Printer Friendly

Appraisal of AY-Phellandrene in Callus Cultures of Momordica charantia L. Cultivars, Jaunpuri and Jhalri.

Byline: Safdar Ali, Alveena Tariq, Muhammad Ajaib, Khalid Mohammed Khan, Shahnaz Perveen and Shazia Shah

Summary:

Secondary metabolite AY-phellandrene was analyze in callus cultures of two varieties of Momordica charantia L. i.e. Jaunpuri and Jhalri. Conditions for seed germination and callus induction were optimized. Seedlings grown under aseptic conditions served as explant sources. 6- Benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented in Murashige and Skoog's (MS) medium were scrutinized as the most suitable combination of plant growth regulators with different concentrations for callus induction in both the varieties. Cotyledon explant of (cultivars) cv. Jaunpuri revealed maximum callus induction with 1.0 mgl-l BAP and 1.5 mgl-1 2,4- D in eight days as compared to internode, apical bud and leaf. Cotyledon and leaf explants of cv. Jhalri responded to 1.5 mgl-l BAP and 1.0 mgl-l 2,4-D in nine days for callus and internode and apical bud with 1.0 mgl-l BAP and 1.5 mgl-l 2,4-D.

Best grown calli from different explants were analyzed through GC-MS for production of secondary metabolites. Along with other secondary metabolites AY-phellandrene was the most prominent secondary metabolites found in in vitro grown callus cultures of both the varieties. The callus cultures of cv. Jaunpuri produced substantial amount of AY-phellandrene i.e. up to 30 percent of the total secondary metabolites as compared to calli from cv. Jhalri explants. The callus cultures of M. charantia can prove the best alternative, rapid and uninterrupted source for natural AY-phellandrene production.

Keywords: Momordia charantia, Terpene, AY-Phellandrene, Callus, Jhalri

Introduction

AY-Phellandrene and a-phellandrene are cyclic monoterpenes and double-bond isomers having similar chemical properties, similar molecular structure and both are insoluble in water, but miscible with other. The a-phellandrene isomer on contact with air at elevated temperature can form hazardous and explosive peroxides but AY-phellandrene is safe to handle. AY-Phellandrene has been isolated from the oil of water fennel and Canada balsam. The enzyme that synthesizes a-phellandrene (92.2%) as a major product with AY-phellandrene (3.8 %) has been found in ginger (Zingiber officinale) [1]. The.AY phellandrene is widely used in perfumes, fragrances and artificial essential oils because of their pleasing aromas. The AY-phellandrene fragrance has been described as slightly citrusy and peppery-minty [2].

The search for new metabolites from plants should be a precedence in existing and future efforts toward viable protection and rational utilization of biodiversity [3]. Plant tissue cultures system has been found to have potential for continuous biomass provision as source material for production of important secondary metabolites as alternative to field grown plants which are limited to seasonal growth [4]. Such system has edge over the field plantation of plants, as not affected by climate changes or threatened nutrients and thus can be controlled better. Plant growth regulators (PGRs) have significant effects on the metabolism of secondary metabolites and are needed for effective induction of secondary metabolite production [5-7]. The plant growth regulators play a major role in the making capability of an in vitro culture as these help to control/enhance production of secondary metabolites [8-9].

The effects of different PGRs on secondary metabolite production have previously been stu died and confirmed in various plants [10-13]. Various tissues and respective callus cultures of M. charantia have been explored and found to contain substantial amount of different secondary metabolites [14]. This study was undertaken for optimization of callus induction and production of AY-phellandrene from callus cultures of M. charantia L. using various PGRs.

Experimental

Plant Material

Seeds of two varieties of Momordica charantia L. i.e. Jaunpuri and Jhalri were provided by Seed Certification Department, near Jain Mandar, Lahore, In vitro Seed Germination.

Seeds of M. charantia were sterilized and used to germinate under aseptic conditions (Tanveer and Ali) [15]. Ten days old lab grown seedlings were used as explant source for callus induction. Explants selected were leaf, stem, cotyledon, internode and apical bud.

Culture Medium Preparation and Sterilization

MS medium (Murashige and Skoog) [16] was used as basal culture medium to raise in vitro cultures. The culture medium was supplemented with different growth regulators i.e. 2,4-D, NAA in combinations with BAP (ranging from 0.5 to 2 mg L- 1) such as 0.5 mg L-1 BAP + 1 mg L-1 2,4-D (designated as BD1), 1 mg L-1 BAP + 1.5 mg L-1 2,4- D (designated as BD2), 1.5 mg L-1 BAP + 1 mg L-1 2,4-D (designated as BD3), 0.5 mg L-1 BAP + 1 mg L-1 2,4-D (designated as BD4). Culture vessels containing medium were autoclaved under 15 lbs/inch2 pressure for 20 minutes.

Explant Culturing, Callus Induction and Secondary Metabolite Production

Different parts of in vitro grown seedlings such as apical bud, cotyledonary leaf, juvenile leaf, internode and root were excised and cultured for callus induction under aseptic conditions. Culture vessels containing explants were incubated in growth room at 26 C 1 C. To avoid the deficiency of nutrients, callus formed was transferred aseptically to fresh medium after every 10 to 15 d. Different parameters studied were callus induction percentage, callus initiation duration, callus colour, callus appearance and Callus index (CI) was calculated.

