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Failure of EMS to induce S-locus mutations in Nicotiana alata (Link and Otto)
Er~ir~l and Experimea~l B0ta~y, Vol. 20, pp. 149 to 155 ~) Pergamon Prem Ltd. 1980. Printed in Great Britain
0098-8472/80/0301-014-9 $02.00/0
FAILURE OF EMS TO INDUCE S-LOCUS MUTATIONS IN NICO TIANA ALA TA (LINK AND O T T O ) K. SREE RAMULU
Association EURATOM-ITAL. Postbox 48, Wageningen, The Netherlands
(Received 5 june 1979; revised 11 October 1979) Sm~E KAMULU K. Failure of EMS to induce S-lo~s rmttations in Nicotiana alata (Link and Otto). ENVIRONMENTAL AND EXPERIMENTAL BOTANY 20, 149-155, 1980.wAttempts were made by EMS treatment of pollen mother cells to induce S-locus mutations in a self-incompatible clone of .Nirotiana alata. While the EMS treatments induced high frequencies of M 2 chlorophyll mutations, no S-locus mutations were induced. The mechanism of generation of S-locus mutations and the action of EMS are discussed.
INTRODUCTION
THE SELF-INCOMPATIBILITY locus in monofactorial gametophydc systems has a tripartite structure: two activity parts control the reaction in the pollen and in the style, respectively, and one specificity part, common to both pollen and style, which determines the specificityJ 31) Research with mutagens, such .as X-rays, fast neutrons and nitroso ethylurethane in Oenothera organensis, Nicotiana alata, Petunia inflata and Lycopersicum peruvianum has shown that these mutagens induced gross chromosomal changes, such as pollen-part mutations accompanied by a centric fragment, or structural changes within the S-complex (pollen-part mutations without a fragment or stylar mutations) leading to self-compatibility, but none generated new incompatibility specific~ties detectable as functional S-alleles in the progenies of trea' ted plants. (8' 16' 17' 2°' 24' 25' 27' 33' 36' 37) Ethyl methanesulphonate (EMS) is a very effective mutagen for both prokaryotic and eukaryotic organisms and is the most widely used chemical mutagen for higher plants. ( 1 ' 6 ' 7 ' 1 2 ' 1 3 ' 2 3 ' 3 0 " 3 5 ' ' 1 " 2 ' 4 3 ' ' $ 6 ) Two previous
studies tls'ls~ whose aim was to induce S-gene mutations after EMS treatments in Nicotiana alata with a monofactorial gametophytic system of self-incompatibility, were unsuccessful. VAN " GASTEL(is) concluded that the absence of Smutations after treatment of inflorescences was due to the fact that the pollen for screening mutations had not been collected from flowers treated at the sensitive stage (PMC). The failure to recover mutations at the S-locus after seed treatment was ascribed to the screening of a population of limited size and/or chimerism in L I and/or L3. ¢Is'Is} In both studies, however, the mutagenicity of the given EMS treatments was not ascertained. Hence, the data were inconclusive regarding the efficiency of EMS to induce S-gene mutations. The present study was undertaken to test the mutagenicity of EMS in the same clone of Nicotiana alata used in previous investigations. Pollen mother cells (PMC) were treated with buffered EMS solutions. Attempts were made to score the effects of EMS on the pollen component of the S-gene because large-scale scoring is possible and no complications are involved in the detection.
149
150
K. SREERAMULU MATERIAI~ AND METHODS
Genotypes The two self-incompatible diploid ( 2 n = 2 x --8) clones of Nicotiana alata (Link and Otto), namely OWL-S2S 3 and OB2-$68 ~ described previously by CARLVCClO et al., ~9) were used as test-material. The plants were grown under controlled conditions (16 hrday, 130001x, 22°C; 8hr night, 17°C; relative humidity 60-70%.
EMS treatment of pollen mother cells Since S-gene mutations induced after the tetrad stage are too late to express themselves in the pollen, c24'39) the EMS treatments were given to premeiotic (close to prophase I) and meiotic cells. The inflorescences of OWL-S2S 3 plants were treated by the tongue-slit method. ~29) EMS was obtained from Eastman Kodak, Co., Rochester, N.Y. (U.S.A.). To avoid toxic effects from acid products which form by EMS hydrolysis, the treatment solutions (16, 24 and 3 2 m M ) were prepared in pH 7 phosphate buffer. In the control plants, the treatments with phosphate buffer. (pH 7) alone did not cause any adverse effects. After the treatments, the tongue-slits were washed with water and continuously supplied with water in tubes until flowering to avoid drying of shoots. Sensitivity as measured by floral development, flowering delay and pollen abortion were established for different developmental stages. PMCs close to prophase I (pre-meiosis) were the most sensitive followed by meiotic and post-meiotic cells. Since 32raM EMS caused poor development of flower buds, premature. flower dropping, great delay in flowering and high percentage of pollen abortion, it was not used in further experiments.
Pollination procedures and detection of EMS effects S-gene mutations. Forty plants of each of the •
1froM and 24raM EMS and control series were analysed. Three to four shoots per plant were treated, and mature pollen was collected from one or two flowers per shoot in pre-meiosis or meiosis at the time of treatment. In the control, only one shoot was treated. The pollen from each flower was applied to the sdgrnas of one or two flowers of untreated plants from the same $2S 3 clone. In all, 794 pollinations w e r e
made from 16 and 2 4 m M EMS. Pollen was applied in monolayer on receptive surface of stigmas. The number of aborted pollen grains was substracted, and the esdmate of the number of pollen grains was made for 794 pollinations by procedures of SR~.~. RAIULU. ~*s) TO prevent abscission of flowers and to induce swelling of the ovaries, 1% naphthalene acetamide in lanoline was applied around the calyx of the flowers immediately after pollination. 13s) Chlorophyll mutations. Chlorophyll mutant phenotypes were scored in the M 2 population derived from the crosses performed with the mature pollen from flowers of $2S 3 plants that were in pre-meiosis or meiosis at the time of EMS treatment on untreated S~S7 plants. Progenies from control series (untreated $6S 7 x untreated .$2S3) were also screened for chlorophyll mutations. Similarly, the progenies derived from bud-pollinations of clone OWL-S2S 3 a n d OB2-$6S 7 were checked for chlorophyll mutations.
