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.

 

The Mutants

Mi 56-251

Bush-Type.  Highly branching due to loss of apical dominance.

Dry seeds irradiated 1955 with 40krad x-rays.  M1 1955.  M2 1956: 38 mutants : 125 normal

plants. (MICKE 1958, 1962)

Mi 56-422

“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)

 

Mi 56-637

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.

 

Mi 58-367

“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)

 

Mi 58-657

“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)

Mi 59-31

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.

 

Mi 59-57

“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.

 

Mi 59-72

“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.

 

Mi 59-97

“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.

 

Mi 59-127

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)

 

Mi 59-159

“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.

 

Mi 59-1518

« 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.

 

Mi 60-407

« unifoliata »Type ?.

Dry seeds treated with neutrons (dose 5). M1 1959.  M2 1960. M3 1961: 7 mutants : 8 other plants.

 

Mi 60-517

“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.

 

Mi 60-587

“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.

 

Mi 60-2525 (= 58-92?)

“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)

Mi 61-654

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)

 

Mi 61-1835 (see also 61-1852)

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)

 

Mi 61-1921

„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

 

Mi 61-2134

Unifoliata-Typ Ia: Single leaflets with petioles. Flowers clustered like cauliflower.

Dry seeds of St.148 irradiated with thermal neutrons (1×10/13n/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.

 

Mi 61-2175

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.

 

Mi 66-325

“tristata”Typ II. Malformed, bulged, necrotic leaves, but normal flowers

Dry seeds irradiated 1965 with fast neutrons.  M2 1966:  1 mutant : 6 normal plants.

 

Mi 66-815

“unifoliata”. Broad roundish leaves with sharp dents.

Dry seeds irradiated with fast neutrons. M1 1964. M2 1966: 1mutant : 37 normal plants.

 

Mi 66-868

“unifoliata” with cauliflower buds.

Dry seeds irradiated with thermal neutrons. M1 1964.  M2 1966: 2 mutants . 69 normal plants.

 

Mi 66-882

Folded leaflets.

Dry seeds irradiated with x-rays. M1 1964.  M2 1966:  8 mutants : 81 normal plants.

 

Mi 66-1007

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)