PROPAGATION BY TISSUE AND ORGAN CULTURING

Most propagative techniques, whether natural or developed by horticul­turists, have been recognized and practiced for centuries. The twentieth century, however, heralded the beginnings of new techniques presently termed tissue culturing and organ culturing. These techniques permit the reproduction of certain species from tiny pieces of plant organs such as embryos, pollen grains, and shoot tips or from undifferentiated plant tissue, usually callus. The piece of organ or tissue is removed from the parent plant under sterile conditions, using precision scalpels, for­ceps, and tweezers (Figures 14-18A, 14-18B, and 14-18C). Immediately after removal, it is transferred to a sterile nutrient material (in either a liquid or gelatinous agar state), usually contained in a clear glass flask, tube, or petri dish.

figure 14-18B. The piece of tip is placed into a container filled with sterile nutrient agar. (Courtesy United States Department of Agriculture)

figure 14-19. A transfer chamber requires an antiseptic environment, a heat source to flame instruments prior to each use, and a supply of sterilized media and containers. (Delmar/Cengage Learning)

Totally sterile (aseptic) conditions are essential throughout the pro­cess to prevent contamination of the culture by microorganisms such as fungi and bacteria. While methods to ensure aseptic conditions vary, the following precautions are typical.

• The transfer chamber is kept free of all contamination. Whether the chamber is a special room used solely for the purpose or a small box­like compartment, it must be rid of all dust, spores, bacteria, and other contaminants (Figure 14-19). Sterilization of small chambers may be accomplished with ultraviolet light (Caution: Ultraviolet light can be blinding and should not be looked at directly.) Larger chambers may be washed down with chlorine bleach or ethyl alcohol.

• All tools, containers, and solutions are kept sterile. They can be made that way initially by subjecting them to high temperatures under pressure in an autoclave (Figure 14-20). During the culturing operation, tools and the openings of containers can be kept sterile by passage through the flame of an alcohol lamp or Bunsen burner.

• All plant tissue is surface sterilized before use, care being taken not to damage the plant tissue. Disinfectants commonly used include Merthiolate, mercuric chloride, calcium hypochlorite, and sodium hypochlorite. Disinfectants include hot water, steam, and formaldehyde.

The nutrient media used for organ and tissue culturing vary with the species of plant being propagated. Agar media are the most com­mon, but liquid media can also be used as long as a filter-paper wick is placed in the tube to hold the tissue or organ piece out of the solution. The media usually contain inorganic mineral elements (both macro and micronutrients), sucrose sugar, growth regulators, and vitamins. Assorted other additives are found in the several media developed by researchers. You may wish to consult texts on plant propagation for

figure 14-20. A laboratory autoclave provides steam heat under pressure to sterilize media, tools, and containers. Here flasks of agar are being set into the autoclave. (Delmar/Cengage Learning)

the formulations of Knop’s solution, the Murashigi and Skoog medium, White’s medium, or Knudson C medium. The media take their names from the scientists who first developed or reported them.

Following a successful transfer from parent plant to the aseptic and nutritious environment of the tube, flask, or petri dish, the piece of plant tissue (termed an explant) enters a period of growth. New tissues and organs are differentiated and a new, complete plant is formed. Not all explants are able to differentiate; they continue to produce callus tissue, rendering the culture useless. The failure of certain species to dif­ferentiate is probably due to our failure to provide the right conditions rather than to any biological limitation, however. The full differentia­tion from callus or organ explant to complete plant may require several aseptic transfers of the developing plant, each time altering the size of the growth container and the composition of the nutrient medium.

Current Applications

Due in part to its complexity and cost, and in part to its difficulty when compared to other methods of plant propagation, tissue culturing still finds its greatest application as a technique for plant breeding, genetic research, and pathological research. However, since the 1970s commer­cial tissue culture has grown in both the number of species propagated and the total number of new plants produced annually. Commercial

production methods seldom begin with single cells, instead using the tips of shoots and roots, as well as tiny pieces of leaves, roots, and stems. Sometimes lateral buds are used.

