plant tissue culture terminology, harmones and media preparation

1. Plant Tissue Culture Terminology :- 

Adventitious---Developing from unusual points of origin, such as shoot or root tissues, from callus or embryos, from sources other than zygotes. 

Agar---a polysaccharide powder derived from algae used to gel a medium. Agar is generally used at a concentration of 6-12 g/liter. 

Aseptic---Free of microorganisms. 

Aseptic Technique---Procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cultures. 

Autoclave---A machine capable of sterilizing wet or dry items with steam under pressure. Pressure cookers are a type of autoclaves.

Callus---An unorganized, proliferate mass of differentiated plant cells, a wound response. 

Chemically Defined Medium---A nutritive solution for culturing cells in which each component is specifiable and ideally of known chemical structure.

 Clone---Plants produced asexually from a single source plant. 

Clonal Propagation---Asexual reproduction of plants that are considered to be genetically uniform and originated from a single individual or explant. 

Contamination---Being infested with unwanted microorganisms such as bacteria or fungi. Culture—A plant growing in vitro.  

Differentiated---Cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo. Modifications of new cells to form tissues or organs with a specific function. 

Explant---Tissue taken from its original site and transferred to an artificial medium for growth or maintenance. 

Horizontal laminar flow unit---An enclosed work area that has sterile air moving across it. The air moves with uniform velocity along parallel flow lines. Room air is pulled into the unit and forced through a HEPA (High Energy Particulate Air) filter, which removes particles 0.3 µm and larger.

 Internode---The space between two nodes on a stem In vitro---To be grown in glass (Latin). Propagation of plants in a controlled, artificial environment using plastic or glass culture vessels, aseptic techniques, and a defined growing medium.

 In vivo---To be grown naturally (Latin) Media---Plural of medium Medium---A nutritive solution, solid or liquid, for culturing cells. 

Micropropagation---In vitro Clonal propagation of plants from shoot tips or nodal explants, usually with an accelerated proliferation of shoots during subcultures. 

Node—A part of the plant stem from which a leaf, shoot or flower originates. 

Passage---The transfer or transplantation of cells or tissues with or without dilution or division, form one culture vessel to another.

 Passage Number---The number of times the cells or tissues in culture have been subcultured or passaged.

 Pathogen---A disease-causing organism. 

Pathogenic---Capable of causing a disease. 

Petiole---A leaf stalk; the portion of the plant that attaches the leaf blade to the node of the stem. 

Plant Tissue Culture---The growth or maintenance of plant cells, tissues, organs or whole plants in vitro.

 Regeneration---In plant cultures, a morphogenetic response to a stimulus that results in the products of organs, embryos, or whole plants.

 Shoot Apical Meristem---Undifferentiated tissue, located within the shoot tip, generally appearing as a shiny dome-like structure, distal to the youngest leaf primordium and measuring less that 0.1 mm in length when excised. 

Somaclonal Variation---Phenotypic variation, either genetic or epigenetic in origin, displayed among somaclones. 

Somaclones---Plants derived from any form of cell culture involving the use of somatic plant cells.

*stages in micropropagation :- 

 Stage I---A step in in vitro propagation characterized by the establishment of an aseptic tissue culture of a plant.

 Stage II---A step in in vitro propagation characterized by the rapid numerical increase of organs or other structures.

 Stage III---A step in in vitro propagation characterized by preparation of propagules for successful transfer to soil, a process involving rooting of shoot cuttings, hardening of plants, and initiating the change from the heterotrophic to the autotropic state. 

Stage IV---A step in in vitro plant propagation characterized by the establishment in soil of a tissue culture derived plant, either after undergoing a Stage III pretransplant treatment, or in certain species, after the direct transfer of plants from Stage II into soil. 

Sterile--- (A) Without life. (B) Inability of an organism to produce functional gametes. (C) A culture that is free of viable microorganisms.

 Sterile Techniques---The practice of working with cultures in an environment free from microorganisms. 

Subculture---See “Passage”. With plant cultures, this is the process by which the tissue or explant is first subdivide, then transferred into fresh culture medium. 

Tissue Culture---The maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function. 

Totipotency---A cell characteristic in which the potential for forming all the cell types in the adult organism are retained. 

Undifferentiated---With plant cells, existing in a state of cell development characterized by isodiametric cell shape, very little or no vacuole, a large nucleus, and exemplified by cells comprising an apical meristem or embryo. 

2. Hormones---

Growth regulators, generally synthetic in occurrence, that strongly affects growth (i.e. cytokinins, auxins, and gibberellins).

Auxin--- A group of plant growth regulators that promotes callus growth, cell division, cell enlargement, adventitious buds, and lateral rooting. Endogenous auxins are auxins that occur naturally. Indole-3-acetic (IAA) is a naturally occurring auxin. Exogenous auxins are auxins that are man-made or synthetic. 

 Examples :- of exogenous auxins included 2,4- Dichloro phenoxy acetic acid (2,4-D), Indole-3-Butyric acid (IBA), α-Naphthalene acetic acid (NAA), and 4-Chlorophenoxyacetic acid (CPA). 

Gibberellins--- A plant growth regulator that influences cell enlargement. Endogenous growth forms of gibberellin include Gibberellic Acid (GA3). 

