Download Plant Tissue Culture

Plant Tissue Culture
Experiment 1: Effect of pH on Nutrition and
Growth of Tissue Cultured Ginger
Introduction
Plant tissue culture is the cultivation of plant cells or tissues on
specially-formulated nutrient media. In appropriate conditions,
an entire plant can be regenerated from each single cell, permitting the rapid production
of many identical plants.
Tissue culture is an essential tool in modern plant breeding. Since it was first developed
in the early 1960s, plant tissue culture has become the basis of a major industry,
providing high-value plants for nurseries. Where a crop (e.g., banana) does not produce
seeds, plants derived from tissue culture are sometimes planted directly in farmers' fields.
However this is rarely done for seed-bearing species - more often tissue-cultured plants
are used to produce the seeds from which crops. Cell culture is also used for the
conservation of those plant varieties that cannot be maintained in a normal seed bank.
The chemical compounds that make up plants are mainly carbohydrates, lipids, proteins
and nucleic acids. These are all organic compounds and contain the elements carbon,
hydrogen and oxygen. Proteins and nucleic acids also contain nitrogen and phosphorus.
Proteins can also contain other elements (eg chlorophyll contains magnesium).
These elements are usually incorporated into tissue culture media as nutrient salts
according to the recipe of Murashige and Skoog. Their availability is affected by pH,
which is known to change over time in culture.
Students will work in groups of 2-3 and will aseptically transfer ginger tissue culture plants
to media containing MS nutrients at 3 pH levels (3, 6 and 9)
Aim
To investigate the effect pH has on nutrient availability on tissue cultured plants as
measured by changes in the plant.
Materials
18 cultured ginger plants
Perspex box
Scalpel
Alcohol burner
Ethanol
Bunsen burner
Swabs (cotton balls)
Ceramic tile
Auto-claved paper
Cling wrap
Tweezers
Specific media (pH 3, 6 and 9)
Lab coat
Face mask
Hair net
Gloves
Permanent markers
18 Plastic tubes with sealable lids
Method
Contamination considerations
The environment used when carrying out the experiment needs to be
completely sterile. Making sure the area, equipment and the students are
totally aseptic is essential to the success of the experiment.
These aseptic techniques ensure there is no contamination and prevents bacterial or germ
growth on the experiment. Using products like Ethanol and heat from the Bunsen burner
assists in sterilizing all utensils preventing contamination.
Step 1. Swab interior of Perspex box liberally with ethanol and cotton buds
Step 2. Sterilize hands with lathering ethanol over them.
Step 3. Sterilize each utensil to be used in the Perspex box by holding it over an open flame.
All utensils that come into contact with the plant are heated by the flame.
1. Working within the Perspex box (laminar flow cabinet) use sterile tweezers to
remove the sealed auto-claved paper and place it on the tile inside the Perspex
box.
2. Collect the Ginger plants of similar size and colour. Inside the Perspex box
remove the ginger plantlets with tweezers and place them one at a time on the
auto-claved paper.
3. Using a sterile scalpel and tweezers measure the plantlet to the desired length and
remove the leaves and roots cutting the plantlet to the desired length. Ensure that
the rhizome remains as part of the plantlet.
4. Using the tweezers place the cut plantlet into a corresponding tube with the correct
media. Ensure the plant is placed securely in the agar and seal the tube. Label
the tube correctly.
6 x pH 3 media
6 x pH 6 media
6 x pH 9 media
5. Repeat the steps 1-5 for each of the ginger plantlets.
6. Place the plants under lights in a controlled environment.
7. Each week plants are to be measured and data recorded including contamination,
height colour etc
8. After 7-8 weeks of growth a deconstructive harvest will take place. This involves
collecting the final set of data and measurements by removing each plant from the
agar tube. These results collected should include weighing the plants (whole plant,
leaves, rhizome etc), final height measurements and photos may be taken.