Pollen fertility Pollen stainability, a measure of pollen abortion induced after EMS treatment, was assessed by tests with Alexander's stain. 12~ The counts were based on 300 pollen grains per flower and 2-3 random flowers per plant in the control and treated series. The fertility of pollen, as ascertained after crosses with a cross-compatible tester-stock, was sufficiendy high. RESULTS
Pollen abortion The data on percentage pollen abortion after EMS treatments of pre-meiotic and meiotic cells are shown in Table 1. Pollen abortion increased with increasing concentration of EMS. The percentage abortion following treatment of premeiotic cells was twice the percentage found after treatment of meiodc buds, suggesting that the pre-meiotic cells (close to prophase 1) are more sensitive than the meiotic cells.
S-gene mutations From the data in Table 2, it can be seen that the rare cases of seed-set did not yield seedlings in any of the EMS-treated series. These results
S-LOCUS MUTATIONS IN aVICOTIANA
151
Table 1. Percentagepollen abortion after EMS treatments of pre-meiotic. (close to prophase I) and meiotic buds in Nicotiana alata
Treatments Control
Stage treated Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds
16 mM
24 mM
% pollen abortion (mean 4- S.E.) 4.2 4- 0.5 4.0+0.4 38.2 + 4.5 18.6_+ 3.2 62.5 4- 5.7 31.8 4- 4.1
For each item about 50 flowers (20 plants) from buds treated at pre-meiosis or meiosis were analysed. indicate that no S-mutations were induced after screening approx. 8 million pollen grains from 794 pollinations. Chlorophyll mutations To verify the mutagenicity of the given EMS treatments (16raM, 24mM), but not to make any comparative analysis of dose effects or of the differential sensitivity of the treated developmental stages, only a limited population was screened. No chlorophyll mutants were observed in the progenies of control (untreated S s S v x u n t r e a t e d S2Sa) and in the progenies from bud-pollinations of $2S 3 and StS 7. These results suggest that the self-incompatible clones were not heterozygous for chlorophyll mutations. Both 1 6 m M and 2 4 r a M EMS treatments were highly effective in inducing chlorophyll mutations (Table 3). From a total of 102 M 1 progenies, 18 segregated for chlorophyll mutations. Sixteen progenies exhibited segregation
ratios that did not deviate significantly from a monogenic ratio (3 normal: 1 mutant), although overall there was some recessive deficit. Two families showed a high deficit of recessives, probably due to low viability and transmissibility of mutated gametes and/or lethality of gametes. The spectrum of chlorophyll mutants included albina, chlorina, xantha and viridis; albina and chlorina occurred in high frequencies. DISCUSSION
The analysis o f the frequency of chlorophyll recessives, a widely used genetic parameter in higher plants, indicates that a mutagenically effective quantity of EMS reached the genetic material, and established the mutagenicity of the given EMS treatments. The results show that of 102 M I plant progenies, 18 segregated for chlorophyll recessives, each segregating M x progeny derived from an independent mutational event in a single pollen grain. After
Table 2. Results of crosses between untreated $2S 3 plants, used as female parents and EMS-treated $2S3 plants in Nicotiana alata
Treatments Control 16 mM 24 mM
Stage treated Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds
Number of pollinations
Number of fruits
Number of seeds
Number of plants
73 81 211 206 193 184
0 1 1 0 2 1
0 1 1 0 3 2
0 0 0 0 0 0
l0 11 17
No. of pollinations* 51$ 735 800
0 0 0
ll 17 35
Pre-meiotic 13 15 35
Meiotic
Stage of treatment
24 32 70
Total no. .
No. of MI progenies~, studied
150. 102 109
Mean progeny size (no. of M2 seedlings analysed per an M t progeny)
M 2 generation
0 3 8
Pre-meiotic
0 2 5
Meiotic
0 5 13
Total no.
No. of M l progenies segregating for chlorophyll mutations
* =Pollen developed fi'om treated PMC's (pre-meiotic and meiotic) ofS2S 3 plants was applied on untreated $6S 7 ~. t = From part of the seeds of crosses, untreated $6S 7 ~¢x treated $2S ~ (treated series) and untreated $6S 7 ~. x untreated $2S 3 lcoutrol). ~.= Derived from bud-pollination of randomly selected M i plants.
Control 16mM 24mM
Treatments
No. of No. of M 1 plants'[" chlorophyll studied mutants
M t generation
Table 3, Frequency of chloropkyll mutatior~ in the M 2 after E,~IS treatments of PMC's in the self-incompatible clone, OWL-S2S 3 0f Nicotiana alata
t,o
S-LOCUS MUTATIONS IN aVICOTIANA
153
Table 4. Fruit and seedyields and frequency of pollen-part mutants after treatments with X-rays and fast neutrons in the self-incompatible clone, OWL-S2S a ofNicofiana alata {from Van Gastel 1976). Irradiation dose
No. of pollinations
No. of seeded fruits
Total no. of seeds
No. of seeds germinated
No. of pollenpart mutants
% pollen abortion
572 472
7 13
22 391"
3 36
1 1
22 50
157
8
311~"
59
2
76
X-rays 300 rad 600 rad Fast neutrons 300 rad
*From these, 247 seeds sown. 1"From these, 110 seeds sown.
