R. Daniel Lineberger, Professor Emeritus
Department of Horticulture
Texas A&M University
College Station, TX 77843
The propagation of plants through tissue culture, micropropagation, was viewed at one time as a research tool to study plant growth and development, growth regulators, and organogenesis. Much useful information was gathered as a result of such research. The impact which micropropagation had on the multiplication of orchids, tropical foliage plants, and selected floricultural crops is now being felt in the nursery crops industry. Rhododendrons, azaleas, mountain laurel, lilacs, and a few deciduous trees are currently being micropropagated. Tissue cultured plants are now accepted without fear of extreme variation, poor vigor, or delay in flowering as was the prior assumption.
ADVANTAGES OF MICROPROPAGATED PLANTS
Certain morphological and physiological characteristics of tissue cultured plants make them superior to conventionally propagated materials. Tissue cultured plants (particularly Amelanchier, Rhododendron, and Betula) tend toward a more branched habit. This tendency results in a young plant which is less “leggy” and salable earlier because of increased fullness. Tissue cultured plants produce cuttings which have increased rooting when rooted by conventional means. This phenomenon is being exploited in the “rejuvenation” of stock plants for cutting propagation. The most obvious advantage inherent to micropropagation is that more plants are produced quicker from a limited number of stock plants (one in some cases). New introductions are available in commercial quantities in a fraction of the time previously required.
THE PROCESS OF MICROPROPAGATION
A brief review of the procedure of tissue culture will clarify the need for specific care and handling. A small piece of the plant to be cloned (the explant) is removed from a healthy, well-maintained stock plant and sterilized in a dilute bleach solution. The source of tissue will vary by species, but shoot tip, leaf, stem, lateral bud, and flower tissues have been used successfully for various plants. Rhododendron, for example, will produce plantlets from flower petal tissue. The sterilized explant is rinsed with sterile water, and placed in aseptically prepared containers on a specially formulated medium. The explant may produce shoot proliferating cultures directly by enhanced lateral bud break, or the tissue may undergo a certain period of unorganized growth (callus) prior to shoot differentiation. The pattern of growth of the cultures is principally determined by the plant growth regulator content of the tissue culture medium (the auxin and cytokinin concentration). Most cultures are established within 4 to 12 weeks depending on the species and in some instances, depending on the cultivar. A shoot proliferating culture is one which can be subdivided (subcultured) to produce divisions which will continue rapid multiplication. Estimates of the rate of multiplication vary and are affected by many factors, but the production of thousands and in some cases millions of plants a year from a single explant has been demonstrated!
MORPHOLOGY OF A MICROCUTTING
The small shoots produced in tissue culture are referred to as “microcuttings”. Microcuttings vary a great deal in size, length and degree of leaf expansion depending upon species, cultivar, and cultural conditions. The tissue culture environment results in the production of shoots at a continuum of stages of development, from ones just recently expanded to those with elongated stems and well expanded leaves. Recognition of the type of microcutting which roots and grows out well is one of the most critical components of a successful system. The determination of the correct sequence of media and/or transfers to optimize the production of ÒnormalÓ microcuttings that root and acclimate in high percentage is important. Thousands of small, spindly microcuttings which root and grow out poorly are worthless in the commercial propagation arena.
CHOICE OF A ROOTING METHOD
Microcuttings are rooted by modifications of two basic methods. In vitro rooting is accomplished by transferring the carefully dissected microcuttings to a medium which is free of growth regulators or has only auxin added as the growth regulator. All manipulations are conducted under sterile conditions, the process requires excessive labor input, and media must be prepared specifically for the rooting stage. Since the cuttings are provided with minerals, vitamins, sugar as a carbon source, and root promoting auxins, many researchers feel this method will allow for rooting of even the most difficult to root plants. This method does allow more flexibility in choosing cuttings since cuttings need not conduct photosynthesis to provide an energy source for rooting. Leaf expansion is therefore less critical.
In most commercial operations, the preferred rooting method is referred to as nonsterile rooting. Microcuttings are harvested (usually under nonsterile conditions by sacrificing whole cultures, but if stocks are low, sterility may be maintained at this stage) and stuck in a clean (but not sterile) mixture of peat moss and perlite or vermiculite, or in preformed foam or peat moss plugs. Cuttings may be stuck in small plastic covered containers and rooted in the lab, or placed directly into flats which are then placed in a mist, fog, or poly tent system. The nonsterile method has given outstanding results with most species tested. The system does require that the microcuttings be of “normal” morphology, have well expanded leaves, and that the rooting medium be free from disease causing organisms. Rooting is accomplished in 3 to 5 weeks, and the microcuttings can be transplanted at this stage and then acclimated or acclimated directly in the rooting flats.
