This study was performed to
reveal the changes in cell division, as a
result of the prolonged period of subculture
on micropropagation medium, of five local
varieties of Solanum tuberosum L.
maintained on in vitro collection at Suceava
Genebank, Romania. For this purpose it was
used the Murashige-Skoog medium (MS-
1962) with addition of 40 g/l sucrose, and 6
mg/l daminozide. The effect of prolonged
period of subculture up to two and 12
months was expressed as mitotic index and
frequency of cells with abnormal division.
Mitotic index ranged from 20.1 to 22.1%
after 12 days, between 15.5 - 17.7% after
two months and between 17.7 - 19.2% after
12 months of subculture. The results
obtained showed that the frequency of
aberrant cells increased with the
preservation time on the in vitro cultures
and their accumulation rate depended on the
genotype. Were identified interphases with
micronuclei, metaphases with retarded
chromosomes, ana-telophases with
chromosomal bridges, retarded
chromosomes and chromosomal fragments,
but their percentage was low in all the
genotypes

Ciobanu, Iustina Brîndușa

Constantinovici, Dana

Creţu, L.

Cercetari Agronomice in Moldova

Ciobanu, Iustina-Brîndușa

ALSE Repository

ASPECTS OF IN VITRO POTATO CYTOGENETICS   71  Cercetări Agronomice în Moldova Vol. XLV , No. 3 (151) / 2012    THE DYNAMICS OF CELL DIVISION IN SOME LOCAL POTATO VARIETIES CULTIVATED IN VITRO ON MICROPROPAGATION MEDIUM  Iustina Brînduşa CIOBANU1*, Dana CONSTANTINOVICI2, L. CREŢU3  *E-mail: iustina_c2004@yahoo.com   Received  May 15, 2012                                                       1 Technical  College “Samuil Isopescu”, Suceava, Romania 2 Suceava Genebank,  Romania 3 University of Agricultural Sciences and Veterinary Medicine, Iaşi, Romania ABSTRACT. This study was performed to reveal the changes in cell division, as a result of the prolonged period of subculture on micropropagation medium, of five local varieties of Solanum tuberosum L. maintained on in vitro collection at Suceava Genebank, Romania. For this purpose it was used the Murashige-Skoog medium (MS-1962) with addition of 40 g/l sucrose, and 6 mg/l daminozide. The effect of prolonged period of subculture up to two and 12 months was expressed as mitotic index and frequency of cells with abnormal division. Mitotic index ranged from 20.1 to 22.1% after 12 days, between 15.5 - 17.7% after two months and between 17.7 - 19.2% after 12 months of subculture. The results obtained showed that the frequency of aberrant cells increased with the preservation time on the in vitro cultures and their accumulation rate depended on the genotype. Were identified interphases with micronuclei, metaphases with retarded chromosomes, ana-telophases with chromosomal bridges, retarded chromosomes and chromosomal fragments, but their percentage was low in all the genotypes.  Key words: Solanum tuberosum L.; Local varieties; Mitotic index; in vitro.   REZUMAT. Dinamica diviziunii celulare la unele varietăţi de cartof, cultivate in vitro pr mediul de propagare. Acest studiu a fost efectuat pentru a evidenţia modificările din diviziunea celulară, rezultate ca urmare a prelungirii perioadei de subcultură pe un mediu de micropropagare, la cinci varietăţi locale de Solanum tuberosum L., menţinute în colecţia in vitro a Băncii de Gene Suceava. În acest scop a fost folosit mediul de micropropagare Murashige-Skoog (MS-1962) cu adaos de 40 g/l zaharoză şi 6 mg/l daminozidă. Efectul prelungirii perioadei de subcultură până la două şi, respectiv, 12 luni a fost exprimat prin indicele mitotic şi prin frecvenţa celulelor cu anomalii ale procesului de diviziune. Indicele mitotic a Iustina CIOBANU, Dana CONSTANTINOVICI, L. CREŢU   72 variat între 20,1 şi 22,1 % după 12 zile, între 15,5 şi 17,7 % după două luni şi între 17,7 şi 19,2% după 12 luni de subcultură. Rezultatele obţinute au arătat că frecvenţa celulelor aberante a crescut odată cu prelungirea perioadei de menţinere a culturilor in vitro, iar rata acumulării acestora a depins de genotip. Au fost identificate interfaze cu micronuclei, metafaze cu cromozomi restanţieri şi ana-telofaze cu punţi cromozomale, cromozomi restanţieri şi fragmente cromozomale, dar procentul lor a fost mic la toate genotipurile.  Cuvinte cheie: Solanum tuberosum L.; varietăţi locale; indice mitotic; in vitro.    