Cytogenetic effects caused by two synthetic auxins: 1-naphtaleneacetic acid (NAA) and indole-3-butyric acid (IBA) as well as by synthetic cytokinin benzyladenine (BA), on root tip cells of ten-day-old European black pine (Pinus nigra Arn.) seedlings were observed. The results showed that auxins induced stronger cytogenetic effects than cytokinin. After treatment with auxins the mitotic activity and the corresponding number of chromosomal aberrations increased in parallel to the concentrations used. Both auxins, NAA and IBA, induced almost the same types of nuclear changes such as spindle failure, chromosome fragmentation, laggards and polyploidy. NAA induced a higher frequency of chromosomal changes than IBA. Also, the recovery period after the application of NAA was much slower as compared to that of IBA. Cytokinin BA showed the opposite effect. After treatment with the highest concentration the mitotic activity was very similar to the control, but after the recovery period the mitotic index increased significantly. This was also the case with a number of aberrant cells.Istraženo je djelovanje dva sintetska auksina: 1-naftalenoctene kiseline (NAA) i indolil-3-maslačne kiseline (IBA) i sintetskog citokinina benziladenina (BA) na meristemske stanice vrška korjenčića 10 dana starih klijanaca crnog bora (Pinus nigra Arn.). Auksini imaju snažnije citogenetsko djelovanje na genom meristemskih stanica od citokinina. Nakon tretmana auksinima mitotska aktivnost stanica je porasla paralelno s upotrijebljenom koncentracijom. Broj aberantnih stanica također se povećavao s koncentracijom auksina. Oba auksina induciraju gotovo iste tipove promjena na razini diobenog vretena i na razini kromosoma. Sintetski citokinin BA je pokazao suprotno djelovanje. Nakon tretmana s najvećom koncentracijom mitotska aktivnost bila je vrlo slična onoj u kontrolnom uzorku. Međutim nakon oporavka značajno je porasla, a također je porastao i broj kromosomskih aberacija