EMS treatment, 1 in 6 pollen grains showed a All previous studies with the same clone of aV. mutation at a chlorophyll locus. I f it is assumed alata have shown that the irradiation treatment that a ' m a x i m u m ' of 1000 loci control the produced fruit and seed yields which gave rise chlorophyll character, it can be expected that to only self-incompatible individuals in the under the present treatment conditions at least progenies. (Is-17) This was attributed to (phy1 in 6000 pollen grains should show a mutation siological) pseudo-compatibility processes inat a given locus, provided that all the loci are duced by treatments with ionizing radiations. equally sensitive to the mutagenic treatment. By The complete absence of seeded (viable) fruits contrast, not a single case of S-locus mutations, after EMS treatment indicates that under the namely pollen-part mutations leading to self- present treatment conditions, EMS induced no compatibility or new S-specificities occurred 'Physiologicals'. As with ionizing radiations, (16' 17, 2s, aa, 36.37) after EMS treatment in spite of screening approx. 8 million pollen grains. The possibility, however EMS failed to induce new S-specificities. This small, that EMS induces pollen-part mutations may suggest that probably new S-allele specifior new S-specificaties, but that EMS or its cities do not result from point mutations at the hydrolytic products prevents their expression, S-locus. However, it should be mentioned that c a n n o t be entirely excluded. in connection with EMS treatment, only one Previous studies on S-locus mutations in N. species has been studied, and further research alata showed that the pollen-part mutations with other incompatible species would be illuinduced by ionizing radiations are accompanied minating in this respect. Inbreeding, the only by a centric fragment; relatively large struc- method which so far has generated new Stural changes are assumed within the alleles(S, al,21, 34,40) does not produce point muS.complex.(15-17, 37--39) By their nature, tations by itself, but leads to other processes pollen-part mutations may be rather rare which result, under favorable conditions, in the events (Table 4). EMS induces in a variety of generation of new S-specificities. The various species a high frequency of mutations, but mechanisms have been extensively discussed by very, few gross chromosomal cha- DE NETTANCOURT(32) and PANDEY.(41) nges.t1,6, v, a4, 19.22,28,30,42,43.44) Further, EMS has also been shown to induce a high proportion Acknowledgement--I am grateful to Prof. J. H. VAN of point mutations (intragenic changes) in higher DER VEZN and Dr. J. SVSZNOAfor critical reading of plants. (a'4'1°) The complete absence of the the manuscript and for suggestions, Prof. H. F. pollen-part mutations at the S-locus of N. alata LINSKENS, Dr. K. K. PAND£V and Dr. D. DE after EMS treatment suggests that, in general, NZTTANCOURTfor their valuable advices and to Mr. P. Dugnuls for excellent technical assistance. these may not be point mutations.
154
K. SREE R A M U L U
REFERENCES
13. GASTEL A. J. G. VAN 0976) Mutability of the self-incompatibility locus and identification of the 1. AARON C. S. and LE~. W. R. (1978) Molecular S-bearing chromosome in Nicotiana alata. Doct. dosimetry of the mutagen ethyl methane sulthesis, Landbouwhogeschool, Wageningen phonate in Drosophila melanogaster spermatozoa: (1976). Agricultural Research Report 852. linear relation of DNA alkylation per sperm cell 16. GASTEL A. J. G. VAN and Nwr'rANCOURT D. DE (dose) to sex-linked recessive lethals. Mutat. Res; (1974) The effects of different mutagens on self49, 27--44. incompatibility in Nicotiana alata (Link and 2. AL~.XAr~roERM. P. (1969) Differential staining of Otto). I. Chronic gamma irradiation. Radiat. Bot. aborted and non-aborted pollen. Stain Techn. 44, 14, 43-50. 117-122. 17. GASTEL A. J. G. VAN and NET:rANcOURT 15. DE 3. AMANO E. (1972) Genetic fine structure analysis (1975) The effects of different mutagens on selfof mutants induced by ethyl methanesulphonate. incompatibility in Nicotiana alata (Link and Otto). In Gamma field symposia (II), Chemical mutagenesis II. Acute irradiations with X-rays and fast neuin Microorganisms and Plants, Tokyo. trons. Heredity 34, 381-392. 4. AMANO E. and SMITH H. H. (1965) Mutations 18. GASTEL A. J. G. VAN, BEEK M. A. and VAN D~a induced by ethyl methanesulphonate in maize. VF.EN J. H. (1973) The effects of EMS treatment Mutat. Res. 2, 344-351. of seed on self-incompatibili/y in Nicotiana alata. 5. ANDERSONM. K., TAYLOR N. L. and DVNCANJ. Incomp. Newslett. Assoc. Euratom-ITAL, Wageningen F. (1974) Serf-incompatibility, genotype identifi3, 62-66. cation and stability as influenced by inbreeding 19. HILLEKER A. J. (1976) Genetic analysis of the in red clover ( Trifolium pratense L.) Euphytica 23, centromeric heterochromatin of chromosome 2 of 140-148. Drosophila melanogaster: Deficiency mapping of 6. AUEReACH C. (1976) Mutation research, problems, EMS-induced lethal complementation groups. results and perspectives, Chapman & Hall, London. Genetics 83, 765-782. 7. AuEs.eACn C. and KILBEY B.J. (1971) Mutation 20. HOFFMANN M. (1971) Induktion und Analyse in eukaryotes. Annu. Rev. Genet., 5~ 163-218. von selbstkompatiblen Mutanten bei Lycopersicon 8. BREWeAV,~RJ. L. and NATARAJANA. T. "(1960) peruvianum (L.) Mill. II. Analyse der selbstkomCentric fragments and pollen-part mutation of patiblen Mutanten. Biol. Zentrabl. 