HORT 201
Plant Propagation
Laboratory Exercise 7
Axillary shoot proliferation; Organogenesis from callus
Reference: Text chapters 17 and 18
Objectives:
1. To gain experience with tissue culture methods used to propagate plants.
2. To learn methods for tissue culture propagation by axillary branching from shoot tips.
3. To learn how to root micro-cuttings form tissue cultures.
4. To learn techniques and procedures for transplanting rooted micro-cuttings from tissue
culture to soil for growing on.
5. To learn methods for tissue culture propagation by organogenesis from leaf blade and petiole sections.
6. To compare organogenesis from leaf blade explants and petiole explants.
Propagation or plant multiplication via tissue culture (an in vitro method of propagation) is typically done through either by: 1) Enhancement of axillary branching or, 2) organogenesis (formation of adventitious shoots with subsequent rooting) or, 3) asexual embryogenesis.
A. Aseptic technique
in plant tissue culture
Fungal spores and other microorganisms are present throughout our environment. When a spore comes in contact with the culture media, conditions are ideal for its germination and rapid growth. The laminar flow hoods operate under positive pressure and are equipped with High Efficiency Particulate Air (HEPA) filters, which are approximately 3 inches thick. The HEPA filters are 99.99% efficient at removing particulate matter 0.3 microns or larger; bacteria and fungal spores are trapped by the filter. If you use proper technique while working inside these hoods, contamination of plant tissue cultures can be virtually eliminated. The following steps will help to minimize the problem:
1. Remove watches and jewelry. Wash your hands in the sink using soap and water.
2. Spray your hands and arms liberally with 70%
ethanol. Do not move your hands into
the hood where the lit Bunsen burner is located until the alcohol has
evaporated off your hands! Rub your hands together to speed
evaporation.
3. Everything that enters the hood must be sterilized first. Spray dissecting tools, baby food jars and other containers with 70% ethanol.
4. Position yourself such that your face is at the edge of the air flow. Keep your head out of the hood. Reach into the hood up to arm's length, but don't place your elbows on the working surface.
5. If it's necessary to cough or sneeze turn your head. Please don't cough or sneeze into the hood. If you contaminate the hood, you may contaminate the cultures of all students who follow you. If you contaminate the hood, please clean it by spraying the hood surface and inside walls with 70% alcohol and wiping down with a clean paper towel.
6. Flame-sterilizing forceps, scalpels and other dissecting tools: Dip the tool in 95% ethanol, and then pass it through the flame very briefly while holding the tool horizontal. Do not hold the tool angled up because you do not want flaming alcohol to run down to your hand. The alcohol will be flamed off in a second or two. Do not hold the tool in the flame. It is not necessary to heat the tool to sterilize it; the flaming is only to burn off the ethanol. It is the ethanol which sterilizes the tool.
Be extremely careful handling alcohol near the open flame! Do not flame a tool if your hands are still wet with alcohol. Carelessness can result in second degree burns!
B. Shoot-tip culture - Enhancement of Axillary
Branching
Although often called meristem culture, shoot-tip culture is a more precise term for this technique since more than just the microscopic apical meristem is generally cultured. Meristems consist of a dome at the very apex of a shoot, with increasingly well-developed primordial leaves at increasing distances from the dome. In the axis of these tiny leaf primordia are the primordial buds which will make the future lateral buds on the expanded shoot. When small shoot tips are cultured, these bud primordia develop as they would have on the intact shoot. However they don't grow out because they're under apical dominance from the apical meristem. Application of exogenous cytokinins effectively breaks apical dominance and induces lateral budbreak in many whole plants. It's not surprising that they have the same effect in tissue culture. The principle of shoot-tip propagation is to multiply shoot tips by culturing them on cytokinin-containing media to encourage several shoots to grow from the original. Auxin also may be necessary to obtain shoot growth; however, many plants apparently synthesize adequate endogenous concentrations. The effectiveness of various cytokinins and auxins, and their optimal concentrations, varies from plant to plant. Shoot-tip culture is also very important in propagation as the technique used to rid pathogens from clones. In this case the smaller the shoot-tip cultured the better the chance of propagating plants free of virus, bacteria or mycoplasm. Frequently, true meristem culture is employed for this purpose.
