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Somatic Cell Genetics While the practical applications of gene-splicing are yet to be realized, cell culture techniques are already proving a valuable too} in crop im- provement. For thousands of years, breeding has been based on genetic diversity and selection of desirable traits. The ability to regenerate plants from cells in culture has given rise to new techniques generally re- ferred to as somatic cell genetics that both increase the supply of genetic diversity and make possible more efficient selection. Selection Millions of cells, each a potential plant, are typically grown in a single flask in cell-suspension culture (see Cell Culture, p. 341. 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 ~ plant wheat, ~ plant I, 800, 000 plants per acre. When my colleagues do a biochemical selec- tion, they plate between 3 anc} 5 million ceils 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 ~ were a corn breeder, that would be the equivalent of 120 to 200 acres." In conventional selection, a breeder applies a herbicide, fungal path- ogen, 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 33
34 GENETIC ENGINEERING OF PLANTS 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. Re- searchers 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 Cal lus cu lture. 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 seeclling 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 prop- agating trees and other sIow-growing species. The drawback is that Cal l us cu Nature 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 celI-suspension or protoplast culture is used. In suspen- sion 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
SOMATIC CELL GENETICS ~ ~ ~~ _ ~\ ~ :~ ~~ ~ ~ :~ .% ~ ~ % .... ? it* ~~,,..,:. . ,.,: ~ -: .::.. ~ .-. :. -. S.-:. .:-::. -,:.,.-.:. - ':. " S:'S a:: ~ SS~ Corn Plants regenerated tram tissue culture. Courtesy of Calgene, I nc. 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 ~osethatability in suspension culture. In general, the plant family So~anaceae including petunia, tobacco, and potatoare the most responsive to ce~-suspension culture; the cereals and legumes are typically very difficult to regenerate. Recent results have been encour- . . . . agi ng: corn can now be regenerated from cel l-suspension cu Itu re, and the list of species is growing yearly. Protop~ast culture is the regeneration of plants from single cells from which the outer wall has been enzymatica~y removed. Because pro- top~asts must be inducer] 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 rarepotatoes and alfalfa are notable exceptions and poorly uncler- stood . I n some cases, regeneration can be ach ieved by start) ng with cells from suspension culture, rather than isolating cells directly from a plant. A concerted research effort is under way, however, because protop~asts are the preferred host ceil for gene-transfer experiments. 35
36 ^1\ Release of Protoplasts From Leaf Cells ,~ Protoplasts Regeneration of Hybrid Plantlets GENETIC ENGINEERING OF PLANTS Fused Hybrid Protoplasts ~ Protoplast From Another .~ I ~ Variety I t\ Cell Wall Regenerates and a Tissue Culture Forms 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 oppor- tunity to fuse Protoplasts from different plants to form hybrids that combine their characteristics. Photograph A shows plantlets of a wild species of clover regenerating from a tissue culture derived from an isolated protoplast. 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 of Kentucky, Lexington. 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 re- form 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
SOMATIC CELL GENETICS 37 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 instruc- tions, which may be incompatible. Researchers have had some success in fusing protoplasts of different species. Yet to date, only hybrid pro- toplasts 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 chIoroplasts or mitochonclria 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 unctifferentiated 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 differ- entiated state releases a pool of genetic diversity. William Scowcroft of the Plant Inclustries 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 phe- nomenon 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 anct 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 wincifall for plant breeders. Scowcroft is optimistic: "l believe that somaclonal variation is accessible and ~ hope that with more knowledge it will be manageable. Most certainly, ~ 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 at-
38 GENETIC ENGINEERING OF PLANTS Somoclonal variation. Plants regenerated from tissue culture often show dramatic var- iation in agriculturally important traits. Although the genetic mechanism for this so- maclonal variation is not fully understood, it can provide a valuable source of genetic diversity for plant breeding. The photographs show somaclonal variants of wheat. Variation in plant height is compared to parent plants on the extreme right and left. Seed head morphology can differ markedly from the parent type shown in the center of the top photograph. Courtesy of William R. Scowcroft, Division of Plant Industry, CSIRO, Canberra, Australia.
SOMATIC CELL GENETICS 39 tension to the primary generants- the plants regenerated clirectly from culture because much of the variation that occurs in them is unstable. Instead, they Took to their progeny to determine if traits are stably transmittecl. The stable traits resulting from somaclonal variation could be caused by a variety of mechanisms, inclucling chromosome breakage and reunion, DNA rearrangement, and point mutations the substi- tution 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, anc! 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 too} for crop improvement. 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 biochem- ical 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 dis- ease 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.