90, 33--41. incompatibility alleles in Petunia. Genetics45, 699- 21. HOOENBOOM N. G. (1972) Breaking breeding 704. barriers in Lycopersi~on. 2. Breakdown of self9. CARLUCJO F., DS Nm'TAr~COURT D. and VAN incompatibility in L. Peruvianum (1.) Mill. Euphytica GASTEL A. J. G. (1974) On a possible involve21, 228-243. ment of chromosome 3 in the formation of self- 22. KONZEL G. (1971) The ratio of chemically incompatibility mutations in Nicotiana alata (Link duced chromosome aberrations to gene mutations et Otto). pp. 41-50. In Polyploidy and induced in barley. A critical study. Murat. Res. 12, 397mutations in plant breeding. IAEA, Vienna. 409. 10. Crxoun~,.Y P. S. and SCHWARTZ D. (1971) Ethyl 23. KONZAK C. F., NILAN R. A., WAONER J. and methanesulphonate-induced mutations of the Sh 1 Fosa~R R. J. (1965) Efficient chemical mutaprotein in maize. Murat. Res. 12, 151-157. genesis. Pages 49-70. In The use of induced mu11. Denward T. (1963) The function of the incomtations in plant breeding. Radiat. Bot. (Suppl.) 5. patibility alleles in red clover (Trifolium pratense 24. LEw~s D. (1949) Structure of the incompatibility L.). Hereditas 49, 189-334. gene. II. Induced mutation rate. Heredity 3, 33912. EHRSNnERG L., GUSTAFSSONA. and LUNDQVXST 355. U. (1961) Viable mutants induced in barley by 25. LEwis D. (1951) Structure of the incompatibility ionizing radiations and chemical mutagens. gene III. Types of spontaneous and induced Hereditas 47, 243-282. mutation. Heredity 5, 399-414. 13. FtsrInEIN L., F L A ~ W. G. and FALK H. L. 26. LEwls D. (1954) Comparative incompatibility (1970) Chemical mutagens. Academic Press, New in angiosperms and fungi. Adv. Genet. 6, 235York. 285. 14. FROESE-GERTZENE. E., KONZAKC. F., NmAN R. 27. LEwis D. and CROVeE L. K. (1954) Structure of A. and H~.XNER R. E. (1964) The effect of ethyl the incompatibility gene. IV. Types of mutation methanesulphonate on the growth response, chin Prunus avium L. Heredity 8~ 357-363. romosome structure and mutation rate in barley. 28. LIM J. K. and SNYDER L. A. (1974) Cytogenetic Radiat. Bot. 4, 61-69. and complementation analyses of recessive lethal
S-LOCUS MUTATIONS IN NICOTIANA mutations induced in the X chromosome of Drosophila by three alkylating agents. Genct. Res.
24,
1-1o.
29. LINs~Ns H. F. (1959) Zur Frag¢ der Entstehung der Abwehrktrper bei der Inkompatibilit~itst. reaktion yon Petunia 2. Mitteilung: Versuche mit radoaktiv markiertem Pollen. Ber. Dtsch. Bot. Ges. 72, 84-92. 30. LUNDQVXSTU. and yon WEz'rsT~x~ D. (1962) Induction of eceriferum mutants in barley by ionizing radiations and chemical mutagens. Hereditas 18, 342-362. 31. NEv'rANCOURTD. DE (i972) Self-incompatibility in basic and applied researches with higher plants. Genetica Agraria 26, 163-216. 32. NEZ'rANCOURT D. Dr. (1977) Incompatibility in angiosperms. Monographs on Theoretical and Applied Genetics 3 (Ed. R. FRANKEL, G. A. E. GALL, H. F. LaNsvd~s), Springer, Berlin. 33. NETTANCOURTD. D~ and ECOCItARD R. (1968) Effects of chronic irradiation upon a selfincompatible clone of Lycoper~icum peruvianum. Theor. Appl. Genet. 38, 289-293. 34. NET'rANCOURTD. ~E, ECOCnAPd)R., PERQUINM. D. G., VAN DER Dlurr T. and WESTERHOF M. (1971) The generation of new S-alleles at the incompatibility locus of L. Peruvianum Mill. Theor. Appl. Genet. 41, 120-129. 35. NI~N R. A. (1972) Mutagenic specificity in flowering plants: facts and prospects. Pages 141151. In Induced mutations and plant improvement. I.A.E.A., Vienna. 36. PANt)EV K. K. (1956) Mutations of selfincompatibility alleles in Trifolium pratense and 7". repens. Genetics 41, 327-343. 37. PANDF.VK. K. (1965) Centric chromosome fragments and pollen-part mutation of the incompatibility gene in Nicotiana alata. Nature (London) 206, 792-795.
155
38. PANDEY K. K. (1967) Elements of the S-gene complex. II. Mutation and complementation at the SI locus in Nicotiana alata. Heredity 22, 255-283. 39. PANDE'¢ K. K. (1970) Elements of the S-gene complex. VI. Mutations of the selfincompatibility gene, pseudo-compatibility and origin of new incompatibility alleles. Genetica 41, 477-5t6. 40. PANDEr K. K. (1970) New self-incompatibility alleles produced through inbreeding. Nature (London) 277, 689--690. 41. PANDEr K. K. (1977) Generation of multiple genetic speeiflcafies: Origin of genetic polymorphism through regulation. Theor. Appl. Genet. 49, 85-93. 42. RtBBELEN G. (1965) EMS efficiency in seeds and gametes of Arabidopsis thaliana. Pages 309-312. In Z. LAI~rDA,ed. Mechanism of mutation and inducing factors. Proc. Syrup. Mutation process, Prague, Czechoslovakia, Academia, Prague. 43. SCnALET A. P. (1978) Interspecific comparison of ethyl meth~nesulphonate-induced mutation rates in relation to genome size. Murat. Res. 49, 313-340. 44. SHARON O. and LIM J. K. (1976) Mutagenic effects of ethyl methanesulphonate on the giantwhite region inserted in the Y chromosome of Drosophila. Genetics 83 (Suppl.), 56-57. 45. SREERAMULtJK. (1978) On some factors influencing estimation of S-gene mutation rates. Incomp. Newslett. Assoc. Euratom-ITAL, Wageningen. Vol. 10, pp. 94-97. 46. VAN DER VEENJ. H. and WmTZ P. (1968) EMSinduced genic male sterility in Arabidopsis thaliana: a model selection experiment. Euphytica 17, 371-377.
