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Producing friable callus for suspension culture in Glycyrrhiza glabra.

Introduction

Many higher plants are major sources of natural products used as pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and pesticides [1], Discoveries of cell cultures capable of producing specific medicinal compounds at a rate similar or superior to that of intact plants have accelerated in the last few years, The possible use of plant cell cultures for the specific biotransformation of natural compounds has been demonstrated. Due to these advances, research in the area of tissue culture technology for production of plant chemicals has bloomed beyond expectations. The major advantages of a cell culture system over the conventional cultivation of whole plants are: (1) Useful compounds can be produced under controlled conditions independent of climatic changes or soil conditions; (2) Cultured cells would be free of microbes and insects; (3) The cells of any plants, tropical or alpine, could easily be multiplied to yield their specific metabolites; (4) Automated control of cell growth and rational regulation of metabolite processes would reduce of labor costs and improve productivity; (5) Organic substances are extractable from callus cultures. friable callus frequently used in tissue culture as a source for suspension culture due produce the secondary metabolites or other plat biotechnology diversities technique, well to many organic and in organic factors have been testes due this importance, but a number of chemical and physical factors like media components, phytohormones, pH, temperature, aeration, agitation, light affecting production of secondary metabolites has been extensively studied. Several Products were found to be accumulating in cultured cells at a higher level than those in native plants through optimization of cultural conditions. At present, production of taxol by various Taxus species cells in cultures has been one of the most extensively explored areas of plant cell cultures in recent years owing to the enormous commercial value of taxol, the scarcity of the Taxus tree, and the costly synthetic process.

Licorice is derived from at least five Glycyrrhiza species. Licorice used as a sweetener as an important drug in oriental medicine there for demand for cultivation of Glycyrrhiza plants which are used as a source for licorice

Has increased, in this aim and to establishment of a rapid in vitro propagation and produce a friable culluse in order to fund for a suspension culture we conducted an experiment as fallow by focusing on plant regulators and their density in an in vitro culture, its 13th century English name was Lycorys, a corruption of glycyrrhiza. Liquorice (Glycyrrhiza glabra) has long been used for both culinary and medical purposes. Used for flavoring and sweetening candies and medical remedies, licorice also has potent effects of its own, particularly for ulcers and adrenal insufficiencies. Whole. It is also used for asthmatic coughs, as an antispasmodic and ulcer remedy, and to cool 'hot' conditions. The roots are unearthed in the autumn of the fourth season. It is grown in India, Spain, Iran, Russia, China & Italy, The herb contains glycyrrhizin, glycyrrhetinic acid, flavonoids, asparagines, iso-flavonoids, and chalcones. Licorice contains the glycoside, glycyrrhizin which has a similar structure and activity as the adrenal steroids. Licorice has an anti-inflammatory activity similar to cortisone and has been found useful for arthritis and allergies. In addition licorice has been used for mild Addison's disease and other adrenal insufficiencies, such as hypoglycemia. Licorice also acts like the hormone, ACTH, causing sodium retention, potassium depletion, and water retention. Excess consumption of licorice can lead to the classic symptoms of hypertension, with edema, increased blood pressure, potassium loss, and muscular weakness. The Deglycyrrhizinated form is most often used to avoid the hypertensive side effects of the glycyrrhetinic acid in whole Licorice, these importance could be a reason for fund an experiment due the potentials for producing friable callus which will be used in a suspension culture or in a bioreactor as an initial clause [1,3,9].

Material and methods

Shot and steams of Glycyrrhiza glabra var. glandulifera were collected from agricultural researching organization of Iran from seed control & certification center and after rinsing with distilled water disinfecting step were continued soaking shots in 0.1% HgCl2 for 8 min then in 75% alcohol for 10 s, followed by three sequential rinse for 1 min in sterile distilled water, from donor plants explants prepare from axillaries buds with 5 [+ or -] 1 mm segments, and used for continues step for callus purpose, The basal medium used was MS medium, supplemented with 3% sucrose and solidified with 0.6% agar, They were placed into conical flasks, each containing 25 ml basal medium with exogenous hormone. The pH of the medium was adjusted to 5.8 prior to autoclaving at 0.15 MPa 121[degrees]C for 15 min. The cultures were incubated under florescent lights with 1500-2000 lux for 12 h photoperiod at 25 [+ or -] 1[degrees]C and 80 [+ or -] 10 relative humidity.

2.1. Callus induction and subculture:

NAA and 2,4-D as auxins and 6-BA as cytokinins were added into MS medium to test their effects on callus formation. The concentration of phytohormone varied from 0.5-2.0 mg/l. Each experiment contained at least 20 replicates and the experiments were repeated three times. Data were documented up to 5 weeks of culture. Well grown callus induced from explants were selected to transfer to MS medium with appropriate hormones for subculture. Cultures were subcultured to the same medium every 15 days. After three times of subculture, the growing states of callus were compared with together.