Dražena Papeš

Mirjana Pavlica

Višnja Besendorfer

English

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Acta Bot. Croat. 50, 31— 36, 1991. CODEN: ABCRA2 ISSN 0365-0588UDC 576.312.32:582.475.4 =  20 Original scientific paperN U C L E A R  C H A N G E S  I N E U R O P E A N  B L A C K  P I N E  S E E D L I N G S  C A U S E D  B Y  G R O W T H  R E G U L A T O R SDRAŽENA PAPES, VIŠNJA BESENDORFER and MIRJANA PAVLICA  (Department o f M olecular B iology, Faculty o f  Science, U niversity o f  Zagreb)R eceived  D ecem ber 20, 1990Cytogenetic effects caused by two synthetic auxins: 1- -naphtaleneacetic acid (NAA) and indole-3-butyric acid (IBA) as well as by synthetic cytokinin benzyladenine (BA), on root tip cells of ten-day-old European black pine (Pinus nigra Arn.) seedlings were observed. The results showed that auxins induced stronger cytogenetic effects than cytokinin. After treatment with auxins the mitotic activity and the corresponding number of chromosomal aberrations incre­ased in parallel to the concentrations used. Both auxins, NAA and IBA, induced almost the same types of nuclear changes such as spindle failure, chromosome fragmentation, laggards and polyploidy. N AA induced a higher frequency of chromosomal changes than IBA. Also, the recovery period after the application of NAA was much slower as compa­red to that of IBA. Cytokinin BA showed the opposite effect. After treatment with the highest concentration the mitotic activity was very similar to the control, but after the recovery period the mitotic index increased significantly. This was also the case with a number of aberrant cells.I n t r o d u c t i o nA number of factors, chemical and physical, contribute to chromo­some instability in cells cultured in vitro as well as in tissues and plants regenerated from them ( B a y l i s s  1980, P a p e s  et al. 1983). The successful establishment of plant cell and tissues cultured in vitro depends mostly on composition of the medium, growth regulator concentrations and culture conditions such as temperature, light and photoperiod.A C T A  B O T . C R O A T . V O L . 50, 1991. 3 1D R A Ž E N A  P A P E S  e t al.It is well known that in cultured tissues ( B a y l i s s  1980) plant growth regulators, which induce proliferation, at the same time cause cytogenetic changes. Plants regenerated from callus or cells in suspen­sion show genetic and phenotypic variabilities ( L a r k i n  and S c o w ­er  o f  t 1981), instability of chromosome structure and variation in chromosome number as reflected by polyploidy and aneuploidy ( B e n -  n i c i  and D ’ A m a t o  1978, D ’ A m a t o  1985). Plant growth regu­lators control the cell division cycle at the molecular level (H o u s s a e t  al. 1990) as specific triggers in one or several steps of cell division ( F o s k e t  et al. 1977). In some species triggering of mitosis of differenti­ated cells may depend on a correct balance of exogenous auxins and cytokinins (C i o n i n i e t a 1. 1978).In this study we investigated the effect of three synthetic growth regulators frequently used in tissue culture of gymnosperms (K o 1 e v - s k a - P l e t i k a p i c  e t  al. 1983) on root tips of European black pine seedlings.M a t e r i a l s  a n d  M e t h o d sBlack pine (Pinus nigra Arn.) seeds from the Institute for Forest Research, Jastrebarsko, were germinated in petri dishes on moist filter paper at room temperature. After ten days the seedlings were treated with various concentrations of two synthetic ayxins, 1-naphtaleneacetic acid (NAA) and indole-3-butyric acid (IBA), and synthetic cytokinin, benzyladenine (BA). The concentrations used varied from 10-3 to HP7 M. The treatment time was 6 h with a period of 7 h recovery in distilled water. Seeds germinated in tap water were used as control (F i s k e s j 6 1988). For each concentration and the corresponding control at least 20 seeds were used. For microscopic studies root tips were cut and fixed in ethanolacetic acid (3:1) for at least 24 h. Slides were prepared using Feulgen squash technique (S h a r m a and S h a r m a 1972). Permanent slides were prepared using liquid carbon dioxide and after drying fqr 1 day the tissue was mounted in Euparal. In order to analyse the mitotic activity and the number of chromosomal aberrations 1500 cells from 20 root tips were scored per concentration and its corresponding control.R e s u l t s1. Effects of auxinsTable 1 shows the mitotic activity and the total chromosome aberra­tions in black pine root tips after 6 h treatment with NAA and IBA. The mitotic activity decreased with the concentration used, while the mitotic activity of the control was lower than that of the treatment with the lowest concentration of NAA and IBA. The number of dividing cells after the recovery period increased slightly with concentration (Tab. 2). The highest number of aberrant cells was observed after treatment with the highest concentration (Hr3 M) of each of the auxins (Tab. 1). Howe­ver, the number of total chromosomal aberrations decreased with the concentration parallel to the mitotic activity. After the recovery period the amount of the total chromosomal aberrations was lower, and also decreased with the concentrations (Tab. 2).32 A C T A  B O T . C R O A T . V O L . 