Somaclonal Variation and Induced Mutations in Crop Improvement (1998)
edited by S.M. Jain, D.S. Brar, B.S. Ahloowalia

P. J. Larkin

The Controversy over Somaclonal Variation

It would be fair to say that the phenomenon of somaclonal variation has been controversial from the beginning. Of course observations of variant plants regenerating from cell and tissue cultures preceded the coining of the terms phenovariants (Sibi, 1976), calliclonal (Skirvin and Janick, 1976), protoclonal (Shepard, 1981), somaclonal (Larkin and Scowcroft, 1981) or gametoclonal variation (Evans et al., 1984). The controversy at first focused on whether the variation existed at all. Some argued that the genetic differences claimed were due to poorly designed experiments, stray pollen or poor controls. Few now doubt that somaclonal variation occurs. Metakovsky et al. (1987) argued that the variation we reported (Larkin et al., 1984) in the patterns of wheat storage proteins in first or second generation grain of regenerated plants was the result of heterozygous material going into culture or stray pollen after culture. More recently, the same authors have published their own evidence that somaclonal variation does in fact occur for wheat gliadins (Upelniek et al., 1995). Our own early work with wheat was in fact done with rigorous controls and painstaking pedigrees of every source plant and every plant regenerated from culture (Larkin et al., 1984). The rigour of the controls was because we anticipated the scepticism, and indeed, carried the burden of our own early doubts.

The controversy shifted to whether somaclonal variation could be useful. The debate in this aspect continues. Indeed, I have had something of a journey on this issue myself. This book will surely make a major contribution to that debate. The authors of the chapters in the second half of the book will themselves be divided on this question, and some will be awaiting further data.

Is Somaclonal Variation Different from Chemical- or Radiation-induced Mutation?

It is still not possible to answer this question with confidence. At the superficial level, there often appears to be a similarity of scope of traits affected. In the cereals the most commonly observed mutations from both methods are those affecting height, awns, head morphology and maturity (heading date). However, this should have been expected, because these are the characteristics of the plants that are most readily observed. In other words, it is a reflection of the common and casual method of screening rather than evidence of a common mechanism or basis.

When screening for mutations becomes more specific and analytical, different outcomes from the two approaches can readily emerge. Sree Ramulu (1982) supplies an excellent example of this. He studied the S-alleles which govern the gametophytic incompatibility in Lycopersicon. Extensive and varied induced mutagenesis had failed ever to produce a new S-allele. However, a very small sample of somaclones (from cultured anthers of L. peruvianum) revealed a number of new S-specificities in both genotypes in the study. Those new specificities were stable and simply inherited.

Another interesting and accessible point of comparison is the relative frequency of dominant mutation. Brock (1979) analysed decades of induced mutagenic studies and estimated that dominant mutants occur at a frequency of 1% of recessives. My scanning of the somaclonal literature suggests the frequency of dominant somaclonal mutations might be closer to 10%.

Somaclonal mutation also appears to be distinguishable by the putative occurrence of homozygous mutants. The examples continue to grow of non-segregating variants arising directly in the primary regenerants (Sibi, 1976; Evans and Sharpe, 1983; Sun et al., 1983; George and Rao, 1983; Larkin et al., 1984; Gavazzi et al., 1987; Kaeppler and Phillips, 1993). Mitotic recombination has been proposed as a possible mechanism of homozygous somaclonal variation. However, a recent study by Xie et al. (1995) failed to demonstrate mitotic recombination at two test loci. Another hypothesis to explain this phenomenon is that culture often involves cell lineages which become haploid (or monosomic) for a few cell generations. If the mutation occurs on a monosomic chromosome which subsequently doubles, it will be in the homozygous condition. Giorgetti et al. (1995) present evidence for the fascinating possibility that cultured plant cells can undergo a form of somatic meiosis to produce haploid cells.

Where Does Somaclonal Variation Come From?

