1. Plant Tissue Culture Definitions and How To’s
A means of propagating plants from tissue pieces – term also used to for organ culture & cell culture.
Cell –
The basic unit of life, all organisms are composed of cells.
Clone –
Plants produced asexually from a single source plant.
Cell division
Growth in cell numbers, reproduction of cells.
Differentiate
Modification of new cells to form tissues or organs with specific functions.
Totipotency –
ability of single cell to develop into an entire plant

2. Botany Callus An unorganized mass of cells. Node
That part of the stem from which a leaf or shoot originates.
internode
internode
Internode
Space between two nodes
petiole
Petiole
Leaf stalk, usually attached at the node.
node
node

3. Meristem Apical meristems located at the tips of roots and shoots
supply cells for the plant to increase in length (grow up for shoots and down for roots)
growth in this direction is known as primary growth
Lateral meristems
located near the periphery of the plant, usually in the vascular tissue.
supply cells for the plant to increase in girth
growth in this direction is known as secondary growth
Annual rings are a way to visualize the work of lateral

4. Botany – influences on Meristem
Hormones
Growth regulators, (G.R.) natural or synthetic chemicals that strongly affect growth
Auxin
G.R. that promotes callus growth, cell division, cell enlargement, adventitious buds and later rooting.
Cytokinin
G.R. that regulates growth & morphogenesis and stimulates cell division.
Gibberellin
G.R. that Influences cell enlargement.

5. Terms of Tissue Culture
Stages of culture
Stage 0 – condition of the stock plant
Stage I – initiation of culture
Stage II – multiplication
Stage III – rooting
Stage IV – return to soil
In vitro
Latin: in glass (in a test tube)
In vivo
Latin: in life (growing naturally).
Subculture
To remove a growing culture from a vessel and either remove certain structures or cut into pieces and return to a new or different medium.
explant
part of the source plant used to initiate a culture.

6. Terms of Tissue Culture
Aseptic
Free of microorganisms
Contamination
An infestation by unwanted microorganisms, such as bacteria or fungi.
Pathogen
A disease-causing microorganism.
Pathogenic
Capable of causing disease
Sterile technique
The practice of working with cultures in an environment free from microorganisms.

7. Equipment
Medium
Liquid or agar-gelled nutrient substance in or on which cultures are grown.
Plural = media
Horizontal laminar flow unit
An enclosed work area that has moving sterile air. Room air is pulled in from the top, and forced through a HEPA (high E Particulate Air) filter
Autoclave
a machine capable of sterilizing wet or dry items with steam under pressure. Syn. pressure cooker.
Agar
A polysaccharide derived from algae used to gel or congeal a liquid. (8g/L)

11. Tie back hair and roll up sleeves.
Put on apron and goggles.
Spray and wipe down table with table cleaner.
Spray scalpel and forceps with alcohol. Place in alcohol jar.
Wash hands up to elbows.
Put on gloves. Rub hand sanitizer onto gloved hands include arm up to elbows.
Wait for Mrs. Fabian to hand out supplies.

12. 8. First culturist, receive plastic bag and lay on table, open side towards you. 9. Helper will spray area in front of bag with alcohol. 10. Culturist will receive petri dish from Mrs. Fabian. Helper will spray outside of petri dish with alcohol. Culturist will place inside of bag. Separate cover from petri dish. One side will be up, other half will be down. 11. Culturist will receive tubes from Mrs. Fabian Helper will spray outside of tubes with alcohol. Culturist will place inside of bag.

13. 12. Culturist will take forceps to prepare to receive explant from Mrs
12. Culturist will take forceps to prepare to receive explant from Mrs. Fabian. Helper will lift lid of sterile water jar while worker dips forceps. Helper will immediately cover sterile water jar. 13. Culturist will receive explant from Mrs. Fabian and place it on the petri dish inside the bag. Return forceps to alcohol jar. 14. Culturist will remove scalpel from alcohol and dip into sterile water. Helper will lift and return jar lid. Use scalpel to cut away any brown areas from explant. Return scalpel to alcohol jar when complete.

14. 15. Culturist will use forceps to transfer explant to agar
15. Culturist will use forceps to transfer explant to agar. Remember to dip forceps into sterile water before touching explant. Helper will help open and close the jar. 16. Insert explant so the node is partially submerged into the agar. 17. Cover tube when complete and leave inside bag. 18. Culturist 1 and Helper switch roles. 19. Slide bag across table. 20. Re-sanitize gloved hands. 21. Spray area in front of bag…