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0ABSTRACTS — KURZE MITTEILUNGEN
Askkll Love : Agamospermy in Ac e t o s a.
Although only relatively few of the many genera of the large family Polygonaceae have as yet been cytogeneticaliy or embryologically investigated, occurrence of agamospermy has been observed in perhaps five or six genera as yet, i.e. Oxyria (Edman, 1929), Atraphaxis (Edman, 1931), Rumex (Dudgeon, 1918), Acetosella (Murbeck, cf. Nokdstedt, 1907; Love, 1943), and Acetosa (Roth, 1906; Love, 1942). Only in the genus Atraphaxis is the agamospermy obligate, and within that genus it is known to be of the type termed apospory (Edman, I. c.; cf. Gustafsson, 1946, 1947). In Oxyria the observations made by Edman (/. c.) are not applicable to all ecotypes of the species, as at least some part of the material from the Scandinavian mountains and Iceland studied by the present writer certainly reproduce — at all events mainly — by sexual methods. Observations made by Dudgeon {I. c.) on Rumex crispus may also be somewhat dubious, although in the experiments performed by the present writer on this species some indications of the occurrence of at least facultative agamospermy in a low frequency of ovules have been obtained. In the arctic-alpine annual species Koenigia islandica, too, indications of the occurrence of agamospermy, perhaps of the aposporous type, are met with in Icelandic material as well as in material from the Scandinavian mountains fixed by Professor Edman and handed over to the present writer. This material, however, has not been completely studied as yet (Love, unpubl.).
In the spring of 1939 the present writer began extensive investigations on the biological behaviour of different genera of Polygonaceae, especially the dioecious genera Acetosella and Acetosa and the Scandinavian representatives of the genus Rumex s. str. (for nomenclature, cf. ltive and Lcjve, 1948). The investigations were performed at the Institute of Genetics of the University of Lund, Sweden, mainly at its substation at Svalof, during the years 1939—1945, but since then they have been worked out at our present institute in Reykjavik.
In 1942 some of the results obtained from the studies of the genus Acetosa were published in a short paper (LOVE, 1942), and its taxonomical groups were described somewhat later on (LOve, 1944; LOve and LOve, 1948). In the paper of 1942 some comments were made as to the occurrence of agamospermy in the material of Acetosa from Scandinavia and Iceland, and it was mentioned that the agamospermy observed must be only of a facultative kind. In 123 of more than 300 isolations made some few seeds, in most cases only 1—10, but in some cases 20—25, were found to develop and give rise to plants, which were all found to have the diploid chromosome number 2n = 14 and two X chromosomes. No plants had flowered when the paper was published in 1942, but the next summer all the plants flowered. They were all found to be female and strictly maternal in their appearance. The plants in question belonged to the species Acetosa arifolia, A. pratensis, and A. thyrsi-flora. The highest frequency was met with in the plants belonging to A. arifolia from Iceland and northern Sweden (2,3 % in 53 % of the plants studied), but the lowest frequency was met with in .4. thyrsiflora from Scania in southernmost Sweden (0,3 % in 21 % of the plants).
In the summers of 1941 and 1942 new isolations were made of female plants from different localities. Practically the same frequency of agamosperm-ous seed setting was found in this material, too. One plant, however, formed an astonishing exception, as the three branches isolated were found to have a complete seed setting in the isolation bags as well as on the branches outside. This plant belonged to the species .4. arifolia and originated from the neighbourhood of Reykjavik, and it looked just like normal female plants from the same locality.
Of course, the seeds obtained after isolation of the female plant in question were suspected to be the result only of an unsuccessful isolation, although no holes were detected in the bags. The seeds were grown out, however, and 200 plants were placed in the experimental fields at Svalof in the spring of 1942. At the same time 100 plants obtained from seeds of unisolated branches of the same female plant were placed in the experimental fields in order to test their sexual and cytological behaviour compared with the first-mentioned progeny.
Only very few of the plants flowered during the summer of 1942, but in the summer of 1943 all the individuals flowered. At flowering time all these individuals were found to be of the same appearance, and not a single male plant was found in the progeny from the isolations or from the unisolated branches of the mother plant. All these 300 individuals were females of the Icelandic ecotype from Reykjavik, which is rather unlike the normal Swedish and Alpine ecotypes of the same species.
Although this evidence might be strong enough to indicate asexual seed setting of this plant and its progeny, the writer made fixations of all the plants in order to obtain embryological material as well as mitotic chromosomes for studies of their karyotype. Unfortunately, however, he has not been able to make complete analyses of the material until now, partly because he moved to Reykjavik and partly owing to lack of experimental fields and apparatus for microscopical work. In the fixations of the flowers no useful material was obtained for embryological analyses, but in the root tips of all the plants the chromosomes could be closely studied and measured. For the cytological methods used, cf. LOve (1943).
