Lab #2 Tissue Culture of Bryophytes
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1 Lab #2 Tissue Culture of Bryophytes In lecture we examined sexual reproduction in bryophytes in some detail. Today, we will examine the ways in which sexual and asexual reproduction are accomplished. There are many types of asexual propagules (Fig. 2-1), which include specialized structures called gemmae, that may be produced in cups (Marchantia polymorpha), on the tips of branches (Aulacomnium androgynum), and amongst leaves (Dicranoweisia cirrata). Fragmentation is a very common means of asexual reproduction. Tissue culture can be used to examine the growth of such propagules. It is important to keep in mind that in culture, plants are grown in artificial conditions so the growth patterns you observe do not necessarily represent those that occur in nature. Plates with media having lowered ph and enhanced nitrogen sulphate are available to simulate environmental acidification and eutrophication. Before you attempt to culture the specimens provided examine them microscopically. You will choose only a few of those provided (or those you have brought to lab). For groups of 2 you will be preparing a total of at least five plates; for groups of 3 you will be preparing a total of at least six plates. Tissue culture techniques will be demonstrated by your instructors. Terms to Know: asexual reproduction cauducous diaspore explant gemmae propagule sexual reproduction 12
2 PROCEDURE 1. Confirm your research group (your group will have its own web area based on name). Submit the group name and list of members to Shona. 2. Discuss which species you would like to work with and the types of explants (explant = piece of plant put into culture). 3. Examine the specimen microscopically. Write a brief description of the general structure of the bryophyte and the component you will be using as an explant. 4. Indicate your specimen selection on the board. 5. Discuss with the members of your group the procedure you plan to follow for each sample. A few pointers: - Keep your work area and utensils as sterile as possible is important; the nutrients in the medium are also favourable for the growth of fungi and bacteria. - The inside surface of Petri dishes and autoclaved vials are sterile surfaces - Metal utensils can be dipped in alcohol then flamed - Cutinized tissue (such as sporophyte) can withstand being dipped in 20% bleach solution for 1 minute. MATERIALS Plant Species and Explant: Species Explant Low ph 7. Enhanced nitrogen Equipment: At your bench: Pipette tube with sterile pipettes, Pipette bulb Sterile water, 5 sterile vials, Black marker, Parafilm strips Sterile forceps (wrapped in aluminium foil) Ethanol dip (for dipping forceps and scalpels) 5 sterile empty Petri dishes, 5 Petri dishes with nutrient agar 2 glass Petri dishes lined with filter paper (to blot specimens) 13
3 At Side Bench: Alcohol burners Matches 20% Bleach solution (with two drops of Tween-20) Scalpels Extra equipment For each specimen outline the procedure used to prepare the specimen and inoculate the Petri dish. If you do more than five samples please add them to the list. You may also use a different format if you like. Specimen 1. 14
4 Specimen 2. Specimen 3. 15
5 Specimen 4. Specimen 5. 16
6 17
7 ADDITIONAL NOTES: 18
8 Tissue Culture Activity (this will continue over the course of the term): Due January 15: On your group s discussion board on the website submit the bryophyte names, types of explants, as well as the plant materials you used and the procedure you followed to inoculate your plates. Due January 20: On your group s discussion board on the website record your observations of the cultures to date. Observations must be posted regularly on your group s discussion board. Observations and conclusions must be finalized by April 8 (this does not mean you leave everything until the end.the grade is based on regularly recorded observations as well as final conclusions). Fig. 2-1 Examples of some vegetative propagules: (A) Dicranum flagellare, (B)Pohlia erecta, (C)Aulocomnium androgynium, (D)Leptobryum pyriforme (rhizoidal), (E)Tayloria serrata (rhizoidal), (F)Calymperes richardii, (G)Encalyptra procera, (H)Syrrhopodon parasiticus. (Schofield, 1985) 19
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