Plant
propagation of mums via tissue culture is not economical for commercial
propagators because stem cutting propagation is so successful. However, tissue
culture is used to obtain virus-free mum stock material. The highly infectious
plant viroid, Chrysanthemum stunt, nearly destroyed the Chrysanthemum industry
in the late 1940âs with infection rates between 50-100%. Even today there is no
chemical control for Chrysanthemum stunt. Tissue culture is used to maintain
virus-free mum stem cutting stocks.
Axillary shoot
proliferation in chrysanthemum:
Stage 1 -
Establishment [each student will establish 4 shoot tips in two baby food
jars]
A. Obtaining mum
explants.
1. Each student needs to take 4 mum cuttings. You should remove leaves until they are too small to break off at their bases, but be careful not to break off the entire apex.
2. Cut off the base so that the shoot tip is ¸ inch long.
B.
Surface-sterilizing your mum explants.
In the crevices, stomatal pores, and throughout the surfaces of all plant material are fungal spores and bacteria which will explosively grow in a tissue culture environment. To prevent fungi and bacteria from overwhelming and killing your explants, your explants must be surface sterilized. But this sterilization must not be so harsh as to kill the plant material.
1. Each group should obtain one baby food jar containing 70% alcohol. Take your baby food jar of 70% alcohol and your groupâs shoot tips to a laminar flow hood, BUT DO NOT PLACE ANYTHING IN THE HOOD YET. Please follow the aseptic techniques described on page 1.
2. In the wire cart next to the hood will be a spray bottle filled with 70% alcohol. Spray your hands and lower arms with 70% alcohol. Then spray the outside of the baby food jar filled with 70% alcohol to sterilize the outside of the baby food jar.
3. Open the baby food jar and place your entire groupâs shoot tips into the alcohol all at the same time and put the lid back on. Place the baby food jar with your shoot tips in the hood. Do not place this handout in the hood because it is not sterile. You may place this handout on the wire cart instead or have another member of your group read the instructions to you.
4. After 45 seconds of immersion in alcohol, transfer the shoot tips using forceps into the baby food jar containing Clorox. Soak the shoot tips in Clorox 5 minutes.
6. Keep the tips in the fourth jar of sterilized water until it is your turn to transfer your 4 tips to a Petri dish for additional cutting.
C. Transferring your explants to mum media.
1. Sterilize your hands with alcohol and air dry before placing them in the hood. In the hood, use flame-sterilized forceps to take 4 shoot tips from the sterile water and place them in a sterile Petri dish that is already in the hood. With a flame-sterilized scalpel cut the tips so the final length is 4-6 mm (1/4 inches).
2. Transfer shoot tips to your baby food jars containing mum media (two shoot tips per jar). This media is designed for Stage I & II cultures and contains 3% sucrose, 0.5 mg/L IAA and 3.0 mg/L Kinetin. Screw the lids securely on the jars and take them out the hood.
3. Label the jar (on the glass, not the lid) with a sharpie. Write your name, species, date, and lab section.
4. The shoot tip cultures will remain in the media for one to three weeks to determine if they are sterile and to allow lateral branches to develop.
Since we are using a media which should both establish and multiply lateral shoots (Stage I and II - high in cytokinins and low in auxin), after 3-4 weeks we should see lateral shoot development.
Axillary shoots from the stage II cultures should be visible in three to four weeks. At that time we will remove 2 axillary shoots (micro-cuttings) and transfer them to jars containing Stage III media that has 3% sucrose, 0.1 mg/L NAA and no cytokinins.
