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

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

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

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.

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.

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.

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)

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.

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.

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.

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…