Annals of RSCB Vol. XVIII, Issue 1/2013

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1 Annals of RSCB Vol. XVIII, Issue /3 CYTOGENETIC EFFECTS INDUCED BY DEPOSIT MYCOFLORA IN PHASEOLUS VULGARIS L. BEANS FROM THE COLLECTION OF SUCEAVA GENEBANK Diana Batîr-Rusu, C. Tudose * SUCEAVA GENEBANK, COLLECTING DEPARTMENT UNIVERSITY AL.I.CUZA IAŞI, FACULTY OF BIOLOGY, DISCIPLINE OF GENETICS Summary Continuing our previous studies regarding the effects induced by deposit mycoflora in various species, the purpose of our work was to study the cytogenetic effects induced by deposit mycoflora in Phaseolus vulgaris L. (n = ) beans from the collection of Suceava Genebank. Cytogenetic effects were studied by means of classical plant chromosomes methods of study. We have observed that the values of the mitotic indexes are decreasing in accordance with the increasing of the storage age and also with the number of mycoflora species that are infesting the beans. Infestation with specific mycoflora produced a relatively large number, statistically significant by comparison to the controls, of interphasic aberrations and also of chromosomal aberrations in mitosis ana-telophase in all studied probes. Comparing our results with similar published data, we can strongly state that the cytogenetic effects induced by deposit mycoflora in Phaseolus vulgaris seeds are similar with those produced by the action of a weak mutagenic agent Key words: Phaseolus vulgaris beans, deposit mycoflora, cytogenetics cristian.tudose@uaic.ro Introduction Continuing our previous studies regarding the effects induced by deposit mycoflora in various species seeds from the collection of Suceava Genebank (Batîr- Rusu and Tudose, 9 a,b), the purpose of our work is to study the cytogenetic effects induced by deposit mycoflora in Phaseolus vulgaris L. (n = ) beans from the above mentioned collection. Cytogenetic effects were studied by means of: calculation of the mitotic index and frequency of mitosis phases, registration of the frequency and types of abnormal interphases and identification of chromosomal aberrations in mitotic ana-telophases, in accordance with classical plant cytogenetics methods. Materials and Methods The biological material consisted of five probes of beans selected from the collection of Suceava Genebank: - The control: 3777, 5 years old, germination ratio %, probe with minimal infestation by mycoflora, 7 - SVGB 4, 6 years old, germination ratio 93%, infested by Penicillium sp., Aspergillus sp. Rhizopus sp., Alternaria alternata, and Stemphylium botryosum - SVGB 46, 6 years old, germination ratio 87%, infested by Penicillium sp., Rhizopus sp., Trichothecium roseum and Trichoderma viride - SVGB 375, 5 years old, germination ratio 93%, infested by Penicillium sp., Cladosporium herbarum, Alternaria alternata, Trichoderma viride and Stemphylium botryosum - SVGB 3775, 5 years old, germination ratio 73%, infested by Penicillium sp. Rhizopus sp., Epicoccum sp. and Trichoderma viride The beans germinated in Petri dishes, on filter paper, wetted with distilled water for all variants till the roots reached -5 mm in length. We calculated the germination percent and the duration of germination for all experimental variants. When the roots

