Classification Of Glycyrrhiza Uralensis Fisch Biology Essay

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Glycyrrhiza Uralensis Fisch has been extensively studied worldwide. There are over 20 species of Glycyrrhiza in the world and 6 species of them are growing in Mongolia, for example, Glycyrrhiza aspera Pall, G.glabla  L, G.inflata Batal, and G. uralensis Fisch.

Table . Classification of Glycyrrhiza Uralensis Fisch

Kingdom

Plantae

Family

Pea

Type

Glycyrhiza.L

Species

Glycyrhiza Uralensis Fisch

Common name

Licorice

 

Botanical features, chemical composition and prevalence of G. Uralensis have been extensively studied. Today many countries s are widely studying classification, prevalence, pharmacology, embryology, physiology and structure of Glycyrrhiza Uralensis Fisch. Glycyrrhiza Uralensis Fisch grows across the 2320 hectare area of Mongolia. Glycyrrhiza Uralensis Fisch is an important herbal medicinal plant and is used as a raw material in the production of pharmaceuticals and cosmetics. It has drought resistance and is cold and salt tolerant. Over 70% of Mongolian Glycyrrhiza Uralensis are in Bayankhongor province. Glycyrrhiza Uralensis Fisch is a perennial herb that has been widely used in traditional medicine in Mongolia as well as in the pharmaceutical industry. In recent years, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural evelopment, natural desertification and uncontrolled and indiscriminate collection in Mongolia. For example, because the roots of Glycyrrhiza Uralensis Fisch are excavated in Bayanhongor province, a 700 hectare area of Glycyrrhiza Uralensis Fisch cannot be grown naturally and due to its indiscriminate collection from natural resources without taking any care for its preservation and propagation, this particular plant is threatened with extinction. Every year 60000 tons of dry root of Glycyrrhiza Uralensis are traded in the global market. The main costumers are Japan, Germany, Czech Republic, Slovakia, England and The United States and the primary suppliers are Turkey, Iran, Russia, China, Afghanistan and Italy (7). Because of plant depletion and the loss of its natural habitat, there is an urgent need to apply in vitro culture methods for micropropagation, conservation and to provide for the increasing demand of Glycyrrhiza Uralensis Fisch.

 Generally, for crude drug preparation, field-grown and naturally grown herbs are used, which are usually infect with bacteria, fungi and insects and difficult to use for crude materials of drugs. And propagation by conventional methods takes a long time for germination, fruit setting and these depend on climate and other conditions as well. Plant tissue culture methods can provide a means to rapid cultivation of valuable, rare and endangered herbal plants. There are many advantages in cultivating Glycyrrhiza Uralensis Fisch in vitro. It's provides clean raw materials for pharmaceuticals and cosmetics, increases the sources of herbal plants can be exported in huge amounts.

Glycyrrhiza Uralensis Fisch became the second most important medicinal plant in the world through its usage and benefits (12). In total, there are 179 different chemical compounds extracted from roots, seeds, leaves and stems of Glycyrrhiza Uralensis Fisch. The roots and shoots of Glycyrrhiza Uralensis Fisch contain 9.1% - 16 % of triperpenic saponin called glycyrrhzin, 1.02% - 2.4% glucose, 1.95% - 3.9% compound flavonoids and 14% - 30% calcium, magnesium salts, substances of pectin and starch (6). Glycyrrhiza uralensis Fisch is used treating in many medical conditions such as dyspnea, enlargement of chest, pneumonia, bronchitis, pregnancy toxicosis, and dryness in mouth, throat, fever and hypomnesia. In addition, it is an ingredient in most black beers and lemonade to which it gives a special taste and color. Salt of glycyrrhinic acid of Glycyrrhiza uralensis Fisch has froth quality. Thus, factories which produce Coca-cola, Terelj and Selenge (Mongolian brand) are using these compounds. The extract of Glycyrrhiza Uralensis Fisch is also widely used in production of chocolate, chewing gum and candy.

Currently, plant cell tissue and organ culture are becoming interesting fields in drug agriculture and food factories for all plants. The reasons are:

Naturally grown plants have many diseases caused by bacteria, fungi, and insects. Thus it's difficult to use them as raw materials in pharmaceuticals.

Most traditional propagation methods depend on climate and take much time until mature plants grows, therefore, they are very expensive.

Because of illegal collection and export, rare and endangered species face destruction of their natural habitat. 

Thus, we need to use modern biotechnological methods of culturing plant cell and tissue which should enable us to conserve and rapidly propagate valuable herbal plants.

1-1. Studied situation

Botanical feature, biology, growth conditions and basis of cultivation of the Glycyrrhiza Uralensis were studied by Dashbaldan (1979) and Dashjamts (1984).