50. 1991.N U C L E A R  C H A N G E S  IN  E U R O P E A N  B L A C K  P IN E  SE E D L IN G SFigs. 1— 4. Chromosomal aberrations in root tip cells of Pinus nigra Arn.after 6 h treatment with NAA, IBA and BA as well as after a 7 h recovery period. 12341. Chromosome stickiness and clumping chromation.2. c-mitosis, metaphase.3. Polyploid cell in anaphase as a result of c-mitosis.4. Chromosome centric and acentric fragments in late prophase.A C T A  B O T . C R O A T . V O L . 50, 1991.N U C L E A R  C H A N G E S IN  E U R O P E A N  B L A C K  P IN E  SEE D LIN G ST able  1. Effects of different concentrations of NAA, IBA and BA on the mitotic acti­vity and frequency of chromosomal changes in root tip cells of a ten-day-old black pine seedlings.Growth Concentration No of cells M. I. Total aber-regulator (M) examined (% ) rant cells (%)i o - 3 1500 24.6 5.8i o - 4 1500 17.6 4.2NAA 10~5 1500 14.5 1.4i o - 6 1500 10.6 1.210“ 7 1500 8.5 0.110-3 1500 24.9 7.2i o * 4 1500 14.7 2.3IBA 10-5 1500 17.8 1.710 -6 1500 14.6 0.81 0 -7 1500 11.2 010-3 1500 9.0 010"4 1500 15.5 1.0BA IO"5 1500 15.0 0.310-6 1500 13.9 0.310-7 1500 13.9 0.3Control 2000 9.5 0Table 2. Mitotic activity and total chromosomal aberrations after a 7 h recovery period.Growth Concentrations No. of cells M. I. Total aber-regulator ( M ) examined (% ) rant cells (%)i o - 3 1500 29.7 3.9i o - 4 1500 17.4 2.8NAA 10-5 1500 15.0 0.610 -6 1500 19.8 0.61 0 -7 1500 1 1 . 8 0.210 -3 1500 26.3 3.110-4 1500 22.4 0.2IBA 10 -5 1500 22.4 0.110 -6 1500 17.3 01 0 -7 1500 12.7 010-3 1500 18.0 0.710-4 1500 15.5 0.3BA I O " 5 1500 20.2 0.210"6 1500 17.4 0.110 -7 1500 15.5 0.2Control 2000 9.5 0A C T A  B O T . C R O A T . V O L . 50, 1991. 33D R A Ž E N A  P A P E S  et al.Both synthetic auxins induced the same types of nuclear changes which were caused by hormonal effects either on the spindle or on chromosomal organization. The most prominent spindle abnormalities were c-mitoses (Fig. 2) and disturbed anaphase. Polyploidy was also ob­served, after a treatment with lower concentrations of NAA and IB A (1(T6 and 1CT7 M) and after a recovery period (Fig. 3). Chromosome frag­mentation (Fig. 4), laggards in anaphase and telophase, anaphase bridges and micronuclei were observed as well. Stickiness was very frequent and it accompanied all the instabilities of spindle and chromosomal organiza­tion (Fig. 1).2. Effects of cytokininAfter treatment with BA, the mitotic activity was lower than after- treatment with auxins. Mitotic activity was similar to that of the control (Tab. 1). However, after the recovery period the mitotic activity incre­ased significantly, especially after treatment with the highest concen­tration (Tab. 2). The number of the total chromosomal aberrations was lower than after the treatment with auxins, although it increased slightly after the recovery period (Tabs. 1 and 2), especially after the treatment with the highest concentration. Laggards in anaphase and telophase were noticed after the recovery period. After treatment with BA micronuclei and anaphase bridges were also observed, while polyploid cells were present only in a small number after the recovery period.D i s c u s s i o nAuxinsData obtained in treatments with NAA and IBA show similarities in mitotic activity (Tab. 1). The mitotic index decreased parallel with the concentrations of either NAA or IBA. But after the treatment with the lowest concentration of NAA or IBA (HP7 M) the mitotic activity was higher than in the control. The synthetic auxins NAA and IBA stimula­ted cell division, which was dose-dependent. After the treatment with the highest concentrations of NAA or IBA (HP3 M) the mitotic activity was 2.5 X higher than in their respective controls. The explanation of these results could be the fact that in tissue culture auxins are almost invari­ably required to promote the initial growth of the meristem, so they stimulate both cell growth and division. Similar results were obtained after the recovery period (Tab. 2). The total chromosomal aberrations occurred in a dose-dependent manner, that is, the highest number of aberrant cells was observed after treatment with the highest concentra­tion of either NAA or IBA, of which NAA had the stronger effect. NAA and IBA induced almost the same types of nuclear changes. The most prominent spindle abnormalities were the c-mitosis and disturbed ana­phases observed also in shallot root tips after treatment with 2,4-D ( P a v l i c a  et  al. 1991). It is well known that synthetic auxins like NAA and 2,4-D used in herbicide concentrations (>  50 ftg/ml), can induce spindle failure and other mitotic abnormalities in intact plants (B a y 1 i s s 1980). Chromosomal fragmentation and laggards at anaphase were also observed. Spindle failure and chromosome laggards may influence the process of polyploidization (D ’ A m a t o  1985), as we also noticed in our experiments.3 4 A C T A  B O T . C R O A T . V O L . 