2 Annals of RSCB Vol. XVIII, Issue /3 reached the length of -5 mm they were fixed in Battaglia for 3 minutes. We have performed fast cytological slides according to Feulgen method (Cimpeanu at al., ). We have studied:. The mitotic index and frequency of mitosis phases. The frequency and types of aberrant interphases 3. The frequency and types of chromosomal aberrations in mitotic ana-telophases (A-T). We have analysed this 3 steps on 5 fresh preparations. For each slide we have studied: microscopic fields (objective 4x) on which we have counted all the cells in interphase, prophase, metaphase, anaphase and telophase; microscopic fields on which were counted all the cells in normal and abnormal interphases and the type of interphasic aberrations and 5 anatelophases were analysed, counting the normal, aberrant ana-telophases and the type of chromosomal aberrations. All data were statistically processed (Fowler and Cohen, 99). Microphotographs were performed with the digital camera of Nikon research microscope with the X objective. Results and Discussions The infestation with fungi generally determined a significant decrease of the mitotic index (Table ). Only for the 4 probe the frequency of dividing cells is 4,3%, a very close value to the mitotic index registered for the control (4,66%) (fig. ). The lowest value of the mitotic index (,%) was observed for the 3775 probe. The values of the mitotic index are proportionally decreasing with the increase of the number of infesting species of fungi and with the storage age of beans. For the majority of probes, and also for the controls, we have recorded a predominance of prophases, followed by metaphases and telophases. Anaphases were recorded only for controls, but in a very small percentage (Table and Figure ). An exception was noted for the probe 3775 where the frequency of prophases was low and metaphases were predominant. The frequency of metaphases, generally low, isn`t decreasing or increasing significantly in comparison with the controls. The frequency of telophases is decreased for all probes in comparison to the controls. % dividing cells Control Fig. : Mitotic index in Phaseolus vulgaris L. studied probes 7

3 Annals of RSCB Vol. XVIII, Issue /3 Division phases % Control Prophases Metaphases Anaphases Telophases Fig. : The frequency of mitosis phases in Phaseolus vulgaris L. studied probes Infestation with fungi induced the increase of interphasic aberrations in all the studied probes (Table and Fig. 3). The highest frequency of aberrant interphases (,94%) was registered for 3775 probe. For the other probes the registered values are similar and quite low, but, in comparison to the very small value of aberrant interphases counted in the control probes (,5%), we can state that the infestation with mycoflora induced a statistically significant increase of interphasic aberrations, without regard to the type of fungi or to the age of probes. The most frequent interphasic aberration is represented by the binucleated cells, which frequency is increasing proportionally with the age of probes and with the number of infesting mycoflora species (Fig. 4). In 4 and 375 probes the infesting mycoflora induced some interphases with one micronucleus and, in a very low percentage, interphases with two micronuclei. Analysing all the mentioned data in comparison with data from literature (Bara, 8; Maniu et al., 5) we can strongly state that the cytogenetic effects induced by deposit mycoflora in Vicia faba beans are similar with those produced by the action of a weak mutagenic agent. %Aberrant interphases Control Fig. 3: Total frequencies of aberrant interphases in Phaseolus vulgaris studied probes 7

4 Annals of RSCB Vol. XVIII, Issue /3 Aberration types% Control Binucleated cells Interphases with one micronucleus Interphases with two micronuclei Fig. 4: Frequency of interphasic aberrations types in Phaseolus vulgaris L.studied probes Analysing the data from table 3 one can observe that both for the control, 46 and 3775 probes were registered chromosomal aberrations in mitosis ana-telophase. In the rest of two probes the number of observed anatelophases was too small to perform statistic analysis. The highest percentage of aberrant ana-telophases (3,5%) was observed for the 3775 probe (Fig. 5). The chromosomal aberrations types show a great variety, being distributed randomly (Fig.6). % Aberrant ana-telophases Control Fig. 5: Total frequency of aberrant ana-telophases in Phaseolus vulgaris L. studied probes 73

