Techniques to Improve Plant Characteristics

Karen Woodbury Hughes, professor and head, Department of Botany, University of Tennessee, Knoxville.

Plant cell and tissue culture techniques hold considerable promise for inducing new genetic variability within a species, and at least a portion of the variability so induced may have commercial potential. These techniques area valuable addition to the battery of procedures available for crop improvement today.

The development of plants with improved vigor, yield, disease resistance, and other desirable traits by either conventional or nonconventional techniques is a common goal of researchers in the plant sciences.

To improve a given crop, however, genetic variability for a desired trait must be available. When a desired trait is not available within a given crop species, the appropriate gene or genes may sometimes be introduced from a related wild species. But in many cases, the wild species and domestic crop cannot be intercrossed. So techniques for creating novel variability within the crop species are needed.

One approach involves the use of plant cell and tissue culture techniques to induce new variability.

Novel Variation Recognized

The phenomenon of novel variation appearing in tissue culture-derived plants was recognized as long as 20 years ago but considered to be undesirable by scientists who were trying to obtain identical clones. Such variants "got in their way." Recently, however, variability from culture has been recognized as having potential value.

Geneticist emasculates cuphea flowers before crossing with other cuphea species as part of a hybridization program.

The general term somaclonal variation is used to describe new genotypes, (genotypes are classes of organisms sharing a specified genetic makeup) different from the source genotype, which appear in plants regenerated from culture. Technically, however, the term should be reserved for cultures derived from somatic (body) cells. The term "gametoclone" has been suggested for plants derived from cultures of gametic tissues.

Somaclonal variability probably arises from two sources, the somatic tissues of the parent plant and the tissue culture process itself. Somehow, genetic changes occur with higher frequency in somatic tissues than they do in meristematic tissues which give rise to the germ line and which are capable of dividing indefinitely. How this happens is not well understood. But tissue culture techniques can be used to induce plant formation from somatic tissues, and this previously untapped source of variability is available to the breeder.

It may be that mobilization of transposable genetic elements causes chromosome breakage and mutations. This could explain, at least in part, the appearance of chromosomal aberrations and point mutations in cultured cells. McClintock has suggested that transposable elements are released under conditions of stress and that placing tissues in culture creates a form of stress.

A second possibility is that the rapid cell division which occurs in culture systems may overwhelm the cells' normal repair mechanisms. Rapid cell divisions also may lead to instability in the mitotic (cell division) apparatus bringing increases in a set of chromosomes (polyploidy) or increase or decrease in a single chromosome (aneuploidy).

Many studies using somaclonal variation to produce new phenotypes involve simply growing plants obtained from tissue culture and examining them for potentially useful changes. In some cases, however, it may be desirable to eliminate the majority of random changes occurring in culture by incorporating a selective agent in the culture medium, thus leaving only the desired mutant for further study.

Examples include selection for herbicide resistance, salt tolerance, toxin resistance, and biochemical pathway mutants.

If the frequency of a desired mutation arising through somaclonal variation is not high enough, the natural variability induced by the culture system can be supplemented with a mutagen, a substance that increases the frequency or extent of mutations. Somaclonal variation includes single gene mutations, chromosomal aberrations, and variation in multigene quantitative traits.

Chromosomal Aberrations

Chromosome aberrations occur in culture systems and in plants regenerated from culture. The extent of chromosomal instability ranges from slight to substantial. Numerous factors contribute to chromosome instability. Yet no characteristic instability for a particular species can be cited.

In some studies, the proportion of chromosomal aberrations is low, in other studies quite high. For example, between 55 and 70 percent of plants regenerated from alfalfa protoclones showed some type of chromosomal aberration, but in other studies few or no chromosomal changes were observed.

Changes in chromosome number observed in culture systems include increases in a set of chromosomes and increases or decreases in a single chromosome. Intrachromosomal changes such as duplications, deletions, and translocations also have occurred.

Explant Process. One source of chromosomal aberrations observed in culture is the explant source itself removing living tissue and placing it in a medium for tissue culture. Chromosome numbers in species are generally constant in tissues such as root tips and reproductive cells. However, differentiated somatic tissues of a plant may have cells that vary in chromosome number. Processes leading to chromosomal duplication include endoreduplication (chromosomes duplicate several times before mitosis) and failure of cytokinesis and endomitosis (repeated nuclear divisions without cytoplasmic division). When these tissues are placed in culture, the various levels of repetition of the basic number of chromosomes become represented in the culture system; however, as the culture ages, this distribution can change significantly.

Media Factors. Media components also can affect the frequency of chromosomal aberrations in a culture system. Media factors such as the plant hormone (auxin) 2,4-D have been implicated in the induction of chromosome aberrations including polyploidy and aneuploidy in some species; however, in studies with alfalfa, there was no correlation between levels of 2,4-D in the culture medium and the frequency of aberrations. Auxin and cytokinin (another growth substance) ratios can affect the proportion of cells with polyploidy but again, different species tend to respond differently. Species and hormone interactions apparently play a role. Coconut milk and yeast extract when added to the medium may increase the frequency of polyploidy. Relatively simple factors such as frequency of subculture, light duration and intensity, and cell density could affect the frequency of chromosomal aberrations, but there is little data in this area.

Age of Culture. Several studies have indicated that the proportion of cells with chromosome aberrations increases with the increasing age of the culture. Often changes accumulate to a level which inhibits regeneration of plants from the culture. Where regeneration does occur, cells with gross chromosomal aberrations are apparently selected against in the differentiating tissues.

There are exceptions. Extended time in culture (up to 9 months) did not significantly increase the proportion of chromosomal aberrations in a study with alfalfa. So there are species differences in response to culture factors. The induction of chromosomal aberrations depends on a number of different and poorly defined factors and may vary from species to species.

Tissue culture systems offer a mechanism for altering the chromosomal makeup of a species. Much of the somaclonal variation appearing from culture is because of changes in the number or organization of the chromosomes. In rice somaclones, potentially useful traits such as thicker stems, larger leaves, and increased grain size were associated with a spontaneous doubling of the chromosomes in culture.