Advances in Botany
Volume 2015 (2015), Article ID 942761, 7 pages
http://dx.doi.org/10.1155/2015/942761

Research Article

Rhizogenesis in Cell Suspension Culture from Mango Ginger: A Source of Isosorbide and n-Hexadecanoic Acid

Chellappan Soundar Raju, Kandhan Varutharaju, Chandrasekaran Thilip, Abubakker Aslam, and Appakan Shajahan

Plant Molecular Biology Laboratory, Department of Botany, Jamal Mohamed College, Tiruchirappalli, Tamil Nadu 620020, India

Received 30 September 2014; Revised 6 February 2015; Accepted 22 February 2015

Academic Editor: Tomotsugu Koyama

Copyright © 2015 Chellappan Soundar Raju et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Curcuma amada Roxb. belongs to the monocotyledonous family Zingiberaceae. It is commonly known as mango ginger and used as a spice and valuable medicine. In this study, adventitious roots of C. amada have been successfully established from cell suspension culture. The highest percentage of adventitious root production was obtained from friable callus derived cell suspension culture. The culture conditions of adventitious root were optimized and the maximum adventitious root production was obtained in half strength MS liquid medium containing 0.3 mg L⁻¹ IBA along with 3% of sucrose after 5 weeks of culture. Among the different initial inoculum density, the best culture condition for root growth occurred at 10 g FW of initial inoculum density. GC-MS analysis revealed that the in vitro raised adventitious roots containing two valuable bioactive compounds, isosorbide and n-hexadecanoic acid. The outcome of the present work will be helpful for the large scale cultivation of adventitious roots for the production of valuable bioactive compounds.

1. Introduction

Curcuma amada Roxb. (mango ginger) belongs to the family of Zingiberaceae which is a unique perennial rhizomatous herb and morphologically resembles ginger and has a flavour of raw mango (Mangifera indica). There are 68 volatile aromas, more than 130 chemical constituents present in the mango ginger rhizome. The aromatic smell raised from C. amada is mainly attributed to the presence of car-3-ene and cis-ocimene compounds, which are used in still food, beverages, cosmetics, and medicines [1–8]. The rhizome is composed (fresh weight basis) of 86% moisture, 0.8% ash, 0.8% total sugars, traces of reducing sugars, 1.4% fibre, 0.1% essential oil, and 6.9% starch and on a dry weight basis, 5.7% ash, 5.8% total sugar, traces of reducing sugars, 10.6% crude fiber, 0.9% essential oil, and 45.6% starch [9]. The C. amada has been reported with high amylase activity that converts starch into simple metabolisable sugars and, in turn, into several valuable aromatic compounds [10]. Due to this metabolic advantage, curcumin-free portion is effective in lowering liver cholesterol in animals [11]. Recently, three terpenoid bioactive compounds (difurocumenonol, amadannulen, and amadaldehyde) were isolated from their rhizomes. They also exhibit potential actions such as antimicrobial, antioxidant, platelet aggregation inhibitor activities, and anticancer property [12]. It also contains antitubercular agent like labdane diterpenoid [13].

In plants, secondary metabolites accumulate in specific or specialized cells, tissues, or organs [14]. In vitro, tissues need dedifferentiation (callus formation) and redifferentiation (rhizogenesis and embryogenesis) process for biosynthesis and accumulation of secondary metabolites [15, 16]. Adventitious root culture is one of the valuable tools, especially cell suspension culture, and adventitious root induction is the best automation process biomass production. The present work reports a simple and reliable procedure for in vitro adventitious root induction from homogenous cell suspension culture of C. amada and examines bioactive compounds using GC-MS analysis.

2. Material and Methods

2.1. Callus Induction

Microrhizome segments were excised from 3-month-old in vitro grown plants [17]. For callus induction, these segments were placed on MS medium [18] with 3.0% sucrose and different concentration of 2,4-D (1.0, 2.0, and 3.0 mg L⁻¹) alone or in combination with BA or Kn (0.25, 0.5 mg L⁻¹). The medium was solidified with 0.8% agar and the pH of the media was adjusted to before solidification. Media were autoclaved at 121°C and 104 kPa for 15 min. Cultures were maintained at °C for 16 hrs photoperiod with 40 μmol m⁻² s⁻¹ light intensity provided by white fluorescent tubes and a relative humidity of 55–65%.