Choijamjts (1987) and Byambasuren (1993) studied water condition of Glycyrrhiza Uralensis Fisch. Tamaki (1973) is first man who made in vitro cultures of Glycyrrhiza tissues. He cultured root fragments of G.glabra on Linsmaier and Skoog's medium (1965) with 2.0 mg/L Indole-3-acetic acid (growth regulator) and 1 % agar

1-2. Goals and Objectives of this research

We investigated appropriate sterilization solution for seeds and a range of additional growth regulators for development of shoot and root of Glycyrrhiza Uralensis Fisch. This investigation was undertaken in the Biotechnology Laboratory of Mongolian State University of Agriculture. 

1. Determining an appropriate solution, concentration and soaking period for the sterilization of seeds

2. Determining a suitable type and concentration of growth regulator (cytokinin) for shoot induction.

3. Defining an appropriate type and concentration of growth regulator (auxin) for root induction.

Chapter II. Materials and methods

II-1. Materials

      The seed samples of wild growing Glycyrrhiza Uralensis Fisch were originally collected from Bayankhongor province.

II-2. Methods

II-2-1 Sterile techniques

The applications of plant cell, tissue culture require sterile techniques. The aseptic or sterile conditions are fundamental for successful tissue culture procedures. All procedures were conducted in a Laminar Flow Cabinet. The cabinets in use for most plant work have a horizontal airflow from the back toward the front.

There are three categories of sterilization. One is preparation of sterile media and containers. The second has to do with obtaining explants materials that are aseptic. The third is maintenance of aseptic conditions of the culture. Contamination usually occurs during transfer to the soil.

II-2-1-1 Sterilization of Laminar Flow cabinet

Before starting, the work area of the Laminar Flow cabinet was exposed to UV light for 15-20 minutes and subsequently wiped down with 96% ethanol.

II-2-1-2 Sterilization of instruments and vessels

Metal instruments were sterilized in a hot air oven for 2-4 hour at 130-170oC. Other plastic instruments and vessels were sterilized by autoclaving at 121oC and 15 kPa (Kilopascal) for 20 minutes. Immediately before use, metal instruments were submerged into 96% ethanol and exposed to the heat of a flame.

II-2-2. Procedures for Media Preparation

Stock solutions

The degree of success in plant cell and tissue culture depends on several important factors including the choice of nutritional components, growth regulators and their appropriate ratio. Stock solutions are concentrated solutions of media chemicals that are prepared ahead of time and used to make several types of media. They may be made in liter quantities of 10 or 100 times the concentration required in the final formula.

Stock solutions were prepared by adding one compound at a time which usually avoided precipitation. Dissolving the inorganic nitrogen sources of the major salts first prevented precipitation between phosphate and calcium sources when added subsequently, which can occur when the pH approaches 6.0. After the salt and compounds were added and dissolved, the pH was adjusted by the addition of an acid or base.

II-2-2-1. Nutrient media preparation

We chose Murashige & Skoog (MS) medium which was invented by plant scientists Murashige and Skoog in 1962. It is the most commonly used medium in plant tissue culture. Seeds of Glycyrrhiza Uralensis Fisch were cultured on ½ MS medium (two times diluted MS medium with distilled water) and shoot tips were cultured on MS medium supplemented with growth regulators.

Growth regulators

Growth regulators, or hormones, are not nutrients, but they influence growth and development. They must be added selectively to culture media. Auxins,cytokinins, gibberellins, and ethylene are among the substances used as growth regulators. Generally, auxins promote cell enlargement and root initiation, whereas cytokinins promote cell division and shoot initiation. In our experiment, we chose 3 kinds of auxins (benzyladenine, kinetin and adenine hemisulfate) for root induction and 3 kinds of cytokinins (indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acetic) for shoot induction.

MS medium was mixed by following order:

1. MS basalt salt mixture

2. Stock solution of Iron-EDTA

3. Stock solution of Vitamin

5. Sucrose (3%)

6. Agar

Components of Murashige & Skoog (MS) basalt salt mixture

Chemicals

g/L

Ammonium Nitrate

1.65

BoricAcid

0.006200

CalciumChloride

0.332200

CobaltChloride

0.000014

CupricSulfate

0.000016

EDTADisodium

0.03725

Ferrous Sulfate•7H2O

0.0278

Magnesium Sulfate

0.1807

Manganese Sulfate•H2O

0.0169

Molybdic Acid•Na2•2H2O

0.000250

PotassiumIodide

0.000830

Potassium Nitrate

1.9

Potassium Phosphate Mono

0.17

ZincSulfate•H2O

0.00537

Total:

4.328g/L

  Stock solution of Iron-EDTA

1. Dissolve 5.57 g of FeSO4 x 7 H2O in 350 ml of distilled water. Apply heat if needed.

2. Dissolve 7.45 g Na2 EDTA in 350 ml of distilled water. Apply heat if needed.

3. When both solutions are dissolved, combine and bring to 1 l final volume.  The chelation reaction is forced to completion by autoclaving. The final stock should be deep golden yellow in color. 

