What a joy life is when you have made a close working partnership with Nature, helping her to produce for the benefit of mankind new. . . fruits in form, size, color, and flavor never before seen on this globe; and grains of enormously increased productiveness, whose fat kernels are filled with more and better nourishment, a veritable store-house of perfect food – new food for all the world’s untold millions for all time to come.
Luther Burbank (1925) Lecture in San Francisco
Introduction
The ability of the plant breeder to create new genetic variation was enormously increased in the mid twentieth century by the invention of tissue culture and the use of growth regulators.62Attempts at wide crossing, as discussed in the previous chapter, were often frustrated by the incompatibility of genomes from relatively distant species. Embryo rescue could sometimes help, but one of the most crucial advances came with the development of chemically induced chromosome doubling, which has been the key to the success of many crop breeding programmes. As well as making possible much wider genetic crosses, chromosome doubling has enabled the use of powerful methods such as somatic hybridisation and haploid breeding, which have been especially useful in developing countries. In the past few decades, the technique of mass propagation has also been of considerable benefit in breeding programmes for tree crops, most of which are too long lived to be accessible to the sorts of approaches developed for the much shorter lived annual crops. The development of methods to prevent seed pro- pagation is another important target for many commercial breeding programmes. Over the past century, new techniques have been devised either to induce fruit or seed sterility, or to prevent seed saving by using hybrid varieties.
In this chapter, we will also see that tissue culture was the key to enabling the transfer of exogenous genes (transgenesis), which is the basis of modern genetic
engineering. Indeed, even today, more than two decades after the first transgenic plants were produced, the efficiency of gene transfer in many crop species is still limited by the capacity of the plants to be cultured and regenerated in vitro, rather than by the ability to transfer exogenous genes per se. Tissue culture has been used in breeding programmes for over 50 years and is now used widely for the improvement of many of our most important crops, including all of the major cereals as well as potatoes, brassicas and even some trees.63Meanwhile, we should remember that the creation of variation is only one of the twin foundations of plant breeding. Breeders also need effective and efficient methods for the identification and selection of variants likely to be agronomically useful. Many traits, such as disease resistance or flour- milling quality, are invisible to the naked eye. There has been striking progress in this frequently overlooked aspect of breeding. Therefore, we will finish the chapter by looking at some of the important developments in screening and selection of the huge numbers of new genetic variants created by all of the above technologies. From gas chromatography and mass spectroscopy to automated sequencing and polymerase chain reaction (PCR), a host of new analytical and screening technologies has enabled breeders to progress from crudely processing a few dozen samples a day to automated, round-the-clock analyses of many thousands of plants in exquisite molecular detail.
Tissue culture technologies Chromosome doubling
One of the most important technologies that has made possible the creation of fertile varieties of interspecific hybrids, such as triticale, brassicas, and many other wide crosses, is the chromosome doubling method. Wide-hybrid plants are often sterile, which means that their seeds cannot be used for further propagation. This sterility is due to the great differences between the sets of chromosomes that these plants inherit from their two divergent parental species. Typically, their chromosomes are so dif- ferent that they are unable to form stable pairs during meiosis (the special form of cell division that precedes formation of haploid pollen and egg cells). However, if the number of chromosomes can be artificially doubled, the hybrid can produce func- tional pollen and eggs and should therefore be fertile. But how could a breeder possibly persuade a cell to double its chromosome number without dividing into two daughter cells? The answer came in 1937 from a rather surprising direction, namely an attractive European meadow flower, Colchicum autumnale (naked lady or autumn crocus), that produced a toxic alkaloid called colchicine.
Colchicine was initially used as a pesticide because of its particular toxicity to insects. Further research soon revealed that this chemical also had the interesting
property of causing the number of chromosomes in a cell to double, without indu- cing cell division. Plant breeders began to experiment with its use for rescuing the fertility of wide hybrids. As a toxin and a mutagen, colchicine did not always give the desired results, and this is still the case today. For example, there are some important crop species, including maize, where its use is not very effective. However, by and large, breeders have been able to minimise the undesirable side-effects of colchicine, while still achieving the desired chromosome doubling in cultured cells and tissues. And despite its toxicity to humans, colchicine is also used to treat several serious diseases, including chemotherapy for some cancers and for inflammatory conditions like chronic gout. As early as the 1940s, colchicine was being used routinely to double chromosome numbers in plants, and thereby to restore fertility to otherwise sterile wide hybrids. Within a few years, the chromosome doubling technique had been applied to more than fifty plant species, including most of the important annual crops and many fruits. Colchicine treatment can also be used to create seedless fruits. Among many commercial fruits developed with the aid of colchicine is the highly popular seedless watermelon. The technology is also widely used in the production of ornamental plants. Colchicine is also important for production of wide crosses and somatic hybrids, as discussed below. More recently, several additional chromosome doubling agents, all of which act as inhibitors of mitotic cell division, have been identified and used successfully in plant breeding programmes.64To date, thanks to colchicine and chromosome doubling technology, dozens of our most important crops have been improved and hundreds of new varieties have been produced around the world.
