Radiation induced mutants of sweet clover (Melilotus albus Desr.)

A. Micke (Vienna, Austria)

Between 1953 and 1974 several series of mutation induction experiments with sweet clover (Melilotus albus Desr.) were carried out at the Max-Planck-Institut für Züchtungsforschung, Forschungstelle Neuhof/Giessen (1953-55) and at the Institut für Pflanzenbau und Pflanzenzüchtung der Universität Göttingen (1956-74) under guidance of  ARNOLD SCHEIBE. The primary aim was the creation of non-bitter breeding stocksfor the development of cultivars of white sweet clover, suitable for European agriculture.

Such attempts had been undertaken in Germany already since the twenties of this century, primarily at the Kaiser-Wilhelm-Institut für Züchtungsforschung Müncheberg upon the initiative of ERWIN BAUR, and after 1945 till about 1958 at the Max-Planck-Institut für Züchtungsforschung Voldagsen (later at Köln-Vogelsang). For literature review and historical details see RUDORF 1943, 1959. The success of chemical mass selection 1928-30 by VON SENGBUSCH in finding spontaneous, simple inherited alkaloid-free lupin mutants (for review see HACKBARTH and TROLL 1959;  GUSTAFSSON and GADD 1965) raised expectations to find glucosid-free sweet clover mutants in a similar way. Although other Melilotus species with low glucosid content had been identified and were used to achieve the goal through species hybridisation with embryo culture (GREENSHIELDS 1954), the expectations to find glucosid-free spontaneous mutants in Melilotus albus were not fulfilled. This failure was probably due to inadequate chemical screening techniques.

SCHEIBE and co-workers, therefore, started in 1953 a programme of experimental mutagenesis with sweet clover, at the same time also looking after the screening technique (MICKE, 1957). Besides some rather uncommon chemical mutagens (SCHEIBE and HÜLSMANN 1957, 1958), x-rays and thermal neutrons were employed as mutagens and numerous of induced mutants of different kind were detected  (MICKE 1955, 1957b, 1958, 1959, 1961b, 1962a, b and c, 1965, 1966a and b, 1968, 1969b, 1991 and unpublished; SCHEIBE and MICKE 1967). In line with the principle aim of the programme to find non-bitter mutants, many published results concern biochemical and genetic aspects of the o-oxy-cinnamic acid glucosid, the compound from which the undesired coumarin derives through enzymatic hydrolysation (KAHNT 1962, 1963;  KAHNT and SCHÖN 1962; MICKE 1957, 1962b; SCHÖN 1961, 1966), others concern more practical plant breeding aspects, particularly the use of induced mutants as hybrids (DANCS 1964; HÜLSMANN and MICKE 1961; MICKE 1961a, 1962/63, 1969a and c, 1974, 1975, 1976; PILLAI 1962; RÖMER 1973, 1974; RÖMER and MICKE 1974). Unfortunately, central-european agricultural interest in sweet clover declined and the programme was discontinued. Until then, 14 induced mutants had been studied and were described in detail (MICKE 1955, 1958, 1962a, 1962/63; SCHEIBE and MICKE 1967) and 83 other mutants had been mentioned in various publications (MICKE 1962b, 1969, 1976; RÖMER 1973; SCHÖN 1966) – see Table 1. The rest of the collection of more than 700 induced mutants was not further studied and no further details were published yet.

Primarily outside Europe, continued agricultural interest in Melilotus was reflected in crossbreeding programmes, resorting mainly to low-coumarin stocks, developed through hybridisation between the bitter species Melilotus albus and M. officinalis and the non-bitter species Melilotus dentatus (as the viability of such hybrids was extremely low, embryo in-vitro culture and embryo grafting had to be employed). In 1965, SMITH and GORZ reviewed sweet clover improvement worldwide and listed just 19 spontaneous mutant traits. Therefore, obviously encouraged by the success of our mutagenesis programme in Göttingen, around 1966 a project on chemical mutagenesis was undertaken at the University of Nebraska, using hydroxylamine, 2-aminopurine, 5-bromouracil and ethyl methanesulfonate (EMS). Only EMS was found to be effective (KLEINHOFS et al. 1968) and results of genetic studies with 7 mutant traits (besides chlorophyll deficiencies) were published between 1969 and 1975 (GENGENBACH et al. 1996, 1970; RONNENKAMP et al. 1973, 1975).