In a previous report, the types of genetic changes associated with culture-induced mutation have been reviewed (Larkin, 1987). These include point mutations, changes to methylation patterns, altered sequence copy number, transposable element movements and chromosomal rearrangements. It is difficult to conceive a single underlying basis for such disparate genetic consequences. Phillips et al. (1994) recently proposed a connection to repeat-induced point mutation (RIP) in Neurospora. According to the RIP hypothesis, newly C-methylated sequences are prone to deamination (to form thymine) which leads to point mutations. The initial C→T transitions can be mismatch repaired with a non-methylated cytosine; or it can be mismatch repaired to give a G→A point mutation. This new methylated A might retain or lose its methylation in subsequent replication. This is particularly interesting in the light of our recent demonstration that gene promoters can be far more active in plant cells with A methylation than without (Graham and Larkin, 1995). It is well known that C methylation can lead to suppression of gene activity. Therefore, the initial change in C methylation can have waves of consequences through subsequent replication, including point mutations and alterations in promoter activity.

At a higher level, much of somaclonal variation probably arises by genetic recombination and chromosomal rearrangement of one sort or another. In other words, it is not so much a phenomenon of creating variation but rather of uncovering variation. We might argue that there is a genetic resource within the plant. Chromosomal rearrangement might bring about new juxtapositions of genes and controlling sequences, thereby silencing previously active genes and activating others. The recent domesticated history of the major crops and their breeding is likely to have already exploited this variation to a considerable extent. Or perhaps a long history of agronomic selection has already come close to optimizing what we may call this internal genotype. If this is true, we might predict that somactonal variation will frequently be deleterious in the highly bred species. An informative recent study in rice by Mezencev et al. (1995) demonstrates the preponderance of deleterious changes but nevertheless the occurrence of some favourable ones. Other rare and favourable somaclonal mutants in major crops include: the Piricularia resistant rice cultivar Dama (Hezsky and Simon-Kiss, 1992); drought-tolerant rice (Adkins et al., 1995); glyphosate-tolerant maize (Racchi et al., 1995).

But other species have a shorter history of conventional breeding, are vegetatively propagated or for other reasons might be considered unsophisticated in breeding terms. Favourable variation has been easier to find in such species, for example: yam, Dioscorea floribunda, with increased diosgenin content (Sen et al., 1991); Japanese mint, Mentha arvensis, with increased herb and oil yield (Kukreja et al., 1991); Indian mustard, Brassica juncea, with low glucosinolates (Palmer et al., 1988); fall armyworm resistance in bermudagrass, Cyanodon dactylon (Croughan et al., 1994); heat- and drought-tolerant dallisgrass, Paspalum dilitatum (Tischler et al., 1993); and a new high-yielding, shattering-resistant Indian mustard cultivar (Katiyar and Chopra, 1995).

It seems likely that the resource of genetic variation exploited by cell culture is available and accessible by other means. Simply the process of intercrossing genetically diverse types may unleash more variation than is evident in the parents themselves. One advantage of writing an Introduction is that I am able to make such a sweeping statement without the burden of proof and simply to be provocative. Episodes of genomic shock have been known to occur by other means such as radiation, environmental extremes or interspecific crossing. It appears that cell culture is a particularly effective means to induce such variation.

Breeders have not extensively exploited somaclonal variation. There are varied reasons for this. One is that there has been little time to assemble well-characterized demonstrations of utility that would be necessary to persuade those associated with plant improvement to invest the time and energy into it. For many too, there is a technological barrier which means they have not had the resources to attempt this. In an environment of limited resources, most breeders prefer to invest, in more established methods. Indeed I have counselled many away from somaclonal variation when the problem before them is one which has obvious solutions by more established methods. In wheat, it makes no sense to search for aluminium tolerance by culture-induced variation, with or without in-vitro selection, when excellent tolerance genes with single dominant effects are already available in the wheat germplasm banks. By contrast, a better case can be made in lucerne (alfalfa) to look for somaclonally induced tolerance because it is very sensitive to aluminium in acid soils, and sources of tolerance appear not to be available by other means.