0098-8472/80/0301-014-9 $02.00/0
FAILURE OF EMS TO INDUCE S-LOCUS MUTATIONS IN NICO TIANA ALA TA (LINK AND O T T O ) K. SREE RAMULU
Association EURATOM-ITAL. Postbox 48, Wageningen, The Netherlands
(Received 5 june 1979; revised 11 October 1979) Sm~E KAMULU K. Failure of EMS to induce S-lo~s rmttations in Nicotiana alata (Link and Otto). ENVIRONMENTAL AND EXPERIMENTAL BOTANY 20, 149-155, 1980.wAttempts were made by EMS treatment of pollen mother cells to induce S-locus mutations in a self-incompatible clone of .Nirotiana alata. While the EMS treatments induced high frequencies of M 2 chlorophyll mutations, no S-locus mutations were induced. The mechanism of generation of S-locus mutations and the action of EMS are discussed.
INTRODUCTION
THE SELF-INCOMPATIBILITY locus in monofactorial gametophydc systems has a tripartite structure: two activity parts control the reaction in the pollen and in the style, respectively, and one specificity part, common to both pollen and style, which determines the specificityJ 31) Research with mutagens, such .as X-rays, fast neutrons and nitroso ethylurethane in Oenothera organensis, Nicotiana alata, Petunia inflata and Lycopersicum peruvianum has shown that these mutagens induced gross chromosomal changes, such as pollen-part mutations accompanied by a centric fragment, or structural changes within the S-complex (pollen-part mutations without a fragment or stylar mutations) leading to self-compatibility, but none generated new incompatibility specific~ties detectable as functional S-alleles in the progenies of trea' ted plants. (8' 16' 17' 2°' 24' 25' 27' 33' 36' 37) Ethyl methanesulphonate (EMS) is a very effective mutagen for both prokaryotic and eukaryotic organisms and is the most widely used chemical mutagen for higher plants. ( 1 ' 6 ' 7 ' 1 2 ' 1 3 ' 2 3 ' 3 0 " 3 5 ' ' 1 " 2 ' 4 3 ' ' $ 6 ) Two previous
studies tls'ls~ whose aim was to induce S-gene mutations after EMS treatments in Nicotiana alata with a monofactorial gametophytic system of self-incompatibility, were unsuccessful. VAN " GASTEL(is) concluded that the absence of Smutations after treatment of inflorescences was due to the fact that the pollen for screening mutations had not been collected from flowers treated at the sensitive stage (PMC). The failure to recover mutations at the S-locus after seed treatment was ascribed to the screening of a population of limited size and/or chimerism in L I and/or L3. ¢Is'Is} In both studies, however, the mutagenicity of the given EMS treatments was not ascertained. Hence, the data were inconclusive regarding the efficiency of EMS to induce S-gene mutations. The present study was undertaken to test the mutagenicity of EMS in the same clone of Nicotiana alata used in previous investigations. Pollen mother cells (PMC) were treated with buffered EMS solutions. Attempts were made to score the effects of EMS on the pollen component of the S-gene because large-scale scoring is possible and no complications are involved in the detection.
149
150
K. SREERAMULU MATERIAI~ AND METHODS
Genotypes The two self-incompatible diploid ( 2 n = 2 x --8) clones of Nicotiana alata (Link and Otto), namely OWL-S2S 3 and OB2-$68 ~ described previously by CARLVCClO et al., ~9) were used as test-material. The plants were grown under controlled conditions (16 hrday, 130001x, 22°C; 8hr night, 17°C; relative humidity 60-70%.
EMS treatment of pollen mother cells Since S-gene mutations induced after the tetrad stage are too late to express themselves in the pollen, c24'39) the EMS treatments were given to premeiotic (close to prophase I) and meiotic cells. The inflorescences of OWL-S2S 3 plants were treated by the tongue-slit method. ~29) EMS was obtained from Eastman Kodak, Co., Rochester, N.Y. (U.S.A.). To avoid toxic effects from acid products which form by EMS hydrolysis, the treatment solutions (16, 24 and 3 2 m M ) were prepared in pH 7 phosphate buffer. In the control plants, the treatments with phosphate buffer. (pH 7) alone did not cause any adverse effects. After the treatments, the tongue-slits were washed with water and continuously supplied with water in tubes until flowering to avoid drying of shoots. Sensitivity as measured by floral development, flowering delay and pollen abortion were established for different developmental stages. PMCs close to prophase I (pre-meiosis) were the most sensitive followed by meiotic and post-meiotic cells. Since 32raM EMS caused poor development of flower buds, premature. flower dropping, great delay in flowering and high percentage of pollen abortion, it was not used in further experiments.
Pollination procedures and detection of EMS effects S-gene mutations. Forty plants of each of the •
1froM and 24raM EMS and control series were analysed. Three to four shoots per plant were treated, and mature pollen was collected from one or two flowers per shoot in pre-meiosis or meiosis at the time of treatment. In the control, only one shoot was treated. The pollen from each flower was applied to the sdgrnas of one or two flowers of untreated plants from the same $2S 3 clone. In all, 794 pollinations w e r e
made from 16 and 2 4 m M EMS. Pollen was applied in monolayer on receptive surface of stigmas. The number of aborted pollen grains was substracted, and the esdmate of the number of pollen grains was made for 794 pollinations by procedures of SR~.~. RAIULU. ~*s) TO prevent abscission of flowers and to induce swelling of the ovaries, 1% naphthalene acetamide in lanoline was applied around the calyx of the flowers immediately after pollination. 13s) Chlorophyll mutations. Chlorophyll mutant phenotypes were scored in the M 2 population derived from the crosses performed with the mature pollen from flowers of $2S 3 plants that were in pre-meiosis or meiosis at the time of EMS treatment on untreated S~S7 plants. Progenies from control series (untreated $6S 7 x untreated .$2S3) were also screened for chlorophyll mutations. Similarly, the progenies derived from bud-pollinations of clone OWL-S2S 3 a n d OB2-$6S 7 were checked for chlorophyll mutations.