50, 1991.N U C L E A R  C H A N G E S IN  E U R O P E A N  B L A C K  P IN E  SE E D L IN G SCytokininsAfter treatment with BA the mitotic activity was lower than that following treatment with auxins. Since cytokinin synthesis takes place in meristematic tissue, mainly in root-tip cells ( S h o r t  and T o r r e y 1972), and the administration of BA on intact roots decreased the mitotic activity, this may have been the consequence of interaction between exo­genous and endogenous cytokinins. After the recovery period we noticed a significant increase of mitotic activity, probably because of a delay in cytokinin activity. We noticed the same trend in the number of aberrant cells. There were laggards, bridges and micronuclei, a sign of chromoso­mal breakage and elimination ( O g u r a  1982). The mechanism of induc­tion of chromosomal aberration could be a consequence of the chemical structure of BA, a derivative of purine, allowing for its possible inte­gration into DNA. Also, plant cells contain a protein which appears to be involved in the initiation of DNA replication ( H o u s s a  e t  al.  1990) and in the regulation of protein synthesis ( Ma a s  and K 1 a m b t 1977). The question thus is, whether BA acts directly or indirectly on nucleic acids. From our results we conclude that, in addition to the well known test-objects Allium cepa and Vicia faba, European black pine is also a very useful test-plant for detection of cytotoxicity.*A c k n o w l e d g e m e n t s :  This study was supported by grants from theCroatian Scientific Research Council, the Federal Project GIBIT and YU-US  Project (YO-FS 104— JB— 14) Improvement of Forest Tree JF 686— 32.R e f e r e n c e sBayliss, M. W., 1980: Chromosomal Variation in Plant Tissue in Culture. International Review of Cytology, Suppl. 11 A, Academic Press, 113—  — 144.Bennici, A., F. D’Amato, 1978: In vitro regeneration of durum wheat plants. I Chromosome numbers of regenerated plantlets. Z. Pflantzenziichtg. 81, 305— 311.Cionini, P. G., A. Bennici, F. D’Amato, 1978: Nuclear cytology of callus indu­ction and development in vitro. Protoplasma 96, 101— 112.D’Amato, F., 1985: Cytogenetics of plant cell and tissue cultures and their regenerants. Critical Reviews in Plant Sciences 3(1\ 73— 112.Fiskesjo, G., 1988: The Allium test —  an alternative in environmental stu­dies: The relative toxicology of metal ions. Mutation Res. 197, 243— 260.Fonket, D. E., M. J. Volk, M. R. Goldsmith, 1977: Polyribosome formation in relation to cytokinin-induced cell division in suspension cultures of Glycine max (L.) Merr. Physiol. Plant 60, 554— 562.Houssa, C., A. Jacqmard, G. Bernier, 1990: Activation of replicon origins as a possible target for cytokinis in shoot meristems of Sinapis. Planta 181, 324— 326.Kolevska-Pletikapic, B., S. Jelaska, J. Berljak, M. Vidakovic, 1983: Bud and shoot formation in juvenile tissue culture of Pinus nigra. Silvae Genetica 32, 49— 5T.Larkin, P. J., W. R. Scowcroft, 1981: Sorraclonal variation —  a novel source of variability from cell cultures for plant cell improvement, Theor. Appl. Genet. 60, 197— 214.A C T A  B O T . C R O A T . V O L . 50, 1991. 35D R A Ž E N A  P A P E S  et al.Maas, H., D. Klambt, 1977: Cytokinin effect on protein synthesis in vivo in higher plants. Planta 133, 117— 120.Ognra, H., 1982: Studies on the genetic instability of cultured tissues and regenerated plants —  effects of auxins and cytokinins on mitosis of Vicia /aba. Proc. 5th Inti. Cong. Plant Tissue and Cell Culture, 433— 434.Papes, D., V. Garaj-Vrhovac, S. Jelaska, B. Kolevska-Pletikapid, 1983: Chro­mosome behaviour in cultured cell populations of higher plants. Kew Chromosome Conference II, George Allen & Unvin, 155— 163.Pavlica, M., D. Papes, B. Nagy, 1991: 2,4-dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells. Mutation Res. 263, 77— 81.Sharma, A. K., A. Sharma, 1972: Chromosome Techniques —  Theory and Practice, University Park Press, Baltimore.Short, K. C., J. G. Torrey, 1972: Cytokinins in seedling roots of pea. Plant Physiol. 49, 155— 160.S A Ž E T A KKROMOSOMSKE PROMJENE U K L IJA N A C A  CRNOG BORA IZA ZV A N E  REGULATORIM A RASTENJADražena Papeš, Višnja Besendorfer i Mirjana Pavlica(Zavod za m olekularnu b io log iju , P rirodoslovno-m atem atički fakultet. Sveučilište u Zagrebu)Istraženo je djelovanje dva sintetska auksina: 1-naftalenoctene kise­line (NAA) i indolil-3-maslačne kiseline (IBA) i sintetskog citokinina ben- ziladenina (BA) na meristemske stanice vrška korjenčića 10 dana starih klijanaca crnog bora (Pinus nigra Arn.). Auksini imaju snažnije cito- genetsko djelovanje na genom meristemskih stanica od citokinina. Nakon tretmana auksinima mitotska aktivnost stanica je porasla paralelno s upotrijebljenom koncentracijom. Broj aberantnih stanica također se po­većavao s koncentracijom auksina. Oba auksina induciraju gotovo iste tipove promjena na razini diobenog vretena i na razini kromosoma. Sin­tetski citokinin BA je pokazao suprotno djelovanje. Nakon tretmana s najvećom koncentracijom mitotska aktivnost bila je vrlo slična onoj u kontrolnom uzorku. Međutim nakon oporavka značajno je porasla, a ta­kođer je porastao i broj kromosomskih aberacija.Dr. Dražena Papeš Departm ent o f  M olecular B iology Faculty o f  Science, U niversity o f Zagreb R ooseveltov trg 6/III P.O.B. 933,Zagreb, Croatia36 A C T A  B O T . C R O A T . V O L . 50, 1991.

Kromosomske promjene u klijanaca crnog bora izazvane regulatorima rastenja

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