As with mutagenesis, it is interesting to speculate on the reception that colchicine technology would get if it were discovered today. Because it was originally developed as a tool in the 1940s, colchicine use is nowadays regarded as part of ‘conventional’ plant breeding. And yet, as breeders have always known, this technology entails the addition of a known toxin and carcinogen to artificially cultured plant cells, in order to disrupt and manipulate their genomic DNA. The resulting chemically induced chromosome doubling does not happen ‘naturally’ (i.e. in the absence of human intervention). The technology involves the use of toxic chemicals to force the pro- duction of artificial hybrids from unrelated species in order to transfer alien genes into crops.65 One suspects that anti-GM campaigners would vehemently condemn colchicine technology as constituting an ‘unnatural intrusion into the integrity of a species’, or some such formulation. That would be a pity. Consider the immense public good that we already know has come out of colchicine technology, not to mention many of the other genetically intrusive manipulations of twentieth century plant breeders. Once again, there are some interesting parallels to be drawn with the contemporary debate on GM technology for crop improvement.
Mass propagation
Another application of tissue culture that had been of great utility for breeders is the mass clonal propagation of certain types of mainly non-annual crops, particularly some of the important, long-lived plantation species. Until relatively recently, very little systematic breeding had been done on such perennial or tree crops, and hardly anything was known about their genome organisations. This is despite the fact that perennial or tree crops include nearly all of our most important sources of edible fruits, nuts and several vegetable oils. Some of the most popular fruit trees include the traditional citruses, such as lemons, limes and oranges, as well as many new citrus crops, such as tangelos, mandarins, satsumas, sweet grapefruit and navel oranges. In recent years, breeders have also started to develop a vast range of new vine-like cultivars of former orchard crops like apples and pears. Popular tree nuts include walnut, almond, pecan, hazel, macadamia, Brazil and pistachio.66Major oil- bearing tree crops include olive, oil palm and coconut. A particular problem with the breeding of trees is their long lifespans compared to the annual crops. For example, an oil palm tree will not bear a useful crop of fruit until it is about seven years old. After that, the trees have a commercial lifetime averaging about 25 years, although an oil palm tree can still produce fruit for over 50 years.
These factors have made it impractical to set up classical breeding programmes for trees, but there is an alternative. Rather than laboriously trying to breed a tree species as if it were a short-lived annual crop like rice, the breeder can use mass clonal propagation as a much faster and cheaper alternative to multiplying up the best genetic stock. Based on traits such as yield, quality and disease resistance, breeders will typically select a few of the best-performing trees, or sometimes just one especially good tree, for propagation. Tissue cuttings, typically of stems or leaves, are then taken from the chosen tree(s) for cultivation in a mixture of nutrients and growth regulators until tiny plantlets are regenerated. The plantlets are subcultured on a massive scale until thousands, or even millions, of new seedlings have been produced. Finally, the batch of plantlets is taken for replanting in the field. In this way, a single elite tree can give rise to an entire plantation, or even a whole series of plantations, in a very short period.
The obvious downside to this technique is that all of the trees from a particular propagation programme could well end up being genetically identical clones. This may be fine if the clones behave exactly like their elite clonal parent, although in the long term their genetic uniformity might still render them dangerously vulnerable to new diseases to which they may have no resistance. However, there is another risk with the use of mass clonal propagation and that is the creation of abnormalities during the tissue culture process itself. As we saw in the previous chapter, tissue
culture of plants can result in somaclonal mutations that are sometimes deleterious, leading to abnormal growth or sterility of the trees after they are planted in the field. Despite the potential drawbacks, mass propagation of clonal lines from a few elite individuals is now commonplace in the breeding of many improved orchard crops, as well as some of the new biomass crops like miscanthus.67 The need for the rapid multiplication of millions of in vitro produced seedlings of many crops has now led to development of high-tech, automated methods for their clonal propagation.68
Clonal propagation has not always been commercially successful, however. In the 1980s, a commercial scheme to mass propagate millions of oil palm plantlets from a superior breeding line foundered when many of the maturing trees were discovered to have a serious abnormality in their floral development that had been induced during tissue culture.69 This so-called ‘mantling’ phenotype led to a failure of fruit formation and, since the major products of the crop are fruit oils, the trees were effectively useless.70In the case of oil palm, the problem was compounded by the fact that fruits do not normally appear on the plant for about five years. This meant that the abnormalities were not discovered until the trees were already established in mature plantations that had been expensively maintained for several years. Although some of these challenges have now been rectified by further research, commercial confidence in clonal propagation has not fully recovered and relatively little planting of clonal oil palm was done during the succeeding decades. It is only in recent years that relatively small-scale clonal propagation programmes have resumed in some of the more advanced plantations.71This episode illustrates some of the problems that can arise from tissue culture. Many of the chemicals used in culturing and regen- erating plants can cause developmental abnormalities, and even mutations. Despite these disadvantages and a few expensive setbacks, tissue culture and mass-propa- gation technology has proved to be an immensely valuable addition to the toolkit of the modern plant breeder. Today, clonal forestry is widely used in the management and improvement of a range of commercial plantation crops, including poplars, eucalypts, acacia and cedar.72
Somatic hybridisation
Somatic hybridisation is used to introduce novel genes into a crop genome from a donor species with which the crop will not normally interbreed.73In this respect, it is similar in its aims to the forms of conventional assisted hybridisation that we have already considered, although, as we will see, somatic hybridisation involves a more radical technological approach. Somatic hybridisation is yet another way of enhancing variation in crop species by importing genes, or even whole chromosomes, from other species that are not related closely enough for normal sexual crossing.