2.2. Initiation of Cell Suspension Culture and Induction of Adventitious Roots

For induction of adventitious roots, ~250 mg fresh mass of different types of callus (nonfriable, semifriable, and friable callus) was transferred to a 150 mL Erlenmeyer flask (each in separate flask). Each flask containing MS liquid medium was supplemented with different concentration of auxins (0.1, 0.2, 0.3, 0.4, and 0.5 mg L⁻¹ IBA or IAA), and then they were placed on orbital shaker at 100 rpm in continuous darkness. MS medium without auxin was used as a control. After one week of culture period, callus responding with root induction (%) was calculated using the following equation:

For biomass production, adventitious roots (~0.5 cm; 35 roots/flask) were transferred in the same media composition and harvested during the 5th week of culture when the biomass reached a maximum level. The suitable auxin was selected for further studies based on the comparison of root length.

2.3. Optimization of Medium Strength, Sucrose Concentration, and Initial Inoculum Density for Adventitious Root Culture

The culture medium was optimized by transferring initial inoculum (~2.5 g FW adventitious roots) to various strengths of MS liquid medium (1/4, 1/2, 3/4, and full strength) and different concentrations of sucrose (1.0, 3.0, 4.5, and 6.0%) for biomass production. For improving adventitious root biomass, optimal inoculum density was standardized using various levels (2.5, 5.0, 10.0, 15.0, and 20.0 g FW) of initial inoculum. Each treatment was carried out three times with seven flasks. Growth ratio (GR) was calculated using the following equation [19]:

2.4. GC-MS Analysis

The adventitious root mass (1.0 g FW) harvested from suspension culture and rhizome of field grown plants was subsequently air-dried for 1 hour and completely ground using pestle and mortar. Extraction was carried out with methanol (10 mL) until ground root changed into white color by sonication. After centrifugation at 8,000 rpm for 15 min, the upper aqueous layer was collected and filtered through a nylon membrane filter and injected into the GC-MS equipment for analysis.

2.4.1. GC-MS Programme

Consider the following:

 column: Elite-5MS (5% diphenyl/95% dimethyl poly siloxane) (Perkin Elmer) 30 m × 0.25 mm × 0.25 μm, film thickness 0.25 μm, equipment: GC Clarus 500 Perkin Elmer, California, USA, carrier gas: 1 mL per min., split: 10 : 1, detector: mass detector Turbomass gold-Perkin Elmer, software: Turbomass 5.2, sample injected: 2.0 μL.

2.4.2. Oven Temperature Programme

Consider the following:

 110°C – 2.0 min: hold, up to 200°C at the rate of 10°C/min: no hold, up to 280°C at the rate of 5°C/min: 9.0 min hold, injector temperature: 250°C, total GC running time: 36 min.

2.4.3. MS Programme

Library used NIST version 2005:

 inlet line temperature: 200°C, source temperature: 200°C, electron energy: 70 Ev, mass scan (m/z): 45–450, solvent delay: 0–2.0 min, total MS running time: 36 min.

2.5. Statistical Analysis

All experimental data were subjected to one way ANOVA followed by statistical significance test. Data were presented as mean, mean ± SE. The mean separations were analyzed by using Duncan’s multiple range test with significance level of (IBM SPSS statistics 19).

3. Results

3.1. Initiation of Cell Suspension Culture and Induction of Adventitious Roots

As the result of the present study, MS medium containing 1.0 mg L⁻¹ 2,4-D in combination with 0.25 mg L⁻¹ BA was found to produce friable callus. Medium containing 1.0 mg L⁻¹ 2,4-D and 0.5 mg L⁻¹ BA was favorable for semifriable callus formation, 2.0 mg L⁻¹ 2,4-D and 0.5 mg L⁻¹ BA were found to produce nonfriable callus (data not shown). To induce adventitious root formation, all three types of callus were transferred to MS liquid medium containing IBA or IAA. Friable callus was suspended easily in single cell manner (Figure 1(a)) and semifriable callus formed cell aggregation. Nonfriable callus settled down in the medium and could not be proliferated into roots (Table 1). Auxins also significantly influenced the adventitious root formation from callus culture. IBA showed higher percentage of root induction than IAA. Maximum percentage (100%) of root formation was obtained from friable callus derived cell suspension in the medium containing 0.2–0.3 mg L⁻¹ IBA. However, maximum root length (7.23 cm) was observed in the medium containing 0.3 mg L⁻¹ IBA (Figure 1(b)). When increasing or decreasing the concentration of IBA to this level, percentage of adventitious root formation gradually decreased.

