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Chapter 3 - media components and preparation

Media Components
The selection or development of the culture medium is vital to success in tissue culture. No single medium will support the growth of all cells, and changes in the medium are often necessary for different types of growth response from a single explant. A literature search is useful for selecting the appropriate medium.provide a useful guide on examining the effect of plant growth regu-lators, salt composition of the basal medium and a statistical analysis of the results. Likewise,can provide a guide for studying the effects of the MS inorganic salts on explant growth. If literature on the plant is not available, the development of a suitable medium is based on trial and error. The approach to developing the medium will depend on the purpose of the cell culture. Many of the media outlined in this manual can serve as useful starting points in developing a medium for a specific purpose, whether it is callus induction, somatic embryo-genesis, anther culture, or shoot proliferation.
Appendixes I and II include useful measurement conversions and a review of solution preparation problems, respectively. A list of suppliers is in given Appendix III.
In general, the medium contains inorganic salts, and organic compounds like plant growth regulators, vitamins, a carbohydrate, hexitols, and a gelling agent. Plant Tissue Culture. Third Edition. DOI: 10.1016/B978-0-12-415920-4.00003-7
Copyright 2013 Elsevier Inc. All rights reserved.
Plant Tissue Culture In addition, the medium can also include amino acids, antibiotics, or natural complexes.
INORGANIC SALTS
The inorganic salt formulations can vary (). have carefully examined the widely used tissue culture media formulations
and have pointed out minor errors in the original publications. outline the inorganic salt compositions of some of the commonly cited
formulations. The Murashige and Skoog (MS) (1962) formulation is the most
widely used ( and will be the major salt formulation
used in these exercises. The MS formulation was developed to insure that no
increases in cell growth in vitro were due to the introduction of additional
salts from plant tissue extracts which were being tested at that time. The MS
formulation insured that the inorganic nutrients were not limiting to tobacco
cell growth and organic supplements such as yeast extract, coconut milk,
casein hydrolysate, and plant extracts were no longer essential sources of the
inorganic salts. The Science Citation Index established the MS 1962 as a cita-
tion classic, as it has been extensively used in many publications on plant
tissue culture. Very few articles in plant science can come close to this highly
cited paper.
The distinguishing feature of the MS inorganic salts is their high content of nitrate, potassium, and ammonium in comparison to other salt formulations. outlines the five MS inorganic salt stock solutions. These salt stocks are prepared at 100 times the final medium concentration, and each stock is added at the rate of 10 ml per 1000 ml of medium prepared. The NaFeEDTA stock should be protected from light by storing it in a bottle that is amber colored or wrapped in aluminum foil. Concentrated salt stocks enhance the accuracy and speed of media preparation.
Salt stocks are best stored in the refrigerator and are stable for several months. Always prepare stocks with glass-distilled or demineralized water and clearly label and date all stocks. Reagent-grade chemicals should always be used to ensure maximum purity. Several salts can be combined to minimize the number of stock solutions. The factors to consider in combining com-pounds are stability and coprecipitability. The nitrate stock will usually pre-cipitate out and must be heated until the crystals are completely dissolved before using. Any stock that appears cloudy or has precipitates in the bottom should be discarded.
PLANT GROWTH REGULATORS
The type and concentration of plant growth regulators used will vary according
to the cell culture purpose. A list of the most commonly used plant growth regu-
lators, their abbreviations, and their molecular weights is provided in
Chapter 3 Media Components and Preparation
TABLE 3.1 Composition of the Five Inorganic Salt Stocks of the Murashige
and Skoog Inorganic Formulation
Concentration (g/liter stock)
Ammonium nitrate (NH4NO3) Potassium nitrate (KNO3) Magnesium sulfate (MgSO4 · 7H2O) Manganous sulfate (MnSO4 · H2O) Zinc sulfate (ZnSO4 · 7H2O) Cupric sulfate (CuSO4 · 5H2O) Calcium chloride (CaCl2 · 2H2O) Potassium iodide (Kl) Cobalt chloride (CoCl233 · 6H2O) PBMo stock
Potassium phosphate (KH2PO4) Boric acid (H3BO3) Sodium molybdate (Na2MoO4 · 2H2O) Ferrous sulfate (FeSO2 · 7H2O) Ethylenediamineteraacetic acid, disodium An auxin (IAA, NAA, 2,4-D, or IBA) is required by most plant cells for division and root initiation. At high concentrations, auxin can suppress morpho-genesis. The auxin 2,4-D is widely used for callus induction: IAA, IBA, and NAA are used for root induction.