In the genus Acetosa subgenus Dioeca sectio Etiacelosae all the male plants are found to have the diploid chromosome number 2n =. 15, three of which are sex chromosomes, 2 F chromosomes and 1 X chromosome. The female plants, however, have only 2n = 14 chromosomes with only two X chromosomes. The chromosomes within this group are rather large and their shape and form are not exactly the same in all individuals of the same species. Therefore, some so-called karyotypes have been described within some of the species (cf. Kihara and Yamamoto, 1931; Ono, 1935; Yamamoto, 1933, 1938; LOve, 1942), and the different karyotypes are found to have a somewhat different distribution in Japan as well as in Scandinavia and Iceland.
The karyotype of the female plant from the neighbourhood of Reykjavik was found to be 10 i + 2 ts + 2 X chromosomes, and the is chromosomes were found to be the second smallest autosome pairs. Exactly this karyotype has not been mentioned by the Japanese workers, as the SAT chromosomes of the
almost corresponding karyotype in Japan are found to be the third longest autosome pair. For the signs of the karyotypes, cf. Love (1943).
All the individuals belonging to the progenies of the above-mentioned female plant were found to have the chromosome number 2n = 14, and in every case the karyotype was found to be the same, i. e. 10 i + 2 is + 2 A', or exactly the karyotype of the original mother plant.
The karyotype mentioned was also found in some other female individuals from the same locality, but those individuals were normal amphimictic females with a relatively high degree of facultative agamospermy (2,3—4,1 % seed setting after safe isolations). In order to test possible differences in length of some of the chromosomes in these individuals and the agamospermous progeny, the chromosomes from five plants of each group were carefully measured in the microscope. They were, however, not found to differ in any way, and therefore it might be suggested that the agamospermy observed is not caused by deletions of large or measurable parts of any of the chromosomes.
As mentioned above, no embryological preparations have as yet been studied. Therefore, it is impossible to decide at present what type of agamospermy is met with in the plants studied by the writer. All the results show, however, that in one of the Icelandic biotypes of Acetosa arifolia obligate agamospermy is met with, and that in a rather high frequency of the material of just this species from the same locality facultative agamospermy is met with besides the normal amphimictic seed formation. Therefore, it might be suggested that both these types of asexual seed setting are genically controlled and that the first plant with obligate agamospermy has been a result of an unusual accumulation of genes for agamospermous seed formation. This suggestion is supported by the relatively high frequency of facultative agamospermy in the material from the same locality.
Summary. — Agamospermous seed formation has been observed in material of the dioecious diploid species Acetosa arifolia, A. pratensis, and A. thijrsiflora. It is clearly facultative and occurs only in a low percentage of the ovules, highest in the northern material of A. arifolia, lowest in the more southern material of A. thyrsiflora.
In one Icelandic plant and its progeny the agamospermy was found to be absolutely obligate, as no seed was formed in the normal amphimictic way. This plant belonged to the Icelandic type of A. arifolia and originated from the neighbourhood of Reykjavik. Its karyotype was quite normal and of exactly the same type as that of the normal amphimictic females from the same locality. The frequency of facultative agamospermy among the other females from the same locality was unusually high.
It is suggested that the agamospermy met with in Acetosa is genically determined and that the obligate agamospermy of the female plant in question and its progeny is the result of an unusual accumulation of genes for this abnormal type of seed formation.
Agamospermy has at present been observed in the following genera of Polygonaceae: Oxyria, Atraphaxis, Rumex, Acetosella, Acetosa, and Koenigia,
although it has not been thoroughly studied in all cases and in most cases it is perhaps only facultative.
Institute of Botany and Genetics, University Institute of Applied Sciences, Reykjavik, Iceland.
Literature cited.
1. Dudgeon, W. 1918. Morphology of Rumex crispus. — Bot. Gaz. 60: 39;:—421.
2. Edman, G. 1929. Zur Entwicklungsgeschichte der Gattung Oxyria Hill., nebst
zytologischen, embryologischen und systematischen Bemerkungen über einige andere Polygonaceen. — Acta Hort. Berg. 9: 165—291.
3. — 1931. Apomeiosis und Apomixis bei Atraphaxis frutescens C. Koch. — Acta
Hort. Berg. 11: 13—es.
4. Gustafsson, ä. 1946. Apomixis in higher plants. Part I. The mechanism of
apomixis. — Acta Univ. Lund, N. F. 2, Vol. 42, No. 3, pp. l—07.
5. — 1947. Apomixis in higher plants. Part II. The causal aspect of apomixis. —
Acta Univ. Lund, N. F. 2, Vol. 43, No. 2, pp. 69—178.