Table 1: Effect of auxins on adventitious root formation from callus via cell suspension culture.

PGRs (mg L⁻¹)
Callus responding with root induction (%)
Root length of FC induced (cm)*

IBA
IAA
NFC
SFC
FC

0.1

—
79.10
88.10
4.46 ± 0.03i

0.2

—
66.66
100
6.84 ± 0.04b

0.3

—
45.88
100
7.23 ± 0.07a

0.4

—
0
91.66
6.37 ± 0.04c

0.5

—
0
83.33
5.42 ± 0.06e

0.1
—
63.88
91.66
4.51 ± 0.04gh

0.2
—
41.66
100
6.46 ± 0.05c

0.3
—
0
88.10
5.66 ± 0.05d

0.4
—
0
72.22
5.07 ± 0.05f

0.5
—
0
61.10
4.63 ± 0.05g

Data was recorded from 5-week-old adventitious root cultures.

All experiments were carried out three times with at least 15 cultures.

*Data represented mean ± SE, mean within a column followed by same letter is not significantly different according to Duncan’s multiple range (DMRT) test, P < 0.05.

NFC: nonfriable callus; SFC: semifriable callus; FC: friable callus.

(a)

(b)

(c)

Figure 1: Adventitious root culture of C. amada via cell suspension culture. (a) Adventitious root induction from friable callus derived cell suspension in MS liquid medium supplemented with 0.3 mg L⁻¹ IBA. (b) Adventitious roots growth in MS liquid medium supplemented with 0.3 mg L⁻¹ IBA after 5 weeks of culture period. (c) Vigorous growth using initial inoculum mass 10 g FW. Scale bars: (a–c) 0.5 cm.

3.2. Optimization of Medium Strength and Sucrose Concentration for Adventitious Root Biomass Production

The present study reveals that MS liquid medium strength and gradient sucrose concentration significantly influenced adventitious root formation. Among various medium strengths and concentrations of sucrose, the highest root biomass (51.60 g FW) production was observed in half strength MS medium supplemented with 3.0% sucrose (Table 2). In contrast, root growth was inhibited when the medium strength or sucrose concentration was increased or decreased to this optimum level.

Table 2: Effect of medium strength and sucrose concentration on adventitious root formation.

Sucrose (%)
Root biomass (g FW)*
Growth ratio

1/4 MS
1/2 MS
3/4 MS
Full MS
1/4 MS
1/2 MS
3/4 MS
Full MS

1.5
18.60 ± 0.33c
27.26 ± 0.28c
22.73 ± 0.40c
14.67 ± 0.29c
7.40
10.91
9.09
5.87

3.0
35.73 ± 0.22a
51.60 ± 0.50a
46.73 ± 0.30a
31.47 ± 0.24a
14.29
20.64
18.69
12.59

4.5
25.93 ± 0.36b
39.00 ± 0.43b
24.00 ± 0.50b
19.53 ± 0.43b
10.37
15.60
9.60
7.81

6.0
13.47 ± 0.27d
23.33 ± 0.57d
17.67 ± 0.27d
10.87 ± 0.35d
5.39
9.33
7.09
4.35

Data was recorded from 5-week-old adventitious root cultures.

All experiments were carried out three times with at least 15 cultures.

*Data represented mean ± SE, mean within a column followed by same letter is not significantly different according to Duncan’s multiple range (DMRT) test, P < 0.05.

3.3. Optimization of Inoculum Density for Adventitious Root Biomass Production

Inoculum density depends on the volume of culture medium and vessel. In the present study, 250 mL Erlenmeyer flasks containing 50 mL medium were used to optimize the inoculum density for achieving maximum root biomass production. On the different initial inoculum density, maximum adventitious root biomass (121 g FW) and growth rate (12.1%) were recorded at 10 g FW of initial inoculum (Figure 1(c)). Further, decrease or increase in inoculum density led to decrease in the biomass production (Table 3).