Developed through basic research and later adapted to commercial methods of tissue and organ culturing, many ornamental plants have found their way to the marketplace as cultivars with improved vigor, new colors, new resistance to disease, greater tolerance to stress, longer bloom periods, and assorted other improvements. Orchids, daylilies, anthuriums, philodendrons, chrysanthemums, carnations, geraniums, gladiola, gloxinias, roses, and rhododendrons have all been improved in one way or another since the mid-1970s using the micropropagation techniques of tissue and organ culture.

Once it was possible to make micropropagation economically profitable, a new commercial industry was born. As of this writing, the major uses of commercial tissue culture are:

• Mass propagation of specific clones, especially those developed by breeders and geneticists who want to rush new plants into the marketplace.

• Perpetuation of parent stock plants that produce desired hybrid seeds.

• Maintaining disease-free germ plasm.

• Keeping plants in year round production by bypassing their natural seasonal cycles.

• Creation of artificial seeds, by cloning embryos in mass and coating them with a protective biodegradable shell containing nutrients, pesticides, and fertilizer. It permits the new plant to be handled like a regular seed, but with all of the desired traits ensured through selective micropropagation.

SUMMARY______________________________________

Plant propagation exemplifies the application of science to the craft of ornamental horticulture. It is a craft that changes continually as new facts are discovered, yet respects the skills and techniques that have evolved over many years of practice.

The medium for propagation must provide support, moisture retention, drainage, aeration, a pest-free environment, and sometimes nutrition. It may be made up of one or more of the following: soil, sand, peat moss, sphagnum moss, perlite, vermiculite, or fired clay. It must be pasteurized before use.

Propagation can occur in greenhouses, hotbeds, cold frames, or lathhouses. The environment must provide proper light, high humidity, warmth, and ventilation. Seed propagation may require a light or dark environment for germination, depending on the species. It may also require scarification or stratification if the seed possesses one or more types of dormancy. Planting techniques for seeds vary depending on whether the species are herbaceous or woody, and whether they are to be transplanted after germination or will be grown at the planting site.

Propagation by cuttings is the most widely used method of asexual plant reproduction. It is quick, easy, and inexpensive. It is made possible by the cell’s ability to revert to an undifferentiated condition, once again initiating the root, stem, and leaf tissues necessary to form a complete

plant. There are different types of cuttings and different methods of propagating them, some capable of producing greater quantities than others. Soon after rooting occurs, the cuttings must be hardened-off to adapt them to more stressful growing conditions.

The technique of grafting necessitates a knowledge of which plants can be grafted, which ones benefit from grafting, and how to make the numerous types of grafts. Grafting is usually done either because the plant benefits from it or because no other propagative method will work. Grafting is possible on both herbaceous and woody plants but is most common on woody plants. The tools needed for grafting are a knife, finger guards, tying materials, grafting wax, and a source of heat. Methods of grafting vary with the type of plant (woody or herbaceous), its size, and sometimes the species.

Budding is a method of grafting that requires somewhat specialized techniques. It involves the removal of a piece of bark on the stock plant and its replacement with a comparably sized piece containing a bud from the scion. There are several different methods of budding.

Layering as a propagative technique permits the stem of a new plant to remain at least partially attached to the parent plant while new roots are initiated. There are different methods of layering. Simple layering, top layering, mound layering, and serpentine layering find frequent use by professionals; air layering enjoys popularity with amateurs.

Tissue and organ culturing are comparatively recent propagative techniques. They permit the reproduction of certain species from tiny pieces of plant organs such as embryos, pollen grains, and shoot tips, or from undifferentiated plant tissue, usually callus. The propagative material is collected under aseptic conditions and transferred to ster­ile nutrient media, usually contained in flasks, tubes, or petri dishes. The piece of tissue then enters a period of growth and new tissues and organs are differentiated to form a complete new plant.

ACHIEVEMENT REVIEW

A. MULTIPLE CHOICE

From the choices given, select the answer that best completes each of the following state­ments.

