Plant Tissue Culture

Question 1

Define Plant Tissue Culture

Answer 1

PTC is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium.
It can propagate complete plants from cells, tissues, organs, meristems, pores, pollen.

Question 2

What is micropropagation?

Answer 2

PTC is widely use to produce clones of a plant in a method known as micropropagation.

Question 3

What are explants?

Answer 3

The plant parts that are cut & removed from parent plants for culturing.

Question 4

Where are explants usually taken from?

Answer 4

Explants are usually taken from young stems, young roots, young leaves and buds which contain meristems.

Question 5

Define meristems

Answer 5

Meristems are areas of actively dividing tissues that consist of undifferentiated cells which can give rise to various organs of the plant and keep the plant growing.

Question 6

What are apical meristems?

Answer 6

Apical meristems are found on the tip of stems and roots.

Question 7

What are shoot apical meristems? (SAM)

Answer 7

Shoot apical meristem gives rise to organs like the leaves and flowers.

Question 8

What are root apical meristems? (RAM)

Answer 8

The root apical meristems provides for future root growth.

Question 9

What are the parts of plants that can be used as explants?

Answer 9

Shoot tip, young leaf, bud. young root, root tip.

Question 10

What is the ability of the plant cells that allow for PTC?

Answer 10

Plant cells have the ability to initiate cell division from almost any tissue and can regenerate lost organs, or undergo different developmental pathways in response to particular stimuli.

Question 11

What concept is PTC based on?

Answer 11

Totipotency.

Question 12

Define totipotency.

Answer 12

It is the ability of a cell or tissue to regenerate a whole plant by cell division.

Question 13

Name the 4 stages of PTC

Answer 13

stage 1: Initiation
stage 2: Multiplication
stage 3: Differentiation
stage 4: Acclimatization

Question 14

Name initiation part 1:

Answer 14

Preparation for sterile environment

Question 15

What part does the autoclave machine and the laminar flow hood play in the preparation of a sterile environment?

Answer 15

Autoclave machine to sterilise culture media and tools

Laminar flow hood to provide a sterile working environment for performing tissue culture work.

Question 16

Name initiation part 2:

Answer 16

Preparation​ of sterile explants

Question 17

Elaborate on the preparation of sterile explants.

Answer 17

Cleaning: Obtain suitable explants from actively growing and dividing regions of the parent plant.
Clean the surfaces of the explants with a soft brush in diluted detergent solution followed by rinsing with tap water
Surface sterilizing: The explants are rinsed with ethanol followed by bleach solution, and finally rinsed off chemicals with autoclaved water 3 times.

Question 18

Name initiation part 3:

Answer 18

Preparation of nutrient medium

Question 19

What should the nutrient medium contain?

Answer 19

The nutrient medium should contain the following:
1. Carbon sucrose (energy)
2. Vitamins for healthy growth
3. Minerals
macro- required in larger amounts 
micro- required in trace amounts.
4. Plant growth Regulators 
5. pH is usually adjusted around 5 to 5.7

Question 20

Name initiation part 4:

Answer 20

Culturing of sterile explants

Question 21

What happens during the culture of sterile explants.

Answer 21

The sterile explants are cut into small pieces and cultured onto nutrient medium.

Question 22

What are the conditions the sterile explants are placed under?

Answer 22

The cultures are placed under cool fluorescent tubes for 12 hours photoperiod a day and at room temperature of 25C

Question 23

Define callus.

Answer 23

A mass of undifferentiated cells that are white and have thin cell walls.

Question 24

What happens during multiplication?

Answer 24

The cultured cells or tissues undergo cell division to differentiate into many whole plants
or
generate a mass of cell known as a callus.

Question 25

Define somaclonal variation.

Answer 25

Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture.

Question 26

Where and how does somaclonal variation occur?

Answer 26

It may occur in plants regenerated from callus.

This may be caused by the mutation of genes during cell division (mitosis) in the formation of the callus.

Question 27

What are some benefits of somaclonal variation?

Answer 27

The mutation may give rise to desirable traits

eg. resistance to herbicide, cold tolerance.

Question 28

What is done to keep the desirable trait that somaclonal variation has given rise to?

Answer 28

It will be propagated using PTC to create a new generation of this new variant.

Question 29

Why can’t tissue culture be kept in the same medium?

Answer 29

This is due to the gradual rise of toxic metabolites, depletion of nutrients and overcrowding.

Question 30

What is then done to plants to avoid the gradual rise of toxic metabolites, depletion of nutrients and overcrowding?

Answer 30

Subculturing

Question 31

Define subculturing.

Answer 31

Subculturing is the practice of subdividing tissues or plantlets before transferring into fresh culture medium.

Question 32

What is happens during subculturing

Answer 32

Nutrients are replenished and toxic wastes are removed. This allows continued growth as well as proliferation of the culture.

Question 33

Why are plant growth regulators added?

Answer 33

They are added into the nutrient medium to regulate morphogenesis in plants, such as to initiate the development of roots or shoots.

Question 34

What are auxins for?

Answer 34

NAA, IAA: Initiate formation of adventitious roots.

Question 35

Cytokinins?

Answer 35

BAP, Kinetin: Stimulate growth of adventitious shoots.

Question 36

Gibberellins

Answer 36

Stimulate shoot elongation.

Question 37

What can subculturing also do? (calli)

Answer 37

Subculturing can also be used to generate a greater quantity of calli before the differentiation stage.

Question 38

Why do plants have to acclimatized?

Answer 38

They have overgrown the jar and need to be transplanted out.
The external environment have substantially lower relative humidity, higher light level, and septic environment that are stressful to micro-propagated plants as compared to in virtro conditions.

Question 39

Why do plants need time to acclimatize to live in the environment outside the flask

Answer 39

This is because they are used to living in the optimum conditions inside the sterile container with optimum temperature, light intensity, rich nutrients, high humidity and a sterile environment free from pathogens.

Question 40

What is done during acclimatisation?

Answer 40

Plants are exposed to a gradual increase in light intensity and lower RH allows plants to harden/acclimatize and increases their chances of survival.

Question 41

What are the applications of PTC?

Answer 41

• Mass production of plants that have desirable traits
• Micropropagation of rare and endangered plant species for conservation
• Production of pathogen-free plants in sterile containers for export to other countries.
• Production of plants from seeds that have very low chances of germinating and growing.
• To cross distantly related species by somatic hybridization / protoplast fusion to create hybrids which are sexually incompatible in nature.

Question 42

What are some of the disadvantages of PTC?

Answer 42

Not all plants can be propagated by PTC
Some plants are prone to somaclonal variation, which makes obtaining genetic clones very difficult
Compared to asexual reproductive methods, PTC is more expensive, requiring special skills and special facilities/equipment.