CHAPTER 4
MATERIALS AND METHODS

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4.1. Materials

4.1.1. Plants

In theory, any part obtained from any plant species can be employed to induce callus tissue, however the successful production of callus depends upon plant species and their qualities. Dicotyledons are rather amenable for callus tissue induction, as compared to monocotyledons; the callus of woody plants generally grow slowly. Stems, leaves, roots, flowers, seeds and any other parts of plants are used, but younger and fresh explants are preferable as explant materials.

Explants obtained must be sterilized using ethanol, sodium hypochlorite and/or other chemicals to remove all microorganisms from the materials and a typical sterilization procedure will be described later as an example.

4.1.2. Media

4.1.2.1. Inorganic Salts

To induce a callus from an explant and to cultivate the callus and cells in suspension, various kinds of media (inorganic salt media) have been designed. Agar or its substitutes is added into the media to prepare solid medium for callus induction.

One of the most commonly used media for plant tissue cultures is that developed by Murashige and Skoog (MS) for tobacco tissue culture (28). The significant feature of the MS medium is its very high concentration of nitrate, potassium and ammonia. The B5 medium established by Gamborg et al. (29) is also being used by many researchers. The levels of inorganic nutrients in the B5 medium are lower than in MS medium. Many other media have been developed and modified and nutrient compositions of some typical media will be described in Table 6. However, it is not always necessary to test many kinds of basal media when a callus is induced. It would be better to use only one or two kinds of basal media in combination of different kinds and concentrations of phytohormones. The most suitable medium composition should be optimized afterwards in order to obtain higher level of products as well as higher growth rate.

4.1.2.2. Carbon Sources

 

Sucrose or glucose at 2 to 4% are suitable carbon sources which are added to the basal medium. Fructose, maltose and other sugars also support the growth of various plant cells. However, the most suitable carbon source and its optimal concentration should be chosen to establish the efficient production process of useful metabolites. These factors depend on plant species and products, therefore it is necessary to optimize the medium compositions including carbon sources in each case. From an economical point of view, the use of more inexpensive carbon sources is appropriate in industry and crude sugars such as molasses have been examined.

Table 6 - Media for Plant Tissue and Cell Cultures (mg/L)

Components Murashige-
Skoog
(1962)
White
(1963
Gamborg
(1968)
Nitsch
(1951)
Heller
(1953)
Schenk -
Hildebrandt
(1972)
Nitsch -
Nitsch
(1967)
Kohlenbach -
Schmidt
(1975)
Knop
(1865)
(NH4)2SO4 - - 134 - - - - - -
MgSO4 7H2O 370 720 500 250 250 400 125 185 250
Na2SO4 - 200 - - - - - - -
KC1 - 65 - 1,500 750 - - - -
CaC12 2H2O 440 - 150 25 75 200 - 166 -
NaNO3 - - - - 600 - - - -
KNO3 1,900 80 3,000 2,000 - 2,500 125 950 250
Ca(NO3)2 4H2O - 300 - - - - 500 - 1,000
NH4NO3 1,650 - - - - - - 720 -
NaH2PO4 H2O - 16.5 150 250 125 - - - -
NH4H2PO4 - - - - - 300 - - -
KH2PO4 170 - - - _ - 125 68 250
FeSO4 7H2) 27.8 - 27.8 - - 15 27.85 27.85 -
Na2EDTA 37.3 - 37.3 - - 20 37.25 37.25 -
MnSO4 4H2O 22.3 7 10 (1 H2O) 3 0.1 10 25 25 -
ZnSO4 7H2O 8.6 3 2 0.5 1 0.1 10 10 -
CuSO4 5H2O 0.025 - 0.025 0.025 0.03 0.2 0.025 0.025 -
H2SO4 - - - 0.5 - - - - -
Fe2(SO4)3 - 2.5 - - - - - - -
NiC12 6H2O - - - - 0.03 - - - -
CoC12 6H2O 0.025 - 0.025 - - 0.1 0.025 - -
A1C13 - - - - 0.03 - - - -
FeC13 6H2O - - - - 1 - - - -
FeC6O5H7 5H2O - - - 10 - - - - -
K1 0.83 0.75 0.75 0.5 0.01 1.0 - - -
H3BO3 6.2 1.5 3 0.5 1 5 10 10 -
Na2M0O4 2H2O 0.25

-

0.25 0.25 - 0.1 0.25 0.25 -
Sucrose
Glucose
30,000
-
20,000
-
20,000
-
50,000
or
36,000
20,000
-
30,000
-
20,000~30,000
-
10,000
-
-
-
Myo-Inositol 100 - 100 - - 1,000 100 100 -
Nicotinic Acid 0.5 0.5 1.0 - - 0.5 5 5 -
Pyridoxine HC1 0.5 0.1 1.0 - - 0.5 0.5 0.5 -
Thiamine HC1 0.1-1 0.1 10 1 1 5 0.5 0.5 -
Ca-Pantothenate - 1 - - - - - - -
Biotin - - - - - - 0.05 0.05 -
Glycine 2 3 - - - - 2 2 -
Cysteine HC1 - 1 - 10 - - - - -
Folic Acid - - - - - - 0.5 0.5 -
Glutamine - - - - - - - 14.7 -

 

4.1.2.3. Vitamins

The basal media described above such as MS medium include myo-inositol, nicotinic acid, pyridoxine HCl and thiamine HCl. Among these vitamins, thiamine is an essential one for many plant cells and other vitamins stimulate the growth of the cells in some cases. The level of myo-inositol in the medium is 100 mg/L which is very high although it is not clear whether such a high level of the vitamin is required.

