318 rubrum leaf sheath is also contributed to the lack of chlorophyll content on the explants. Synthesis

319 of chlorophyll in the presence of light-driven loss of the callusing ability of leaf sheath was

320 observed by Sengar et al. (2011). Low chlorophyll contents in the cells naturally increase the

321 morphogenetic potentials of the explants. Besides, Bhattacharya and Sen (1980) demonstrated the

322 totipotency characteristic of leaf sheath cells. In vitro conditioning or wounding of totipotent plant,

323 cells will instruct the dedifferentiation process to drive the formation of an undifferentiated cell

324 mass called callus (Sugimoto et al., 2011).

325 Thin roots exposed to picloram-induced lateral root formation (Figures 3E and F) and

326 incubation at 4 and 8 mg/L of concentration demonstrated insignificant callogenesis frequency

327 (above 70%) by producing pale white to brownish compact callus (Figure 3F) in the range of 0.09

328 g and 0.43 g, respectively (Table 1). Lateral rooting on root segments of Z. officinale var. rubrum

329 by picloram is stimulated by auxin's ability on root induction from the pericycle of roots. The

330 observation is backed by auxin's interaction with receptors localized to the pericycle layer (Da

331 Costa et al., 2013) to activate root hair and lateral root initiation (Ishimaru et al., 2018) like root

332 developmental pathway driven by ectopic expression of root meristem regulators (Xu et al., 2018).

333 Mendoza and Kaeppler (2002) established that picloram-enriched media stimulates roots and

334 rootlike structures on the induced callus.

335 Thick root exhibited callogenesis upon exposure to 4 and 8 mg/L picloram (Figure 3H) by

336 producing a callus mass of 0.06 g and 0.17 g (Table 1). Explants treated with 0.5, 1, 2 mg/L

14
337 picloram demonstrated no morphogenetic changes (Figure 3G) similar to the lack of callogenesis

338 potential observed on root segments in the presence of 2,4-D either alone or in combination with

339 naphthalene acetic acid (NAA), indole acetic acid (IAA) or Indole-3-butyric acid (IBA) by Khan

340 et al. (2017). Supplementation of picloram was unable to induce callogenesis response on leaf

341 explant which exhibited decolouration and turned brown during 12 weeks of dark incubation

342 (Figure 3D). Browning of Z. officinale var. rubrum leaf explants could have been caused by

343 endogenous polyphenols content (Nurwahyuni et al., 2020) or phytotoxic nature of picloram

344 contributed by increased mobility and persistent within the cells (Lemma et al., 2019). Miri

345 (2020) reported that Z. officinale leaf explants formed green-red compact and friable calli on MS

346 medium enriched with 1 mg/L 2,4-D, Dicamba, or 6-benzyladenine (BA) (Miri, 2020) while

347 Somashekar et al. (2018) observed inability of 2,4-D either alone or in combination with 6-

348 benzylamino purine (BAP) on inducing callus from leaf explants. According to Ikeuchi et al.

349 (2013), plants inherit a robust mechanism that inhibits unnecessary callogenesis to maintain their

350 tissue organization.

351 Callus initiation and frequency of callogenesis of Z. officinale var. rubrum are significantly

352 influenced by the concentration of exogenous picloram and the type of explant. Based on the result,

353 Z. officinale var. rubrum leaf sheath incubated on a callus induction medium enriched with 8 mg/L

354 picloram demonstrated rapid callogenesis. Therefore, callus derived from leaf sheath explant was

355 used for the subsequent experiments.

356

357 3.2. Establishment of the callus growth curve

358
359 Callus growth kinetics was studied for 60 days at 5 days interval in terms of proliferation

360 capacity defined by FW and DW. Zingiber officinale var. rubrum callus proliferation increased

15
361 relative to picloram concentration and cultivation period (Figure 4). Increasing the concentration

362 of picloram has proven to uphold Z.officinale var. rubrum callus growth. For example, fresh and

363 dried biomass growth of Callus 8 on the 60th day was recorded to be 3-fold higher than that of 2

364 mg/L picloram-treated callus (Table 2).