For example, if the crop species and the donor species are not closely related, it may not be possible to get the pollen of one of the species to fertilise eggs of the other. In such cases, even embryo rescue techniques are of no avail because there is no fertilisation and hence there are no embryos to rescue. An analogy in animals would be attempting to hybridise a species like a mouse with a human – because the sperm and eggs of these two species cannot fuse, such a hybridisation is normally impos- sible, even in vitro. However, the development of sophisticated microinjection and cell fusion techniques in the 1960s and 1970s allowed researchers to fuse whole cells or parts of cells to create a new composite cell.
In the case of plants, it is possible to take a cell from one species and to fuse it with another cell from a totally unrelated species. When this happens, the nuclei from the two different species will also fuse to create a hybrid nucleus that contains both sets of parental genes. From such hybrid cells, new adult hybrid plants can be regener- ated. Scientists had been attempting to fuse plant cells since the beginning of the twentieth century. As early as 1909, German botanist E. Kuster was able to fuse plant cells whose cellulose walls had been removed (i.e. protoplasts), although the products did not survive further culture. The first report of interspecific hybridisa- tion via protoplast fusion in two species of Nicotiana was published in 1972.74 In addition to fusing two complete cells together, it is also possible to perform what is termed an asymmetric cell fusion. This technique involves the use of micro-dissection to transfer part of the nucleus of one cell into another cell. In this way, it is possible to transfer a small number of chromosomes from, say, a wild species that is unrelated to a particular crop into a cell from the crop plant. The resultant asymmetric hybrid cell can be treated with colchicine to induce chromosome doubling, hence stabilising the distinctly odd new genome. The next challenge is to coax the hybrid cells to divide and then to differentiate into a new adult hybrid plant.75
Since the 1970s, protoplast fusion has been used to create new types of plant from combinations of unrelated species, and the technique has also been very useful in several areas of basic research.76In at least one case, cultured human cells were fused with tobacco protoplasts, which demonstrated the potential for using somatic hybridisation to transfer genetic information between virtually any species of eukaryotic organism.77 Somatic hybridisation was introduced into crop breeding programmes in the early 1980s and has so far been used to create several new commercial varieties of potato and oilseed rape. The technique has been attempted with many other crops but the main technical hurdle at present is the instability of the new genome combination created by the fusion of chromosomes from two dis- similar species. To a great extent, somatic hybridisation has been replaced over the past decade by transgenesis, with its greater precision, fewer problems with genome instability and higher overall success rate. However, transgenesis is only of use when
there is a known gene(s) to be transferred. Many useful traits are controlled by unknown sets of genes and can only be transferred into a crop by adding an entire donor genome, or at least a substantial portion thereof. In such cases, we come back to the various forms of hybridisation as the only recourse for the breeder.
In recent years, breeders have started to return in greater numbers to explore the potential of somatic hybridisation, especially in fruit crops like the citrus group.78 The reasons for this development are threefold. First, it has become obvious that transgenesis is by no means a quick and easy option for variation enhancement in crops, especially perennial species like trees. Second, tissue culture and molecular marker techniques have improved considerably over the past decade, which has increased the rate of success in the regeneration of genetically stable progeny from somatic hybridisation programmes. Third, somatic hybridisation is not regarded by regulatory authorities around the world as ‘genetic manipulation’ (as in GM or transgenic). This means that varieties produced by this technology are not subject to the same burden of regulatory approval and testing as transgenic varieties, which has created a business opportunity for breeders in private sector companies, who possess the necessary cell culture expertise to exploit these hybrids commercially. An example of such a company is Green Tec GmbH, which is a spinoff from a noted public research institute in Germany, the Max Planck Institute for Plant Breeding Research. The advantages of somatic hybridisation over transgenic technology are summarised on the Green Tec website as follows:
The somatic hybrids created by our technology are not considered as GMOs (genetically modified organisms) and are not regulated according to the genetic engineering directives of the EU. Therefore, the market approval is not restricted by lengthy and costly investigation imposed for genetically engineered food and feed varieties but only by normal variety