Plant Biotech (sterilisation + micropropagation)

Question 1

What are the sterilisation methods for equipment and cultures?

Answer 1

Dry heat, wet heat, membrane filtration, chemical sterilisation, radiation sterilisation

Question 2

What is sterilised by dry heating and why?

Answer 2

Carried out in sterilisation oven
Used for glassware, cutting instruments, metal apparatus (stuff that can sustain high heat)
Cannot be used for plastics (will melt), cell culture media (proteins will denature), alcohol / detergents (will evaporate

Question 3

What is sterilised by wet heating and why?

Answer 3

Used for liquids (like culture medium w/o proteins), some labware (hard plastics)

Cannot be used for oils that can’t be mixed with water, metals (rusting), dry powder (will get wet)

Question 4

What is sterilised by membrane filtration and why?

Answer 4

Filters so smol so that microorganisms cannot pass through

Used for liquids with heat-sensitive chemicals like proteins, amino acids, antibiotics (sterilised using this mtd before adding to culture medium)

Question 5

What is sterilised with chemical sterilisation and why?

Answer 5

to disinfect surface of explants (kill contaminants but minimise plant tissue death so theres the need to consider conc and duration of sterilisation)

for large surface areas + large equipment (using lethal gas to kill contaminants like ethylene oxide)

Question 6

What is sterilised with radiation sterilisation and why?

Answer 6

uses UV light to sterilise things like laminar flow hood
causes DNA damage and interferes with cellular metabolism

Question 7

What are the stages of micropropagation?

Answer 7

Stage 0: prep of donor plants
Stage 1: establishment of primary cultures
Stage 2: proliferation of shoots
Stage 3: rooting of shoots
Stage 4: acclimatisation and plating out

Question 8

What is the aim and procedure of stage 0 (prep of donor plants)

Answer 8

Aim: obtain explants that will respond readily in culture
Procedure:
Select high quality explants that wld reduce contamination
- picking leaves that are healthy + disease free (preferably aerial explants)

Would want to choose plants grown in glasshouses that aint watered overhead

Question 9

What is the aim and procedure of Stage 1 (establishment of primary cultures)

Answer 9

Aim: get clean cultures to initiate growth in vitro

Procedure:
Need to do surface sterilisation (refer back to Q5)
Disinfectants that are commonly used for sterilisation r sodium hypochlorite (bleach), 70% ethanol.
Rinse explant with sterile water to remove disinfectant

Question 10

What is the aim and procedure of Stage 2 + 3
(proliferation of shoots den roots)

Answer 10

Stage 2:
Aim: increase no. of plantlets by subculturing
Procedure:
Using PGRs to induce growth of shoots / callus (cytokinin!!)
Shoots / callus are cut into smaller pieces and subculture with same media

Stage 3:
Aim: to produce complete plantlets (shoots n roots)
Procedure:
Increasing auxin ratio to induce rooting to form full plantlet (done in vitro with media)

Question 11

What is the aim and procedure of Stage 4 (acclimatisation) (meaning is being accustomed to a new climate)

Answer 11

Aim: obtain highest possible survival chances of plantlets when transferring plants to the natural growing environment
Procedure:
Have to prevent plantlets from dying cuz they haven’t fully develop + vulnerability to infection, drying, heat, temp shock.
Can prevent dying by acclimatising plantlets by:
- having plantlets placed in sterilised soil + covered with a plastic bag (encouraging growth of proper root systems)
- Keeping plantlets under shade (acclimatise plants to light conditions)
- Keeping humidity at 90-100% den back to 70% (acclimatise plants to humidity)