Mutation
Breeding
Mutations in Plant
Breeding
by Alexander Micke
Plant breeding using mutations.
Breeding has been practiced since the
early human civilization and selection
was the first method of breeding, adding the criteria of suitability for man’s
use (e.g. larger seed, better taste, easier harvestability) to those of natural
adaptation, fitness and offspring. It has been said, that the ultimate source
of all heritable variation to select from are mutations. But such a statement
leaves open, where the genes to start with and the genetic code came from. Recombination
of genes can provide additional genetic variation, if differences exist not
only between various genes, but also in form of alleles of particular genes of
prospective recombinants. Such alleles derive from mutations. Using mutants in
cross breeding requires no in depth knowledge about mutations, because the
mutated trait is the object of desire. But when the mutant trait is not
inherited as expected, the breeder may begin to think about the actual
mutational event, that led to the mutant phenotype.
There are different definitions of the
term „mutation“ and this may create the impression, that the term is somewhat
woolly. Definitions range from „a sudden phenotypic change in a character of an
individual, not due to crossing or segregation“ up to „an alteration in the
macro-molecule of DNA“ (where it remains open, whether the alteration leads to
a change in gene function or not). Included under the term „mutation“ is also
the augmentation of genetic material through
nucleotide or gene copies, through additional individual chromosomes, as
well as through the multiplication of whole genomes towards polyploidy. In
order to speak more clearly about mutations and their potential for crop
improvement, it would seem desirable to have different terms at least for (a) the phenotypic alteration and (b) the various underlying molecular and
numerical changes. But in any case, a mutation has to be phenotypically
expressed to be selectable, all other mutations are only of scientific
interest.
We have today a relatively good
understanding of the processes of mutation induction by UV, by ionizing
radiation and also by certain chemical mutagens, but the causes of
„spontaneous“ mutations are still to some extent uncertain (HALL 1990;
RASMUSSON and PHILLIPS 1997). In particular, the kind and rate of mutations
during the millions of years of evolutionary history are subject to discussion,
as current mutation rates can hardly explain the progress of past
evolution (OHNO 1970; KIYOSAWA and
NOMURA 1988; KOINANGE et al. 1996; KENRICK and CRANE 1997).
Stability vs. differentiation.
In 1865, Gregor MENDEL showed, that
characters (traits) of individuals must be based on controlling elements
(genes), that are passed-on intact and unchanged from one plant generation
to the next. But these controlling
elements are also passed-on intact and unchanged from cell generation to cell
generation during the individual plant’s development, i.e. from mother cell to
daughter cells in every somatic cell division (based on the mechanism of
mitoses). MENDEL should be forgiven for
not paying attention to this matter. Even today we still have a problem to
comprehend the ontogenetic development from a single „totipotent“ cell (zygote)
into a complex organism with different tissues, consisting of specialized cell
types, forming organs with special functions, and at the same time maintaining
an intact „germ line“ for potentially „totipotent“ generative cells. Long after
MENDEL the question emerged, how this ontogenetic differentiation could be
reconciled with the stability of the heritable elements postulated by MENDEL
and confirmed by the discovery of chromosomes as carriers of genes, the
meticulously conservative DNA-replication, the course of events during mitosis
and meiosis etc. Now we begin to learn about factors determining the
ontogenetic fate of particular meristematic cells (GOODRICH et al., 1997)
or e.g. the co-ordination between
embryo and endosperm during seed development (RAY, 1997).
In spite of booming in-vitro culture business we have
difficulties to clone differentiated cells as such, i.e. with maintaining their
specialized functions. This should be extremely useful for the industrial
production of particular compounds. Still we don’t know really what distinguishes
a differentiated cell from a „totipotent“ (embryonic) cell, what is involved in
the de-differentiation of „explant“ cells leading to the grwoth of callus, or
what is going on when callus cells eventually become organogenic. Isn’t it
still surprising, that the profound processes of differentiation leave the
genome essentially unaffected, so that by a de-differentiation „totipotent“
cells can be obtained again? Evolution apparently made provisions for high
genetic flexibility during ontogenesis and especially for performance under the
challenge of environments (a plant cannot migrate!), but at the same time
established reliable systems to preserve genetic integrity for subsequent
generations. The unnatural conditions of tissue and cell culture, on the other
hand, demand apparently too much from the control system and thus result in
genetic instability (PHILLIPS et al. 1994).