365 Z. officinale var. rubrum callus growth kinetic exhibited a sigmoidal curve with variation

366 in the growth pattern influenced by picloram concentration (Figure 4). The present study displayed

367 a lag phase of 20 days (Figure 4) accompanied by an insignificant increase of callus biomass in all

368 the tested picloram concentrations (Table 2) due to the gradual adaptation of the callus to the

369 environment. During the lag phase, cells synthesise and mobilise proteins and specific metabolites

370 in the absence of cell multiplication (Asrori et al., 2020). However, at a longer incubation period,

371 FW biomass increased distinctly in response to higher picloram concentration (Table 2).

372 Callus 2 exhibited a weak sigmoidal curve with 20 days of lag phase and a subsequent fall

373 on day 40 before establishing a linear phase from day 50 to 60 (Figure 4A). Callus 2 exhibited

374 insignificant biomass increment in the range of 0.13 to 0.44 g during the lag phase which rose to

375 0.59 g and 0.82 g on days 25 and 30, respectively with a sharp proliferation, however, decreased

376 to 0.31 g on day 40 (Figure 4A). A steady upsurge in Callus 2 proliferation above 1g was observed

377 from day 45 onwards which marked a 1.95 g biomass gain on day 60. Contrarily, the dry weight

378 DW of the proliferated callus remained at 0.10 g from days 50 to 60, suggesting that callus growth

379 is stagnant (Table 2).

380 Callus 4 presented lag (until 20 days), exponential (25 to 40 days), linear (45 to 55 days),

381 and decelerating (day 60) growth phases. The exponential phase of Callus 4 demonstrated an

382 insignificant rise in proliferation by recording FW mass in the range of 0.90 to 1.43 g (Figure 4A).

16
383 However, FW (1.81 g) and DW (0.15 g) of Callus 4 biomass increased on day 45. Significant

384 difference demonstrated by Callus 4 FW from day 50 to 60, was nullified by the insignificant DW

385 (0.21 to 0.25 g) recorded during the last 15 incubation days, suggesting a stagnant growth which

386 is indicated by plateau condition (day 55 and 60) on Figure 4B.

387 Five growth phases including lag (15 days), exponential (20 to 35 days), linear (40 to 55

388 days), and deceleration (day 60) were detected for the proliferation of Callus 8 (Figure 4A). Callus

389 8 ended shortened lag phase with a sharp increase of biomass gain at the beginning of the

390 exponential phase on day 20 (0.94 g). The transitory stage is validated by doubling up the FW on

391 day 25 (2.00 g) in comparison to insignificant lag phase mass gain. Although the growth of Callus

392 8 was not statistically significant during 25 to 35 days of the exponential phase (Table 2), the

393 proliferation was on the rise on days 45 and 50 before reaching deceleration and plateau during

394 days 55 and 60 (Figures 4A and B). The predominant meristematic nature of callus cells (Coimbra

395 et al., 2019) maximises cell division during the exponential phase (Santos et al., 2017), and

396 increases biomass. Furthermore, in addition to the continuous production of primary metabolites

397 (Damayanti et al., 2020), the diameter and volume of the callus cells increase due to cell

398 enlargement (Bhatia, 2015) during the exponential phase which resulted in an increment of

399 biomass. However, in terms of DW, the growth of Callus 8 from day 45 to 60 was denoted

400 insignificant (0.31 to 0.33g) indicating a plateau condition. Insignificant DW may suggest higher

401 water content on the enlarged callus cells which also contributed to the significant FW of the callus.

402 The callus growth curve aid in identifying the timeline for the highest callus biomass

403 indicating subculturing period as the behaviour of callus cells differs at each phase of kinetics

404 growth (Daffalla et al., 2019). Z.officinale var. rubrum callus demonstrated a sigmoid pattern with

405 varied growth phases in response to picloram concentration (Figures 4A and B). The lag phase is

17
406 a transient non-replication period for cells to adapt to the environmental stress while producing

407 cellular components and metabolites to prepare for the upcoming cell division process (Bertrand,

408 2019; Burgos-Zazueta et al., 2021). The duration of the lag phase varies based on the stress level

409 experienced by the initial callus cells. For example, Z.officinale var. rubrum callus proliferated on

410 a medium supplemented with 2 and 4 mg/L picloram demonstrated 20 days of lag phase while 8

411 mg/L picloram reduced the transition period to 15 days.