1. The propagation medium must be_____ .

a. unpasteurized b. pasteurized

2. The medium must_____ moisture.

a. repel b. retain

3. Fired clay, sand, or perlite can improve the of the media.

a. drainage b. moisture

retention

4. The best sand for use in propagation

media is_____ sand.

a. fine b. quartz

5. Propagation of seeds requires that the medium contain.

a. nutrients b. agar

B. TRUE/FALSE

Indicate if the following statements are true or false.

1. Propagation structures must exclude all light.

2. Intermittent mist systems are useful in the prevention of wilting in cuttings.

3. The application of a soil drench can reduce damping-off of seedlings.

4. Temperature controls the rate of root and shoot development in cuttings.

5. In a cutting bed, the rooting medium should be cooler than the air temperature.

6. Most cuttings form better root systems in acidic rather than neutral propagating media.

7. High light intensity is necessary for vigorous root formation on cuttings.

8. Grafting wax keeps moisture outside the graft union.

9. High humidity discourages the formation of callus tissue in grafts.

10. Once roots have formed on cuttings, the propagation chamber should be ventilated.

C. DEMONSTRATIONS

1. Collect or purchase seeds of several herbaceous and woody species common to your region of the country. Precondition as necessary. Prepare a suitable soil for germination and fill separate greenhouse flats for each species to be planted. Plant the seeds in furrows and cover. Water the flats either by immersing them in water (if the seeds are fine) or by sprinkling gently. Cover with sheets of plate glass during the day and remove at night. Monitor the day and night temperatures as closely as conditions permit.

For added interest and learning, vary the planting techniques. Treat some seeds with fungicide before planting and leave others untreated. Apply a soil drench to some flats and not to others. Keep records of the percentage of seeds that germinate, develop damping-off disease, and develop into healthy plants.

2. Plant 100 seeds of the same species of flowering annual in each of two plug sheets. (Use regular greenhouse flats if plug sheets are not available.) Maintain the same growing conditions for each sheet (watering, low-concentration N fertilizer), but grow one sheet at 80° F day and 60° F night temperatures and the other sheet at 70° F day temperatures and

75° F night temperatures. After both sheets are showing approximately the same percentage of germinated seeds:

a. count the number of seedlings in each sheet.

b. measure the length of each seedling and calculate an average seedling length per sheet.

c. compare the results and draw a conclusion if one is possible.

3. Using Table 14-3 (A Guide to Propagating Cuttings) as a reference, take several different types of cuttings from local plants. The season of the year and the species of the plant will determine the types of cuttings taken. Propagate them as indicated in the table. Keep records of the percentage of cuttings that do and do not develop roots. You may want to use different media to compare their effects on the number and quality of roots formed, and the number of days required for rooting.

4. Practice making some grafts. Use as many different techniques as the available plants and conditions allow. With Table 14-4

(A Guide to Grafting Techniques) as an aid, select and prepare stocks and scions from compatible species. For beginners who have no knowledge of species compatibility, the stock and scion may be of the same species for practice purposes. If grafts do not unite, have them checked by a teacher or commercial propagator to determine the reason.

5. Practice budding on roses or fruit trees, using either greenhouse or landscape plants. Try T-buds, I-buds, and patch buds. Evaluate which ones worked most successfully and why.

6. Using some large garden shrubs as stock plants, layer some branches using the techniques of simple and tip layering.

If pendulous shrubs are available, try a serpentine layer as well. Initiate the layering in the spring and separate the plants in the fall. For comparison, wound the buried stems of some branches and leave other unwounded. Mark the

branches so that a count of successfully rooted plants can be made and the techniques compared.

7. If a large stalky foliage plant is available for use, make an air layer. When the roots are visible within the plastic wrapping, sever the plant.

D. SHORT ANSWER

Answer the following questions as briefly as pos­sible.

1. Define tissue and organ culturing.

2. Why is sterility of the transfer chamber and the growth container so essential?

3. In what physical states do nutrient media for culturing commonly occur?

4. What tools are used to excise and transfer the explants?

5. What are the two major benefits to commercial propagators from tissue or organ culturing?