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PLANT & TISSUE CULTURE - C3 (Media Preparation) Cheat Sheet (DRAFT) by

Brief summary of Chapter 3 (Media Preparation) of Plant and Tissue Culture Subject

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Culture Medium Components

Media for Plant Cultures

1. Macron­utr­ients (mM concen­tra­tions)
N, P, K, Ca, Mg, S
2. Micron­utr­ients (µm concen­tra­tions)
Fe, B, Cu, Mn, Zn, Mo, I, Co
3. Carbon Source
sucrose, glucose, other sugar
4. Vitamins
thiamine, biotin, pantot­henic acid, nicotinic acid, pyrido­xine, folic acid, ascorbic acid, tocoph­erol, myo-in­ositol
5. Complex Organic Supple­ments
coconut water, banana powder, yeast extract, peptone, potato homogenate

Macron­utr­ients

Micron­utr­ients

Carbon and Energy Source

Every living organism needs to have a source of energy in order to complete all the vital processes within the organism, and therefore each medium needs sugars as a source of carbon and energy.
The preferred carboh­ydrate in plant cell culture media is sucrose.

Vitamins

Vitamins work as an assistant in enzymatic systems.
They are required in very small amounts.
Thiamine (B1), is more commonly used in plant tissue cultures and other vitamins such as nicotinic acid, pyridoxine (B6) etc.

Plant Hormones / Growth Regulators

-involved in the regulation of growth and organized organ develo­pment of plant tissues directly or indirectly
-inter­actions of auxin and cytokinin are considered to be the most important regulation to induce organ develo­pment in the cultured tissues
-requi­rement of hormones (gibbe­rellin, abscisic acid, ethylene) which will help to induce the develo­pmental response in cultures depends on the type of explant and species to be cultured
-addition of growth regulators in the culture media also depends on the goal of the culturing process
-i.e. gibber­ellin, ethylene, and abscisic acid are not required for organ develo­pment or cell prolif­eration in culture

Five Classes of Plant Hormon­es/­Growth Regulators

1. Auxin
2. Cytokinin
3. Gibber­ellin
4. Ethylene
5. Abscisic acid

1. Auxins

-Naturally occurring: indole­-3-­acetic acid (IAA)
-Synth­etic: 2,4-di­chl­oro­phe­nox­yacetic acid (2,4-D), 1-naph­tha­len­eacetic acid (NAA), idole-­3-b­utyric acid (IBA)
-Funct­ions:
a) Cell division and differ­ent­iation (with cytokinin)
 
b) Shoot and root apical dominance
 
c) Parthe­nocarpy in some species
 
d) Abscission of fruits in other species
 
e) At high concen­tration will kill plant as herbicide
*Parth­eno­carpy is the natural or artifi­cially induced production of fruit without fertil­ization of ovules, which makes the fruit seedless
*Absci­ssion is the shedding of various parts of an organism, such as a plant dropping a leaf, fruit, flower

2. Cytokinin

-Naturally occurring: zeatin
-Synth­etic: kinetin, benzyl­ami­nop­urine (BAP) and adenine
-Synthesis from adenine in root tips, embryos, young fruits, leaves in all plants
-Funct­ions:
a) Growth and develo­pment (in combin­ation with auxin)
 
b) Delay senescence (Plant senescence is the process of aging in plants)
 
c) Break apical dominance

Collab­oration of Auxin and Cytokinin

3. Gibber­ellins

-Naturally occurring: gibber­ellic acid-3 (GA3), GA4, GA7 (more than 90 different GA recogn­ized)
-Synth­esized from mevalonate shoot and root apices, embryos, cotyle­dons, fruits, tubers
-Function:
a) stem elongation and flowering
 
b) effects on seed germin­ation (breaking seed dormancy)
 
c) promotes cell division in combin­ation with IAA
 
d) Improves fruit set, fruit growth, fruit maturation and fruit ripening
 
In tissue culture, GA's (gibbe­rellic acids) are supple­mented in some operations and avoided in others.
-For example, the presence of GA's in media can inhibit organ develo­pment (root and shoot formation) and somatic embryo­gen­esis.
-However, gibber­ellic acids are necessary to induce normal callus growth. Similarly, gibber­ellic acids can inhibit the merist­emoid initia­tion; the catch is the merist­emoid initiation is required for the growth and develo­pment of organs that are already formed.