Recently, however, mutations in Melilotus albus are looked for again with revived interest, this time because sweetclover appears suitable as a leguminous model species for molecular genetics and gene engineering (COOK et al. 1997). For this reason an effort is now made by us to list and describe the most interesting of the so far unpublished mutants observed in our Melilotus mutagenesis programme between 1953 and 1969 (Table 2).

Table 1: (still to be inserted)

Table 2: (still to be inserted)

Address of author: Prof. Dr. Alexander Micke, Salmannsdorfer Str.94, A-1190 Vienna (Austria)

Website: www.plantmutations.com

e-mail: alexander.micke@plantmutations.com



Model legumes get the nod.

Plant Cell 9,275-280.

DANCS, L. (1964)

Untersuchungen über die Anbauwürdigkeit cumarinreicher und cumarinarmer Steinkleeformen (Melilotus albus)im Vergleich mit Luzerne.

Z.Acker- und Pflanzenbau 120,17-46.


Genetic studies of induced mutants in Melilotus alba.  II.Inheritance and complementation of chlorophyll-deficient mutants.

Crop Science 10,154-156.


Genetic studies of induced mutants in Melilotus alba.  I.Short-internode dwarf, curled leaf, multifoliate leaf, and cotyledonary branching.

Crop Science 9,607-610.


Embryology of interspecific crosses in Melilotus.

Can.J.Bot. 32,447-465.

GUSTAFSSON, A. and I.GADD (1965)

Mutations and crop improvement. II.The genus Lupinus (Leguminosae).

Hereditas 53, 15-39.

HACKBARTH, J. and H.-J. TROLL (1959)

Lupinen als Körnerleguminosen und Futterpflanzen.

Handb.d.Pflanzenz. 2.Aufl. Bd.4. p.1-51. Parey, Berlin.

HÜLSMANN, G. und A.MICKE (1961)

Untersuchungen über den günstigsten Zeitpunkt der Samenernte beim weißen Steinklee.

Angew.Botanik 35,181-183.

KAHNT, G. (1962)

Isolierung des trans- und cis-o-Oxyzimtsäureglucosids aus Steinkleeblättern (Melilotus albus) und Umwandlung der trans-Form in die isomere Verbindung in vivo und in vitro durch Sonnenlicht.

Naturwiss. 49,207-208.

KAHNT, G. (1963)

Veränderungen des Glucosids der o-Oxyzimtsäure in den Blättern des weißen Steinklees (Melilotus albus) während der Ontogenese der Pflanze.

Angew.Botanik 37,131-161.

KAHNT, G. und W.J.SCHÖN (1962)

Zur quantitativen Analyse des Cumarins und des Glucosids der Cumarinsäure in Blättern von Melilotus albus.

Angew.Botanik 36,33-49.


Mutation induction in Melilotus alba annua by chemical mutagens.

Crop Science 8,631-632.

MICKE, A. (1955)

Die Auswirkungen einer Röntgenbestrahlung lufttrockener Samen von Melilotus albus Desr. auf die Bestrahlungsgeneration und deren Nachkommenschaften.

Dissertation zur Erlangung des Doktor-Grades, Justus Liebig -Universität Gießen, BRD.

MICKE, A. (1957a)

Eine vereinfachte Methode zur Prüfung von Steinklee-Individuen auf Cumarin.

Der Züchter 27,179-181.

MICKE, A. (1957b)

Über die Auslösung isotomer Sproßgabelungen bei Melilotus albus durch Röntgenbestrahlung der Samen.

Angew. Botanik 31,106-116.

MICKE, A. (1958)

Mutationszüchtung beim weißen Steinklee (Melilotus albus) mit Hilfe von Röntgenstrahlen.