9. After each plant is removed and measured discard the plant and remove the agar
from each tube.
Plant Tissue Culture
Experiment 2: Effect of Light and Sucrose on
Growth of Tissue Cultured
Ginger
Introduction
Plant tissue culture is the cultivation of plant cells or tissues on
specially-formulated nutrient media. In appropriate conditions, an entire plant can be
regenerated from each single cell, permitting the rapid production of many identical
plants.
Tissue culture is an essential tool in modern plant breeding. Since it was first developed
in the early 1960s, plant tissue culture has become the basis of a major industry,
providing high-value plants for nurseries. Where a crop (e.g., banana) does not produce
seeds, plants derived from tissue culture are sometimes planted directly in farmers' fields.
However this is rarely done for seed-bearing species - more often tissue-cultured plants
are used to produce the seeds from which crops. Cell culture is also used for the
conservation of those plant varieties that cannot be maintained in a normal seed bank.
Plants are autotrophic; via photosynthesis they use carbon dioxide and water to make
carbohydrates and lipids. Plants in tissue culture are unable to photosynthesize
effectively so tissue culture media almost always has sucrose added as a readily
available source to stimulate growth and development. Tissue cultured plants can
therefore be grown as heterotrophic organisms.
Students will work in groups of 2-3 and will aseptically transfer ginger tissue culture plants
to media containing MS nutrients and some media with added sucrose.
Aim
To investigate the effect of light and sucrose on the growth and development of tissue
cultured plants as measured by changes in the plant.
Materials
24 cultured ginger plants
Perspex box
Tweezers
Scalpel
Alcohol burner
Ethanol
Bunsen burner
Swabs (cotton balls)
Ceramic tile
Auto-claved paper
Cling wrap
Foil
24 Plastic tubes with sealable lids
Specific media (MS media and MS media + sucrose)
Lab coat
Face mask
Hair net
Gloves
Permanent markers
Method
Contamination considerations
The environment used when carrying out the experiment needs to be
completely sterile. Making sure the area, equipment and the students are
totally aseptic is essential to the success of the experiment.
These aseptic techniques ensure there is no contamination and prevents bacterial or germ
growth on the experiment. Using products like Ethanol and heat from the Bunsen burner
assists in sterilizing all utensils preventing contamination.
Step 1. Swab interior of Perspex box liberally with ethanol and cotton buds
Step 2. Sterilize hands with lathering ethanol over them.
Step 3. Sterilize each utensil to be used in the Perspex box by holding it over an open flame.
All utensils that come into contact with the plant are heated by the flame.
1. Working within the Perspex box (laminar flow cabinet) use sterile tweezers to
remove the sealed auto-claved paper and place it on the tile inside the Perspex
box.
2. Collect the Ginger plants of similar size and colour. Inside the Perspex box
remove the ginger plantlets with tweezers and place them one at a time on the
auto-claved paper.
3. Using a sterile scalpel and tweezers measure the plantlet to the desired length and
remove the leaves and roots cutting the plantlet to the desired length. Ensure that
the rhizome remains as part of the plantlet.
4. Using the tweezers place the cut plantlet into a corresponding tube with the correct
media. Ensure the plant is placed securely in the agar and seal the tube. Label
the tube correctly.
12 x MS media (6 to be kept in light, 6 in dark)
12 x MS + sucrose media (6 to be kept in light, 6 in dark)
5. Repeat the steps 1-5 for each of the ginger plantlets.
6. Those plants that are to be kept in full light are to be placed under lights in a
controlled environment and those that are to be kept in the dark are to be carefully
wrapped in alfoil and placed in the same controlled environment.
7. Each week plants are to be measured and data recorded including contamination,
height colour etc
8. After 7-8 weeks of growth a deconstructive harvest will take place. This involves
collecting the final set of data and measurements by removing each plant from the
agar tube. These results collected should include weighing the plants (whole plant,
leaves, rhizome etc), final height measurements and photos may be taken.