Gene expression: dominance vs. recessivity.
MENDEL
discovered another phenomenon, relevant for the concept of mutations: He observed differences in strength of gene
expression and ascribed this to the
occurrence of „alleles“ at the gene locus: the stronger ones he called
„dominant“, the weaker ones he called „recessive“ (at some gene loci multiple
alleles can occur). MENDEL described
the phenotypes observed, when alleles of one or two genes interacted in the same (heterozygous)
individuum, and he stated the segregation ratio of phenotypes found upon
self-pollination of the heterozygous plant. He did not look into possible
reasons for the different strength of expression. He also did not mention any observation he might have made about
interactions between genes at different loci, influencing the kind and level of
their phenotypic expression (epistasis, pleiotropy).
A few years after MENDEL found the fundamental principles of
inheritance, Friedrich MIESCHER (1871) discovered the desoxyribonucleic acid
(DNA). Although he himself did not realize the genetic role of this compound,
these events marked the beginning of a most fascinating research century,
focussing at the chemical basis of
inheritance, the genetic code, the structure of chromosomes, the analysis
of bio-chemical pathways and their
control, the possibility for far-reaching genetic manipulations („gene
engineering“), and approaching
eventually the understanding of „life“. However, in spite of all progress in
molecular genetics, we still cannot
fully answer the seemingly rather simple questions: What distinguishes
different alleles and what causes the difference between dominant and recessive
gene expression? The answers should be most relevant for induced mutations and
the judgement of their breeding value, because the majority of induced
mutations act like changes from a dominant allele into a recessive one, or
represent deletions, which in crossing experiments mimic recessive alleles. The
question gets more complicated by the functional hierarchy among genes and the
location/time/stage-specific regulation of gene expression by other genes: A
silenced dominant gene would probably act like its recessive or null-allele;
the mutation of a dominant inhibitor gene to its recessive or null-allele may
allow one or more (previously silent) genes to express as dominant alleles.
Artificially induced mutations.
The pioneering experiments by MULLER
(1927, 1928) have shown, that it is possible to knock down the stability of
genes. This new possibility was looked upon with great optimism. MULLER himself
thought even about breeding better human beings, but the plant breeder STADLER
was sceptical about the usefulness of genetic alterations induced e.g. by
x-rays. The scepticism resulted from experiments with cereals (STADLER 1930,
1932), where he was not satisfied with an impressive quantity of heritable variation,
but rather looked for the kind of changes, that might lead towards crop
improvement. He did not observe any dominant mutations and suspected, that a
recessive one was „simply the destruction of a gene“. He advised students to
better make use of „natural“ genetic variation for profitable crop improvement.
When after World War II funds became
available for „peaceful uses of atomic energy“, many young researchers in
developed as well as developing countries embarked on the fashionable
technology of using radiation and radioactive isotopes for inducing mutations,
often without paying attention to the rather modest results obtained between
1930 and 1945 by using radiations as well as some chemical mutagens. It was
confirmed, that mutagen treatments harm the delicate molecular structure of
genes, and hardly produce the minor changes in nucleotides, which could be
expected to lead to meaningful codes for different (or even new) enzymes
(STADLER and ROMAN 1948). Some chemical mutagens were found to act less
destructive and to cause a much higher proportion of minor changes. However,
even these lead to missense and nonsense mutations and did not contribute much
to crop improvement.