ACCLIMATION OF ROOTED MICROCUTTINGS
Microcuttings are very sensitive to changes in the physical environment, and the success or failure of the whole operation may hinge on the acclimation process. Microcuttings are poorly adapted for growth in the greenhouse environment at the time they are removed from the rooting containers, whether these were held in the laboratory or in the mist or fog area. Microcuttings rooted and grown in very high humidity have stomates which have been shown to be “sluggish”, responding to decreased relative humidity too slowly to prevent desiccation of the cutting. This problem is compounded by the apparent lack of a well developed, waxy cuticle, the barrier to water loss over the entire plant surface. Research has shown that the cuticle is present, but not functional to the extent that exists on seedling or even cutting propagated material.
Microcuttings must be acclimated to increased light intensity in much the same manner as acclimation to decreased relative humidity. If too much leaf surface is injured by direct transferal to higher light intensity (sun-burning), the vigor of the cutting will be reduced markedly because the plant is too small to have enough stored starch to force a new flush of growth.
The following protocol has been successful for a number of woody species (Amelanchier, ÔHally JolivetteÕ cherry, birch, elm, apple, crabapple, and others). Rooted microcuttings are transplanted to plug trays, sectioned flats, or peat moss pots into a commercially prepared medium (usually a peat moss and perlite or peat-vermiculite medium; it is important that the medium be fine textured, similar to a seed starting mix). This operation must be accomplished quickly, and care must be taken to fog or mist the microcuttings to Òwater them inÓ. Transplanted flats are carried to the mist bed just as soon as they are filled. Microcuttings which are allowed to wilt during the transplanting process show poor recovery, and poor transplant survival! The mist cycle is set for a duration of 4 or 6 seconds with the interval set at 4 or 6 minutes. Bottom heat is beneficial. The mist area must be shaded with heavy shade, with a light exclusion of at least 60%. Microcuttings are left in the mist + shade for 5 to 7 days (some species may require 2 or 3 more days if there is any sign of wilting upon removal) and they are transferred to a shade house(shade greater than 50% light exclusion) for an additional 7 days. Microcuttings usually begin a flush of growth near the end of the time in the shade house. Modifications of this procedure can be made for fog or poly tent rooted microcuttings, but the transition from high to low relative humidity and low to high light must be gradual. Microcuttings which are forced into a growth cessation by improper acclimation procedures have low survival percentages, and tend to grow out slowly and nonuniformly.
SEASONAL FLUCTUATIONS IN ACCLIMATION EFFICIENCY
Micropropagation is unique among propagation methods in that seasonality has little influence on the production of microcuttings in the lab. Most woody species, however, do display seasonal differences in acclimation efficiency and subsequent growth in the greenhouse. Microcuttings acclimated between February and September establish well in the greenhouse and generally grow out rapidly and uniformly. Microcuttings acclimated between October and January acclimate adequately, but usually do not put on the first growth flush uniformly, and generally go into a semidormant condition which requires a cold treatment to break. Several techniques have been used in attempting to break this cycle. HPS lighting to supplement natural daylengths to 18 hours of light per day, constant feed fertilization, and incandescent light breaks have all failed to coax lagging microcuttings into active growth. We have concluded that light intensity, rather than absolute daylength, must be governing the early growth response of the acclimated microcuttings. The current recommendation is to schedule microcuttings to grow out in the greenhouse between February and September to avoid nonuniform growth following acclimation.
SHOULD PRODUCTION NURSERIES PURCHASE SHOOT PROLIFERATING CULTURES?
The concern over whether production nurseries should purchase proliferating cultures directly from tissue culture labs has arisen on many occasions recently. Shoot proliferating cultures are a convenient, compact means of transporting cuttings, they are ÒcleanÓ, and are available to a much lower unit cost since the cost of the rooting and acclimation stages are not included.
The current recommendation is that production nurseries unfamiliar with rooting of tissue cultured materials not attempt rooting and acclimation of microcuttings without first conducting limited trials to gain experience in the procedure.
Consultation with individuals who have developed rooting and acclimation techniques has revealed that the first few experiments usually result in less than acceptable results. Individuals not familiar with the sensitivity of microcuttings to shifts in relative humidity, light intensity, and the importance of cutting selection generally will not experience the level of success necessary to make the process commercially viable. Once the tissue cultured plants have formed the first growth flush (rooted liner), they could be handled as any transplant and would not require any increased skill level from the grower.
Micropropagation is rapidly becoming a standard method in the nursery crops industry, much as it has in the foliage plants industry. The success or failure of the technique at the commercial level is often not in the lab, but rather in the greenhouse at the level of rooting and acclimation of microcuttings. Microcuttings are very sensitive to changes in environmental conditions in the early stages, but respond just as any other propagule after the first growth flush. Production nurseries should purchase tissue cultured plants at the rooted liner stage, unless a suitable system has been developed to root and acclimate microcuttings from the shoot proliferating culture stage.