Selection

Millions of cells, each a potential plant, are typically grown in a single flask in cell-suspension culture (see Cell Culture, p. 34). This offers a tremendous potential to use biochemical agents to identify and select useful variants, saving both the space and the time of screening whole plants in the field. As Stephen P. Baenziger, a plant breeder with the Agricultural Research Service, explained, "When I plant wheat, I plant 1,800,000 plants per acre. When my colleagues do a biochemical selection, they plate between 3 and 5 million cells in a petri dish. That single petri dish with an area of about 6 square inches is the equivalent to one-and-a-half to two-and-a-half acres of wheat plants. If I were a corn breeder, that would be the equivalent of 120 to 200 acres."

In conventional selection, a breeder applies a herbicide, fungal pathogen, or other selective agent directly to plants in the field. In cellular-level selection, the breeder simply douses the cells in culture with the herbicide or other selective agent, screening millions of cells at one time. The resistant cells are those that live. They would then be regenerated to see if the trait is still expressed in the whole plant. If the regenerated plant retains resistance, then its progeny must be evaluated to see if the trait is stably inherited.

Not all traits can be selected as easily as herbicide resistance, where the trait itself is the selective agent. For other characteristics, such as height, there is no direct biochemical assay at the cellular level. Researchers are looking for other biochemical markers that will enable them to select such traits in culture.

CELL CULTURE

There are three methods for regenerating plants from cells in culture: callus, cell-suspension, and protoplast culture. The most reliable of these is callus culture. Through experiments with agricultural species began just a few years ago, many can now be routinely regenerated from callus culture. In this approach, a tiny piece of tissue is snipped from a seedling shoot or other appropriate plant part and placed in a petri dish containing the plant hormones auxin and cytokinin, along with organic and inorganic nutrients. The cells grow and divide, forming a mound of undifferentiated cells called a callus. When transferred to a regeneration medium, the cells in the callus differentiate into roots and shoots, which then grow into plants.

Since hundreds to thousands of plants can be regenerated from one piece of tissue, callus culture offers a means of cloning far more plants in less time than is possible using conventional vegetative propagation. Indeed, since the 1960s, callus culture has been used in the mass cloning of orchids and other horticultural plants that are difficult or costly to propagate otherwise. It is also a promising technique for propagating trees and other slow-growing species. The drawback is that callus culture is labor-intensive and expensive. For that reason, it is not yet being used commercially for the propagation of any agricultural crops.

Most crop improvement schemes involving genetic engineering hinge on the ability to regenerate plants from single cells, not clumps of tissue. For that, either cell-suspension or protoplast culture is used. In suspension culture, a piece of callus is agitated in a flask containing a liquid medium. The callus breaks apart into single cells or clumps of two or

Figure

Corn plants regenerated from tissue culture. Courtesy of Calgene, Inc.

more cells. These cells then regenerate either by forming roots and shoots or else by forming somatic embryos, which then differentiate into entire plants.

Achieving regeneration from single cells is far more difficult than starting from a clump of tissue. Many species that readily regenerate from callus lose that ability in suspension culture. In general, the plant family Solanaceae—including petunia, tobacco, and potato—are the most responsive to cell-suspension culture; the cereals and legumes are typically very difficult to regenerate. Recent results have been encouraging: corn can now be regenerated from cell-suspension culture, and the list of species is growing yearly.

Protoplast culture is the regeneration of plants from single cells from which the outer wall has been enzymatically removed. Because protoplasts must be induced to re-form their cell walls and then proceed through callus and somatic embryogenesis, this culture technique is more complicated than the other two. Successes in crop plants are rare—potatoes and alfalfa are notable exceptions—and poorly understood. In some cases, regeneration can be achieved by starting with cells from suspension culture, rather than isolating cells directly from a plant. A concerted research effort is under way, however, because protoplasts are the preferred host cell for gene-transfer experiments.

Figure

Protoplast fusion. Isolated protoplasts from some plants such as petunia, potato, and clover can easily be cultured and regenerated into whole plants. This offers the opportunity to fuse protoplasts from different plants to form hybrids that combine their (more...)

Figure

Photograph A shows plantlets of a wild species of clover regenerating from a tissue culture derived from an isolated protoplast.

Figure

Photograph B is a hybrid protoplast created by fusing a protoplast of red clover with one from the wild species. The darker regions of this hybrid protoplast are from red clover. Courtesy of Glenn B. Collins and Jude Grosser, Agronomy Department, University (more...)