However, with international
coordination and some financial assistance by IAEA and FAO from 1964 onwards it
could be convincingly demonstrated,
that ionizing radiations and also certain chemicals, when handled properly,
could induce many useful alterations in the genomes of crop plants. Records
maintained by the Joint FAO/IAEA Division in Vienna show, that ca. 2000 crop
cultivars with one or more useful
traits from induced mutations (mainly from x- and gamma-rays) were released
worldwide over a period of 35 years. Included in these records are some
outstanding examples of cultivars (e.g. „Diamant“ and „Trumpf“ in barley; „IRAT
13“, „Yuanfengzao“ and „Calrose 76“ in rice; „Lumian 1“ and „NIAB 78“ in
cotton; „Pervenets“ in sunflower; „Star Ruby“ in grapefruit), which had a
remarkable economic impact (Anonymous 1991; MICKE et al. 1980, 1985; FAO/IAEA
Mutation Breeding Newsletters 1972 - 1997). Inspite of the number of induced
mutants recognized as valuable crop cultivars and used successfully in cross
breeding, of course „new genes“ in the strict sense could not be produced (MICKE
1991). Consequently, one would expect much interest in the question, what kind
of molecular alteration in the chromosomal or cytoplasmic DNA, or what kind of
structural/numerical alteration in the genome happened in those successful
mutant cultivars. However, so far there were not many efforts to bring into
accord the destruction caused by mutagens and the good performance of so many
induced mutants. The answers to this question should be very relevant for any
future investment into mutation breeding and gene engineering, to give a reliable forecast of what can be
expected.
Induced mutations occur more or less
randomly in the genome, even their target cannot be directed. Only one of the
(two or more) alleles of a locus is affected, inheritance is almost ever
recessive, therefore homozygosity is normally required for proper expression.
Accordingly, results were more often useful in self-pollinating plant species.
On the other hand, mutant heterosis has been repeatedly reported (MICKE 1968, 1969, 1976; RÖMER and MICKE 1974;
MALUSZYNSKI et al. 1987, 1989) and specific mutations e.g. concerning male
sterility (DASKALOV and MICHAILOV 1988) or grain quality traits (RÖBBELEN 1990)
proved useful in cross-pollinating species. In vegetatively propagated crops,
which usually are heterozygous and therefore could be improved also by
deletions uncovering existing alleles, success has been tremendous, but mainly
in ornamental plants (BROERTJES and VAN HARTEN 1988).
Today, mutation breeding is not anymore based
only upon classical physical mutagens like x- or gamma rays or classical
chemical mutagens like EMS or NMH, but also upon variation that occurs during in-vitro culture and has been termed
„somaclonal variation“ (SKIRVIN, R.M. 1978; OONO et al. 1984; NOVAK et al.
1988; NOVAK 1991; SUKEKIYO and KIMURA 1991). It was pointed out already, that
the term „mutation“ is rather woolly defined, but the term „somaclonal
variation“ is even worse! Initially, it described the unexpected, sudden
occurrence of more or less heritable variants in the somatic or generative
offspring of in-vitro cultured plant
material (HEINZ and MEE 1969). In the meantime, it became known, that a wide
array of alterations in nuclear and cytoplasmic genetic elements contribute to
the observed phenotypic variation, and
that many of them are of epigenetic nature
(D’AMATO 1986; PHILLIPS et al. 1994). Initially directors of tissue
culture laboratories provoked the interest of plant breeders and sponsors by
promising new and particularly useful genetic variation (OONO 1978; LARKIN and
SCOWCROFT 1981; CHALEFF 1983; EVANS and SHARP 1983), but soon one learned, that uncontrollable occurrence of
„somaclonal variation“ could ruin valuable genetic stocks (GÖBEL et al.
1986). An interesting feature of
somaclonal variation was the repeatedly reported occurrence of ‘homozygous
mutants’ (e.g. OONO et al. 1984). At least some of them were later identified
as a kind of „Dauermodifikation“, resulting from gene inhibitions, that could
pass through meioses and were stable for several generations (OONO 1985). It
seems, that also here more research is needed, to clarify the underlying causes
of new variation before its use in crop breeding.