Pollen fertility Pollen stainability, a measure of pollen abortion induced after EMS treatment, was assessed by tests with Alexander's stain. 12~ The counts were based on 300 pollen grains per flower and 2-3 random flowers per plant in the control and treated series. The fertility of pollen, as ascertained after crosses with a cross-compatible tester-stock, was sufficiendy high. RESULTS
Pollen abortion The data on percentage pollen abortion after EMS treatments of pre-meiotic and meiotic cells are shown in Table 1. Pollen abortion increased with increasing concentration of EMS. The percentage abortion following treatment of premeiotic cells was twice the percentage found after treatment of meiodc buds, suggesting that the pre-meiotic cells (close to prophase 1) are more sensitive than the meiotic cells.
S-gene mutations From the data in Table 2, it can be seen that the rare cases of seed-set did not yield seedlings in any of the EMS-treated series. These results
S-LOCUS MUTATIONS IN aVICOTIANA
151
Table 1. Percentagepollen abortion after EMS treatments of pre-meiotic. (close to prophase I) and meiotic buds in Nicotiana alata
Treatments Control
Stage treated Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds
16 mM
24 mM
% pollen abortion (mean 4- S.E.) 4.2 4- 0.5 4.0+0.4 38.2 + 4.5 18.6_+ 3.2 62.5 4- 5.7 31.8 4- 4.1
For each item about 50 flowers (20 plants) from buds treated at pre-meiosis or meiosis were analysed. indicate that no S-mutations were induced after screening approx. 8 million pollen grains from 794 pollinations. Chlorophyll mutations To verify the mutagenicity of the given EMS treatments (16raM, 24mM), but not to make any comparative analysis of dose effects or of the differential sensitivity of the treated developmental stages, only a limited population was screened. No chlorophyll mutants were observed in the progenies of control (untreated S s S v x u n t r e a t e d S2Sa) and in the progenies from bud-pollinations of $2S 3 and StS 7. These results suggest that the self-incompatible clones were not heterozygous for chlorophyll mutations. Both 1 6 m M and 2 4 r a M EMS treatments were highly effective in inducing chlorophyll mutations (Table 3). From a total of 102 M 1 progenies, 18 segregated for chlorophyll mutations. Sixteen progenies exhibited segregation
ratios that did not deviate significantly from a monogenic ratio (3 normal: 1 mutant), although overall there was some recessive deficit. Two families showed a high deficit of recessives, probably due to low viability and transmissibility of mutated gametes and/or lethality of gametes. The spectrum of chlorophyll mutants included albina, chlorina, xantha and viridis; albina and chlorina occurred in high frequencies. DISCUSSION
The analysis o f the frequency of chlorophyll recessives, a widely used genetic parameter in higher plants, indicates that a mutagenically effective quantity of EMS reached the genetic material, and established the mutagenicity of the given EMS treatments. The results show that of 102 M I plant progenies, 18 segregated for chlorophyll recessives, each segregating M x progeny derived from an independent mutational event in a single pollen grain. After
Table 2. Results of crosses between untreated $2S 3 plants, used as female parents and EMS-treated $2S3 plants in Nicotiana alata
Treatments Control 16 mM 24 mM
Stage treated Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds Pre-meiotic buds Meiotic buds
Number of pollinations
Number of fruits
Number of seeds
Number of plants
73 81 211 206 193 184
0 1 1 0 2 1
0 1 1 0 3 2
0 0 0 0 0 0
l0 11 17
No. of pollinations* 51$ 735 800
0 0 0
ll 17 35
Pre-meiotic 13 15 35
Meiotic
Stage of treatment
24 32 70
Total no. .
No. of MI progenies~, studied
150. 102 109
Mean progeny size (no. of M2 seedlings analysed per an M t progeny)
M 2 generation
0 3 8
Pre-meiotic
0 2 5
Meiotic
0 5 13
Total no.
No. of M l progenies segregating for chlorophyll mutations
* =Pollen developed fi'om treated PMC's (pre-meiotic and meiotic) ofS2S 3 plants was applied on untreated $6S 7 ~. t = From part of the seeds of crosses, untreated $6S 7 ~¢x treated $2S ~ (treated series) and untreated $6S 7 ~. x untreated $2S 3 lcoutrol). ~.= Derived from bud-pollination of randomly selected M i plants.
Control 16mM 24mM
Treatments
No. of No. of M 1 plants'[" chlorophyll studied mutants
M t generation
Table 3, Frequency of chloropkyll mutatior~ in the M 2 after E,~IS treatments of PMC's in the self-incompatible clone, OWL-S2S 3 0f Nicotiana alata
t,o
S-LOCUS MUTATIONS IN aVICOTIANA
153
Table 4. Fruit and seedyields and frequency of pollen-part mutants after treatments with X-rays and fast neutrons in the self-incompatible clone, OWL-S2S a ofNicofiana alata {from Van Gastel 1976). Irradiation dose
No. of pollinations
No. of seeded fruits
Total no. of seeds
No. of seeds germinated
No. of pollenpart mutants
% pollen abortion
572 472
7 13
22 391"
3 36
1 1
22 50
157
8
311~"
59
2
76
X-rays 300 rad 600 rad Fast neutrons 300 rad
*From these, 247 seeds sown. 1"From these, 110 seeds sown.