412 In the present study, FW of callus proliferated on 4 and 8 mg/L picloram exhibited a

413 deceleration phase at day 60. Generally, a decrease in cell growth is caused by continued

414 consumption of the limiting nutrient. Cell division and cell expansion may reduce during the

415 deceleration phase (Rawat et al., 2020). According to Setiaji et al. (2020), during deceleration, the

416 rate of cellular division is reduced due to the depletion of nutrients, and the saturation of cells with

417 phenol-containing metabolites may cause the death of cells. However, no signs of senescence such

418 as callus browning were observed at the end of the incubation period of the present study.

419 The DW of Callus 2, Callus 4, and Callus 8 remained at 0.1, 0.2, and 0.3g, respectively

420 from day 50 to 60 which may designate reduced cell division and cell expansion leading to plateau

421 condition as highlighted by Castro et al. (2008) and Rawat et al. (2020). Eventually, cells enter the

422 stationary phase and transition to the death phase in the absence of a replenished growth medium.

423 Hence, it is suggested that Callus 2, Callus 4, and Callus 8 should be subcultured on days 50, 55,

424 and 45, respectively as the highest DW was recorded on these days.

425

426

427

18
428 3.3 Histological analysis of primary and subcultured callus

429

430 Primary callus develops on the parenchyma cells of the mesophyll tissue that recommenced

431 meristematic activity. Histological analysis of Zingiber officinale var. rubrum primary callus

432 induced on leaf sheath explant confirmed the presence of friable mass with non-embryogenic

433 nature (Figure 5). Globular units composed of organized small isodiametric cells and pigmentation

434 indicating embryogenic competence were missing on the primary callus and sub-cultured callus of

435 Z. officinale var. rubrum. Irrespective of the picloram concentration, both primary and sub-

436 cultured callus mass exhibited non-embryogenic characteristics such as an unorganized mass of

437 irregularly shaped cells with relatively smaller nuclei (yellow arrow) and highly vacuolated large

438 parenchymatic cells (Figure 5A to F) as reported by Suraiya and Alina (2018) on rice callus.

439 Furthermore, loosely connected non-embryogenic callus cells were observed by Ijaz et al. (2019)

440 on rice calli. Similarly, the non-embryogenic callus of Z. officinale var. rubrum disintegrates easily

441 as they were loosely packed granular in texture (Figure 5D) and presented intercellular space

442 (yellow star).

443

444 3.4 Antioxidant potentials of Zingiber officinale var. rubrum extracts and detection of related
445 compounds by GCMS

446
447 DPPH scavenging activity demonstrated by cultivated leaf sheath (CLS, in vitro leaf sheath

448 IVLS, and callus (C) extracts of Zingiber officinale var. rubrum increased in a concentration-

449 dependent manner (Table 3). The effectiveness of extracts at 1mg/ml concentration against DPPH

450 inhibition was recorded in the order of CLS (88.87%) > C2 to C8 (71.45 – 82.30%) > IVLS

451 (45.91%). Furthermore, CLS marked the lowest EC50 concentration besides the highest TPC and

19
452 TFC. EC50 of CLS, IVLS, C2, C4, and C8 were estimated to be 0.208, 1.324, 0.786, 0.295, and

453 0.295 mg/mL, respectively (Table 4).

454 Therefore, the DPPH free radical scavenging activity of Z. officinale var. rubrum extracts

455 is closely linked to TPC and TFC. Plant extracts rich in phenolic compounds exhibit greater free

456 radical scavenging activity (Goswami et al., 2020; Ng et al., 2020) since they act as redox

457 participants such as reducing agents, hydrogen donors, and singlet oxygen quenchers to perform

458 an antioxidant activity (Manssouri et al., 2020). The synergistic antioxidant action of 9,12-

459 Octadecadienoic acid (Z, Z)-methyl ester (30.66%) and hexadecanoic acid (15.43%) in CLS

460 extract (Table 5) with other compounds possibly produced the highest free radical scavenging

461 activities. Aggarwal et al. (2018), Mazumder et al. (2020), and Shah et al. (2020) have reported on

462 the antioxidant properties of these compounds.