Table 3: Effect of initial inoculum density on adventitious root formation.

Initial inoculum density (g FW)
Root biomass (g FW)*
Growth ratio

2.5
51.60 ± 0.50e
20.64

5.0
87.20 ± 0.34b
17.44

10.0
121.00 ± 0.78a
12.10

15.0
78.87 ± 0.52c
5.20

20.0
64.27 ± 0.54d
3.21

Data was recorded from 5-week-old adventitious root cultures.

All experiments were carried out three times with at least 15 cultures.

*Data represented mean ± SE, mean within a column followed by same letter is not significantly different according to Duncan’s multiple range (DMRT) test, < 0.05.

3.4. GC-MS Analysis

The essential oil components were found to be varied between rhizome of field grown plants and in vitro raised adventitious roots (Tables 4 and 5). Out of 29 peaks which were detected from rhizome, 14 peaks were identified (Figure 2(a)) and out of 21 peaks detected from adventitious roots, 3 peaks were identified (Figure 2(b)) with their respective compounds. Interestingly, the in vitro raised adventitious roots showed only three compounds in detectable relative percentage of peak area. This was not the case with rhizome, where other compounds were also found in larger proportion. Among those three compounds detected in samples of adventitious roots, the isosorbide and 1-buten-1-ol, 2-methyl-4-(2,6,6,-trimethyl-1-cyclohexen-1-yl)-, formate, (E)- exhibited higher peak area when compared to samples of rhizome. Relative peak area of n-hexadecanoic acid was less in samples from adventitious roots than rhizome.

Table 4: Phytochemical profile of field grown rhizome of C. amada.

Number
RT
Name of the compound
Molecular formula
Molecular weight
Peak area (%)*

1
4.44
Isosorbide
C₆H₁₀O₄
146
9.52

2
9.95
Cyclopropanementaol, 2-methyl-2-(4-methyl-3-pentenyl)
C₁₁H₂₀O
168
2.86

3
11.29
1-Buten-1-ol, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, formate, (E)-
C₁₅H₂₄O₂
236
11.90

4
12.74
1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-, (Z,E)-
C₁₅H₂₄
204
4.29

5
12.97
n-Hexadecanoic acid
C₁₆H₃₂O₂
256
14.29

6
13.17
2,6,10,14-Hexadecatetraenoic acid, 3,7,11,15-tetramethyl-, methyl ester, (E,E,E)-
C₂₁H₃₄O₂
318
0.95

7
13.31
Hydroxydehydrostevic acid
C₂₀H₃₀O₃
318
2.86

8
18.24
4-Hexen-1-0l, 6-(2,6,6-trimethyl-1-cyclohexenyl)-4-methyl-, (E)-
C₁₆H₂₈O
236
18.57

9
21.95
1-Heptadec-1-ynyl-cyclopentanal
C₂₂H₄₀O
320
6.19

10
22.53
Acetic acid, 1-[2-(2,2,6-trimethyl-bicyclo [4,1.0]hept-1-yl)-ethyl]-vinyl ester
C₁₆H₂₆O₂
250
2.86

11
22.87
2H-Pyran, 2-(7-heptadecyyloxy) tetrahydro-
C₂₂H₄₀O
336
3.81

12
25.47
2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-
C₁₆H₂₆O
410
6.19

13
25.75
Squalene
C₂₂H₄₀O₂
410
5.71

14
31.79
Dihydrotachysterol
C₂₈H₄₆O
398
10.00

Relative intensity.

Table 5: Phytochemical profile of in vitro raised adventitious roots of C. amada.

Number
RT
Name of the compound
Molecular formula
Molecular weight
Peak area (%)*

1
4.43
Isosorbide
C₆H₁₀O₄
146
65.71

2
11.29
1-Buten-1-ol, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, formate, (E)-
C₁₅H₂₄O₂
236
20.00

3
12.95
n-Hexadecanoic acid
C₁₆H₃₂O₂
256
12.86

Relative intensity.

(a)

(b)

Figure 2: GC-MS spectrum from methanol extract of C. amada. (a) Field grown rhizome. (b) Cell suspension induced adventitious roots.