Stock solution of Vitamins

The stock solution of Vitamins must be stored in freezer (-20)

      Vitamins (100x) mg/100ml

Nicotinic acid      50

Tiamin HCl         50

Pridoxin HCl       10

Mio-inozitol        10000

 Protocol

1. Always establish a step by step routine for preparing a medium. The routine will insure that the medium is identical in composition each time.

2. Add each component according to the list of ingredients and check it off on the list as it is added. Each ingredient must be completely dissolved before adding the next. If 1L of medium is required, start with 400 ml distilled water in beaker placed on magnetic stirrer

3. Measure each component carefully by using the correct sized pipette or graduated cylinder and the proper range balance.

4. After all the ingredients have been added and dissolved, with the exception of the gelling agent, add water to the just below the final volume

5. Check and adjust the pH of the medium to the required value by adding either 0.2 M KOH or 0.5 N HCl while stirring. Before autoclaving MS medium, the pH must be between 5.6-5.8

6. After adjusting the pH, bring the solution to the final volume.

7. Autoclaving the media at 121at 15 kPa for 20 minutes. Allow sufficient air volume in the flask to prevent the media from boiling over.

8. Cool the medium, and swirl the solution gently as it cools.

9. If filter sterilized compounds are to be added to the medium, the appropriate amount of the compound is added to autoclaved media when cooling but not yet solidified, warm to the touch but not hot. Mix thoroughly by swirling the solution. Make sure that the volume of the autoclaved medium plus the volume of the added filter sterilized compound sums to the final volume of the prepared medium.

10. When sufficient cool, dispense the medium into labeled, sterile containers using aseptic techniques.

II-3. Strategies of plant cell and tissue culture

            Stage 1: Differentiating explants from donor plant (Isolation)

            Stage 2: Establishment of the aseptic culture (Inoculation)

            Stage 3: Multiplication of explants (Incubation)

            Stage 4: Re- establishment of plant (Subculture)

            Stage 5: Preparation for re-establishment of plant in soil. 

Isolation - In vitro culture conditions require the use of explants, which are small plant or organs or tissues detached from the whole plant. Isolation is the process of separating explants from sterilized plant material. For the seeds, we should directly inoculate without isolation. The chosen plant material should be put on sterilized filter paper or glass and explants cuts by a sterilized blade and pincers.

All processes such as sterilization of the plant material, separation of explants and inoculation should be made in a Laminar Flow Cabinet.

Inoculation - Placing explants on the growing medium is called an inoculation. Inoculation should be made in a Laminar Flow cabinet. If inoculations are made in a test tube or flask, which contain medium, they should be placed horizontally. It is one way of preventing to growth of microorganisms. How to place the plant tissue on the medium depends on the culture type. For example: seeds and meristem tips should be placed on the surface of the medium.

Incubation - After placing explants on the medium, growing it in the appropriate conditions called incubation. Explants are inoculated in the cultivating room where we are able to control light, temperature, aeration and moisture.

 Subculture - Process of a plantlet that grew on medium to transfer new prepared medium is called subculture. If medium depleted or dried or medium is contaminated, subculture can be made. Sometimes the medium surface gets black spots, because of the secretion of toxic compound from the explants after several weeks inoculating on the medium. In this case it is necessary to transfer to new medium.

 

Chapter-III Results

III-1. Determining an appropriate solution concentration and soaking period for the sterilization of seeds

The sterilization of plant material is the most important stage in plant tissue culture. This stage (appropriate solution concentration and soaking period) can directly affect on final results of tissue culture. There are several kinds of sterilization solutions used for seeds. We chose sodium hypochloride ( NaOCl ) and Dimercury (I) dichloride ( HgCl ) for Glycyrrhiza Uralensis Fisch seeds.

1. Rinsed seeds in tap water

2. Washed seeds for 5 minutes by submerging in water with few drops of liquid detergent.

3. Rinsed in tap water to remove the soap. Covered the seeds with 70% ethanol, and shake for 30 seconds.

4. Covered seeds with a solution of NaOCl or HgCl. Concentrations and Periods showed in Table-1

5. After that rinsed in distilled water, 3 times.

After the seed sterilization, we cultured seeds on 1/2 MS medium (two time diluted MS medium with water) and observed contamination and survival of seeds.