Callus Index (CI)

CI (Callus index) was calculated by the following formula (Khosh and Singh) [17].

Equations

n = Number of explants initiating callus; G = Visual callus rating of initiated explants;

N = Total number of explants planted

Analysis of Secondary Metabolites by GC-MS

The oils were obtained through Soxhlet Apparatus from M. charantia field grown plant parts and respective calli from in vitro grown seedling explants. GC-MS analyses of the extracted oils were carried out on Shimadzu GC-2010 series gas chromatograph interfaced with mass spectrometer (QP 2010A), Kyoto Japan equipped with DB-5MS column (30 m A- 0.25 mm i.d thickness: 0.25 m), J and W (Scientific, Folsom, CA, USA) equipped with split/splitless injector. All mass spectra were acquired in electron impact (EI) mode with ionization voltage 70 eV.

Ion source and quadruple temperatures were 200C, transfer line temperature was 220C and helium at 1 mL min-1 constant flow. Initial column temperature was 50 C fixed for 5 min, programmed to 230 C at 5 C min-1; held for 5 min at 200 C, injector temperature 200 C and 2 L sample was injected using SGE microliter syringe. The spectrum was scanned with acquisition mass range 50 to 600 m/z, scan rate 1.6 scans s-1.

Experimental Design and Statistical Analysis

The experiment was conducted as Completely Randomized Design (CRD) with three replications. The results were reported as mean [] standard error (SE).

Results and Discussion

The seeds employed for germination produced healthy seedlings in in vitro (Fig. 1). Among plant growth regulators (PGRs) tested for callus induction, BAP (6-benzylaminopurine) in combination with 2,4-D (2,4-dichlorophenoxyacetic acid) successfully induced callus from all the explant types. Medium BD2 (1 mg/L BAP + 1.5 mg/L 2,4-D) supported in vitro cultures positively to produce callus in different explants employed for callus induction except juvenile leaf of both the cultivars where least amount of callus was produced taking longer duration i.e. 11 days for callus induction initiation with callus induction percentage of 25 and callus index of 50 (Table 1 and 2). Callus index of Jaunpuri cultivar was 150, whereas callus index of Jhalri cultivar was 75.

Cotyledonary leaf explant of cv. Jaunpuri generated better callus in shorter duration as compared to its counterpart in cv. Jhalri in particular and apical bud and internode in general with BD2 medium (Table 1 and 2) whereas callus indices were lower in BD3 medium. The callus initiation duration in cotyledon explant of cv. Jaunpuri was 8 days with 100% callus induction with callus index of 400 (Table 1). It was observed that time taken for the initiation of callus formation in apical bud was longer than the internode with approximately comparative callus indices (Table 1 and 2).

Table-1: Response of tissue types of Jaunpuri to different PGRs for callus induction.

Medium Code###MS Medium + PGRs###Explant Type###Callus Induction Callus Initiation###Callus Index###Characteristics of Callus

###(mg/L)###(%age)###(Days)

###BD2###2,4-D 1.5 +###Apical bud###50 11.10###12 0.91###200###Green

###BAP 1###Cotyledonary###100 15.0###08 0.72###400###Yellowish green

###Leaf

###Juvenile leaf###25 09.31###11 0.61###50###Yellowish green

###Internode###50 11.11###09 0.82###200###Green

###BD3###2,4-D 1 +###Apical bud###50 12.01###13 1.04###150###Green

###BAP 1.5###Cotyledonary###100 15.7###10 0.94###300###Yellowish green

###Leaf

###Juvenile leaf###50 11.11###08 0.82###150###Green

###Internode###25 08.98###11 0.98###100###Green

Table 2: Response of tissue types of Jhalri to different PGRs for callus induction.

Medium###MS Medium + PGRs###Callus Induction###Callus Initiation

###Explant Type###Callus Index###Characteristics of Callus

Code###(mg/L)###(%age)###(Days)

BD2###2,4-D 1.5 +###Apical bud###75 12.31###11 0.28###255###Brownish green

###Cotyledonary

###BAP 1###100 15.72###10 0.19###300###Yellowish green

###leaf

###Juvenile leaf###25 10.15###11 0.21###50###Yellowish green

###Internode###75 12.19###10 0.24###150###Green

###BD3###2,4-D 1 +###Apical bud###75 13.01###12 0.75###200###Green

###Cotyledonary

###BAP 1.5###100 16.13###09 0.52###325###Yellowish green

###leaf

###Juvenile leaf###25 09.91###09 0.49###75###Green

###Internode###50 10.13###11 0.64###100###Green

Different explants from Jaunpuri and Jhalri cultivars responded differently to BAP and 2,4-D (different concentrations) in MS medium in in vitro cultures to produce callus cultures. On the whole cv. Jaunpuri performed better in BD2 medium with respect to callus induction and cv. Jhalri seems to take edge over Jaunpuri in BD3 medium for the same (Table 1 and 2). The callus cultures of both cultivars were greener in colour in BD3 medium as compared to BD2 (Fig. 1).