INTRODUCTION  In order to reduce the frequency of subculture, the growth rate of potato plantlets in vitro may be restricted by various methods including manipulation of nutrients in the culture medium, incubation in a lower temperature, and light intensity and a shorter photoperiod (Westcott, 1981a,b; Estrada et al., 1983; Ishige, 1995; Siddiqui et al., 1996; Lopez-Delgado et al., 1998; Gopal et al., 2005). In any program of germplasm conservation, which is using tissue culture methods, the stored genotypes must remain sufficiently stable, knowing that sometimes undesirable aspects such as genetic instability and loss of morphogenetic potential could appear (Denton et al., 1977; Ghiorghiţă et al., 2005). A parameter used successfully in cytogenetic studies is the mitotic index. It is reflecting the genotype response to the action of various factors and it is an important criterion in the cell division rate assessment (Ghiorghiţă, 1999; Cîmpeanu et al., 2002; Ciobanu et al., 2011).   MATERIALS AND METHODS  The research has been initiated using minicuttings prelevated from local populations of potato SVGB 14376, SVGB 15079, SVGB 15102, SVGB 15140 and SVGB 15446, maintained as plantlets on in vitro collection of Suceava Genebank by slow growth.  The experiments were conducted on micromultiplication medium (M14) based on Murashige-Skoog (MS-1962) recipe, supplemented with 1 mg / l K, 0.02 mg/l NAA, 40 g/l sucrose and 6 mg/l daminozide. The high quantity of kinetin added to the medium allowed a better survival of the inoculums in the storage conditions during the experiment, knowing the positive effect of this hormone to increase tolerance in seedlings at low temperatures and long periods of subculture (Kotkas, 2004). The five local varieties of potato have been investigated in terms of cytogenetic after three periods of cultivation in vitro on the micropropagation medium, respectively: 12 days, 2 months and 12 months. There were three different experimental versions and the results obtained at 12 days after inoculation was considered the control. For the study of mitosis, after 12 days, were used the root meristems obtained by placing the minicuttings on solid micropropagation medium and the culture flasks were placed at a temperature of 20-220C, photoperiod 16/24 hours, with a light intensity of 2500 lx. The biological material used to study the mitosis at 2 and 12 months was collected from root of plantlets ASPECTS OF IN VITRO POTATO CYTOGENETICS   73 maintained under micropropagation medium at temperatures between 6-120C, photoperiod 10/24 hours, with a light intensity of 1000 lx . After 12 days, 2 and 12 months, respectively, after inoculation, root tips of 10-12 mm were collected and fixed in Carnoy solution for 48 hours at 50C. It was followed by hot hydrolysis with HCl 1N at 600C for 25 minutes and the material was colored with Carr solution. It was used the fresh squash type method to examine the specimens under the optical microscope Olympus CX 41. The images were done with the camera included in the microscope.   RESULTS AND DISCUSSION  The mitotic index. The effect of the cultivation period and genotype, on the dynamics of cell division in five varieties of Solanum tuberosum L. studied, is presented in Table 1 and Fig. 1. The data inserted in Table 1 present the dynamics of mitotic index which is decreasing, compared to the 12 days control, in the plantlets cultivated for an extended period on micropropagation medium. The bigger reduction was registered from 12 days to 2 months. At the end of this subculture period it was noted the variety SVGB 15140 with a maximum of mitotic index, the difference from the control (2.9%) is less than in case of the other varieties analyzed. The changes in mitotic activity of root meristems of the samples grown for 12 months on M14 medium, regardless of variety, reflect the start of adaptation to the preservation conditions, the number of cells in division was positively influenced, and therefore, the mitotic index values are higher than those of 2 months. An explanation of this development could be that cells in the roots began to divide to produce secondary roots, leading to a relatively large number of cells in mitosis. The results were validated by the statistical relationship between the subculture period and the number of cells in division (Fig. 1). The analyses of limit differences are reflecting distinct or very significant decreases statistically assured in the samples at 2 and 12 months; it was an exception for the genotype SVGB 15079 with smaller differences statistically insignificant. The variety SVGB 15079 was noted at 12 months because it was the only genotype having the number of cells in division higher compared to the control, but the difference is also insignificant. It has to be emphasized, however, that in four from five of the analyzed varieties the average values of this parameter, after 12 months, was lower than control.      