EMS treatment, 1 in 6 pollen grains showed a All previous studies with the same clone of aV. mutation at a chlorophyll locus. I f it is assumed alata have shown that the irradiation treatment that a ' m a x i m u m ' of 1000 loci control the produced fruit and seed yields which gave rise chlorophyll character, it can be expected that to only self-incompatible individuals in the under the present treatment conditions at least progenies. (Is-17) This was attributed to (phy1 in 6000 pollen grains should show a mutation siological) pseudo-compatibility processes inat a given locus, provided that all the loci are duced by treatments with ionizing radiations. equally sensitive to the mutagenic treatment. By The complete absence of seeded (viable) fruits contrast, not a single case of S-locus mutations, after EMS treatment indicates that under the namely pollen-part mutations leading to self- present treatment conditions, EMS induced no compatibility or new S-specificities occurred 'Physiologicals'. As with ionizing radiations, (16' 17, 2s, aa, 36.37) after EMS treatment in spite of screening approx. 8 million pollen grains. The possibility, however EMS failed to induce new S-specificities. This small, that EMS induces pollen-part mutations may suggest that probably new S-allele specifior new S-specificaties, but that EMS or its cities do not result from point mutations at the hydrolytic products prevents their expression, S-locus. However, it should be mentioned that c a n n o t be entirely excluded. in connection with EMS treatment, only one Previous studies on S-locus mutations in N. species has been studied, and further research alata showed that the pollen-part mutations with other incompatible species would be illuinduced by ionizing radiations are accompanied minating in this respect. Inbreeding, the only by a centric fragment; relatively large struc- method which so far has generated new Stural changes are assumed within the alleles(S, al,21, 34,40) does not produce point muS.complex.(15-17, 37--39) By their nature, tations by itself, but leads to other processes pollen-part mutations may be rather rare which result, under favorable conditions, in the events (Table 4). EMS induces in a variety of generation of new S-specificities. The various species a high frequency of mutations, but mechanisms have been extensively discussed by very, few gross chromosomal cha- DE NETTANCOURT(32) and PANDEY.(41) nges.t1,6, v, a4, 19.22,28,30,42,43.44) Further, EMS has also been shown to induce a high proportion Acknowledgement--I am grateful to Prof. J. H. VAN of point mutations (intragenic changes) in higher DER VEZN and Dr. J. SVSZNOAfor critical reading of plants. (a'4'1°) The complete absence of the the manuscript and for suggestions, Prof. H. F. pollen-part mutations at the S-locus of N. alata LINSKENS, Dr. K. K. PAND£V and Dr. D. DE after EMS treatment suggests that, in general, NZTTANCOURTfor their valuable advices and to Mr. P. Dugnuls for excellent technical assistance. these may not be point mutations.
154
K. SREE R A M U L U
REFERENCES
13. GASTEL A. J. G. VAN 0976) Mutability of the self-incompatibility locus and identification of the 1. AARON C. S. and LE~. W. R. (1978) Molecular S-bearing chromosome in Nicotiana alata. Doct. dosimetry of the mutagen ethyl methane sulthesis, Landbouwhogeschool, Wageningen phonate in Drosophila melanogaster spermatozoa: (1976). Agricultural Research Report 852. linear relation of DNA alkylation per sperm cell 16. GASTEL A. J. G. VAN and Nwr'rANCOURT D. DE (dose) to sex-linked recessive lethals. Mutat. Res; (1974) The effects of different mutagens on self49, 27--44. incompatibility in Nicotiana alata (Link and 2. AL~.XAr~roERM. P. (1969) Differential staining of Otto). I. Chronic gamma irradiation. Radiat. Bot. aborted and non-aborted pollen. Stain Techn. 44, 14, 43-50. 117-122. 17. GASTEL A. J. G. VAN and NET:rANcOURT 15. DE 3. AMANO E. (1972) Genetic fine structure analysis (1975) The effects of different mutagens on selfof mutants induced by ethyl methanesulphonate. incompatibility in Nicotiana alata (Link and Otto). In Gamma field symposia (II), Chemical mutagenesis II. Acute irradiations with X-rays and fast neuin Microorganisms and Plants, Tokyo. trons. Heredity 34, 381-392. 4. AMANO E. and SMITH H. H. (1965) Mutations 18. GASTEL A. J. G. VAN, BEEK M. A. and VAN D~a induced by ethyl methanesulphonate in maize. VF.EN J. H. (1973) The effects of EMS treatment Mutat. Res. 2, 344-351. of seed on self-incompatibili/y in Nicotiana alata. 5. ANDERSONM. K., TAYLOR N. L. and DVNCANJ. Incomp. Newslett. Assoc. Euratom-ITAL, Wageningen F. (1974) Serf-incompatibility, genotype identifi3, 62-66. cation and stability as influenced by inbreeding 19. HILLEKER A. J. (1976) Genetic analysis of the in red clover ( Trifolium pratense L.) Euphytica 23, centromeric heterochromatin of chromosome 2 of 140-148. Drosophila melanogaster: Deficiency mapping of 6. AUEReACH C. (1976) Mutation research, problems, EMS-induced lethal complementation groups. results and perspectives, Chapman & Hall, London. Genetics 83, 765-782. 7. AuEs.eACn C. and KILBEY B.J. (1971) Mutation 20. HOFFMANN M. (1971) Induktion und Analyse in eukaryotes. Annu. Rev. Genet., 5~ 163-218. von selbstkompatiblen Mutanten bei Lycopersicon 8. BREWeAV,~RJ. L. and NATARAJANA. T. "(1960) peruvianum (L.) Mill. II. Analyse der selbstkomCentric fragments and pollen-part mutation of patiblen Mutanten. Biol. Zentrabl. 