4. Discussion

Adventitious root culture is one of the valuable biological tools for feasible production of bioactive compounds without depending on field grown parent plants and abiotic and biotic factor effects [20, 21]. In the present study, a promising adventitious root induction system was successfully developed for mango ginger, which is an important aromatic rhizomatous plant. Among the different qualities of callus, friable callus responds more favorably for adventitious root formation when compared with semifriable and nonfriable callus. Prakash et al. [22] also reported that the friable callus seems to be one of the most suitable starting materials for induction of organogenesis in C. amada. It may be probably due to the presence of more physiological active cells which are more powerful than the cells in semifriable callus and nonfriable callus [21]. The results of exogenous auxin treatment indicate that 0.3 mg L⁻¹ IBA was the optimum for adventitious root formation more than IAA. A similar phenomenon was also found in W. somnifera [21], Morinda citrifolia [23], and Periploca sepium [24]. The year round availability of adventitious root culture can solve the problem of seasonal availability of mango ginger.

In plant cell/organ culture, sucrose is an important balanced carbon source and plays a vital role in the synthesis of cell constituents as substrate to provide energy for cell growth [25]. It promotes cell growth by hydrolysis of invertase and sucrose synthase acts as building blocks and regulates osmotic potential [26, 27]. In the present study, 3% sucrose was suitable for adventitious root growth in terms of biomass production. Lower concentration cannot provide enough energy and high sucrose concentration exhibited negative effect in root primordial induction.

The concentration of salts in the MS medium significantly contributes to biomass production and phytochemical accumulation in cultured cells and tissues [28]. Wu et al. [29] proposed that the interactions among the nutritional salts enhance the availability of ions to the roots and thereby promoting the root growth and phytochemical production. The present study confirmed that the optimization of MS salt concentration is very essential for adventitious root production and half strength MS medium is the best for optimal root primordial induction and growth in C. amada. The same phenomenon was also documented in root culture of Zingiberaceae member Alpinia galanga [30]. Further it was observed that when increasing the MS salt strength in the medium, root biomass production was reduced. It indicated that high MS salt concentration promoted a stress condition and reduced the growth of adventitious roots. Determination of optimal inoculum density is a prerequisite for enhanced production of secondary metabolite from in vitro root biomass [19, 31, 32]. In W. somnifera, optimal level of initial inoculum density is 15 g FW. The increase or decrease level of inoculum density inhibits root biomass production [21]. In the present study, maximum root biomass production in C. amada was obtained when inoculum density was at 10 g FW.

The in vitro raised adventitious roots contained two compounds in higher proportion and one on par with field grown rhizome. This offers new avenue for scaling up of the two identified compounds such as isosorbide and n-hexadeconoic acid [33, 34]. Isosorbide, being valuable derivative of glucose, can be used for further conversions into several chemicals like green solvents, fuels, fuel additives, and so forth [33]. n-Hexadeconoic acid is also very useful in the production of cetyl alcohol which is used in food and cosmetic industry [34]. Similar attempts have been made by other investigators [35]. The present study successfully mimics the levels of two bioactive compounds produced by field grown rhizome. Reports in related species (C. longa) have achieved this similarity between ex vitro plants and in vitro raised plants that are established ex vitro [35].

In conclusion the present investigation opens up a new route for large scale production of active compounds, isosorbide and n-hexandeconoic acid, from homogenous cell suspension mediated adventitious root culture of C. amada. To the best of our knowledge, this is the first report of in vitro isosorbide and n-hexadeconoic acid production from adventitious root cultures. Further, the results obtained in the present study might be useful in further research on biotransformation and production of these secondary metabolites of C. amada in large scale.

Abbreviations

MS: | Murashige and Skoog medium
2,4-D: | 2,4-Dichlorophenoxy acetic acid
BA: | 6-Benzyladenine
Kn: | Kinetin
IBA: | Indole-3-butyric acid
IAA: | Indole-3-acetic acid
GC-MS: | Gas chromatography-Mass spectrometry
FW: | Fresh weight.

Conflict of Interests

The authors declare that they have no conflict of interests regarding the publication of this research paper.

Acknowledgments

The authors acknowledge the University Grants Commission, New Delhi, for financial support by Major Research Project grant to Dr. A. Shajahan (F. no. 42-946/2013). The authors also thank DST, Government of India, for providing facilities through FIST program and University Grants Commission, New Delhi, for its support through “College with potential for excellence” programme.

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