4. Ethylene

-naturally occurring gaseous hormone that plays a role in fruit ripening, senesc­ence, and leaf abscission
-Function:
a) wound responses
 
b) causes thickening of stems and leaf abscission (aging)
 
c) reduces advent­itious shoot formation
 
d) control fruit ripening in climac­teric fruit
 
e) inhibits the growth and develo­pment of the plants in the culture at a higher concen­tra­tion; but enhances the responses of plants towards auxin at lower concen­tra­tions
*Silver nitrate (AgNO3) has anti-e­thylene activity

5. Abscisic acid

-maintains bud and seed dormancy, inhibits cell wall acidif­ication and slows cell elongation
-promotes somatic embryo­genesis at a lower concen­tra­tion; but halts the develo­pmental response of cultures at a higher concen­tration
-Used in agricu­lture where seed dormancy is important
-Function:
a) water stress response
 
b) seed protein synthesis
 
c) seed dormancy
 
d) seed germin­ation (in combin­ation with gibber­ellins)
 
e) enhance somatic embryo­genesis

Support Matrices

1. Agar
-commonly used gelling agent in plant tissue culture
-mixture of polysa­cch­arides derived from red algae
-Agarose is often used when the impurities in agar are not desired, such as in protoplast and anther culture
-contain:
a) 70% agarose (gelling component) (polymer of altern­ating D-gala­ctose and 3,6anh­ydr­oga­lac­tose)
 
b) 30% agarop­ectin (non-g­elling fraction, polymer of sulphated D-gala­ctose units)
 
2. Gellan Gums
-gelling agent used for plant tissue and cell culture
-produces a high transp­arent gel, which allows better observ­ation (inspe­ction of contam­ina­tion) of root growth compared with conven­tional agar gel
-polymers from glucose, glucoronic acid and rhamnose units
-not only easier for root inspec­tion, but allow better root growth than agar

Antibi­otics

-plants are sensitive to many antibi­otics
-use of antibi­otics to prevent contam­ination is uncommon in plant tissue culture
 
1. Cefotaxime
kill Agroba­cterium but not the plant cell
2. Kanamycin
kill plant cell
 
-Trans­genic plant carrying the kanamycin selectable marker would survive on medium containing kanamycin
 
-Non transgenic plant would not survive Plant Tissue Culture (PTC)
 

Step-B­y-Step Guide to Prepare Medium

Pre-Pr­epa­ration
1. Prepare 1 L of MS agar medium with 1 mg/L 2,4-D
a) Prepare all the stock solutions
 
b) Measure 800 mL of deionised water into a 2 L beaker
 
c) Put in a magnetic stirrer and start stirring
 
d) Add 50 mL of MS macro, 5 mL of MS micro, MS ferum and MS vitamin
 
e) Weigh 30 g of sucrose
 
f) Add into the mixture, stir until dissolve
 
g) Add 200 μL of 2,4-D
 
h) Adjust pH to 5.5
 
i) Top up medium to 1 L
2. Weigh 5 g of agar
1a) Autoclave, then pour medium
 
1b) Pour medium into a 2 L bottle.
 
1c) Add agar into medium in the bottle. Swirl to mix
 
1d) Autoclave medium. Swirl after autocl­aving
 
1e) Allow medium to cool
 
1f) Pour medium into containers in a laminar air flow cabinet
 
2) Dispense, then autoclave
 
2a) Add agar into medium in the 2 L beaker
 
2b) Dissolve agar in a microwave oven
 
2c) Dispense medium into containers
 
2d) Autoclave all the containers with medium
 
2e) Allow to cool
3. Allow medium to cool and incubate for at least 3 days to 5 days before use.
-if contam­inants present, it would appear during this period