4.1.2.4. Phytohormones

Phytohormones or growth regulators are required to induce callus tissues and to promote the growth of many cell lines. As an auxin, 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneaceic acid (NAA) is frequently used. The concentration of auxins in the medium is generally between 0.1 to 50 µM. Kinetin or benzyladenine as a cytokinin is occasionally required together with auxins for callus induction at concentrations of 0.1 to 10 µM. Other derivatives of auxin and kinetin are also used in some cases. Since each plant species requires different kinds and levels of phytohormones for callus induction, its growth and metabolites production, it is important to select the most appropriate growth regulators and to determine their optimal concentrations. Gibberellic acid is also added to the medium if necessary.

4.1.2.5. Organic supplements

In order to stimulate the growth of the cells, organic supplements are sometimes added to the medium. These supplements include casamino acid, peptone, yeast extracts, malt extracts and coconut milk. Coconut milk is also known as a supplier of growth regulators.

4.2. Methods

4.2.1. Preparation of Media

To prepare the medium, many researchers mix the stock solutions which were made previously since the medium compositions are generally complicated (30).

For example, MS medium is prepared as follows:

a.    MS-Micronutrient stock solution (store in freezer)
       Ingredient mg/100 ml
       H3BO3 620
       MnSO4 4H2O 2230
       ZnSO4 7H2O 860
       Na2MoO4 2H2O 25
       CuSO4 5H2O 2.5
       CoCl2 6H2O 2.5

b.    Vitamins (store in freezer)
       Vitamins mg/100 ml
       Nicotinic acid 100
       Thiamine HCl 1,000
       Pyridoxine HCl 100
       Myo-Inositol 10,000

c.    Calcium chloride
       CaCl2 2H2O 15 g/100 ml

d.    Potassium iodide (store in amber bottle in refrigerator)
       KI 75 mg/100 ml

e.    2,4-D (2.2 mM)
       Dissolve 50 mg 2,4-D in 2 to 5 ml ethanol, heat slightly and gradually dilute to 100 ml with water. (Store in refrigerator).

f.     NAA (2.8 mM)
       Prepare the same as 2,4-D above.

g.    Kinetin (1 mM)
       Dissolve 21.5 mg of kinetin in a small volume of 0.5 N HCl by heating slightly and gradually diluting to 100 ml with distilled water. (Store          in refrigerator) Similar procedures can be used for other cytokinins.

A certain volume of each stock solution is mixed and an appropriate carbon source is added to the mixture. After pH is adjusted to around 5.5 with 0.2 N K0H or 0.2 N HCl, distilled or deionized water is added to the mixture up to the certain volume required. Agar (0.6 to 1.0% wt/vol) is added for a solid medium.

The medium thus prepared is distributed into vessels such as Erlenmyer flasks (for example, 50 ml of the medium in a 300 ml volume Erlenmyer flask) and sterilized by using an autoclave at 120 C for 15 minutes. The sterilization conditions should be varied based on the volume of the medium and the size of the vessel.

4.2.2. Callus Induction

Explants are sterilized with 2% sodium hypochlorite solution and/or 70% ethanol solution. The period of time for submerging the plant materials in these solutions depends upon plant species, their parts and age. For example, a piece of stem of tobacco plant (approximately 3 cm in length) is submerged in 70% ethanal solution of 2-3 minutes and then in 1.2% sodium hypochlorite solution for 10 minutes. The explants should be rinsed with sterilized water.

The stem or any other part of plants thus sterilized is cut to approximately 1 cm in length using a sterilized scalpel and each piece is transferred with tweezers to a solid medium in a flask or a petri-dish. The plant material is incubated aseptically at around 25 C on the solid medium for several weeks or more and a callus is produced. The callus is subcultured by transferring a small piece to fresh solid medium. After several subsequent transfers, the callus becomes soft and fragile.

4.2.3. Suspension Culture

The growth rate of the suspension cultured cells is generally higher than that of the solid culture. The former is more desirable particularly in production of useful metabolites in a large-scale. A piece of the callus is transferred to a liquid medium in a vessel such as an Erlenmyer flask and the vessel placed on a rotary or reciprocal shaker. The culture conditions depend on plant species and other factors, but in general, the cells are cultivated at 100 r.p.m. on a rotary shaker at 25 C; some researchers are fond of much slower, or faster speeds. By subculturing for several generations, a fine cell suspension culture containing small cell aggregates and single cells is established. The time required to establish the cell suspension culture varies greatly and depends on the tissue of the plant species and the medium composition. The cells in suspension are also used for a large-scale culture with jar-fermentors and tanks.

4.2.4. Scaling-up

For commercialization, it is necessary to progress through several stages increasing the volume at each stage until the requisite bioreactor size is attained. In theory, it is anticipated that such large scale suspension cultures will be suitable for industrial production of useful plant chemicals such as pharmaceuticals and food additives, in a manner similar to that of microbial fermentation. Generally speaking, the culture period in plant cell cultures is longer than that in microbial cultures, and it is crucial to protect against microbial contamination.

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