9. After each plant is removed and measured discard the plant and remove the agar
from each tube.
Plant Tissue Culture
Experiment 3: Effect of Hormones on Growth of
Tissue Cultured Ginger
Introduction
Plant tissue culture is the cultivation of plant cells or tissues on
specially-formulated nutrient media. In appropriate conditions, an entire plant can be
regenerated from each single cell, permitting the rapid production of many identical
plants.
Tissue culture is an essential tool in modern plant breeding. Since it was first developed
in the early 1960s, plant tissue culture has become the basis of a major industry,
providing high-value plants for nurseries. Where a crop (e.g., banana) does not produce
seeds, plants derived from tissue culture are sometimes planted directly in farmers' fields.
However this is rarely done for seed-bearing species - more often tissue-cultured plants
are used to produce the seeds from which crops. Cell culture is also used for the
conservation of those plant varieties that cannot be maintained in a normal seed bank.
Natural plant hormones and their synthetic relatives have enormous benefits for tissue
culture of plants. Plant hormones increase productivity and control of stages of
development during culture. There are two broad classes of hormone used in culture;
cytokinins (eg BAP) to control shoot proliferation and multiplication and auxins (eg IBA) to
control root development.
Students will work in groups of 2-3 and will aseptically transfer ginger tissue culture plants
to media containing MS nutrients and some media with high or low levels of the hormone
cytokinin.
Aim
To investigate the effect of cytokinin on the growth and development of tissue cultured
plants as measured by changes in the plant.
Materials
18 cultured ginger plants
Tweezers
Scalpel
Alcohol burner
Ethanol
Bunsen burner
Swabs (cotton balls)
Ceramic tile
Auto-claved paper
Cling wrap
18 Plastic tubes with sealable lids
Specific media (MS media, MS media + low level cytokinin and MS media + high level cytokinin)
Lab coat
Face mask
Hair net
Gloves
Permanent markers
Perspex box
Method
Contamination considerations
The environment used when carrying out the experiment needs to be
completely sterile. Making sure the area, equipment and the students are
totally aseptic is essential to the success of the experiment.
These aseptic techniques ensure there is no contamination and prevents bacterial or germ
growth on the experiment. Using products like Ethanol and heat from the Bunsen burner
assists in sterilizing all utensils preventing contamination.
Step 1. Swab interior of Perspex box liberally with ethanol and cotton buds
Step 2. Sterilize hands with lathering ethanol over them.
Step 3. Sterilize each utensil to be used in the Perspex box by holding it over an open flame.
All utensils that come into contact with the plant are heated by the flame.
1. Working within the Perspex box (laminar flow cabinet) use sterile tweezers to
remove the sealed auto-claved paper and place it on the tile inside the Perspex
box.
2. Collect the Ginger plants of similar size and colour. Inside the Perspex box
remove the ginger plantlets with tweezers and place them one at a time on the
auto-claved paper.
3. Using a sterile scalpel and tweezers measure the plantlet to the desired length and
remove the leaves and roots cutting the plantlet to the desired length. Ensure that
the rhizome remains as part of the plantlet.
4. Using the tweezers place the cut plantlet into a corresponding tube with the correct
media. Ensure the plant is placed securely in the agar and seal the tube. Label
the tube correctly.
6 x MS media
6 x MS media + low level cytokinin
6 x MS media + high level cytokinin
5. Repeat the steps 1-5 for each of the ginger plantlets.
6. Place the plants under lights in a controlled environment.
7. Each week plants are to be measured and data recorded including contamination,
height colour etc
8. After 7-8 weeks of growth a deconstructive harvest will take place. This involves
collecting the final set of data and measurements by removing each plant from the
agar tube. These results collected should include weighing the plants (whole plant,
leaves, rhizome etc), final height measurements and photos may be taken.
9. After each plant is removed and measured discard the plant and remove the agar
from each tube.