6. Kihara, H. and Yamamoto, Y. 1931. Karyomorphologische Untersuchungen an
Rumex acetosa L. und Rumex montanus Desf. — Cytologia 3: 84—118.
7. LÖVE, A. 1942. Cytogenetic studies in Humex. III. Some notes on the Scandi-
navian species of the genus. — Hereditas XXVIII: 289—296.
8. — 1943. Cytogenetic studies on Rumex subgenus Acetosella. — Diss. Lund
1943; Hereditas XXX: 1—130.
9. — 1944. The dioecious forms of Rumex subgenus Acetosa iii Scandinavia. —
Bot. Notiser 1944:237—254.
10. Löve, A. and Löve, D. 1948. Chromosome numbers of northern plant species. —
Icel. Univ. Inst. Appl. Sei., Dept. Agric. Rep. Ser. B, No. 3: 1—131.
11. Nordstedt, O. 1907. Apogami hos Rumex. — Bot. Notiser 190 7 : 238.
12. Ono, T. 1935. Chromosomen und Sexualität von Rumex Acetosa. — Sei. Rep.
Tohoku Imp. Univ. (4th Ser.) (Biol ), 10:41—210.
13. Roth, F. 1906. Die Fortpflanzungsverhältnisse bei der Gattung Rumex. — Verh.
naturh. Ver. preuss. Rheinl. Westf., Bonn, 63 : 327—360.
14. Yamamoto, Y. 1933. Karyotypes in Rumex acetosa and their geographical
distribution. — Jap. Journ. Genet. 8: 204—274.
15. — 1938. Karyogenetische Untersuchungen bei der Gattung Rumex. VI. — Mem.
Coll. Agric. Kyoto Imp. Univ. 43 (Genet. Ser. 8): l—39.
Bengt Kihlman: The effect of purine derivatives on chromosomes.
It has been shown that caffeine and theophylline are able to induce mutations in the ascomycete Ophiostoma maltiannulatum (Fries and Kihlman, 1948) and chromosome changes in Allium (Kihlman and Levan, 1949). The research on the cytological effect was extended to include 15 purine derivatives. The test object was as before root-tips of Allium Cepa, kindly provided by Dr. Albert Levan of Lund.
Most of the purines have been synthezised by the author. The ethyl
xanthines only were obtained from the Research Laboratory of the N. V. Organon through the courtesy of Professor Sune HekgstrOm, Lund.
The particular stress in this investigation is laid on the effect on the chromosomes. Other changes which the purines induce in the root meristem will only be briefly mentioned.
The toxic effect of the purines increases when methyl groups are substituted by other alkyl groups.
The allvl group is less toxic than the propyl group.
Primary effect Secondary effect
The lowest cone, tes-
Piirine derivatives ted where the effect is "I" AA/mmol
, , purine
to be seen «¡0 AA/mmol purine ~"/i"ÄA/iiiinol
mmol »/0 purine calfeinc
1,7-diinethylhj'poxan thine ...... 0 0 + +
theophylline .................. + + 1,0 33
caffeine ...................... 0 0,04 3,0 100
8-nielhoxvcaffeine ............. 0,2 0,004 4,8 160
8-ethoxvcaffeine ............... 0,2 0,005 6,2 207
8-propyloxycaffeine ........... 0,2 0,005 3,3 110
8-allyloxvcaffeine .............. 0,2 0,005 6,0 200
l-elhvltheohromine ............ + + + +
1,3-dietliylxanthine ............ + + + +
1,3,7-1 riethylxan thine .......... -f -f -r +
1,3,7-trimethyIuric acid ........ + + + +
l,3,7,!Metramethyluric acid..... 0,5 0,01 19,5 650
!)-ethyt-l,3,7-triinethyluric acid .. 5,0 0,12 2,2 73
9-allyM,3,7-trimethyluric acid . . + + 1,0 33
1,3,7,'.Mctramethyl-4,5-d ¡me thoxy-
ditivdrouric acid ............ 0 0 0 0
+ means that primary or secondary changes, respectively, are too weak to be expressed in numerals.
% AA means the percentage of abnormal anaphases.
Later 8-chlorocaffeine was also tested. The secondary effect proved to be intermediary between the effect of the 8-ethers of caffeine and tetramefhyluric acid, or, in numerals, 10 % AA/mmol, and 333 per cent of the caffeine effect. The threshold of the primary effect lies between 0,5—'J,2 ninnil (0,()l—0,005 per cent).
In the same way the c-mitotic tendencies increase when methyl groups are substituted by other alkyl groups and the water solubility decreases. This fact is in agreement with observations made by Lev an and Ostekuben (1943).