Auxin stocks are usually prepared by weighing out 10 mg of auxin into a 200-ml beaker, adding several drops of 1 N NaOH or KOH until the crystals are dissolved (not more than 0.3 ml), rapidly adding 90 ml of double-distilled water, and increasing the volume to 100 ml in a volumetric flask ). Auxins can also be dissolved in 95% ethanol and diluted to volume; however, ethanol is toxic to plant tissues. The K-salts of auxin are more soluble in w Plant Tissue Culture TABLE 3.2 Inorganic Salt Formulation of Several Commonly Used Basal
Salts for Plant Tissue Culture in Milligrams per Liter of Medium
Ca(NO3)2 · 4H2O 300 cN6, . Chapter 3 Media Components and Preparation
TABLE 3.3 Common Plant Growth Regulators Used in Plant Tissue Culture
Plant growth regulator
Indole-3-acetic acid Naphthaleneacetic acid Indole-3-butyric acid Make IAA stocks fresh weekly; IAA is degraded within a few days by light () and within several hours to a few days by plant tissues Auxins are thermostable at 110–120°C for up to 1 h ). However, IAA is destroyed by low pH, light, oxygen, and peroxides (NAA and 2,4-D, which are synthetic auxins, are more stable than IAA, which is the naturally occurring auxin.
Cytokinins (kinetin, BA, zeatin, and 2iP) promote cell division, shoot proliferation, and shoot morphogenesis (). Thidiazuron (TDZ; N-phenyl-N1-1,2,3-thiadiazol-5-ylurea) has cytokinin activity and is commercially used as a cotton defoliant. Thidiazuron has been effective in low concentrations to stimulate shoot formation (. Cytokinin stocks are prepared in a fashion similar to that for auxin stocks, except that 1 N HCl and a few drops of water are used to dissolve the crystals (). Gentle heating is usually required to completely dissolve crystals. Double-distilled water is rapidly added to avoid the crystals' falling out of solution. Bring the stock up to the desired volume in a volumetric flask. Cytokinin stocks can be stored for several months in the refrigerator. There can be some photochemical degradation in long-term experiments ). Plant Tissue Culture Cytokinins (kinetin and zeatin) are thermostable; no breakdown products were detected after 1 h at 120°C ; 2iP and BA are stable for 20 min at 100°C.
Because it can inhibit callus growth and auxin-induced adventitious root formation, gibberellin (GA3) is infrequently used in plant cell culture ( However, it is useful in studies on morphogenesis. Stock solutions of GA3 can be prepared by dissolving the crystals in water and adjust- ing the pH to 5.7. At an alkaline pH, GA is converted to an inactive isomer and in an acid pH and high temperature, GA3 is also converted to biologically inac- tive forms ). Solutions of GA3 are not thermostable, and 20 min at 114°C reduces GA3 activity by more than 90% ( ). Stock solutions should be made up fresh before addition to the medium by filter sterilization.
Abscisic acid (ABA), a plant hormone involved in leaf and fruit abscission and dormancy, is useful in embryo culture. Abscisic acid is heat stable but light sensitive. The partial conversion of the 2-cis isomer of ABA to the 2-trans iso-mer of lower biological activity occurs in the light (). Stock solutions can be prepared in water.
Vitamins have catalytic functions in enzyme reactions. The vitamin considered important for plant cells is thiamine (B1). Other vitamins, nicotinic acid (B3) and pyridoxine (B6), are added to cell culture media, as they may enhance cel- lular response. Vitamin stocks are best stored in a freezer and can be made up such that 10-ml aliquots are used per liter of medium prepared. The  vitamin stocks used in these exercises contain 5 mg of nicotinic acid and 5 mg pyridox-ine-hydrochloride per 100 ml of water. The thiamine stock has 40 mg thiamine-hydrochloride  in  1000  ml. Other common vitamin formulations are those of with in milligram-per-liter medium: 0.5 nicotinic acid, 0.1 pyridoxine-hydrochloride, and 0.1 thiamine-hydrochloride; B5 Gamborg (with in milligram-per-liter medium: 100 inositol, 1.0 nicotinic acid, 1.0 pyridoxine-hydrochloride, and 10.0 thiamine-hydrochloride; with in milligram-per-liter medium: 100 inositol, 0.5 nicotinic acid, 0.5 pyridoxine-hydrochloride, and 0.1 thiamine-hydrochlo-ride. Most workers add vitamin stock solutions to the medium before autoclav-ing; however, for specific studies on vitamins, they should be filter sterilized ( CARBOHYDRATES
Green cells in culture are generally not photosynthetically active and require a
carbon source. Sucrose or glucose at 2–5% (w/v) is commonly used in cell cul-
ture. Other carbohydrate sources, such as fructose and starch, can also be used.
Chapter 3 Media Components and Preparation
Lower levels of a carbohydrate may be used in protoplast culture, but much higher levels may be used for embryo or anther culture.