Z.Pflanzenzüchtg. 39,419-437.

MICKE, A. (1959)

Wachstumsförderung bei Melilotus albus durch schwache Neutronenbestrahlung trockener Samen.

Naturwiss. 46,407-408.

MICKE, A. (1961a)

Mutation work in Melilotus alba.

In: Report of the Ninth Sweetclover Improvement Conference.

Madison, Wisconsin, USA. (H.J.GORZ and W.K.SMITH, edit.)

University of Wisconsin. p.10-11.

MICKE, A. (1961b)

Comparison of the effects of x-rays and thermal neutrons on viability and growth of sweet clover (Melilotus albus) after irradiation of dry seeds.

In: Effects of Ionizing Radiation on Seeds. Proceedings of an Internat. Symposium, Karlsruhe (FRG). p.403-410.

IAEA, Vienna.

MICKE, A. (1962a)

Eine bitterstoffreie Mutante bei Melilotus albus nach Bestrahlung von Samen mit thermischen Neutronen.

Naturwiss. 49,332.

MICKE, A. (1962b)

Genetische Untersuchungen beim weißen Steinklee (Melilotus albus).

I. Über die Vererbung der Fähigkeit zur Bildung von Cumarinsäureglykosid.

Z.Pflanzenzüchtg. 48,1-13.

MICKE, A. (1962c)

Morphological, physiological and genetical effects of X-rays and thermal neutrons in Melilotus albus.

Paper presented at the Second International Congress of Radiation Research, Harrogate, Yorkshire UK, 5-11 August 1962.

MICKE, A. (1962/63)

Genetisch-züchterische Arbeiten beim weißen Steinklee.

Z. Acker- und Pflanzenbau 116, 354-360.

MICKE, A. (1965)

Unusual reaction of sweet clover seeds to x-radiation at different moisture contents and to post-irradiation storage.

Nature  205, 1126.

MICKE, A. (1966a)

Modification of radiosensitivity in seeds of different plant species by secondary factors.

In: Mechanism of Mutation and Inducing Factors.

Proc. of the Symposium on the Mutational Process, Prague (CSSR), 9-11 August 1965. p.27-32.

Academia Prague. Publishing House of the Czechoslovak Academy of Sciences.

MICKE, A. (1966b)

Der Einfluß modifizierender Faktoren auf die Wirkung ionisierender Strahlen bei pflanzlichen Samen.

Z.Pflanzenzüchtg. 55, 29-66.

MICKE, A. (1968)

Modification of radiosensitivity and of genetical radiation effects.(Final Report IAEA Res. Contract No.396)

In: IAEA Research Contracts – Eighth Annual Report. Technical Report Series No.85. p.42-43.

IAEA, Vienna.

MICKE, A. (1969a)

Utilization of induced mutants as hybrids.

In: Proc. Fifth Congress of the European Association for Research on Plant Breeding EUCARPIA, Milano (Italy) 1968 (Edit. G.C. CHISCI, G.HAUSSMANN). p.262-268.

Genetica Agraria.

MICKE, A. (1969b)

Zur Frage der mutagenen Wirkung wiederholter Röntgenbestrahlungen.

Naturwiss. 56, 40.

MICKE, A. (1969c)

Improvement of low yielding sweet clover mutants by heterosis breeding.

In: Induced Mutations in Plants. Proc. of a FAO/IAEA Symposium, Pullman (USA) 1969. p.541-550.

IAEA Vienna.

MICKE, A. (1974)

Heterosis bei Kreuzung von Mutanten derselben Ausgangsform.

In: Bericht über die Arbeitstagung 1974 der Arbeitsgemeinschaft der Saatzuchtleiter, Gumpenstein (Österreich). p.314-328.

Verlag der Bundesversuchsanstalt für alpenländische Landwirtschaft Gumpenstein.

MICKE, A. (1975)

Zur Wahl von induzierten Mutanten für die Kreuzungszüchtung.

In: Bericht über die Arbeitstagung 1975 der Arbeitsgemeinschaft der Saatzuchtleiter, Gumpenstein (Österreich). p.249-260.