In conformity to the methylxanlhines the alkylated oxvpurines so far investigated suppress the cell wall formation, although the effect is not always so obvious as, for instance, that of caffeine. Tetramethyl-4,5-dimethoxydi-hydrouric acid, which lacks the double bond between the carbon atoms 4 and 5, is however quite inactive.
The physiological or primary changes, i.e. the »stickiness» and »pseudo-chiasma» (Levan and Tjio, 1948) phenomena were analysed after a treatment of four hours. As appears from the accompanying table, tetramethyluric acid and the 8-ethers of caffeine induce primary chromosome changes in four to ten times more diluted solutions than caffeine. The stickiness phenomenon induced by these compounds is also of a more extreme type, and usually results in anaphase bridges between all of the separating chromosome arms. Tetra-methyl-4,5-diniethoxydihydrouric acid is completely inactive, dimethylhypo-xanthine and the ethylxanthines are practically inactive, whereas theophylline, 9-allyltrimethyluric acid and 1,3,7-trimethyluric acid induce a weak effect.
The structural or secondary chromosome changes were analysed after a treatment of 24 hours followed by 1—4 days in water. The slight solubility in water of the purine derivatives has sometimes rendered the investigation more difficult (viz. propyloxycaffeine, allyloxycaffeine) or impeeded it completely (theobromine).
The analysis is performed in such a way that the anaphases with structural chromosome changes are counted and expressed in per cent of the total number of anaphases on the slide. Each substance is tested in a series of concentrations and the percentage of abnormal anaphases is determined per mmol of purine. Since the effect is usually not proportional to the concentration, the highest value thus ohtained is taken as an expression of the strength of the effect. As a comparison, the effect of each substance is then expressed in per cent of the caffeine effect calculated in the same way. This method of analysis is not exact, the standard error being about ± 10 %. In such a comparative investigation the method is suitable, however.
It appears from the table, that the 8-ethers of caffeine and especially tetramethyluric acid, again, are much more effective than caffeine, while tetra-methyl-4,5-dimethoxydihydrouric acid is totally inactive, trimethyluric acid and dimethylhypoxanthine practically so.
When further comparing caffeine with 1-ethy¡theobromine and with 1,3,7-triethylxanthine, theophylline with 1,3-diethylxanthine and finally tetramethyluric acid with 9-ethyltrimethyluric acid it will be seen that the substitution of a N-bound methyl group with an ethyl group considerably reduces the effect. When several methyl groups in this way are substituted by ethyl groups the purine derivative is practically inactive. On the other hand, it seems that the length of the carbon chain in the oxygen bound alkyl groups of the 8-ethers of caffeine does not in a higher degree influence the effect. The effect seems further to increase with an increasing N-methylation and with an increasing number of electronegative substituents in positions 6 and 8.
An explanation of these results is possibly to be found in an investigation by Weil-Maliierhe (1946). He analysed the solvent effect of a great number of natural and synthetical purine derivatives on int. al. 3,4-benzpyrene. Among these purines are also such as have been tested in the present investigation. A comparison shows that a striking concordance exists between the solubiliz-ing power, on the one hand, and the strength of the effect on the chromosomes, on the other. The only exception, 1,3,7-trimethyluric acid, may also be explained from the investigation by Weil-Malherbe, showing that purines with acidic functions are strongly inactivated when the acidic group is ionized. He
further points out that such purines in the buffered medium of the cell are likely to be ionized. Hence it is to be expected that they are inactive there.
So far as it can be estimated from the rather limited number of purine derivatives hitherto tested, the same rules are valid with regard to the solvent effect as well as to the cytological one. The latter is thus also favoured by those molecular-constitutional factors which, according to Weil-Malherbe, favour both the polarizability and the hydrophilic character of the molecule.
A detailed account of the results of the experiments will be published when the comparative investigation of the effect of purine derivatives on chromosomes and micro-organisms is finished.
Institute of Physiological Botany, University of Upsala, May 28, 1949.
Literature cited.
1. Fries, N. and Kihlman, U. 1948. Fungal mutations obtained with methyl
xanthines. — Nature 162 : 573.
2. Kihlman, B. and Levan, A. 1949. The cytological effect of caffeine. — Hereditas
XXXV: 109.
3. Lk.van, A. and Tjio, J. H. 1948. Induction of chromosome fragmentation by
phenols. — Hereditas XXXIV: 453.
4. Levan, A. and Ostergren, G. 1943. The mechanism of c-mitotic action. Ob-
servations on the naphthalene series. — Hereditas XXIX: 381.
5. Weil-Malherbe, H. 1946. The solubilization of polycyclic aromatic hydro-
carbons by purines. — Biochem. Journ. 40: 351.
