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  Section: Biotechnology Methods » Tissue Culture Techniques
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Many Dimensions of Plant Tissue Culture Research

Tissue Culture Techniques
  Tissue Culture Methods
  Plant Tissue Culture
  Plant Tissue Culture (Cont.)
  Many Dimensions of Plant Tissue Culture Research
  What is Plant Tissue Culture?
  Uses of Plant Tissue Culture
  Plant Tissue Culture demonstration by Using Somaclonal Variation to Select for Disease Resistance
  Demonstration of Tissue Culture for Teaching
  Preparation of Plant Tissue Culture Media
  Plant Tissue Culture Media
  Preparation of Protoplasts
  Protoplast Isolation, Culture, and Fusion
  Agrobacterium Culture and Agrobacterium — Mediated transformation
  Isolation of Chloroplasts from Spinach Leaves
  Preparation of Plant DNA using
  Suspension Culture and Production of Secondary Metabolites
  Protocols for Plant Tissue Culture
  Sterile Methods in Plant Tissue Culture
  Media for Plant Tissue Culture
  Safety in Plant Tissue Culture
  Preparation of Media for Animal Cell Culture
  Aseptic Technique
  Culture and Maintenance of Cell Lines
  Trypsinizing and Subculturing Cells from a Monolayer
  Cellular Biology Techniques
  In Vitro Methods
  Human Cell Culture Methods

The 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 Micropropagation

“Micropropagation” is the term that 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 also 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. 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 or the differentiation of shoots directly from the callus, 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 2 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 1 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 Culture
In 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 Resistance

Perhaps 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 that are visible, but for each of these that 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 that 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. 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 Plants
Another 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. These are allowed to enlarge and proliferate under the sterile conditions of the in vitro culture with the resulting plantlets tested for presence of the pathogen (a procedure called indexing). Cultures that reveal the presence of the pathogen are destroyed, while those that 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 Hybridization
The ability to fuse plant cells from species that 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 that 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 at one extreme, to discovering a yellow-flowered African violet at 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. 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.

Plant tissue culture research is multidimensional. 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 that can have
a profound impact on the nursery industry.

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