463 In the present study, 32% of scavenging activity by CLS was contributed by 125 ug/mL

464 extract (Table 3) while Ghasemzadeh et al. (2010) reported that 45 ug/mL extract was sufficient

465 to induce the same level of activity. Variations in phytochemical and biological performances are

466 influenced by the origin of organ, environmental and developmental factors experienced by the

467 cultivated Z. officinale var. rubrum plants. The dynamics of secondary metabolite accumulation in

468 medicinal plants are affected by developmental stage and environmental stresses (Li et al., 2020)

469 and in response to the signaling molecules which trigger the biosynthesis of the compounds

470 (Ashraf et al., 2018). In comparison to CLS (0.208 mg/ml), Mustafa et al. (2019) demonstrated

471 that IC50 of 27.97 µg/mL by the highly potent rhizome extract of Z. officinale var. rubrum to

472 scavenge DPPH.

473 Callus extracts demonstrated higher DPPH scavenging activity and significantly lower

474 EC50 values than IVLS extract although comparatively lower than CLS extract (Table 4). Even

20
475 organs of cultivated Z. officinale Rosc. exhibited higher free radical scavenging activity in

476 comparison to callus extracts (Pawar et al., 2015; Ali et al., 2018) which may require intermission

477 by endogenous application of elicitors like glycine, yeast, and salicylic acid extract to upregulate

478 the synthesis of secondary metabolites by in vitro cultures. However, identified phytoconstituents

479 such as 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-Pyran-4-one (DDMP), 2-methoxy-4-

480 vinylphenol, 2,4- bis(1,1-dimethylethyl) and ethyl.alpha.-d-glucopyranoside (Table 5) may have

481 enhanced the antioxidant activity of the callus extracts as they are known to be strong antioxidants.

482 Antioxidant activity of DDMP (Li et al., 2019), 2-methoxy-4-vinylphenol (Rubab et al., 2020;

483 Nanok and Sansenya, 2021), 2,4- bis(1,1-dimethylethyl) (Shah et al., 2020) and ethyl.alpha.-d-

484 glucopyranoside (Sivakumar, 2019) has been proven scientifically. Antioxidant activities of C2,

485 C4, and C8 extracts show that Z. officinale var. rubrum callus cultures will be a viable system for

486 the pharmaceutical industry to ensure in vitro production of secondary metabolites towards

487 assisting the management of oxidative stress-associated disorders. For example, the content of

488 beta-sitosterol was the highest in callus extracts compared with leaf sheath extracts while

489 stigmasterol was only present within in vitro cultures and not found in CLS extract (Table 5).

490 Hence, Z. officinale var. rubrum callus cultures can be scaled up for the production of

491 medically important phytosterols such as beta-sitosterol and stigmasterol. Commonly used

492 anticancer agents demonstrate severe side effects apart from increasing resistance of target cancer

493 cells (Kowalczyk et al., 2022). Secondary metabolites synthesized by callus cultures will ease the

494 process of generating plant-based constituents in the fight against cancer. At the same time, scaled-

495 up synthesis of secondary metabolites via callus cultures may also contribute to the availability of

496 critical medicines such as antivenom agents. For example, the antivenom activity of b-Stigmasterol

497 has been reported against abundant phospholipase 2 protein of cobra and viper venom (Chakkinga

21
498 Thodi et al., 2022). In short, Z. officinale var. rubrum callus cultures which are independent of

499 geographical and climatic factors could serve as a viable, sustainable, incessant, and most

500 importantly economical mode of secondary metabolites production.

501 MeOH-assisted extraction produced the highest yield for C2 (42.18%) while IVLS

502 produced the least yield (17.36%) (Table 4). MeOH was chosen on the basis that it extracts

503 bioactive components including phenolics, flavonoids, alkaloids, and terpenoids besides being

504 reported to increase the extraction yield for Onosma sieheana (6.5%) (Binzet et al., 2019),

505 Aesculus hippocastanum extract (8.9%) (Amiri et al., 2019), Severinia buxifolia (Truong et al.,

506 2019), and Strobilanthes crispus (5.92%) (Gul et al., 2020).

507 Based on our observation, the TPC level was recorded as higher than TFC in all the tested

508 extracts (Table 4). Based on the shikimic acid pathway, phenolic acids, hydroxycinnamic acid,

509 lignans, and flavonoids are derivatives of phenolic compounds (Ramawat, 2009). Accumulation

510 of phenolic components such as cinnamic acid may suppress phenylalanine ammonia-lyase

511 enzyme activity which down-regulates flavonoid biosynthesis (Cheng et al., 2009). Therefore,

512 reduced TFC level in Z. officinale var. rubrum extracts could be contributed to the accumulated

513 phenolic components.

