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Plant tissue culture

Plant tissue culture or micro propagation is a broad term used for the Invitro cultivation of plant, seeds and parts like organs, embryos, tissues, single cells, protoplasts. This process of cultivation is done in laboratories using nutrition media under aseptic conditions.  Highly mature plant cells and cells that can be differentiated have the ability of Totipotency. Totipotency is the ability of plant cells, where a cell can change to a meristematic state and can be differentiated into a whole plant. This property of plant cells has an advantage over Animal cell tissue culture.  In 1902, Gottlieb Haberlandt cultivated isolated plant leaf cells on a nutrition media, hence he is regarded as the Father of plant tissue culture.

General steps involved in Tissue culture

Plant Cell culture 

In-Plant cell culture, the cells are isolated from the parent tissue by mechanically scraping of the cells or by enzymatically disaggregation of cells by using enzymes as pectinase or use of macerozyme which can degrade lamella and weakens the cell wall. These plant cells can be cultured by either batch culture of suspension culture. Cell culture has a major advantage in the production of secondary metabolites.

Requirements of a plant cell or tissue culture laboratories

The following are the requirements for plant tissue culture

1. Area for preparation of culture medium: well equipped with required glassware, and laminar airflow, deep freezer, and autoclave
2. A sterile room for tissue culture practice that is, distribution of medium, transfer of explants, etc.
3. Various apparatus, instruments, and glassware.
4. Culture media

Culture media

Culture or nutrition media plays a very important role in developing in-vitro plant tissue culture. Thus, in-vitro plant growth and morphogenesis depend on the culture media used. As plant growing In-vitro cannot synthesize their own food material for their growth hence composition of nutrition or culture media should contain the same nutrients required for the whole plant.

Composition of media

The composition of culture media depends on

1. The plant species used
2. Type of plant material: e.g. organs, tissues, cells, protoplast, etc.
3. The media used is either solid (solid-state) or liquid (liquid state), based on the requirements of the specific culture system.

Types of culture media based on different components

Whites medium: used for root culture

MS medium: used for organogenesis

B5 medium: used for callus culture, cell suspension and Protoplast culture

N6 medium: used for cereal anther culture

Nitsch’s medium: used for anther culture

Constituents of media

Several elements are supplied in media for plant nutrition and to for their physiological function. The culture media contains the following basic elements

1. Inorganic nutrients: macro elements like calcium (cell membrane and cell wall synthesis), nitrogen (Component of proteins), Potassium (inorganic Cation), sulfur (a component of amino acids), and magnesium (a component of chlorophyll). Micronutrients like Zinc (for photosynthesis), Iron (a component of cytochrome), Boron, Manganese (cofactor for enzymes), Molybdenum (a component of certain enzymes), and copper (involves in an electron transport mechanism).
2. Carbon and energy source: sucrose is widely used in culture media because it can convert into glucose and fructose.
3. Organic nutrients: Vitamins like thiamine, riboflavin, niacin, pyridoxine, folic acid, biotin, ascorbic acid, PABA, Vit-E are used for good growth of cells. Amino Acids include, L-glutamine, L-asparagine, L-arginine, L-cysteine are used as cell growth stimulators.

Activated charcoal is used to remove toxic compounds generated by cultured plants. Antibiotics are used to prevent microbial growth in culture medium e.g. streptomycin or kanamycin

1. Growth regulators: phytohormones such as auxins, cytokinins, gibberellins, and abscisic acid are used to promote growth, differentiation, and development of plants.
2. Solidifying agents: Agar, gelatin, and other gelatin agents like polyacrylamide pellets, agarose, are used to solidify culture media
3. The pH of medium: pH of 5.0 to 6.0 (less than 5 or more than 6 plants stops growing) is optimum for most tissue cultures.

Applications of Plant tissue culture

1. Production of monoclonal antibodies, human interleukin-6, Edible vaccine, secondary metabolites,
2. Production stress-tolerant crops in agriculture
3. Production of biofuel and biodiesel
4. Producing nutrition-rich plants

MCQ

1. who is the father of plant tissue culture___________

a) Alexander Fleming

b) Gottlieb Haberlandt

c) Kary Mullis

d) Leeuwenhoek

2. ___________ is the advantage of plant tissue culture over animal tissue culture

a) Plant culture require less time

b) Plant tissues are easily available

c) Totipotency of the plant cell

d) Plant tissue culture can be easily maintained with minimum requirements

3. Laminar airflow is used for the following reasons except:

a) Preparing media

b) Transferring explants

c) Aseptic transfer

d) For culture growth

4. Selection of culture media depends on________

a) Type of plant species used

b) Time for preparation of culture media

c) Cost for preparation

d) Maintenance of culture media

5. ___________is the type of Cell culture

a) Organ culture

b) Protoplast culture

c) Callus culture

d) Explant culture

6. Cell culture is used for ___________

a) Production of enzymes

b) Production of Secondary metabolites

c) Production of vaccines

d) Production of monoclonal antibodies

7. The following are the plant material used for tissue culture EXCEPT:

a) Tissues

b) Cells

c) Protoplasts

d) Flower

8. Zinc as micronutrient is used in culture media for _____________

a) Protein synthesis

b) DNA replication

c) Enzyme synthesis

d) For photosynthesis

9. Calcium used In nutrition media is the main component of ________part of plant cell

a) Mitochondria

b) Endoplasmic reticulum

c) Golgi bodies

d) Cell wall and cell membrane

10. Activated charcoal is used in nutrition media to _____________

a) Absorb toxic substances

b) Absorb moisture

c) Absorb elements

d) Absorb microbes

Answer Key 

1. b
2. c
3. d
4. a
5. b
6. b
7. d
8. d
9. d
10. a

Reference

1. Satyanarayana, biotechnology, 1^st e.d. Kolkata, books & allied (p) ltd publishers, 2005  page: 497- 522
2. p. vyas, v.k. dixit, pharmaceutical biotechnology, New Delhi, cbs publishers and distributors, 2008, page: 227-234
3. j. Nair, introduction to biotechnology and genetic engineering, 1^st e.d. New Delhi, infinity science press llc, 2008, page 682-683