Verlag der Bundesversuchsanstalt für alpenländische Landwirtschaft, Gumpenstein.

MICKE, A. (1976)

Hybrid vigour in mutant crosses – Prospects and problems of exploitation studied with mutants of sweet clover.

In: Induced Mutations in Cross Breeding. Proc. of FAO/IAEA Advisory Group, Vienna (Austria) 1975. p.199-218.

IAEA Vienna.

MICKE, A. (1991)

Induced mutations for crop improvement.

In: Biotechnology and Mutation Breeding. Proc. Gamma Field Symposia No.30. Inst.of Radiation Breeding, NIAR, MAFF Ohmiya-machi, Ibaraki-ken, Japan. p.1-21.

PILLAI, K.M. (1962)

Vergleichende Untersuchungen über den Verlauf der Stoffproduktion und der Entwicklung bei bitterstoffreichem und -freiem Steinklee und Luzerne.

Dissertation, landw.Fakultät d.Univ.Göttingen. pp.64

RÖMER, F.W. (1973)

Heterosis und Kombinationseignung in diallelen Kreuzungen von strahleninduzierten Steinklee-Mutanten.

Z.Pflanzenzüchtg. 70,323-348.

RÖMER, F.W. (1974)

Bedeutung der Selbstungsanteile bei der Ermittlung der allgemeinen Kombinationseignung von Steinklee-Mutanten nach einem Polycross.

Z.Pflanzenzüchtg. 71,151-169.

RÖMER, F.W. and MICKE, A. (1974)

Combining ability and heterosis of radiation-induced mutants of Melilotus albus DES.

In: Polyploidy and Induced Mutations in Plant Breeding. Proc. of two meetings organized by FAO/IAEA and EUCARPIA, Bari (Italy) 1972. p.275-276.

IAEA Vienna.


Genetic studies of induced mutants in Melilotus alba.  IV.Inheritance and complementation of six additional chlorophyll-deficient mutants.

Crop Science 15, 187-188.


Genetic studies of induced mutants in Melilotus alba.  III.Foldet leaflet, elongated stem, and short-petiole dwarf.

Crop Science 13, 320-321.

RUDORF, W. (1943)

Steinklee, Melilotus albus DESR., Melilotus officinalis (L.) DESR., und andere Arten.

Handb.d.Pflanzenz. 1.Aufl. Bd.3. p.260. Parey, Berlin.

RUDORF, W. (1959)

Steinklee, Melilotus albus DESR., und Melilotus officinalis (L.) DESR.

Handb.d.Pflanzenz. 2.Aufl. Bd.4.  p.218-238. Parey, Berlin.


Über das Auftreten bitterstoffarmer Pflanzen von Melilotus albus in der C2-Generation nach Behandlung mit mutagenen Chemikalien.

Naturwiss. 44,17-18.


Mutationsauslösung durch Chemikalien beim Steinklee (Melilotus albus).

Z.Pflanzenzüchtg. 39,299-324.

SCHEIBE, A. and MICKE, A. (1967)

Experimentally induced mutations in leguminous forage plants and their agronomic value.

In: Induzierte Mutationen und ihre Nutzung (Induced Mutations and their Utilization) Erwin-Baur-Gedächtnisvorlesungen IV. Gatersleben 20-24 June 1966. p.231-236.

Akademie-Verlag Berlin.

SCHÖN, W.J. (1961)

About the analysis of cinnamic acid derivatives in Melilotus albus.

In: Report of the Ninth Sweetclover Improvement Conference. Madison, Wisconsin, USA. (H.J.GORZ and W.K.SMITH, edit.) University of Wisconsin. p.11-12.

SCHÖN, W.J. (1966)

Über die stereospezifische ß-Glucosidase in bitterstoff-freien Mutanten bei Melilotus albus.

Angew.Botanik 40,38-54.

SMITH, W.K. and H.J.GORZ (1965)

Sweetclover improvement.

Advances in Agronomy 17,163-231.

Academic Press, New York.