Examples of Released Somaclonal Cultivars

Despite the relatively short time since their first description, somaclonally derived mutants are finding their way to the market and to agriculture. The company FreshWorld Farms has been marketing a tomato with altered colour, taste, texture and shelf life since 1993. American Cyanimid is expected to release an imidazolinone herbicide-resistant maize in the next couple of years. There is newly released germplasm of bermudagrass, Cyanodon dactylon, called Brazos-R3, with increased resistance to fall armyworm compared to its donor genotype Brazos (Croughan et al., 1994).

The recent example that has most caught my imagination is that of the Lathyrus sativus somaclone which no longer accumulates neurotoxin in the grain (Mehta et al., 1994; Yadav and Mehta, 1995). This species is wonderfully adapted to harsh and dry environments in India and can become a crucial food source to desperate farmers in bad seasons. New somaclonal variant lines are now available which have negligible levels (0.03%) of these harmful toxins compared to the parental seed (0.3%). In addition, the new cultivar has increased seed yield and is earlier maturing.

The accumulating examples of useful somaclonal variants and new cultivars derived from them give testimony and some confidence that this approach will make an ongoing contribution to plant improvement. In a few cases, a methodological comparison was set up in which somaclonal variation delivered the desired mutants while chemical or gamma treatments did not (e.g. Gavazzi et al., 1987). On the other hand, Sala et al. (1990) found the frequency of salt- and drought-tolerant tomato mutants similar between the chemical mutant combination EMSIMNU and somaclonal variation. Upelniek et al. (1995) found a higher frequency (2.07% cf. 0.69%) of mutations affecting wheat grain gliadins from nitrosoethylurea than from cell culture.

Introgressing Alien Genes

'In particular, the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species' (Larkin and Scowcroft, 1981). It is because of our conviction that recombination and rearrangement are at the heart of much somaclonal variation, that our own work shifted to exploiting it in the introduction of alien genes. If cell culture induces non-homologous recombination within a genome, then in the presence of alien chromosomes, it might also enhance alien gene introgression. The alien chromosomes or chromatin might be introduced by wide sexual crossing or by protoplast fusion.

One of the distinguishing features of this approach is that the researcher sets up a genotype (alien chromosome addition line) to target a specific and predesigned improvement by rearrangement. The post-culture screening of regenerant progeny is also specifically designed to identify or select the desired variant.

Barley Yellow Dwarf Virus (BYDV)

BYDV is a serious viral disease of small grain cereals worldwide. Its importance to wheat has often been underestimated due to the subtlety of its primary symptoms, and the fact that it renders wheat susceptible to a number of secondary root and foliar diseases. No adequate resistance has been identified in wheat germplasm itself. We were able to find a number of sources of resistance in related perennial grasses, notably Thinopyrum intermedium, commonly called intermediate wheatgrass (Brettel et al., 1988). This species can be crossed to wheat, but its chromosomes do not recombine meiotically with the wheat genomes. By intercrossing the two species, a line was produced carrying all the wheat chromosomes and only one alien chromosome — the one carrying the virus resistance gene. This was placed into cell culture. After plant regeneration and analysis over a number of generations, eight independent families out of 1200 have been shown to carry the resistance on recombinant wheat/Thinopyrum chromosomes (Banks et al., 1995).

Molecular probes which allow recognition of T. intermedium chromatin in wheat background, suggest that the translocations analysed to date are still associated with a block of alien chromatin, and can be followed through a breeding programme using restriction fragment length polymorphisms (RFLP) or randomly amplified polymorphic DNA (RAPD). Nevertheless, the segment of alien chromosome carrying the resistance gene is smaller than the arm of the chromosome from which they derive, at least in some of the recombinants (Banks et al., 1995; Hohmann et al., 1996). After backcrossing to recurrent wheat cultivars, it was shown that there was no apparent yield or quality loss associated with the recombinants, and therefore no impediment to using this resistance for new varieties of wheat.