Sugars undergo caramelization if autoclaved too long and will react with amino compounds (Maillard reaction). Caramelization occurs when sugars are heated, degrade, and form melanoidins, which are brown, high-molecular-weight compounds that can inhibit cell growth. A yellow to light brown color of an autoclaved medium is an indication that it was in the autoclave too long. The medium should be discarded.
HEXITOLS
The hexitol myo-inositol has been found to be important in tissue cultures
(. Myo-inositol is an interesting hexi-
tol involved in cyclitol biosynthesis, storage of polyhydric compounds as
reserves, germination of seeds, sugar transport, mineral nutrition, carbohydrate
metabolism, membrane structure, cell wall formation, hormonal homeostasis,
and stress physiology Myo-inositol is also consid-
ered as a growth enhancer in vitro and may be a carbohydrate source, but some
feel it has vitaminlike action. Mannitol and sorbitol are hexitols, which are good
osmotica for protoplast isolation.
GELLING AGENT
Many tissue culture experiments are conducted on some type of stationary support
and a gelling agent is most commonly used. However, stationary supports can
include filter paper, cotton, cheesecloth, vermiculite, and special membrane rafts
with a liquid medium. The type of agar used to gell the medium can affect the
response of your experiments ). If the agar is unwashed or not
purified, it will generally discolor the medium because it contains various impuri-
ties. Since agar is a product derived from seaweed, it can have physiological activ-
ity on the plant tissue. Sometimes dramatic differences in explant response can be
observed by changing the brand of agar used. To minimize problems from agar
impurities, purchase washed or purified agars. Gelrite is transparent in appearance
and is a polysaccharide produced as a fermentation product from a Pseudomonas
species , and it is consistent in its composition. The exercises
described in this manual use TC agar, Difco-Bacto agar, or Gelrite.
When melting agar over a hot plate or flame, keep the agar in motion either with a magnetic stir bar on the hot plate or by agitating the flask by hand. Use a heat-resistant glove on your hand because the flask can get very hot. The agar must be kept in motion or it will burn on the bottom of the flask. The agar must be completely dissolved before it is dispensed into the culture containers. The agar is dissolved when, after the Erlenmeyer flask is agitated, the medium that sheets off the interior glass surface does not have small granules of visible agar. Plant Tissue Culture Remove the flask from the heat immediately because excessive heat after this point will cause the medium to boil out of the flask. Do not melt 1 liter of medium in a 1-liter Erlenmeyer flask; use a 2-liter Erlenmeyer flask to prevent media from boiling over. The medium is then dispensed in measured amounts in the culture container, which is capped and autoclaved. A dispensing burette can be used to accurately fill the culture container; however, because these are easily broken and are expensive, students should fill one container with the measured amount of water and use this as a guide to hand-fill the remaining containers. It helps to pour the hot medium from the Erlenmeyer flask into a 400- to 600-ml beaker before pouring it into the culture containers. Commercial cell culture laboratories use automatic media-dispensing equipment to rapidly fill culture containers.
The agar can also be melted in the autoclave in a foil-capped Erlenmeyer flask for 15 min at 121°C, 15 psi. When cool to the touch, the medium is dis-pensed aseptically into sterile containers within a transfer hood. If this method is used, the medium can be maintained in a water bath at 40°C to prevent it from solidifying before it is dispensed into the sterile containers.
When agar is not used, liquid media can be agitated on some type of a shaker. As mentioned earlier, explants can also be cultured on stationary liquid media usually on some type of a support like filter paper or membrane rafts. discusses the use of stationary liquid medium for micropropagation. Interactions of the gelling agent concentration on the nutritional availability, hyperhydricity, and propagation rates are presented.
More recently bioreactor systems have been used to culture plant explants. The bioreactor is a sterile environment, and allows for exchange of the culture medium, as well as regulating air supply, pH and temperature. Bioreactor systems have been of high interest in commercial mass propagation of ornamentals to reduce labor costs ( Debnath, 2009; ).
AMINO ACIDS
Amino acids and amines can be very important in morphogenesis. All l-forms of amino acids are the natural forms detected by the plant; l-tyrosine can con-tribute to shoot initiation ), l-arginine can facilitate root-ing, and l-serine can be used in microspore cultures to obtain haploid embryos. Amides, such as l-glutamine and l-asparagine, sometimes significantly enhance somatic embryogenesis.
Casein hydrolysate, an enzymatic digest of milk protein (do not use the acid digest of milk proteins), was a common ingredient in many early media formu-lations as it provided a mixture of amino acids to enhance tissue response. Since enzymatic digests can result in slight differences in amino acid composition, addition of specific amino acids is preferred since it will more precisely define the ingredients in the medium. Today it is more common to examine individual amino acids for desirable tissue response.