514 In comparison to callus extracts, IVLS extract demonstrated the lowest free radical

515 scavenging activity but contained higher TFC (2.67 mg QE/g dry extract) and equivalent TPC

516 (Table 4) in addition to 9.45% phytol (Table 5). Furthermore, palmitic acid (11.53%), phytol, and

517 phytosterols such as beta-sitosterol (30.36%) and stigmasterol (9.89%) (Table 5) in IVLS may

518 have been involved in a scavenging activity. Manisha et al. (2018) and Abdollahnezhad et al.

519 (2021) have emphasized the antioxidant characteristics of beta-sitosterol and stigmasterol.

520

22
521 4. Conclusion
522
523 Leaf-sheath explant cultured on ½ strength MS medium supplemented with 8 mg/L picloram

524 under complete darkness produced the optimal condition for Zingiber officinale var. rubrum callus

525 induction by forming a non-embryogenic friable mass. Callus growth kinetics and subculture

526 duration of proliferated callus mass influenced by the picloram concentration as day 45 for callus

527 grown on 8 mg/L picloram while day 50 for 2 and 4 mg/L picloram grown callus. Callus extracts

528 exhibited mediatory free radical scavenging activity by the synergistic effect of 4H-Pyran-4-one,

529 2,3-dihydro-3,5-dihydroxy-6-methyl-, 2-methoxy-4-vinylphenol (phenol), phenol, 2,4- bis (1,1-

530 dimethylethyl), ethyl alpha-d-glucopyranoside, methyl linoleate, and beta-sitosterol.

531

532 Author contributions

533 Pavallekoodi Gnasekaran, Suganthi Appalasamy, Zuraida Abdul Rahman, Dwi Kusuma Wahyuni,

534 Maheswaran Solayappan, Nelson Jeng Yeou Chear and Sreeramanan Subramaniam designed and

535 conducted the experiments, and analysed the data. Sreeramanan Subramaniam, Vanitha

536 Mariappan, Jasim Uddain and Chew Bee Lynn conceptualised and supervised the research.

537 Pavallekoodi Gnasekaran, Suganthi Appalasamy and Sreeramanan Subramaniam wrote the

538 manuscript.

539

540 Declaration of Competing Interest

541 The authors declare that they have no known competing financial interests or personal

542 relationships that could have appeared to influence the work reported in this paper.

23
543 Ethical statement

544 The results in the Manuscript are the author's original work. The authors confrm that this

545 manuscript has not been previously published and is not currently under consideration by any other

546 journal. Additionally, all of the authors have approved the contents of this paper and have agreed

547 to the journal’s submission policies

548

549 Acknowledgments

550 UMK Rising Star 2021 Grant (R/STA/A0800/01350A/007/2021/ 00937)

551
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31
Table 1: Callus induction and callogenesis frequency of Zingiber officinale var. rubrum explants on picloram enriched callus induction
medium.

Leaf sheath Thick root Thin root

Picloram
(mg/L)
Biomass (g) Frequency (%) Biomass (g) Frequency (%) Biomass (g) Frequency (%)

0 NIL NIL NIL NIL NIL NIL

0.5 NIL NIL NIL NIL NIL NIL

1 NIL NIL NIL NIL NIL NIL

2 1.74 ± 0.08c 77.08 ± 4.29ba NIL NIL NIL NIL

4 2.37 ± 0.05b 85.42 ± 3.13ba 0.06 ± 0.02** 54.17 ± 11.36c 0.09 ± 0.02** 79.86 ± 4.18ba

8 3.68 ± 0.07a 93.75 ± 2.76a 0.17 ± 0.09* 67.36 ± 9.19cb 0.43 ± 0.02* 73.61 ± 6.57cba

*Different letters within column indicate statistically significant differences (p < 0.05) using Duncan Multiple Range Test. (n=9)

32
Table 2: Proliferation of callus relative to the number of incubation days.

Picloram concentration (mg/L)