Results and discussions

3.1. Callus induction and subculture:

According to table. I the explants of Glycyrrhiza glabra on MS medium free of growth regulators, no calli were induced but When 2,4-D was used into MS medium, all explants formed white callus and induction rates increased with the increase of concentration of 2,4-D. It was found that the addition of high concentration of 6-BA into the MS medium in combination with 2,4-D had prompt effect on callus formation, The maximum induction rate was recorded as 96% in MS medium with 0.5 mg/l 2,4-D and 2 mg/l 6-BA with a light compact structure. However, MS medium with single 6-BA induced no callus in none of explants, although the combination of NAA and 6-BA could induce callus formation in explants, but callus turned brown. As previous primary experiments indicated that only high concentration of BA in combination with 2, 4-D or NAA in medium was relatively more suitable for subculture, the well grown callus were Selected for subculture on MS medium with 2.0 mg/l 6-BA in combination with 0.5-2 mg/l 2, 4-D or with 0.5-2.0 mg/l NAA to test their growing state. 40 days later after the callus were subculture on the MS with 6-BA and 2,4-D, they turned from green to white and became compact, while callus cultured on MS medium with 6- BA and NAA became loose, and spongy which was more suitable for continuous subculture and suspension culture. Therefore, MS medium supplemented with 0.5-2.0 mg/l NAA and 2.0 mg/l 6-BA was used for subsequent experiments. Through testing, it was found that the MS medium containing 2 mg/l 6-BA and 0.5 mg/l NAA had the maximum effect and was decided for subculture and cell suspension culture [5,11,10].

4. Conclusion:

By the base of this experiment due producing a friable callus for our aim its seems that explants of Glycyrrhiza glabra on MS medium by the maximum induction rate was recorded as 96% in MS medium with 0.5 mg/l 2,4-D and 2 mg/l 6-BA with a light compact structure but it's not a suitable for callus induction, using a hormonal dense with 1.0 mg/l NAA and 2 mg/l 6-BA by 91.6 induction rate with a Loose, spongy, friable structure its seems that by increasing the level or the dense of NAA as an auxins the potential for producing a friable callus decreases and this could shows the interaction between cytokines and auxins and the importance kind and role of this hormones' due producing a friable Loose, spongy, friable callus for a suspension culture which use in bioreactors.

References:

[1.] Balandrin, M.J. and J.A. Klocke. 1988. Medicinal, aromatic and industrial materials from plants. In Y.P.S. Bajaj (ed.), Biotechnology in Agriculture and Forestry. Medicinal and Aromatic Plant, vol. 4. Springer-Verlag, Berlin, Heidelberg, pp. 1-36.

[2.] BHMA 1983 British Herbal Pharmacopoeia, BHMA, Bournemouth.

[3.] Bradley, P.R. (ed.) 1992 British Herbal Compendium, Volume 1, BHMA, Bournemouth

[4.] Grieve, M. 1931 A Modern Herbal, (ed. C.F. Leyel 1985), London.

[5.] Goldstein, W.et al. In "Plant Tissue Culture as a Source of Biochemical's" Ed.Stable, Ed. stable, E.J., 191-234 (1980) C.R.C. Press Raton, Florida.

[6.] Staba, E.J., In "Proc. 5th Intl.cong. Plant Tissue Cell culture", Ed.Fujiwar, A., P.25 (1982) Maruzen Co., Tokyo.

[7.] Street, H.E. (Ed), In "Plant Tissue and Cell culture ", Blackwell Scientific Publ., London (1973).

[8.] Wash LK, Bernard, JD. Licorice-induced pseudoaldosteronism. Am J Hosp Pharm. 1975;32(1):73-74.

[9.] White L, Mavor S. Kids, Herbs, Health. Loveland, Colo: Interweave Press; 1998:22, 35.

[10.] Yeung. C.E, Trevor A and Thorpe., Invitro fertilization and Embryo culture.Dept of Biology Calgary, Alberte, Canada Arase Y, et al. The long term efficacy of glycyrrhizin in chronic hepatitis C. Cancer. 1997;79:1494-1500.

[11.] Zenk, M.H., In "Frontiers of Plant Tissue Culture 1978", Ed. Thorpe, T.A., p.1 (1978). Univ. of Calgary.

(1) Ali Parsaeimehr, (2) Badrossadat mousavi

(1) PhD student at the field of plant Biotechnology at the National Academy Of science institute of G.S.Davtyan Institute of Hydroponics Problems.

(2) Ms at the field of plant Biotechnology at Shiraz agriculture institute.

Corresponding Author Ali Parsaeimehr, PhD student at the field of plant Biotechnology at the National Academy Of science institute of G.S.Davtyan Institute of Hydroponics Problems.
Table 1: The induction of callus from variable Plant Growth Regulators
and interaction of them

                                     induction
No     plant growth regulator (mg/l) rate (%)

       2,4-D     NAA       6-BA

1      000                           0
2      000                           0
3      000                           0
4      0.5       0         0         12.2
5      1.0       0         0         22.6
6      2.0       0         0         40.4
7      0         0.5       0         6.4
8      0         1.0       0         16.2
9      0         2.0       0         30.3
10     0.5       0         0.5       33.6
11     1.0       0         0.5       28.8
12     2.0       0         0.5       19.8
13     0.5       0         1.0       46.2
14     1.0       0         1.0       24.4
15     2.0       0         1.0       35.2
16     0.5       0         2.0       96.8
17     1.0       0         2.0       92.6
18     2.0       0         2.0       89.8
19     0         0.5       0.5       82.8
20     0         1.0       0.5       78.6
21     0         2.0       0.5       68.6
22     0         0.5       1.0       82.6
23     0         1.0       1.0       72.3
24     0         2.0       1.0       68.6
25     0         0.5       2.0       95.4
26     0         1.0       2.0       91.6
27     0         2.0       2.0       87.6

No     Growing state

1      --
2
3
4      Hard , compact, pale
5
6
7      loose, spongy
8
9
10     compact, hard, granular
11
12
13
14
15
16     light compact
17
18
19     Loose, spongy, friable
20
21
22
23
24
25
26
27

IAA:Indol-3-acetic acid

NAA:1-Naphthalene acid

BA: Benzyladenine
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Title Annotation:Original Article
Author:Parsaeimehr, Ali; mousavi, Badrossadat
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:May 1, 2009
Words:1933
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