Protoplast Fusion

Under appropriate conditions protoplasts from different plants can be induced to fuse in culture, combining their genetic information to create a new hybrid. The recombinant protoplast can then be induced to reform a cell wall, proliferate, form callus, and regenerate. This technique can be used to fuse protoplasts of the same species or of different species. It is the latter possibility that has engendered the most excitement: the creation of entirely new hybrids from species that cannot be crossed sexually. It is also the most speculative. It would combine within one cell wall two complete—and different—sets of developmental instructions, which may be incompatible. Researchers have had some success in fusing protoplasts of different species. Yet to date, only hybrid protoplasts from closely related species have been induced to regenerate from culture. In addition, fusion lacks the precision of gene-splicing, in which a specific gene can be transferred to create a carefully tailored plant. One approach to both of these problems is to delete part of the genome in one of the two protoplasts. For example, cytoplasmic traits—those controlled by the DNA in chloroplasts or mitochondria—could be transferred separately by using a donor protoplast from which the nucleus was removed.

Somaclonal Variation

Cell culture is also making available a new, unanticipated source of genetic diversity. It was originally assumed that plants regenerated from the same clump of tissue would be identical. Yet many of the plants arising from undifferentiated cells in culture are strikingly different from each other and the parent plant from which the culture was derived.

In some as yet unknown way, the process of culturing cells—of going from a differentiated state to an unorganized state and back to a differentiated state—releases a pool of genetic diversity. William Scowcroft of the Plant Industries Division of CSIRO in Australia likens it to a genetic earthquake moving through the genome, rearranging the genetic information. The exact cause of this somaclonal variation, as it is called, is uncertain, although theories abound. What is clear is that the phenomenon is ubiquitous, occurring in rice, corn, wheat, barley, potato, alfalfa, rape, and other species, and affecting many agronomically useful traits. In several species, for instance, the somaclonal variants include resistance to diseases: sugarcane has developed resistance to eyespot disease, Fiji virus, downey mildew, and smut; potatoes to late and early blight; corn to Southern corn leaf blight; and oil seed rape to vitricular disease.

If it can be harnessed, if useful variants can be selected from culture and used for breeding, this variation could be an unexpected windfall for plant breeders. Scowcroft is optimistic: ''I believe that somaclonal variation is accessible—and I hope that with more knowledge it will be manageable. Most certainly, I believe it is applicable for the real world of plant breeding.''

Scowcroft and his colleagues are looking for useful variants arising from the culture of wheat. According to Scowcroft, they pay scant attention to the primary generants—the plants regenerated directly from culture—because much of the variation that occurs in them is unstable. Instead, they look to their progeny to determine if traits are stably transmitted. The stable traits resulting from somaclonal variation could be caused by a variety of mechanisms, including chromosome breakage and reunion, DNA rearrangement, and point mutations—the substitution of a single nucleotide base. At this stage, however, research is just beginning on the causes of somaclonal variation. The amount of variation appears to be affected by several controllable factors, including length of time the cells are in culture, the genotype, the medium, and the culture conditions. An understanding at the molecular level of the factors that control the stability or instability of the plant genome would provide another powerful tool for crop improvement.

Figure

Somaclonal variation. Plants regenerated from tissue culture often show dramatic variation in agriculturally important traits. Although the genetic mechanism for this somaclonal variation is not fully understood, it can provide a valuable source of genetic (more...)

Scowcroft and colleagues have tracked useful variants through several generations. Some plants differ in just one trait; others have multiple changes. One of the most unexpected, and welcome, findings is that stable variation occurs in multigene traits, such as height and maturation date, as well as in single-gene traits. In wheat, they have found a variation in height, color, number of side shoots (tillers), and the shape of the awns that surround the grains. Variation also occurs in biochemical characteristics, such as the production of alpha amylase enzyme and in the seed storage proteins. Many of these are potentially useful.

Scowcroft's research group is already attempting to use somaclonal variation in wheat improvement. Specifically, they are screening cells in culture for traits that would be useful in no-till farming, which is becoming increasingly prevalent as a means of conserving soil. Some existing cultivars are poorly suited for no-till farming. Desired new traits include rapid establishment, herbicide tolerance, winter habit, and disease resistance. Somaclonal variation may also provide genotypes suited for tropical environments—genotypes able to tolerate heat or acidic soils containing harmful levels of aluminum and manganese.