90, 33--41. incompatibility alleles in Petunia. Genetics45, 699- 21. HOOENBOOM N. G. (1972) Breaking breeding 704. barriers in Lycopersi~on. 2. Breakdown of self9. CARLUCJO F., DS Nm'TAr~COURT D. and VAN incompatibility in L. Peruvianum (1.) Mill. Euphytica GASTEL A. J. G. (1974) On a possible involve21, 228-243. ment of chromosome 3 in the formation of self- 22. KONZEL G. (1971) The ratio of chemically incompatibility mutations in Nicotiana alata (Link duced chromosome aberrations to gene mutations et Otto). pp. 41-50. In Polyploidy and induced in barley. A critical study. Murat. Res. 12, 397mutations in plant breeding. IAEA, Vienna. 409. 10. Crxoun~,.Y P. S. and SCHWARTZ D. (1971) Ethyl 23. KONZAK C. F., NILAN R. A., WAONER J. and methanesulphonate-induced mutations of the Sh 1 Fosa~R R. J. (1965) Efficient chemical mutaprotein in maize. Murat. Res. 12, 151-157. genesis. Pages 49-70. In The use of induced mu11. Denward T. (1963) The function of the incomtations in plant breeding. Radiat. Bot. (Suppl.) 5. patibility alleles in red clover (Trifolium pratense 24. LEw~s D. (1949) Structure of the incompatibility L.). Hereditas 49, 189-334. gene. II. Induced mutation rate. Heredity 3, 33912. EHRSNnERG L., GUSTAFSSONA. and LUNDQVXST 355. U. (1961) Viable mutants induced in barley by 25. LEwis D. (1951) Structure of the incompatibility ionizing radiations and chemical mutagens. gene III. Types of spontaneous and induced Hereditas 47, 243-282. mutation. Heredity 5, 399-414. 13. FtsrInEIN L., F L A ~ W. G. and FALK H. L. 26. LEwls D. (1954) Comparative incompatibility (1970) Chemical mutagens. Academic Press, New in angiosperms and fungi. Adv. Genet. 6, 235York. 285. 14. FROESE-GERTZENE. E., KONZAKC. F., NmAN R. 27. LEwis D. and CROVeE L. K. (1954) Structure of A. and H~.XNER R. E. (1964) The effect of ethyl the incompatibility gene. IV. Types of mutation methanesulphonate on the growth response, chin Prunus avium L. Heredity 8~ 357-363. romosome structure and mutation rate in barley. 28. LIM J. K. and SNYDER L. A. (1974) Cytogenetic Radiat. Bot. 4, 61-69. and complementation analyses of recessive lethal
S-LOCUS MUTATIONS IN NICOTIANA mutations induced in the X chromosome of Drosophila by three alkylating agents. Genct. Res.
24,
1-1o.
29. LINs~Ns H. F. (1959) Zur Frag¢ der Entstehung der Abwehrktrper bei der Inkompatibilit~itst. reaktion yon Petunia 2. Mitteilung: Versuche mit radoaktiv markiertem Pollen. Ber. Dtsch. Bot. Ges. 72, 84-92. 30. LUNDQVXSTU. and yon WEz'rsT~x~ D. (1962) Induction of eceriferum mutants in barley by ionizing radiations and chemical mutagens. Hereditas 18, 342-362. 31. NEv'rANCOURTD. DE (i972) Self-incompatibility in basic and applied researches with higher plants. Genetica Agraria 26, 163-216. 32. NEZ'rANCOURT D. Dr. (1977) Incompatibility in angiosperms. Monographs on Theoretical and Applied Genetics 3 (Ed. R. FRANKEL, G. A. E. GALL, H. F. LaNsvd~s), Springer, Berlin. 33. NETTANCOURTD. D~ and ECOCItARD R. (1968) Effects of chronic irradiation upon a selfincompatible clone of Lycoper~icum peruvianum. Theor. Appl. Genet. 38, 289-293. 34. NET'rANCOURTD. ~E, ECOCnAPd)R., PERQUINM. D. G., VAN DER Dlurr T. and WESTERHOF M. (1971) The generation of new S-alleles at the incompatibility locus of L. Peruvianum Mill. Theor. Appl. Genet. 41, 120-129. 35. NI~N R. A. (1972) Mutagenic specificity in flowering plants: facts and prospects. Pages 141151. In Induced mutations and plant improvement. I.A.E.A., Vienna. 36. PANt)EV K. K. (1956) Mutations of selfincompatibility alleles in Trifolium pratense and 7". repens. Genetics 41, 327-343. 37. PANDF.VK. K. (1965) Centric chromosome fragments and pollen-part mutation of the incompatibility gene in Nicotiana alata. Nature (London) 206, 792-795.
155
38. PANDEY K. K. (1967) Elements of the S-gene complex. II. Mutation and complementation at the SI locus in Nicotiana alata. Heredity 22, 255-283. 39. PANDE'¢ K. K. (1970) Elements of the S-gene complex. VI. Mutations of the selfincompatibility gene, pseudo-compatibility and origin of new incompatibility alleles. Genetica 41, 477-5t6. 40. PANDEr K. K. (1970) New self-incompatibility alleles produced through inbreeding. Nature (London) 277, 689--690. 41. PANDEr K. K. (1977) Generation of multiple genetic speeiflcafies: Origin of genetic polymorphism through regulation. Theor. Appl. Genet. 49, 85-93. 42. RtBBELEN G. (1965) EMS efficiency in seeds and gametes of Arabidopsis thaliana. Pages 309-312. In Z. LAI~rDA,ed. Mechanism of mutation and inducing factors. Proc. Syrup. Mutation process, Prague, Czechoslovakia, Academia, Prague. 43. SCnALET A. P. (1978) Interspecific comparison of ethyl meth~nesulphonate-induced mutation rates in relation to genome size. Murat. Res. 49, 313-340. 44. SHARON O. and LIM J. K. (1976) Mutagenic effects of ethyl methanesulphonate on the giantwhite region inserted in the Y chromosome of Drosophila. Genetics 83 (Suppl.), 56-57. 45. SREERAMULtJK. (1978) On some factors influencing estimation of S-gene mutation rates. Incomp. Newslett. Assoc. Euratom-ITAL, Wageningen. Vol. 10, pp. 94-97. 46. VAN DER VEENJ. H. and WmTZ P. (1968) EMSinduced genic male sterility in Arabidopsis thaliana: a model selection experiment. Euphytica 17, 371-377.