The importance of gene regulation: inhibition
vs. activation.
During ontogenesis, particular gene products are needed for a
particular phase (e.g. the early embryonic development), but hey have to be
suppressed during other phases. The original „totipotency“ of meristematic cells
has to be restricted for initiating the formation of the different,
functionally specialized tissues. Does this happen by activating genes that
were inhibited in embryonic cells or by inhibiting genes that were active
during early phases of ontogenesis?
From tumor research it is known, that
in the adult human body certain cells may regain their „totipotency“ by the
mutation or deletion of a so-called „onco-gene“ (SVOBODA 1994). These genes in
reality are inhibitory genes, because their dominant alleles repress
„totipotency“ and thereby permit the development of differentiated somatic
tissues and the function of specialized organs (STANBRIDGE 1990). A recessive
mutation in one of such „onco-genes“ was found to activate more than 15 other
genes. Also in plants the transition from a „totipotent“ zygote to specialized
somatic cells requires the inhibition of a number of genesand the activation of
others. Augmentation of certain genomic portions helps to meet the requirements
for specific cell functions.
Ultimately, forming a plant organism with its highly specialized tissues
and organs should result from a delicate interplay of gene inhibition and gene
activation. To assure, that in a particular cell and at a particular time only
the needed genes function, requires a highly sophisticated epigenetic system of
gene regulation with ability to respond quickly to signals from outside and
inside (OHNO 1970; CLARK 1997).
Shouldn’t there be a relationship
between the transient epigenetic regulation of gene activity for ontogenetic
development and for somatic metabolic performance in already differentiated
tissues, and the mechanisms of a (truly heritable) control of gene expression
by „regulator genes“? Various mechanisms of gene regulation are known (e.g. CLARK 1997). Obviously many factors
can play a role as triggers, such as light and temperature, influences by
neighbouring tissue and the position in the organism, chemical concentration
gradients and special signals. Regulation may be by transcriptional or
post-transcriptional inhibition (GALLIE 1993), it may be cell specific (EDWARDS
and CORUZZI 1990), tissue specific, or concern the whole organism (GALLIE and
BAILEY-SERRES 1997), it may be the result of parental imprinting or of allelic
interaction (MATZKE and MATZKE 1993).
For the control of gene expression by regulatory genes it seems relevant
to keep in mind, that this interaction should be subject to recombination
during meiosis, unless regulation is via the promoter region of the gene, or
the regulatory gene is very tightly linked to the controlled gene(s).
A rather well studied example of organ
specific genetic regulation is the control of seed endosperm composition, where genes for storage compounds
(proteins, starches, lipids etc.) function only during seed formation (GOTTSCHALK
and MÜLLER 1982). Here regulation is also concerned with the coordination
between embryo, endosperm and maternal tissue (RAY 1997). Well studied is also
the regulation of anthocyanin formation e.g. in maize (JAYARAM and REDDY 1990). An example of genetic regulation concerning
the whole organism would be the photoperiodic reaction: A single gene may
decide, whether and when flowering is induced by a particular photoperiod
(MICKE 1979) and this is a crucial decision, whether under natural conditions or
in farmers field. Resistance against pathogens is another example (often
studied but not so well understood). So-called ‘resistance genes’ recognize
attack by a pathogen and signal activation to a cascade of host plant genes for
defending the organism (e.g. JÖRGENSEN 1988, 1992). Formation of cell wall
appositions, “suicide” of already infected cells („hypersensitivity“), toxin
degradation, blockade of pathogen migration are defence measures handled
„non-specifically“ by a number of ‘downstream’ genes („polygenic resistance“).
But these genes have to be switched on by
other genes, which are able to recognize specific ‘pathotype’
attributes. These ‘watchtower-genes’ are a very important acquisition for a
plant, because it would be too costly (and probably create physiological
problems) to keep all defence genes against all potential pathogen attacks
active all life time. Unfortunately, the pathogens have acquired the known
„gene-for-gene“ camouflage possibilities to trick out the plant’s ‘watchtower-genes’.
The interesting questions are: In
which way are genes activated? How is their activity inhibited? How can a mutation interfere with these
activation and inhibition processes?
Every gene
must possess a „promoter“-sequence, without which the gene will never be
transcribed. The promoter can be blocked, apparently also be mutated to be less
promoting, its duplication may cause co-repression, but what initiates or
terminates the promoter activity? There
are external and internal triggers, but any message first has to find a
receptor inside the cell, which then will produce a signal to activate and
coordinate gene activities within a tissue, within an organism, at a given
time, during a particular ontogenetic stage etc. These signals and their way of
transmission are still quite a mystery.
From artificial organogenesis under the influence of hormones in culture media
one can learn something about somatic regulation of ontogenesis, but the in-vitro environment is rather
a-typical. We know really still very little about regulation under natural
conditions by systems, which reside in the genome, are inherited, and form a
genetic network.
Thinking about induced mutations in
this context, one has to keep in mind, that any new mutation is a single cell
event, and therefore is irrelevant as long as the mutation is not
phenotypically expressed in a sizeable tissue, or is part of the germ-line.
Since the effect of mutagens is mainly gene inactivation or deletion, removal of an inhibition as well as the
elimination of activation should be possible by a mutation in a dominant
regulator gene. In the early days, some researchers were interested to revert
recessive mutations to normal functioning dominant alleles. Many ‘revertants’
were obtained, but these were not true ‘backmutations’. They were recessive or
semi-dominant mutations in other loci and on other chromosomes (REDEI 1969).
Apparently recessive mutations can suppress the original defect of recessive
mutations (TULEEN et al. 1966). Nowadays, mutations are being used to dissect
the network of genes. By eliminating so far unknown genes, their normal
functions are identified. In fact, progress in plant genetics as well as plant
physiology depends to a large extent on the induction of recessive or
„null-allele“- mutations in the model plant Arabidopsis
thaliana (ANONYMOUS 1995).
Which genes are dealt with in evolution and
in crop improvement?
Plant breeders in the past used to
ascribe a particular trait of a plant or the performance in the field to a
particular gene or group of genes, which are to be dealt with in crossing like
MENDEL demonstrated more than 100 years ago. It was not taken into
consideration, that genes looked at by the breeder indeed are either objects of
regulation or are regulator genes themselves and that the genes introduced from
parents may all be accumulated in the
offspring, but do not function all the time in all the tissues, and would
require particular provoking environmental conditions or triggers from outside/inside
to be expressed.
All living beings, according to the
degree of relatedness, have most of their genes in common. These are first of
all the fundamental genes evolved some hundred million years ago, which are
required for the basic cell compounds, such as amino acids, proteins, sugars,
starches, fatty acids and for the basic cell metabolism , concerning energy,
growth, coordination, multiplication etc. (OHNO 1970). Related species share
most of the genome of a common ancestor, from which they started separation may
be 50 million years ago. The genomes of cultivars belonging to the same species
of course may differ only in the alleles of a few dozen genes. Geneticists discovered a striking syntheny among plant
species, which means, that e.g. in the chromosomes of grasses like rice, barley
and maize there are groups of identical genes in much the same order of
sequence. Relevant differences mainly resulted from duplications and
translocations within the genome of a common ancient cereal progenitor (KURATA
et al. 1994; MOORE et al. 1995). Most of the molecular polymorphism detected
within genes seems to be neutral, i.e. not altering gene function. For a
breeder, however, only genes with functional polymorphism are of interest.
These will not be the essential „structural genes“, but also not the basic
„regulator genes“, which coordinate the standard cell and organism functions.
It also will not be those genes that determine cell walls or turgor, stomata or
photosynthesis, the development of vessels or the transport of nutrients,
because manipulations in those genes can hardly be tolerated by any plant.
Consequently, the breeder will be able to manipulate only the kinf of genes,
which influence the course of events during ontogenesis of a crop plant and
those, which are involved in the interaction of this organism with a particular
environment. Breeding will be able to influence the timing of processes, the
distribution of resources, the partitioning of products and a number of
morphological traits. One should note, however, that all these characteristics also fall within the
range of the plant’s epigenetic regulation flexibilities!
One may conclude, that plant breeders
deal only with a certain portion of „regulator genes“, and this would also refer to experimental
mutagenesis. It must be admitted, that
in reality there might not exist a rigid separation between „structural“ and
„regulatory“ genes. Among the former, some may be coding for a product of
little importance for the survival of the individuum (e.g. flower pigment) and
they could perhaps possess other temporary regulatory roles in the genetic
network. The really essential „structural“ and „regulatory“ genes would be
expected to belong to the extremely „conservative“ group of genes which exhibit
a minimum of deviations (alleles) and so be practically exempt from
manipulation by recombination. This group of indispensable genes probably
became during the course of evolution well protected against mutations
(spontaneous as well as induced), e.g. by duplications, or by particularly
effective repair systems.
If one realizes this rather restricted
potential manipulation of genomes by plant breeding, one may wonder, what
happened during natural evolution. It seems, that valuable genetic systems once
developed, were so well protected, that they became virtually „immortal“ (OHNO
1970). Recently the opinion gains ground that much of the natural evolution was
based on altered regulations rather then on making new genes (SHUBIN et al.
1997). A most prominent example of how evolution (and domestication) may have
worked, is the development of maize from its ancestor theosinte: One single
regulator gene (tb1) was responsible
for this phenotypically large step(DOEBLEY et al. 1997).
Do epigenetic mechanisms play a role in evolution and plant improvement?
Heritable alterations of regulatory
genes by mutations have certainly played a crucial role during evolution. Their
effect, however, to some extent resembles the epigenetic control of gene
activities in cell metabolism and in the numerous organism functions during
life time.
Evolution usually is thought to be based
upon preformed random genetic variation, from which the fittest portion
is selected “by nature”. However, epigenetic variation in regulatory
systems would seem to be the faster, more effective and more flexible way for a
plant to respond as an organism to edaphic or climatic changes in the
environment, to temporary abiotic stresses, to competition by other plants, to
attack by pathogens and pests, while at the same time maintaining and
accurately replicating the accumulated most valuable ‘structural’ as well as
‘regulatory’ genes. In contrast to single cell mutations, which become relevant
eventually in the next generation, epigenetic alterations almost
immediately are expressed in tissues, organs or whole plants, and therefore are
selectable in the same generation. Natural selection of better adaptable
individuals and populations therefore can act without delay, favouring their
survival or number of progeny. Thus
epigenetic processes (although by definition not heritable) should have a
profound effect on what goes into the next generation and therefore should have
a tremendous influence on evolution. Could this influence be measured in
comparison to the rather slow and vague process of accumulating spontaneous
(undirected!) mutations, which require phenotypic establishment in part of a
population before they can make a contribution of a better fitness, adaptation
or competitiveness. The plant breeder, on the other hand, does not appreciate
epigenetic variation, and when selecting desired phenotypes, routinely performs
progeny tests to sort out the heritable variation from non-heritable.
Is there perhaps another connecting point
between mutations and epigenetic regulation? The possibility should not be
bluntly rejected (CULLIS 1986; PREM DAS and MESSING 1994). SCHWOCHAU and HADWIGER already in 1969 reported,
that certain chemicals acting as mutagens were also unspecific inducers of gene
activity. They assumed, that these chemical mutagens attack preferentially the
promoter region of genes, because the rest of the gene may be covered by
histones and only the regulator site readily accessible.
The idea of inheritance of acquired
characteristics was discussed many times and was vehemently rejected. With
current advances in molecular genetics the question may be brought up again
from a different angle: Do epigenetic regulatory changes have a chance,
somehow, to be transferred into the genome? Some epigenetic phenomena have
already been found to be transmittable into the next generation, e.g. parental
imprinting. Should nature really have
thrown away the chance to take advantage in evolution of the striking
flexibility, a plant population achieves through epigenetic adaptation of
metabolic processes, of the plant architecture, of resistance etc. ? If so,
epigenetic regulations at least gave a chance of survival and through this
another generation of time for a required adaptation by the generally accepted
genetic means such as mutation, selection and recombination.
What is
the basis for beneficial induced mutations?
There were a number of futile attempts
to link the successful performance of crop plant mutants to the mutagen and the
applied dose by which they were originally induced, but there are hardly any
investigations about the molecular changes of genes in the genomes of improved
mutant cultivars, and only few concerning specific crop plant mutants.
Mutation experiments on the seed quality
of cereals induced by various mutagens may shed some light on the issue. AMANO
and co-workers studied carefully the changes induced in the waxy loci of rice and maize using EMS,
UV, thermal neutrons, and gamma-rays. They looked at the expression of the waxy gene in pollen and in the endosperm
(AMANO 1981, 1985; YATOU and AMANO 1991). They have confirmed a destructive
effect of all the mutagens used, but the kind and degree of destruction
differed between the mutagens applied, from impairment of proper code
transcription (mainly by EMS) to total inactivation or even complete deletion
of the locus. Another interesting insight can be derived from studies on the
effect of chemical mutagens on a particular locus in barley (Mla12), responsible for a „specific“
resistance to Erysiphe graminis
(JÖRGENSEN 1996). Mla12 is a dominant
gene whose function is to recognize attack by the mentioned fungus. The recognition
is restricted to a particular product of the corresponding fungal „avirulence“
gene. Mutagenesis was tried to alter the dominant, resistance activating allele
into a recessive or the null-allele and to see what happens. The (somewhat
unexpected) results were: (a) mutants with mutations in the Mla12 locus causing reduced, but not
completely lost ability to recognize the corresponding specific pathogen type;
(b) mutations outside this locus, which reduced gradually the defence
effectiveness. These mutations apparently occurred in genes (activated by the
intact dominant Mla12 gene) whose
proper functioning is required for performing an effective defence. (For
incomprehensible reasons the mutated genes were named suppressor (sup), although they were originally active defence
genes, now to some extent damaged by a mutation).
Accepting it as a fact, that
conventional mutagens have destructive effects on genetic material (MALUSZYNSKI
et al. 1995), one could postulate, that
those successful mutants mentioned before (e.g. listed in the FAO/IAEA Data
Base) carry alterations only in genes, where even a „null-allele“ causes
tolerable changes in the plant’s ontogenesis and physiology. But this does not
make it plausible, why they became useful from the point of view of a crop
breeder or farmer.
If one looks into the literature or at
one’s own experiments, one would be able to list numerous examples of induced
or spontaneous mutants, which could be explained by an alteration or complete
loss of a regulation (e.g. MICKE et al. 1980; YAMAGATA 1981; GOTTSCHALK and
WOLFF 1983; DONINI et al. 1984; KONZAK 1984; MICKE 1988). Some specific examples could be listed as
follows:
- loss of daylength sensitivity in rice,
cotton, castor bean (GUSTAFSSON and LUNDQUIST 1976; MICKE 1979; DONINI et al.
1984)
- loss of apical dominance leading to profuse
branching in sweet clover (MICKE 1958; SCHEIBE and MICKE 1967)
- altered control over ontogenetic
development, leading to short culm and increased tillering in cereals
(MALUSZYNSKI et al. 1986)
- loss of control of root nodulation towards
non-, inefficiently or abundantly nodulating pea, bean, chickpea, faba bean,
soybean etc. (JACOBSEN and FEENSTRA
1984; CARROLL et al. 1985; PARK and BUTTERY
1989; etc.)
- altered control over endosperm composition in cereals (GOTTSCHALK
and MÜLLER 1983; IIDA et al. 1993; VON WETTSTEIN 1995; HABBEN and LARKINS 1995)
- altered control over compounds deposited as
reserves in cotyledons of oil seeds (GREEN and MARSHALL 1984; RÖBBELEN 1982)
and of grain legumes (TAKAGI et al.
1989; BHATTACHARYYA et al. 1993)
- loss of control over regular leaf and
floral organ development in sweet clover (SCHEIBE and MICKE 1967), in faba bean
and in pea (GOTTSCHALK 1971)
- improved resistance of barley to powdery
mildew by loss of Ml-o regulated
inhibition of callose apposition (JÖRGENSEN 1992; MICKE 1992)
- increased resistance to rusts by deletion
of inhibitor genes in wheat (KERBER and GREEN 1980; DYK 1982; WORLAND and LAW 1991; WILLIAMS et al. 1992)
- altered control over deposition or
recycling of secondary metabolic products in lupin (MICKE and SWIECICKI 1988), in sweet clover (MICKE 1962
a and b; SCHÖN 1966), in rape seed (RÖBBELEN 1990).
Pleiotropy (often seen as a typical attribute of induced mutations) and its
modification through crosses (GAUL et al. 1968) could reasonably well be
explained by mutational changes in genes functioning as regulators for several
other genes, but should then be distinguished from „downstream“ effects of
mutant genes, acting „early“ in metabolic pathways.
Conclusions
It seems unlikely, that any of the
induced mutations used successfully for variety development affected essential
structural genes common to almost all forms of living creatures and preserved
over millions of years. It seems also unlikely, that the mutations concern any
other gene, that has assumed during the course of evolution an essential
position in the gene network of the particular species, because such mutations
would have resulted in loss of vigor and probably death (e.g. „chlorophyll
mutations“). The mutations used by plant breeders most likely concern those
genes, which are involved in genetic/environmental interaction, regulating the
response to triggers by signalling activation or inhibition to other genes
responsible for particular reactions (MICKE 1996). These regulating genes
should be the ones categorized usually as „major genes“ because of their
predominating role, in contrast to regulated genes which should be many, each
of relatively minor effect on the phenotype (usually classified as minor genes
or polygenes).
Considering the use of in-vitro culture techniques in mutation
breeding, the following might be concluded: All genetic alterations caused
eventually by physical or chemical mutagens can be expected also from
‘somaclonal variation’. In addition, one may expect activation of transposons,
but this also suspected for some chemical mutagens. ‘Somaclonal variation’ will
be compounded with epigenetic effects. This makes the use for crop improvement
more difficult, particularly in asexually propagated plants, since crossing and
reselection would be required to sort out desired mutations from other alterations.
In-vitro selection is handicapped by
a-typical gene expression. Use of haploids derived from anther culture has
found its best application in the „doubled-haploids-technique“ , which leads
faster to homozygosity for more effective selection.
Experimental mutagenesis has now a more
restricted, but better defined place among the methods of applied genetics. In
addition to the use of induced mutations in crop impropvement, there is now a
most valuable use in fundamental genetics and plant physiology. This will
benefit breeders through more effective strategies for crop improvement.
However, if a breeder wants „new“ genes with „new“ gene products, he would
definitely have to resort to gene engineering
and use mutagen treatments as a supplementary tool..
Literature:
see Mutation breeding references
The full
text of this review was published in “Breeding in Crop Plants: Mutations & In
Vitro Mutation Breeding”; Editors Bahar A. Siddiqui and Samiullah Khan;
Kalyani Publishers, Ludhiana (India) 1999. p. 1-19. ISBN 81-7663-104-3.
A good
source of useful information and publications on using induced mutations for
crop improvement would be the Plant Breeding and Genetics Section of the
Joint FAO/IAEA Division, P.O.Box 5, A-1400 Vienna (Austria) http://www.org/programmes/nafa
Address of the author: Dr. Alexander Micke, Salmannsdorfer Str. 94, A-1190 Vienna (Austria).