Thus, we have exploited the somatic chromosomal recombination occurring in culture to achieve the transfer of a useful disease resistance gene from an alien chromosome to a recipient crop chromosome. Without this step of culture-enhanced recombination, the resistance does not recombine meiotically with wheat. As a consequence, the BYDV resistance can now be deployed for wheat improvement and new wheat cultivars are being developed.

Asymmetric Lucerne Somatic Hybrids

Surprisingly, few authors have noted the potential synergy between somatic hybridization and somaclonal variation. Protoplast fusion brings together chromosomes of disparate species, albeit for a brief time, in a cell culture environment. Prior irradiation of one parent (the donor) may bias the chromosome loss to the other parent (recipient), but it may be the cell culture environment which enhances the desired prospect of introgressing genes from the donor into the recipient chromosomes. We are currently attempting to exploit this using lucerne (Medicago sativa) as the recipient and Lotus pedunculatus as the donor. The characteristics of interest in Lotus are foliar condensed tannin (for bloat-safety in grazing ruminants) and acid soil tolerance. A collection of over 4000 asymmetric hybrid plants have been produced following the technical approach of our pilot study (Li et al., 1993). These plants have a general morphology like the lucerne parent, though many plants have some degree of morphological variation. RAPD analysis of a sample of plants demonstrated that most have some Lotus DNA present (Stoutjesdijk, Larkin and Sale, unpublished and 1995). Although, the screening for tannins and for aluminium tolerance is continuing, some positives have already been identified. The crucial aspect of this work will be the stability of the desired trait. If the governing genes are still on a Lotus chromosome, they might be expected to be unstable. However, if the genes are now on lucerne chromosomes as a result of culture-induced somatic recombination, then stable genotypes should be recoverable. Other examples of this approach include somatic hybrid derived plants of Brassica (Liu et al., 1995) and potato (Xu and Pehu, 1993).


Chemical- and radiation-induced mutations as a means to plant improvement were controversial for at least four decades. As we saw earlier, it is now possible to catalogue many hundreds of mutant-derived cultivars that have stood the scrutiny of merit testing and registration in many countries. It remains to be seen, whether the much more recent approach of somaclonal variation will be able also, with time, to catalogue its contribution in similar terms.

We have been able to describe a number of somaclonal variants at the chromosomal and molecular level. However, we are only speculating as to the chain of causal events which led to these genetic changes. This book will give further examples of molecular characterization and speculation regarding causes. It remains to be clarified, how this phenomenon fits into the bigger biological picture of the plant genome and its extraordinary plasticity. This book is a compendium of much of the current understandings and examples which should serve as a basis for students wishing to explore the broader biological picture.

Mutations induced by chemicals, radiation or culture have perhaps receded from the centre stage of plant improvement because of the advent of genetic engineering. It is already possible to isolate a gene, modify it specifically in vitro, and return it to the original plant species to achieve a desired mutation. A good example is the research under way to improve barley's utility for beer brewing by increasing the heat stability of β-glucanase (McElroy and Jacobsen, 1995). The protein structural requirements for heat stability have been defined (Fincher, 1994), the native barley gene has been cloned, and the necessary codon changes have been performed by site-directed mutagenesis. These genes are now being transformed into barley. This more heat‑stable glucanase is expected to survive the kilning and mashing, and is anticipated to reduce the viscosity of the wort and reduce the glucan hazes in the beer by degrading the glucan polymers. Such experiments are the ultimate in directed mutagenesis. A number of plant resistance genes have recently been cloned. The study of these genes is expanding our understanding of the genetic basis of host plant resistance to diseases and pests. We might anticipate experiments in the next decade in which resistance genes are manipulated in vitro to alter their specificity and effectiveness.

However, the current technical difficulties and regulatory constraints have delayed major contributions from genetic engineering for plant breeding. While the power of molecular biology will certainly and eventually impact mightily on crop improvement, it would seem mutagenesis will continue to pay its way in this endeavour upon which so much of our future welfare depends.