From Aggie Horticulture, Texas A&M AgriLife Extension, Texas A&M University System
by R. Daniel Lineberger, Professor of Horticulture

The Methodology Of Tissue Culture

This article first appeared in The Buckeye Nurseryman, September, 1980, Columbus, OH.

Much has been written and more has been said about the applications of plant tissue culture to the nursery industry. The subject is still poorly understood by a majority of plant propagators. Tissue culture is often looked upon as being practical only for propagation of those plants which are difficult to propagate by conventional methods. This notion is quickly being laid to rest. An industry is emerging in various areas throughout the country which is taking advantage of technological advances in the realm of tissue culture of woody plants. This technology suggests that propagation by tissue culture is indeed applicable to the "difficult to propagate" species, and equally as important, it may offer economic advantages for some species which are considered relatively "easy to propagate".
The intent of this communication is to briefly describe the contemporary techniques of tissue culture of woody plants, and to review the advantages and limitations of tissue culture as a propagation method.

The Methodology of Tissue Culture

The variety of techniques which can be used to get plant development in vitro (that is, by techniques such as tissue culture) is considerable and completely dependent upon the species in question. Single cells of leaf tissue can regenerate whole plants, as can shoot tips, leaf pieces, root pieces, lateral buds, or stem sections. Not all of these methods are applicable to woody plants, and indeed, not all have been applied to a commercial level to any plants.

Shoot tip culture is the method in widest use for the mass propagation of woody species. An actively growing shoot tip is surface sterilized and placed on a defined culture medium under sterile conditions. The culture medium contains inorganic and organic salts (macronutrients, micronutrients and vitamins) as well as an energy source (sucrose or table sugar), growth regulators, and agar )to gel the medium). If the growth regulators are appropriately balanced, the shoot tip elongates, lateral buds break and begin growth, and adventitious shoots are also produced on the stem piece. This rapid proliferation of shoots results in masses of shoots being produced from a single shoot tip. Up to a hundred shoots may be produced in as little as eight to twelve weeks from a single tip. The number of shoots produced and the rapidity of shoot proliferation varies between species, and in some cases, between cultivars of a single species. Shoots are removed from the cultures at regular intervals and a portion of the mass is replaced on fresh media to continue proliferation. The small shoots which are removed are then rooted in a separate medium, either a sterile gelled medium or a peat-perlite medium (in much the same fashion as conventional woody cuttings).

Shoots produced through tissue culture are generally easy to root, even though the same cultivar may be difficult to root by cutting propagation. The resulting rooted shoot is referred to as a "plantlet" because of its miniature size, but these can generally be grown in the greenhouse at a rapid rate and with a high degree of survival. Techniques for rooting tissue cultured shoots are currently receiving a great deal of research attention as are methods for establishing these shoots into the greenhouse environment.

The Tissue Culture Laboratory

Most propagators would probably list the expense of setting up a tissue culture laboratory among the reasons for not getting involved with this technology. A lab does cost more than a mist bed! A more severe limitation is without doubt the absence of personnel skilled in the techniques of tissue culture. An individual who has had prior experience with tissue culture methodology can successfully operate a laboratory with a bare minimum of equipment. An autoclave or sterilizer, an accurate means of weighing chemicals, a small inventory of glassware, and chemical stocks or prepackaged media are all that is required to initiate cultures. The laboratory can be enlarged, more equipment added, and additional personnel hired as the need for such arises. The most critical factors are the ingenuity and experience of the laboratory manager.

As a tissue culture laboratory becomes operational, procedures other than the actual culturing process become critical. Maintenance of disease and insect free stock plants with a low titer of bacterial and fungal contamination is critical. Also important is the care and handling of the thousands of plantlets prior to their transfer to the field or containers. The ability to grow off the plantlets ultimately will limit production, rather than any limitation due to the propagation phase.

Tissue Culture Propagation of Woody Species

 Major advances in the mass propagation of woody plants have been made over the past ten years. Because of the somewhat variable response of species and cultivars to the in vitro environment, much research was (and still is) needed to define the cultural conditions required by ornamental species. As an example of the variability encountered, we have observed that seedling shoot tip cultures of crabapple produce as few as one or as many as sixty-four shoots per explant after three months in tissue culture. Similar variations in the tendency for multiple shoot production have also been noted with adult clonal material of different apple cultivars.

The number of woody plant species which have been clonally propagated through tissue culture is increasing at a rapid rate, and in fact, most commercially important ornamental species have been studied. Perhaps the best publicized of the early research on woody plants involved apples, Douglas fir, and rhododendrons. An extensive commercial scale production program for apple scion cultivars and rootstocks has developed in England and in several European countries.

Using the successes of the apple, rhododendron, and maple programs as a basis, many recent advances have been made which will facilitate the mass propagation of other woody species. While it is true that cultivar differences account for lack of success with some plants, a growing body of evidence suggests that taxonomic families behave somewhat alike in tissue culture. A large number of deciduous and evergreen Rhododendron species share common cultural requirements, the most notable of which is the requirement for the same cytokinin for adventitious shoot formation. A similar relationship exists between the trees and shrubs of the Rosaceae (including crabapples, pears, plums, and hawthorns). Most rosaceous species will likely be mass propagated on media very similar to that found to be optimal for apples. 

The anomalies alluded to by the crabapple seedling responses to tissue culture and the emerging similarities between the requirements of rosaceous trees and shrubs demonstrate that much research is needed to define the conditions necessary for commercial scale tissue culture production programs. As is often found to be the case with propagation systems, those systems described as optimal in the laboratory would likely need modification to be considered economical by the industry.

Impact of the Tissue culture Industry

As is always the case for any emerging technology, the strongest supporters for the adoption of that technology are likely those involved in its development. Having acknowledged these biases, one must now consider the advantages and limitations of tissue culture propagation, and what its economic impact on the nursery industry will be. 

Not every nursery will OPERATE a tissue culture laboratory. The trend toward specialization of nursery operations as either "propagators" or "producers" is likely to continue in the future. Operation and management of a tissue culture facility requires personnel and physical facilities which are quite different from those required by a container or field production nursery. Previously in this report where the establishment of the laboratory was considered, the relatively small initial investment in physical facilities and equipment was discussed. It should be reemphasized at this point, that the training and quality of the laboratory manager is more important than the laboratory itself, and that this expertise is not currently available among contemporary nursery propagators. 

Not every nursery will NEED a tissue culture laboratory. Commercial operations specializing in mass propagation of nursery crops can likely produce a sufficient number of plants to satisfy the market. 

As with any other technology, the success of tissue culture within the nursery industry will be determined by economic factors. Can the plantlets be produced in sufficient numbers as a cost to compare with traditional propagation? Will the propagules fit into the established production sequence in the nursery industry? Are the propagules true-to-type? These answers must be obtained on a species by species, cultivar by cultivar basis.

Publication from Aggie Horticulture®
The information, as it is presented on this Website, does not represent an endorsement
by the State of Texas or any State agency.

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Publication from Aggie Horticulture®

Lineberger, R. Daniel "The Methodology of Tissue Culture." Aggie Horticulture®, Texas A&M AgriLife Extension. 1980. Web. 24 May 2014.

Published 20 July 2015 KJ
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