Cytokinin--- A group of plant growth regulators that regulate growth and morphogenesis and stimulate cell division. Endogenous cytokinins, cytokinins that occur naturally, 

Eg:- include zeatin and 6-γ,γ-dimethylallylaminopurine (2iP). Exogenous cytokinins, cytokinins that are man-made or synthetic, include 6-furfurylaminopurine (kinetin) and 6-benzylaminopurine (BA or BAP)

3. media composition and types of media :- 

• 1. Murashige and Skoog medium (MS) ...
• 2. Linsmaier and Skoog medium (LS) ...
• 3. Gamborg medium (B5) ...
• 4. Nitsch and Nitsch medium (NN) ...
• 5. White's Medium. ...
• 6. Quoirin & Lepoivre medium (QL)

PREPARATION :- 

MAJOR COMPONENTS OF MS MEDIA

The media involves the following four major components:

1. Inorganic nutrient: It includes mineral salts that are important for the growth and development of the plants. It is categorized into two groups: Macronutrients (Calcium, magnesium, nitrogen) and micronutrients (copper, iron, and zinc).
2. Organic nutrient: It mainly includes vitamins and amino acids, required for the growth and differentiation of the cultures.
3. Growth hormones: It includes auxins, cytokinins, and gibberellins. It is essential for the growth and development of tissues and organs.
4. Gelling agents: It includes agar and gelatin. It provides support to the cultures for their establishment.

1. MS media preparation and stocks 

1. Micronutrient Stock (100X)

• Take 400 ml double-distilled water in a 1L beaker, then weigh the salts given in the table below and dissolve it in the water.
• Transfer the solution to the 1L volumetric flasks, and make up the volume to 1L. Pipette out 10 ml of the solution to make 1L MS media.

Salts	Concentration - 100X (mg/l)
MnSO4.4H2O	2230
ZnSO4.4H2O	860
H3BO3	620
KI	83
Na2MoO4.2H2O	25
CuSO4.5H2O	2.5
COCl2.6H2O	2.5

2. Iron Stock (20x)

• Take 80 ml double-distilled water in a 100 ml beaker, weigh the components given in the table and dissolve it completely (in the same order as given in the table).
• Transfer the solution to a volumetric flask of 100 ml and makeup to the final volume.
• Pipette 5 ml of the stock solution for 1L of MS media.

Components	Concentration-20X (mg/100 ml)
Na2EDTA	672
FeSO4.7H2O	556

3. Vitamin Stock (100x)

• Take a 100 ml beaker and add 50 ml double-distilled water in it. Weigh the vitamins given in the table below and dissolve it completely in the water.
• Transfer the solution to a volumetric flask of 100 ml and makeup to the final volume.
• Pipette out 1 ml of the vitamin stock solution for 100ml of MS media.
• Add vitamins after the media is autoclaved to protect it from heat degradation. Vitamins can be sterilized by ultrafiltration technique.

Components	Concentration-100X (mg/100ml)
Glycine	20
Nicotinic acid	5
pyridoxine.HCl	5
Thiamine.HCl	1

NOTE: Discard the vitamin solution after 30 days.

3. Cytokinin Stock (100X)

• Weigh “10mg kinetin” and dissolve it into a few drops of 1N HCl.
• Add a few ml of double-distilled water to the above solution and transfer it to the 100 ml volumetric flask and makeup to the volume.
• Store the stock in the refrigerator.
• Use 1 ml of the stock for 1L of the MS media.

Steps of the Preparation

Take 400 ml double-distilled water in a 1L beaker. Weigh the macronutrients given in the table below, and dissolve them completely into the water.

Macronutrients	Concentration-100X (mg/L)
(NH4)NO3	1650
KNO3	1900
CaCl2.2H2O	440
MgSO4.7H2O	370
KH2PO4	170

1. From the prepared stock solutions, pipette out 5ml iron, 10 ml micronutrient, and 1ml kinetin to the 1L beaker of the media.
2. Weigh 100 mg Myo-inositol and dissolve it in the previous mixture.
3. Weigh 10 mg IAA and dissolve it into a few drops of 1N NaOH. Then, transfer the solution to the previous mixture.

NOTE: The stock of IAA is not prepared because of its oxidative degradation.

1. Add 800 ml of double-distilled water in the beaker and adjust the pH of the media to 5.7.
2. Transfer the prepared solution to a 1L volumetric flask and make up the final volume to 1L.
3. Keep the solution in the refrigerator.

Final steps

1. Sterilize all the equipment andglass culture jars used for the tissue culture process.
2. Weigh 0.8g of supreme grade agar and 3.0g reagent-grade sucrose and transfer them to 250 ml Erlenmeyer flask.
3. Add 100 ml of the stored MS media, in the flask and seal the cap with aluminum foil.
4. Sterilize the flask with the media.
5. After sterilizing the media for 15-20 minutes, add 1 ml vitamin solution.
6. Swirl the flask for the dissolution of the vitamin, agar, and sucrose into the media, before pouring it into the culture bottles.
7. Pour the media into culture jars and store them in the refrigerator for 1 hour, before the culturing process.

Now, your culture bottles are ready for the tissue-culture processes.

Here is the handy chart of the MS media recipe for your experiments:

Components	Concentration for all-100X (mg/L) except For iron (20X): (mg/100ml) For vitamin: mg/100ml
1. Macronutrients	(NH4)NO3	1650
KNO3	1900
CaCl2.2H2O	440
MgSO4.7H2O	370
KH2PO4	170
2. Micronutrients	MnSO4.4H2O	2230
ZnSO4.4H2O	860
H3BO3	620
KI	83
Na2MoO4.2H2O	25
CuSO4.5H2O	2.5
COCl2.6H2O	2.5
3. Iron (20X) (mg/100ml)	Na2EDTA	672
FeSO4.7H2O	556
4. Vitamins (100X)(mg/100ml)	Glycine	20
Nicotinic acid	5
pyridoxine.HCl	5
Thiamine.HCl	1
5. Cytokinin (mg/100ml)	Kinetin	100
Myo-inositol	10
IAA	10
Sucrose	10
Agar	30,000