5 Annals of RSCB Vol. XVIII, Issue /3. One bridge One micronucleus % A-T aberrations Two micronuclei One bridge and two micronuclei Tripolar A-T Control Tripolar A-T with one expulsed Fig. 8: Frequency of chromosomal A-T aberrations in Phaseolus vulgaris L.studied probes We have observed and counted many simple chromosomal aberrations such as: one bridge, one micronucleus, two micronuclei, tripolar ana-telophases, expulsed chromosomes and some complex chromosomal aberrations such as: one bridge with two micronuclei, tripolar A-T with expulsed chromosomes, etc. The highest frequency was registered for tripolar ana-telophases and micronuclei. The increase of the number of aberrations in ana-telophase is statistically significant reported to the controls; comparing our results with similar published data (Bara, 8; Maniu et al., 5) we can strongly state that the cytogenetic effects induced by deposit mycoflora in Phaseolus vulgaris L. (n=) beans are similar with those produced by the action of a weak mutagenic agent. Conclusions. The values of the mitotic indexes are decreasing in accordance with the increasing of the storage age and also with the number of mycoflora species that are infesting the beans.. Infestation with specific mycoflora produced a relatively large number, 74 statistically significant by comparison to the controls, of interphasic aberrations and also of chromosomal aberrations in mitosis ana-telophase in all studied probes. 3. Comparing our results with similar published data, we can strongly state that the cytogenetic effects induced by deposit mycoflora in Phaseolus vulgaris L. seeds are similar with those produced by the action of a weak mutagenic agent. References Băra C.I.,- Radiaţia ultravioletă - factor mutagen fizic. Efecte ale iradierii cu UV la fasole (Phaseolus vulgaris L.), Ed. Universităţii Al.I.Cuza, Iaşi,3-4, 7 86, 8. Batîr-Rusu D., Tudose C., Cytogenetic effects induced by deposit mycoflora in Vicia faba L. beans from the collection of Suceava Genebank, Analele Ştiinţifice ale Universităţii Al. I. Cuza Iaşi, Secţiunea Geneticã şi Biologie Molecularã. TOM X, fasc.3: 7-4, 9. Batîr-Rusu D., Tudose C., Cytogenetic effects induced by deposit mycoflora in Zea mays L. beans from the collection of Suceava Genebank, Analele Ştiinţifice ale Universităţii Al. I. Cuza Iaşi, Secţiunea Geneticã şi

6 Annals of RSCB Vol. XVIII, Issue /3 Biologie Molecularã. TOM X, fasc.4: 49-54, 9 Cîmpeanu M., Maniu M., Surugiu C.I., Genetica metode de studiu, Ed. Corson, Iasi,. Fowler J., Cohen L., Practical statistics for field biology, Open University Press, New York, 99. Maniu M., Truta E., Tudose C., Maniu C., Efecte citogenetice induse de tratamentul singular şi combinat cu Oltisan extra şi UV-B la linia DH 74- de orzoaică de primavară, Analele Societatii Nationale de Biologie Celulara C. Craciun, A. Ardelean (editors), VII, :- 4, Maniu M., Tudose C., Tudose M., In vivo mutagenesis induced by sodium azide in DH-9 double haploid line of barley, Romanian Biotechnological Letters, (4):55-6, 5. 75

7 Annals of RSCB Vol. XVIII, Issue /3 Table : Frequency of mitoses and of mitosis phases in Phaseolus vulgaris L. studied probes Probes Total studied cells Total interphases Total mitoses Total prophases Total metaphases Total anaphases Total telophases Nr. Nr. % Nr. % Nr. % Nr. % Nr. % Nr. % Control 95, ,66 7 3,33 8,86,5 9, , ,3 97 3, 3, , , ,53 68,4 3, , ,37 79,63 5,73 3, , ,79 34,,43 9, , Probes Table : Frequency of interphasic aberrations in Phaseolus vulgaris L. studied probes Aberration types Total Total normal Interphases studied Total aberrant cells interphases Binucleated cells with one cells micronucleus Nr. Nr. % Nr. % sx Interphases with two micronuclei ± Nr. % Nr. % Nr. % Control ,75 5,5,6 4,, ,55 43,45,3 35,8 7,3, ,48 4,5,3 4, ,3 49,68,3 48,64, ,6 54,94,4 5,86,4,4 Probes Table 3: Frequency of chromosomal aberrations in mitosis ana-telophase (A-T) in Phaseolus vulgaris L. studied probes Types of chromosomal aberrations in mitosis A-T Total Tripolar A-T Studied Total abnormal One Two One bridge normal Tri-polar with A-T A-T One bridge micronucleunuclenuclei micro- + micro- A-T A-T expulsed chromosome 76 Expulsed chromosome Nr Nr % Nr % Nr % Nr % Nr % Nr % Nr % Nr % Nr %

8 Annals of RSCB Vol. XVIII, Issue /3 Control 98 99,,,5 - -, ,5 5,5,5, - -,5, ,5 7 3,5,5, - -,5,5,5,5 77