Number
2 4 8
of
incubation Fresh Dry Fresh Dry Fresh Dry
days weight weight weight weight weight weight
(g) (g) (g) (g) (g) (g)
0.13 ± 0.01 ± 0.23 ± 0.01 ± 0.23 ± 0.01 ±
5
0.01h 0.00 f 0.045 i 0.002 h 0.026 g 0.004 g
0.23 ± 0.02 ± 0.28 ± 0.03 ± 0.31 ± 0.03 ±
10
0.02gh 0.00 ef 0.04 hi 0.00 gh 0.02g 0.00 fg
0.33 ± 0.03 ± 0.40 ± 0.03 ± 0.53 ± 0.05 ±
15
0.01 fg 0.00 de 0.10hi 0.00 fgh 0.04 fg 0.01 f
0.44 ± 0.04 ± 0.47 ± 0.05 ± 0.94 ± 0.08 ±
20
0.02 f 0.00 cd 0.21 h 0.01 efg 0.16 f 0.01 e
0.59 ± 0.04 ± 0.64 ± 0.06 ± 2.00 ± 0.16 ±
25
0.03 e 0.01 cd 0.18 gh 0.01 efg 0.20 e 0.01 dc
0.82 ± 0.06 ± 0.90 ± 0.06 ± 2.26 ± 0.14 ±
30
0.02 d 0.00 bc 0.24fg 0.01 ef 0.34 de 0.02 d
0.70 ± 0.06 ± 1.22 ± 0.10 ± 2.30 ± 0.17 ±
35
0.06 de 0.01b 0.06 ef 0.00 d 0.12 de 0.01 c
0.31 ± 0.05 ± 1.43 ± 0.09 ± 2.68 ± 0.18 ±
40
0.02 fg 0.00 b 0.16 e 0.01 de 0.07 d 0.00 c
1.03 ± 0.06 ± 1.81 ± 0.15 ± 4.39 ± 0.33 ±
45
0.10 c 0.01 b 0.05 d 0.01 c 0.16 c 0.01 a
1.11 ± 0.10 ± 3.21 ± 0.25 ± 6.16 ± 0.31 ±
50
0.06 c 0.00 a 0.10 c 0.03 a 0.09 b 0.01 a
1.48 ± 0.10 ± 4.89 ± 0.21 ± 7.58 ± 0.31 ±
55
0.04 b 0.00 a 0.04 b 0.00 b 0.08 a 0.01 a
1.95 ± 0.10 ± 5.48 ± 0.22 ± 7.29 ± 0.32 ±
60
0.07 a 0.01 a 0.14 a 0.01 ab 0.25 a 0.01 a
Callus maintained on half-strength MS media supplemented with 2,4, 8 mg/L
picloram. Different letters indicate statistically significant differences (p < 0.05) using
Duncan Multiple Range Test. (n=5)

33
Table 3: Percentage of DPPH scavenging of the methanolic extracts of cultivated leaf sheath, in vitro leaf sheath, and callus cultures of
Zingiber officinale var. rubrum.

Concentrations (mg/mL)

Extracts 2 1 0.5 0.25 0.125 0.0625 0.03125

Cultivated LS 83.81 ± 0.5 b 88.87 ± 0.53 a 80.08 ± 0.53 b 52.98 ± 1.69 f 32.00 ± 0.14jk 20.48 ± 0.24n 16.86 ± 0.63nopq

in vitro LS 65.67 ± 0.11 d 45.91 ± 00.17 gh 29.77 ± 0.88 jk 20.65 ± 0.29 n 15.80 ± 0.34 opqr 11.91 ± 0.53 rs 9.35 ± 0.17 s

Callus 2 91.26 ± 0.40 a 71.45 ± 0.33 c 47.41 ± 1.69 g 32.72 ± 0.63 ij 21.04 ± 0.19 mn 13.13 ± 0.29 qrs 11.3 ± 1.55 rs

Callus 4 92.77 ± 0.45 a 80.47 ± 0.7 b 58.77 ±0.54 e 36.95 ± 1.02 i 25.21 ± 0.77 lm 19.48 ± 0.59 no 12.91 ± 1.43 qrs

Callus 8 92.45 ± 0.15 a 82.3±1.96 b 59.2 ± 0.79 e 41.78 ± 0.59 h 27.8 ± 0.42 kl 17.99 ± 0.3 nop 13.71 ± 1.05 pqrs

*Different letters within column indicate statistically significant differences (p < 0.05) using Duncan Multiple Range Test.

34
 Table 4: Total yield, EC50 values for 2,2-diphenyl-1-picrylhydrazyl scavenging activities, total flavonoid content and total phenolic
content of methanolic extracts of cultivated leaf sheath, in vitro leaf sheath, and callus cultures of Zingiber officinale var.
rubrum.

