The Many Dimensions of Plant Tissue Culture ResearchR. Daniel Lineberger
Professor of Horticulture
Texas A&M University
College Station, TX 77843
Webmaster of Aggie Horticulture (http://aggie-horticulture.tamu.edu/)
INTRODUCTIONThe practice of plant tissue culture has changed the way some nurserymen approach plant propagation. In the recent past, the applicability of this technology to the propagation of trees and shrubs has been documented. Some firms have established tissue culture facilities and commercial scale operations are presently in operation for the mass propagation of apples, crabapples, rhododendrons, and a few other selected woody species. The intent of this research update is to briefly examine "what is being done" and to explore "what can be done" with regard to the tissue culture of ornamental plants. Such a consideration necessarily includes an overview of tissue culture as a propagation tool. The major impact of plant tissue culture will not be felt in the area of micropropagation, however, but in the area of controlled manipulations of plants at the cellular level in ways which have not been possible prior to the introduction of tissue culture techniques.
THE ART AND SCIENCE OF MICROPROPAGATIONOf all the terms which have been applied to the process, "micropropagation" is the term which best conveys the message of the tissue culture technique most widely in use today. The prefix "micro" generally refers to the small size of the tissue taken for propagation, but could equally refer to the size of the plants which are produced as a result.
Micropropagation allows the production of large numbers of plants from small pieces of the stock plant in relatively short periods of time. Depending on the species in question, the original tissue piece may be taken from shoot tip, leaf, lateral bud, stem or root tissue (Fig. 1). In most cases, the original plant is not destroyed in the process -- a factor of considerable importance to the owner of a rare or unusual plant. Once the plant is placed in tissue culture, proliferation of lateral buds and adventitious shoots (Fig. 2) or the differentiation of shoots directly from callus (Fig. 3), results in tremendous increases in the number of shoots available for rooting. Rooted "microcuttings" or "plantlets" of many species have been established in production situations and have been successfully grown on either in containers or in field plantings. The two most important lessons learned from these trials are that this methodology is a means of accelerated asexual propagation and that plants produced by these techniques respond similarly to any own-rooted vegetatively propagated plant. Micropropagation offers several distinct advantages not possible with conventional propagation techniques. A single explant can be multiplied into several thousand plants in less than one year. With most species, the taking of the original tissue explant does not destroy the parent plant. Once established, actively dividing cultures are a continuous source of microcuttings which can result in plant production under greenhouse conditions without seasonal interruption. Using methods of micropropagation, the nurseryman can rapidly introduce selected superior clones of ornamental plants in sufficient quantities to have an impact on the landscape plant market.
PLANT IMPROVEMENT THROUGH TISSUE CULTUREIn introducing this research update, it was mentioned that the major impact of tissue culture technology would not be in the area of micropropagation, but rather in the area of controlled manipulations of plant germplasm at the cellular level. The ability to unorganize, rearrange, and reorganize the constituents of higher plants has been demonstrated with a few model systems to date, but such basic research is already being conducted on ornamental trees and shrubs with the intent of obtaining new and better landscape plants.
SELECTION OF PLANTS WITH ENHANCED STRESS OR PEST RESISTANCEPerhaps the most heavily researched area of tissue culture today is the concept of selecting disease, insect, or stress resistant plants through tissue culture. Just as significant gains in the adaptability of many species have been obtained by selecting and propagating superior individuals, so the search for these superior individuals can be tremendously accelerated using in vitro systems. Such systems can attempt to exploit the natural variability known to occur in plants or variability can be induced by chemical or physical agents known to cause mutations.
All who are familiar with bud sports, variegated foliage and other types of chimeras have an appreciation for the natural variability in the genetic makeup or expression in plants. Chimeras are the altered cellular expressions which are visible, but for each of these which are observed many more differences probably exist but are masked by the overall organization of the plant as a whole. For example, even in frost-tender species, certain cells or groups of cells may be frost hardy. However, because most of the organism is killed by frost, the tolerant cells eventually die because they are unable to support themselves without the remainder of the organized plant. Plant tissues grown in vitro can be released from the organization of the whole plant through callus formation. If these groups of cells are then subjected to a selection agent such as freezing, then those tolerant ones can survive while all those which are susceptible will be killed. This concept can be applied to many types of stress as well as resistance to fungal and bacterial pathogens and various types of phytotoxic chemical agents. The goal of selecting such resistant cell lines would be to reorganize whole plants from them which would retain the selected resistance (Fig. 4). Current research in this area extends across many interests including attempts to select salt tolerant lines of tomato, freezing resistant tobacco plants, herbicide resistant agronomic crops, and various species of plants with enhanced pathogen resistance. Imagine, if you will, the impact of a fireblight-resistant Bartlett pear, a clone of pin oak for alkaline soils, or a selection of southern magnolia hardy to zone 4!