C. Organogenesis from
callus ö African violet (Saintpaulia)
Each student will establish: 2 leaf explants in one baby food jar
2 petiole explants a second baby food jar
Organogenesis is the formation of organs, namely shoots and roots. In organogenesis, an explant is used to obtain callus from which organs - shoots and/or roots - differentiate. Organ differentiation is controlled by the ratio of cytokinins to auxins in the medium. Minimum concentrations of both are necessary for callusing; however, if cytokinin levels are higher than auxin levels, shoot formation is favored. These shoots can often be easily rooted as small softwood or herbaceous stem cuttings. Many species will produce callus when given equivalent concentrations of auxin and cytokinins, however some species will instead produce shoots and roots simultaneously with equivalent auxin and cytokinin concentrations. African violet is such a species.
The African violet is an extremely popular flowering houseplant. Although it can easily be propagated from leaf/petiole cuttings, it also can be propagated via tissue culture. Micropropagation is a very successful means to commercially mass-produce this plant. We will compare the ability of leaf explants and petiole explants to generate shoots and roots.
When petiole or leak blade sections are placed on Murashige and Skoog medium containing specific levels of auxin and cytokinin, they form callus which can then differentiate into organs. Our media for African violet has 3% sucrose, 0.5 mg/L benzylamine purine (BAP) and 0.5 mg/L NAA.
1. Cut one leaf including at least a ¸ inch of petiole from an African violet plant. Rinse the leaf in a beaker of water that is next to the plants to remove any dust. If you have a large leaf, reduce the leaf blade size by cutting off the top half of the leaf blade, but make sure that the remaining leaf blade is large enough so that later in this lab procedure you will be able to cut two squares (1cm x 1cm) from the leaf blade.
2. Combine the 3 leaves from your lab group and surface sterilize them just as the mum shoot tips were surface sterilized. Follow steps in part B above. Keep the leaves in the fourth baby food jar of sterile water until it is your turn to transfer your leaf to your Petri dish for cutting.
3. Sterilize your hands with alcohol and air dry before placing them in the hood. In the hood, remove your leaf from the water rinse with flame-sterilized forceps and place it in a new sterile Petri dish. With a flame-sterilized scalpel cut two sections of petiole, each 3 mm (¹ inch) long and cut from the leaf blade two squares (1cm x 1cm) with each square containing a part of a leaf vein.
4. Using flame-sterilized forceps, transfer your two leaf explants into 1 baby food jar containing African violet media. Flame-sterilize your forceps again and transfer the two petioles to the other jar of African violet media by inserting the petiole vertically into the media so it is inserted about halfway into the agar. Whether the distal or proximal end is up has little effect in this environment. Cap both jars securely and remove them from the hood.
5. With a sharpie, label your jars on the glass (not the cap) with your name, date, species and lab section.
6. All cultures will be available for your observation in the following weeks. Your instructors can tell you where they will be kept.
Part D:
Score rooting of Chrysanthemums stem cuttings and herbaceous placed in
Zone 16 three weeks ago. Mums have been moved to Zone 21. Leaf cuttings are
still in Zone 16
1. Score the mums using the data sheet provided to your lab group to record your data. Turn in your data at end of lab. You may pot up any cuttings you wish to keep and place on bench where potted up plants are kept.
2. Check
your leaf cuttings for rooting by gently pulling on the cutting. If there is
resistance, score the cutting as rooted. Use the data sheet provided to your
group to record your data. Turn in at end of lab. If all the cuttings in a
market pack have rooted, please move the market pack to Zone 21 to the bench
designated for your lab section. When shoots form on the leaf cuttings, you may
pot up what cuttings you wish to keep
Part E: Score germination rates of Redbud
and Honeylocust seeds planted three weeks ago and placed on heating mats in
Zone 21
Use the data sheet provided to your
group to record your data. Turn in at end of lab. You may pot up any seedlings
you wish to keep and place on the Zone 21 bench where potted up plants are
kept.