On evaluation of AY-phellandrene from GC- MS analyses (Fig. 2a-2d) of field grown plant parts and callus cultures of different explants of two varieties of Momordica charantia L. i.e. Jaunpuri and Jhalri other metabolites were also detected. According to GC-MS analyses (Fig.2a-2d) AY- phellandrene was the most prominent secondary metabolite found to be present in highest percentage in all field grown plant parts and respective calli from in vitro grown seedling explants. AY-Phellandrene and other secondary metabolite of both varieties produced under the influence of 2,4-D + BAP in MS medium were similar under similar concentrations but generally cv. Jaunpuri contained high % age of AY- phellandrene than cv. Jhalri (Table 3, Fig. 3). The use of plant cell culture systems increased in current years is due to better understanding of the secondary metabolite pathway in economically important plants.

Conclusion

In this investigation it was found that AY- phellandrene from calli of in vitro seedling explants were comparable to the related plant parts from field grown plant tissues of both the cultivars of Momordica charantia L. i.e. Jaunpuri and Jhalri with minor differences. AY-Phellandrene can be produced round the year through in vitro cultures, by passing the seasonal pressure, cultivation practices and vegetative growth period of M. charantia and as an alternate secondary metabolite production through in vitro biomass production.

References

1. H. J. Koo and D. R. Gang, Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues, PLoS. One, 7, e51481 (2012).

2. S. W. L. Jacobs and J. Pickard, Plants of New South Wales, ISBN 0-7240-1978-2 (1981).

3. J. D. Phillipson, Products from Plant Tissue Culture. B. V. Charlwood, and M. J. C. Rhodes (eds.), Oxford: Clarendon Press, p. 1 (1990).

4. S. R. Ramachandra and G. A. Ravishankar, Biotransformation of Protocatechuic Aldehyde and Caffeic Acid to Vanillin and Capsaicin in Freely Suspended and Immobilized Cell Cultures of Capsicum frutescens, Journal of Biotechnology, 76, 137 (2000).

5. J. J. Zhong, Y. Bai and S. J. Wang, Effects of Plant Growth Regulators on Cell Growth and Ginsenoside Saponin Production by Suspension Cultures of Panax quinquefolium, Journal of Biotechnology, 45, 227 (1996).

6. K. O. Al-Sane', R. A. Shibli, N. M. Freihat and M. K. Hammouri, Cell Suspension Culture and Secondary Metabolites Production in African Violet (Saintpaulia ionantha Wendl.). Jordan Journal of Agricultural Sciences, 1, 84 (2005).

7. H. P. Shilpashree and R. Ravishankar, In vitro Plant Regeneration and Accumulation of Flavonoids in Hypericum mysorense, International Journal of Integrative Biology, 8, 43 (2009).

8. W. G. W. Kurz and F. Constabel, Plant Cell Cultures, a Potential Source of Pharmaceuticals, Adv. App. Microbiol., 25, 209 (1979).

9. E. J. Staba, Plant Tissue Culture as a Source of Biochemicals, CRC Press, Boca Raton, Florida, p. 32 (1980).

10. A. Ikuta and H. Itokawa, Berberine and other Protoberberine Alkaloids Callus Tissue of Thalictrum minus, Phytochemistry, 21, 1419 (1982).

11. A. J. Nair, P. R. Sudhakaran, J. R. Madhusudana, and S. V. Ramakrishna, Berberine Synthesis by Callus and Cell Suspension Cultures of Coscinium fenestratum, Plant Cell, Tissue and Organ Culture, 29, 7 (1992).

12. P. J. Weathers, G. Bunk and M. C. McCoy, The Effect of Phytohormones on Growth and Artemisinin Production in Artemisia annua Hairy Roots, In Vitro Cellular and Development Biology- Plant, 41, 47 (2005).

13. T. Khan, D. Krupadanam, and S. Y. Anwar, The Role of Phytohormone on the Production of Berberine in the Calli Cultures of an Endangered Medicinal Plant, Turmeric (Coscinium fenestratum L.), African Journal of Biotechnology, 7, 244 (2008).

14. S. Ali and A. Tariq, Analysis of Secondary Metabolites in Callus Cultures of Momordica Charantia cv. Jaunpuri, Biologia (Pakistan), 59, 23 (2013).

15. H. Tanveer, S. Ali and M. R. Asi, Appraisal of Secondary Metabolites in in vitro Cultures of Citrullus colocynthis (L.) SCHARD. Science International, (Lahore), 14, 528 (2012).

16. T. Murashige and F. Skoog, A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Culture, Physiol. Plants, 15, 437 (1962).

17. M. Khosh and K. Singh, Callus Induction and Culture of Roses, Scientia Horticulturae, 17, 361 (1982).
COPYRIGHT 2015 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Aug 31, 2015
Words:2329
Previous Article:Casearia Tomentosa: A Potential Antimicrobial and Antioxidant Source.
Next Article:Preparation of a Rechargeable Battery Using Waste Protein from the Fish Scales.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters