Developmental misregulation in sweet clover (Melilotus)
by induced mutations.
by
Alexander Micke (Vienna, Austria)
During the last century since the late
twenties, the tool of ionising radiations and later on also “radiomimetic”
chemicals has been applied by numerous geneticists and plant breeders, with the
aim to obtain new useful genetic variation for the improvement of crops. After
a period of nearly 50 years, these efforts have given rather unexpected good
results in terms of a great number of crop cultivars (MICKE et al. ). While the ultimate aim was crop
improvement, numerous publications described what else has been observed during
the course of the extensive studies in terms of effects of applying different
types of radiation and chemical mutagens on seed germination, plant development
and survival, on plant physiology and morphology, on cytology and chromosomes
etc. (Refer e.g. to IAEA publications 1961-1990). To some extent, these studies
stimulated fundamental research and thus contributed to the enormous
advancement of genetics (molecular genetics, genomics) during the past 30
years. While much of the pioneering ‘mutation breeding’ work sank into
oblivion, molecular geneticists discovered, that experimental mutagenesis is
the method of choice for analysing the role of particular genes in plant
metabolism and developmental pathways. The research interest has been focussed
on a few model crops like rice, maize, pea, rape, cotton, but in particular
upon Arabidopsis thaliana as a botanical ‘guinea-pig’. As a model for
leguminous species (and particularly for studying their symbiotic nitrogen
fixation) Melilotus alba (= M. albus
Desr.), the white flowering sweet clover, was found very suitable
( ) and new mutation induction
projects were initiated ( ). It would seem interesting and worthwhile, to
confront and compare the impressive new findings with some of the results
obtained and observations made long before modern molecular techniques became
available.
Within the frame of large scale mutation
induction experiments aiming at the genetic improvement of white sweet clover (Melilotus
albus Desr.) as a forage crop, a rather high number of mutants obtained
were of no interest for the plant breeder, but should be interesting and
perhaps of great value from the point of view of understanding ontogenetic
development in plants and its genetic regulation. Among the mutants observed
many were affected in leaf colour (mainly chlorophyll/chloroplast defects),
others in rather complex characters such as plant vigour or plant architecture.
Other mutants, however, displayed rather distinct alterations in organs, such
as leaves or flowers. These should be more amenable for the interpretation of
steps in the genetic regulation of development and differentiation of plant
organs and therefore are presented and discussed in the following. A few
examples were mentioned and displayed before (SCHEIBE and MICKE 1967), but most
of them so far were not described or published elsewhere, just because they
were not considered to be of potential use for crop improvement.
Sweet clover in all leaves except the
cotyledons and the primary leaf displays a typical trifoliate leaf structure,
common also to many other Leguminosae, and shows the very typical
papilionaceous flower structure, common to the family of the Papilionaceae.
About 20-100 flowers (each 5-8 mm long) are arranged in 2-15 cm long monopodial
racemes, emanating from leaf angles (Fig.1-4).
Initially it was a surprise, that such
apparently very conservatively protected characteristics, which are
identification marks of the species, can easily be broken up and altered by
mutational events (LAMPRECHT and GOTTSCHALK). The great majority of mutants
observed showed a segregation pattern close to monomeric recessive. This would
probably best be explained by assuming the inactivation or deletion of
particular “major” genes, which possess certain crucial regulatory functions in
processes of differentiation and are inhibiting/promoting distinct steps in
sequential processes of ontogenetic development. Many such processes have been
identified already in other plant species, and some are paralleled in animal
species ( ). In elder publications, frequently ‘pleiotropic mutations’ were
mentioned ( ), meaning that a simply
inherited alteration affected several “plant characters”.
Several of our mutants showed leaf a
reduction of the leaflet number of the normal trifoliate-type,
eventually (but not always) leading to distinct unifoliata - types. The
alteration of leaf development sometimes involves elongated petioles, but
sometimes their loss, causing ‘sessile’ leaves. In the majority of cases, an
altered leaf structure was reflected also in an altered structure of floral
organs (raceme, calyx, corolla, carpel, anthers).
Material
and methods.
The original sweetclover material used in
these experiments was taken from a natural local collection in Germany,
described elsewhere ( ). Dry seeds
were irradiated with x-rays or neutrons (fast, thermal), in some experimental
series also in combination. The seeds were sown immediately or after a storage
period of up to two years. They were transplanted generally in the open field.
At flowering time, a few branches per plant were protected by paperbags against
undesired cross-pollination. Plants derived from irradiated seeds (M1-plants)
were selfed in this way and their seeds harvested separately. The second
generation (M2) was sown as plant progenies in greenhouses for observing
seedling characters (and for testing the coumarin-content). Afterwards they
were transplanted into the open field. Cultivation as plant progenies facilitated the discovery of mutants and the
identification/elimination of accidental (never completely avoidable)
contaminants. As a rule, the occurrence of more than one variant of the same
type per M2-progeny was taken as indication for an induced mutation. The
inheritance pattern of such mutations was confirmed ( if possible by
selfpollination) from the segregation of heterozygotes in the next (M3-)
generation. In most cases the segregation
was found to be close to a 1:3 ratio, although frequently there was a
numerical deficit of the recessive mutants. Some crosses by hand and (in
isolating greenhouses) with the help of honey or bumble bees have been made in
order to study the identity of certain mutations, evaluate the F1-vigour
(heterosis?), and probe the possibility to combine several mutant traits in one
genotype ( ). A rather detailed
photo-documentation and a seed collection had been established. The seeds,
however, have mostly lost their germinability during the 30-40 years passed.
Bush-Type. Highly branching due to loss of apical
dominance.
“tortuosa”-type. All shoots, branches, leaves
and flower racems bent, curved or crooked.
Dry seeds
irradiated 1955 with 40krad x-rays. M1
1955. M2 1956: dark green dwarfs. M4
1958: bulged leaves, bent shoots 28 mutants : 40 normal plants.
(RÖMER
1973)
Bush-type.
Better growth than 56-251, still some apical dominance.
Dry seeds
irradiated 1955 with 40krad x-rays. M1
1955. M2 1956 7 mutants : 18 normal plants.
“unifoliata” Type I; larger sessile leaflets (no
petioles), no nyctinasty; cauliflower-like sterile flowers.
Dry seeds
irradiated 1956 with 40 krad x-rays. M1 1957.
M2 1958: 2 mutants : 50 normal plants.
M3 1959 from
normal looking sister plants: 2 mutants : 16 normal plants.
Figure 1: St.148 (left),
mutant (right)
Figure 2: Mutant Mi
58-367
Figure 3: Shoot tips
St.148 (left), mutant Mi 58-367 (right)
“waxless”.
Small dark green leaflets, borders bent.
Dry seeds
irradiated 1956 with x-rays. M1 1957.
M2 1958: 3 mutants : 27 normal plants.
(RÖMER 1973)
“unifoliata”Type
II. Partly unifoliata, partly oak-type. Only central leaflet sessile.
Malformed flowers.
Dry seeds
irradiated with x-rays. M1 1957. Morphologically malformed infertile M1-plant
vegetatively propagated. M2 1959(+1961) : 4 mutants : 33
normal plants.
“unifoliata”Type II. Partly unifoliata with sessile leaves, partly
oak-type. More growth than 59-31. Filled flowers? Fertile?
Dry seeds
irradiated with x-rays. M1 1957. Morphologically malformed infertile M1-plant
vegetatively propagated. M2 1959 (+ 1961): 3 mutants : 69 normal
plants.
“unifoliata”Type II.
Like 59-31, but
darker,smaller leaves; flowers with doubled perianth, 3-4 clustered.
Dry seeds
of St.148 irradiated with x-rays (40-50 krad). M1 1957. Morphologically
malformed infertile M1-plant vegetatively propagated (cuttings). Seeds
harvested 1958 in the greenhouse. M2 1959: 3 mutants : 36 normal plants. In the same progeny also some xantha/chlorina
type mutants.
“unifoliata” Type II. Oak-type.
Dry seeds
irradiated with x-rays. M1 1957.
Morphologically malformed infertile M1-plant vegetatively
propagated. M2 1959: 6 mutants : 47 normal plants.
“unifoliata” Type
II. Leaves somewhat
like oak-leaves; pinnately lobed, not trifoliate. Bushy growth.
Chlorophyll-defect viridis.
Dry seeds
irradiated 1957 with x-rays. M1 1957. Morphologically malformed infertile
M1-plant vegetatively propagated. M2 1959/M3 1961: 7 mutants : 45 normal
plants.
(RÖMER 1973)
“unifoliata” Type II. Like 59-57.
Dry seeds
irradiated with x-rays. M1 1957.
Morphologically malformed infertile plant vegetatively propagated. M2 1959: 4 mutants : 34 normal plants.
« unifoliata »Type IIa. Vigorous.
Dry seeds
irradiated with 60 krad x-rays. M1
1958, seedlings treated with 0,1% colchicine.
M2 1959 : 2
mutants : 7 normal plants.
« unifoliata »Type ?.
Dry seeds
treated with neutrons (dose 5). M1 1959.
M2 1960. M3 1961: 7 mutants : 8 other plants.
“unifoliata”Type II. Irregular type. Leaflets with short petioles.