Table . Seed sterilization

Sterilization solution

Concentration

(%)

Sterilization period (min )

Number of seeds

Contamination (%)

Surviving percentage (%)

Sodium hypohloride

0.5

20

30

0

70

1

15

30

0

100

2

10

30

20

80

HgCl

0.1

5

30

20

80

0.1

15

30

10

90

0.1

20

30

0

80

Table 2 shows that sterilizing seeds of Glycyrrhiza Uralensis Fisch with 1% sodium hypochloride for 15 minutes was the most appropriate concentration and period. However sterilizing seeds of Glycyrrhiza Uralensis Fisch with 0.1 % HgCl for 20 minutes had no contamination, seed survival was decreased and it means that concentration was over dosed.

III-2. Determining suitable type and concentration of growth regulator for shoot induction

Table . The types and concentration of Cytokinins in MS medium

Medium number

Volume

/mg/

Type of growth regulator

Amount of growth regulator

Medium -MS-I

200

Adenine Hemisulfate /AdSO4/

2 mg/l

Medium - MS-II

200

Benzyladenine /BA/

2 mg/l

Medium - MS-III

200

Kinetin

1 mg/l

Medium - MS-III

200

Kinetin

2 mg/l

Medium - MS-V

200

Control

(no cytokinins)

-

In order to determine appropriate concentration of cytokinin, we prepared 5 different MS medium for shoot induction. We cultivated 5 plantlets on each medium and observed height, shoot multiplication and survival after cultivation for 45 days.

Table . The influence of the growth regulators (cytokinins) in shoot induction

Medium

number of shoot

Average height of the shoot /after 45 days/ cm

Average amount of shoot

Temperature /0С/

Humidity

%

Survival

%

Medium-MS-I

5

6,5

5

22

65

100

Medium- MS-II

5

6,0

5

22

65

100

Medium- MS-III

5

3,5

5

22

65

40

Medium- MS-III

5

4,0

4

22

65

100

Control

5

2,0

3

22

65

60

Control

MS-IV

MS-III

MS-II

MS-IIn this experiment, the height of shoots cultured in the control medium was the shortest. The experiment shows that growth regulators improve growth and survival. MS-I and MS-II were the most effective on the shoot height and survival. 3Figure . Comparison of height of plantlet growing on the MS medium with different cytokinins

III-3. Define appropriate type and concentration of growth regulator for the root induction

In order to determine suitable type and concentration of auxin, we prepared 10 different MS media. Nine MS media contained 3 different auxins with 3 different concentrations for each. We cultivated seven plantlets on the each medium in the test tube and observed root length and survival of plantlet after cultivation of 30 days. Table-3 shows the result of determining influence of the auxins with different concentrations.

Table . Influence of growth regulators (Auxins) on root induction

Growth regulator

/auxins/

Amount of growth regulator / MS medium 1L

Repeating number

/Number of culture in test tube /

Survival

/%/

Root length of plantlet /Average root length of plantlet after 30 days /

Indole-3-acetic acid

IAA

0,5 mg/l

7

42,8

1,0

IAA

1 mg/l

7

42,8

0,8

IAA

2 mg/l

7

42,8

2,4

1-Naphtaleneacetic acid

NAA

0,5 mg/l

7

57,1

2,8

NAA

1 mg/l

7

85,7

3,0

NAA

2 mg/l

7

85,7

2 ,1

Indole-3-butyric acid

IBA

0,5 mg/l

7

42,8

0,9

IBA

1 mg/l

7

71,4

1,8

IBA

2 mg/l

7

57,1

1,8

Control/ without hormone

-

7

28,5

0,3

The results show the development of root of plantlets cultured on control medium were poor and some of them died. The plantlets cultured on MS having auxins had better to rooting than plantlet cultured on control medium. Among different media, the root of plantlets grown in media with NAA (1mg/l) were tallest and average root length after 30 days was 2.7 cm longer than the roots of plantlets grown on control medium.

Conclusion

Sterilizing seed of Glycyrrhiza Uralensis Fisch with 1% sodium hypochloride for 15 minutes was most the appropriate concentration and soaking period.

Shoot induction. Kinetin (1 mg/l) in the MS medium was sufficient for multiplication, but it did not promote elongation of shoot, and hence shoots on MS medium with Adenine Hemisulfate (2mg/l) and Benzyladenine (1mg/l) were 4.0-4.5 cm (average height) higher than control medium.

Rooting. Best rooting response (85.7%) was observed on MS medium with NAA (1mg/l) (Table 3). Multiple roots (average 3.0 cm in length) developed in 30 days. The roots of plantlets which cultured on MS medium supplemented with NAA (1mg/l) were 2.7 cm longer than cultured on control medium after 30 days.

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