TISSUE CULTURE AND PATHOGEN FREE PLANTSAnother purpose for which plant tissue culture is uniquely suited is in the obtaining, maintaining, and mass propagating of specific pathogen-free plants. The concept behind indexing plants free of pests is closely allied to the concept of using tissue culture as a selection system. Plant tissues known to be free of the pathogen under consideration (viral, bacterial, or fungal) are physically selected as the explant for tissue culture. In most cases, the apical domes of rapidly elongating shoot tips are chosen (Fig. 5). These are allowed to enlarge and proliferate under the sterile conditions of in vitro culture (Fig. 6) with the resulting plantlets tested for presence of the pathogen (a procedure called indexing). Cultures which reveal the presence of the pathogen are destroyed, while those which are indexed free of pathogen are maintained as a stock of pathogen-free material. Procedures similar to these have been used successfully to obtain virus-free plants of a number of species and bacteria-free plants of species known to have certain leaf spot diseases. The impact of obtaining pathogen-free nursery stock can only be speculative, since little research documenting viral, bacterial, or fungal diseases transmitted through propagation of woody ornamentals is available.
SOMATIC HYBRIDIZATIONThe ability to fuse plant cells from species which may be incompatible as sexual crosses and the ability of plant cells to take up and incorporate foreign genetic codes extend the realm of plant modifications through tissue culture to the limits of the imagination. Most such manipulations are carried out using plant "protoplasts". Protoplasts are single cells which have been stripped of their cell walls by enzymatic treatment. A single leaf treated under these conditions may yield tens of millions of single cells, each theoretically capable of eventually producing a whole plant. This concept has fueled speculation as diverse as the possibilities of obtaining nitrogen-fixing corn plants on the one extreme to discovering a yellow-flowered African violet on the other extreme.
The observation that has provided the impetus for most of this research is that when cells are stripped of their cell walls and brought into close contact, they tend to fuse with each other (Fig. 7). This "somatic hybridization" is not subject to the same incompatibility problems that limit traditional plant breeding strategies. It is conceivable then that one could hybridize a Juneberry with a crabapple or a plum, but the fundamental research required to demonstrate such an event has yet to be conducted.
The potential use of somatic hybridization to bring about novel combinations of genetic material has been demonstrated in the genera Petunia and Nicotiana. Research funded in part by the Horticultural Research Institute at the University of Wisconsin is investigating the feasibility of using such techniques with woody species. Brent McGown and co-workers have succeeded in obtaining naked cells from tissue cultures of Betula and Rhododendron, but as of yet, they have neither obtained plants from single cells not achieved cellular fusion. However, further research in this area promises to have a tremendous impact on our concepts of woody plant diversity. Just as remarkable as the idea of fusing plant protoplasts is the idea of incorporating foreign genetic material into the genetic code of plant cells. Such transformations have been carried out in the so-called "gene-splicing" experiments where the information for making insulin was incorporated into bacteria. Not only is the desired information transmitted to succeeding generations of bacteria, but the bacterial cultures become synthesizers of insulin as well. Plant cells can be made to take up foreign genetic codes, but evidence that this can be transmitted into the daughter cells and serve the intended function is lacking. What if, for example, the genetic information for accumulating a very high sugar content is incorporated into a clone of sugar maples? One could think of enough what ifŐs in this category to fill several volumes!
SUMMARYPlant tissue culture research is multi-dimensional. While most nurserymen have been introduced to the techniques and advantages of micropropagation, few have ventured to use it as a propagation tool. The applicability of micropropagation for woody trees has been demonstrated as feasible since all aspects of the technology have confirmed the fact that trees produced by this method look like and grow like their counterparts produced by traditional methods of cloning.
Other dimensions of tissue culture research have been less well publicized. The potential for selecting pathogen free plants, for selecting stress-tolerant and pathogen-resistant clones of plants, and the novel genetic combinations to be achieved through somatic hybridization are all lines of research which can have a profound impact on the nursery industry.