Chapter 3 Media Components and Preparation
ANTIBIOTICS
Because of excessive contamination problems with certain plant explants,
many workers have incorporated fungicides and bactericides in the culture
medium (. examines antibiotics, and
their action, origin, and antibiotic resistance. Generally, these additions
have not been very useful because they can be toxic to the explant, and the
contaminant can reappear as soon as the fungicides or bactericides are
removed.
Transformation experiments using Agrobacterium make it necessary to incorporate antibiotics into the medium. Several antibiotics have been found not to be toxic to the explant and, at the same time, control or eliminate the Agro-bacterium; commonly used antibiotics are timentin, carbenicillin (500 mg/liter), cefotaxime (300 µg/ml) and augmentin (250 mg/liter). The antibiotics are soluble in water, should be made up fresh, and should be added to the medium after autoclaving by filter sterilization.
NATURAL COMPLEXES
Many other additions to nutrient media serve various purposes. Antioxidants are
sometimes used if there is excessive browning of the explant, and they retard
oxidation of the explant. Examples of antioxidants are citric acid, ascorbic acid,
pyrogallol, and phloroglucinol. Sometimes when there is excessive tissue dis-
coloration of the medium and explant, absorbents are used. Two absorbents are
polyvinylpyrrolidone (PVP) and activated charcoal (0.1–0.3%). Use activated,
acid-neutralized charcoal.
Some good references on activated charcoal are and . Recently presented data on the effect of the source of activated charcoal on coconut embryogenic callus. suggests that activated charcoal may promote morphogenesis by absorbing inhibitory compounds and reducing toxic metabolites, phenolic exudation and accumulation. Activated charcoal releases substances naturally present in the charcoal that may promote growth. It also adsorbs vitamins and plant growth regulators from the medium, and may gradually release them to the explant. There may also be a beneficial effect related to darkening the medium.
A natural complex can be used when a defined medium fails to support a particular growth response. Natural complexes added to the medium gener-ally make the medium undefined, since variations in growth-promoting or inhibitory compounds in these complexes are to be expected. Some exam-ples and general concentrations of these natural complexes are coconut endosperm (CM, 10–20% v/v; ), yeast extract (YE, 50–5000 mg/liter), malt extract (500 mg/liter), tomato juice (30%), orange juice (3–10%), banana (150 g/liter), potato extract (, Plant Tissue Culture casein hydrolysate (30–3000 mg/liter, use enzyme digest), and fish emulsion (1 tsp/liter).
Much has been published on the compounds present in coconut milk, which is the liquid endosperm from Cocos nucifera L. A discussion in 1998 on the Plant-tc listserve (see also) evaluated the chemical composition and biological properties of coconut water including the sugars, vitamins, minerals, amino acids and phytohormones. Coconut water is the liquid from immature coconuts. As the coconut matures, the liquid turns to a jellylike substance called coconut milk. Coconut "water" is what is used as coconut "milk" in tissue culture. The coconut milk is boiled to precipitate the protein, cooled, filtered, and stored frozen until use. It is generally difficult to obtain coconut milk from coconuts in the grocery store, as they are mature and the endosperm is solid, or is real coconut milk. Coconut milk can be purchased from most chemical suppliers.
MEDIA pH
The pH of plant tissue culture media is generally adjusted to pH 5.5 to 6. Below
5.5, the agar will not gel properly and above 6.0, the gel may be too firm
(. Media pH generally drops by 0.6 to 1.3 units after autoclaving
(Cultures of some plant tissues cause a pH drop over time
that is attributed to the production of organic acids or nitrogen examined media pH as influenced by the inorganic salts, carbohy-
drate source, gelling agent, activated charcoal, and medium storage method. All
of these factors influenced the pH.
Adjust the medium pH with 1.0 or 0.1 N HC1 or NaOH by using a medicine dropper while keeping the medium stirred. Always adjust the pH before adding the agar.
Outline the medium to be prepared and check off the ingredients as they are added to the flask in which the medium is being prepared. Keep records on the date of preparation and use.
In media preparation always use glass-distilled water—never tap water or tap-distilled water. Some water ( 500 ml for final 1-liter volume) must always be in your flask before the stock solutions are added; otherwise, concentrated stocks will coreact and precipitate out.
Never pour excess stocks back into the original stock solution container and never put excess sucrose or agar back into the original container. Always clean up spills around balance and work areas.
Packaged powders of the MS salts and other media are available, eliminating the need to prepare stocks and measure ingredients. The suppliers of pre-pared plant tissue culture media are listed in Appendix III. Follow the manu-facturer's directions for their use.
Chapter 3 Media Components and Preparation
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