Total yield DPPH EC50 TPC TFC

Extracts
(%) (mg/mL) (mg GAE/g dry extract) (mg QE/g dry extract)

Cultivated leaf sheath 38.79 0.208 ± 0.002a 191.26 ± 7.75 a 4.54 ± 0.29 a

in vitro leaf sheath 17.36 1.324 ± 0.005e 49.29 ± 6.34 b 2.67 ± 0.25 b

Callus (2mg/L Picloram) 42.18 0.786 ± 0.010 d 53.68 ± 3.55 b 1.37 ± 0.30 c

Callus (4mg/L Picloram) 30.32 0.295 ± 0.008 c 69.21 ± 15.55 b 1.40 ± 0.10 c

Callus (8mg/L Picloram) 36.28 0.275± 0.011b 51.33 ± 14.85 b 2.00 ± 0.03 c

*Different letters within column indicate statistically significant differences (p < 0.05) using Duncan Multiple Range Test.

35
 Table 5: Relative content of chemical compounds identified in the extracts of Zingiber officinale var. rubrum

Retention Correlation Percentage Maximum

Peak (%)a of extracts
time Identified compound
label
(min) CLS IVLS C2P C4P C8P
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
1 5.82 ND ND 16.29 6.23 10.00
(DDMP)
2 7.16 2-methoxy-4-vinylphenol (Phenol) ND ND 4.72 6.97 9.28
3 8.43 Phenol, 2,4- bis(1,1-dimethylethyl) 4.05 3.10 9.86 6.49 5.01
Ethyl .alpha.-d-glucopyranoside
4 9.22 ND ND 23.54 47.92 100
(Phenolic glucoside)
15.4
5 10.72 Hexadecanoic acid, methyl ester (Palmitic acid ) 11.53 9.80 6.32 10.55
3
Benzenepropanoic acid, 3,5-bis (1,1-dimethylethyl)-4-
6 10.86 3.04 2.21 2.99 2.31 3.78
hydroxy-, methyl ester
9,12-Octadecadienoic acid (Z,Z)- methyl ester (Methyl 30.6
7 11.56 6.30 13.39 7.90 15.82
linoleate) 6
8 11.63 Phytol ND 9.45 ND ND ND
9 18.17 Vitamin E ND ND ND 2.19 5.57
10 21.17 Campsterol ND ND ND ND 4.55
11 21.71 Stigmasterol ND 9.89 3.59 ND 9.49
12 22.94 beta-sitosterol 7.18 30.36 20.74 15.15 39.31
(ND - Not detected; CLS – Cultivated leaf sheath; IVLS – In vitro leaf sheath; C2P, C4P, C8P – Callus grown on 2, 4, 8 mg/L Picloram)
a
Percentage of peak area relative to the largest peak of Ethyl alpha-d-glucopyranoside, which was set at 100%.

36
 List of Figures

Figure 1: Calibration curve of gallic acid.

Figure 2: Calibration curve of quercetin.
Figure 3: Picloram induced callus formation on various explants of Z. officinale
var. rubrum after 12 weeks of observation. Leaf sheath (A) Control, (B)
2 mg/L picloram, and (C) 8 mg/L picloram. Thin root (D) 2 mg/L picloram
and (E) 8 mg/L picloram. Thick root (F) 2 mg/L picloram, (G) 4 mg/L
picloram. Leaf (H) 8 mg/L picloram. Bar represents 1 cm.
Figure 4: Growth curve of Zingiber officinale var. rubrum callus cultures over
60 days. (A) Average fresh weight and (B) Average dry weight. Callus mass
proliferated on half-strength MS media supplemented with 2, 4, 8 mg/L
picloram. Different letters indicate statistically significant differences (p <
0.05) using Duncan Multiple Range Test. (n=5)
Figure 5: Histological sections of primary and subcultured callus of Zingiber
officinale var. rubrum. Callus induced at (A) 2 mg/L picloram, (C) 4mg/L
picloram and (E) 8 mg/L picloram. 4 Weeks old subcultured callus on MS
supplemented with (B) 2 mg/L picloram, (D) 4mg/L picloram and (F) 8
mg/L picloram. Star – Abundant airspace; Arrow – Nucleus.

37
Figure 1

Figure 2

38
Figure 3

39
Figure 4

40
Figure 5

41