Open flowers.
Dry seeds
irradiated with neutrons (dose 20). M1 1959.
M2 1960: 4 mutants : 44 other
plants.
“unifoliata”.
Narrow grey-green
leaflets with long petioles, some leaflets like little boats.
Dry seeds
orradiated 1959 with 40 krad x-rays. M1
1959. M2 1960: 1 mutant : 4 normal plants. M3
1961 : 3 mutants : 54 normal plants.
“unifoliata-type
III”. Single oval shaped leaflets, sessile = no petioles. Flowers normal.
M1 ? M2 ?
M3 1960:
(SCHEIBE
& MICKE 1967)
Mi 61-44
Partially „unifoliata“,
often incomplete separation of leaflets (pinnately lobed)
Dry seeds
not irradiated with x-rays, but 2 years stored.. M2 1961: 16 mutants : 30 normal plants.
(SCHEIBE & MICKE 1967)
„unifoliata
IIa”. Leaflets with petioles, central leaflet often sessile, frequent
“cups”.no or deformed sterile flowers.
Dry seeds
irradiated with x-rays. M2
1961: 5 mutants : 15 normal plants.
(SCHEIBE &
MICKE 1967)
“rosetta”. „unifoliata II“. Single leaflets with petioles.
“Filled” flowers, with many petals, roselike, clustered, sterile.
Dry seeds
irradiated 1959 with thermal neutrons (dose 10). M1 1960. M2 1961: 1 mutant : 20 normal plants.
(SCHEIBE & MICKE 1967)
„tristata“.
Enlarged bubbled
leaves, drooping on long petioles, sterile. Similar to rugose mutant observed
by GOPLEN (1967)?
Dry seeds
irradiated with thermal neutrons (dose 10) in 1959. M1 1960. M2 1961: 3 mutants : 33 normal. M3 1961 : 9 mutants : 45 normal
plants
Unifoliata-Typ Ia: Single leaflets with
petioles. Flowers clustered like cauliflower.
Dry seeds
of St.148 irradiated with thermal neutrons (1x10/13n/cm³) in 1959 and
additionally with 40 krad x-rays in 1960. M1 1960.
M2 1961 3 mutants : 14 normal plants. Flowers look like small cauliflowers, somewhat
clustered on the racemes, completely sterile.
M3 1962 from an open-pollinated normal looking sister plant: segregation 3 unifoliata : 12
partially unifoliata : 10 normal trifoliate.
“unifoliata”
with Ginkgo-shaped leaves. Normal petioles. Sterile. Small plant
Dry seeds
irradiated with thermal neutrons (dose 10) in1959 and with 40 krad x-rays in
1960. M1 1960. M2 1961:
1 mutant : 29 normal plants. M3 1962 : Sisterplants segregate for reduced leaflet nr. With large
leaves, no flower buds.
“tristata”Typ II. Malformed, bulged, necrotic
leaves, but normal flowers
Dry seeds
irradiated 1965 with fast neutrons. M2 1966:
1 mutant : 6 normal plants.
“unifoliata”.
Broad roundish
leaves with sharp dents.
Dry seeds
irradiated with fast neutrons. M1 1964. M2 1966: 1mutant : 37 normal plants.
Dry seeds
irradiated with thermal neutrons. M1 1964.
M2 1966: 2 mutants . 69 normal plants.
Folded
leaflets.
Dry seeds
irradiated with x-rays. M1
1964. M2 1966: 8 mutants : 81 normal plants.
Leaves
hanging down, funnel-shaped leaflets, loose flowers, stamens with separate
filaments, two pistils.
Dry seeds
irradiated with fast neutrons. M1 1964. M2 1966: 12 mutants : 77
normal plants.
References.
ANONYMUS
(Blüten aus lauter Kelchblättern)
Nature 405, 200.
BOWMAN, J.L., ALVAREZ, J., WEIGEL, D., MEYEROWITZ, E.M. and SMYTH, D.R. (1993)
Control of
flower development in Arabidopsis thaliana by APETALA 1 and interacting
genes.
Development
119, 721-743.
OKADA, K (1997)
Genetic
dissection of floral development using Arabidopsis mutants.
Gamma Field
Symposia No.36, Institute of Radiation Breeding NIAR, MAFF, Japan. p.1-12.
SCHEIBE, A. and A. MICKE (1967)
Experimentally
induced mutations in leguminous forage plants and their agronomic value.
In: Induced Mutations and their Utilization. Proc. Erwin-Baur-Gedächtnisvorlesungen IV, Gatersleben (Germany) 1966. Akademie-Verlag Berlin. p.231-236.
RÖMER (1973)