Iustina CIOBANU, Dana CONSTANTINOVICI, L. CREŢU   74 Table 1. The effect of genotype and period of subculture on mitotic index in five varieties of Solanum tuberosum L. grown on micropropagation medium M14  Mitotic index / Subculture period Genotype 12 days 2 months 12 months SVGB 14376 22,1% 16,4% 19,2% SVGB 15079 20,1% 17,0% 17,7% SVGB 15102 21,9% 16,1% 18,8% SVGB 15140 20,6% 17,7% 19,1% SVGB 15446 20,3% 15,5% 18,4%  684745 762ooo661ooo760o744oo736oo673ooo905Mt834Mt991Mt726Mt858oo697ooo1006Mt0200400600800100012002 days2 months12 months2 days2 months12 months2 days2 months12 months2 days2 months12 months2 days2 months12 monthsSVGB 14376 SVGB 15079 SVGB 15102 SVGB 15140 SVGB 15446Number of cell division .DL 5% -56DL 1% -85DL 0,1% -136DL 5% -53DL 1% -80DL 0,1% -129DL 5% -50DL 1% -76DL 0,1% -122DL 5% -59DL 1% -89DL 0,1% -143DL 5% -41DL 1% -63DL 0,1% -100   Figure 1 - The influence of genotype and cultivation period on the cell number in division in root meristems of the five local varieties of Solanum tuberosum L. inoculated on medium M14  The frequency of cells with chromosomal aberrations. The relationship between the cultivation period of the explants on medium M14 and average frequency of cells with chromosome aberrations in root meristems of studied potato varieties is shown in Fig. 2. Compared to the control, the biggest increases were recorded after 12 months of culture in varieties SVGB 14376 (with 200%) and SVGB 15102 (with 75%), the differences having distinct significations for the first genotype, and without statistical signification for the second. In case of variety SVGB 15140 the aberrant cells appeared in identical numbers after 2 and 12 months of subculture on M14 medium (0.39%). ASPECTS OF IN VITRO POTATO CYTOGENETICS   75  5.003.00o5.00Mt3.003.003.00Mt7.005.004.00Mt5.004.004.00Mt9.00**4.003.00Mt0123456789102 days2 months12 months2 days2 months12 months2 days2 months12 months2 days2 months12 months2 days2 months12 monthsSVGB 14376 SVGB 15079 SVGB 15102 SVGB 15140 SVGB 15446Average number of cells with chromosomal abearation   DL 5%-3,3DL 1%-4,9   DL 0,1%-7,9DL 5%-2,8DL 1%-4,3   DL 0,1%-6,9DL 5%-3,2DL 1%-4,8   DL 0,1%-7,7DL 5%-1,3DL 1%-2,0   DL 0,1%-3,1DL 5%-1,9DL 1%-2,9   DL 0,1%-4,7  Figure 2 - The number of cells with chromosomal aberrations in root meristems of five local varieties of Solanum tuberosum L. inoculated on medium M14  A special behavior was registered in the variety SVGB 15446 because the average frequency of cells with chromosome aberrations after 2 months of subculture was lower than in control version with 40%, the difference being statistically significant. In other cases the analyzed, differences from control were small and statistically uninsured. The microscopic analysis revealed the following types of chromosome aberrations: interphase with micronuclei, metaphases with retarded chromosome, ana-telophases with chromosomal bridges, retarded chromosomes and chromosomal fragments (Table 2). The micronuclei were present in cell interphases of control cultures in very small percentage (0.10 to 0.13%) to four of the five local varieties and in potato varieties SVGB 15079 and SVGB 15102 after 2 and 12 months, respectively, after the inoculation on the medium. Retarded chromosomes were found in all the experimental variants, including control, where they recorded the lowest frequency (between 0.10 to 0.29%). Some aspects of chromosomal aberrations recorded after different periods of subculture on micropropagation medium M14 are presented in Fig. 3÷8. Also, chromosome bridges were registered in all experimental variants, but in subunit proportions; an exception was the variety SVGB Iustina CIOBANU, Dana CONSTANTINOVICI, L. CREŢU   76 15079, at 2 months of subculture, to which this type of aberrations was not met. Most ana-telophases with bridges were registered in the variety SVGB 14376 after 12 months after inoculation. Ana-telophases with fragments were not present in the control variant, being present only in genotypes SVGB 14376 and SVGB 15102 after 12 months of cultivation on the culture medium M14.  Table 2 - The frequency of chromosomal aberrations types in root meristems of five local varieties of Solanum tuberosum L. inoculated on M14 medium, after different in vitro culture periods   Aberrations types Genotype Subculture  period Cells in division Metaphases with retarded chromosome A-T with simple bridges A-T with double bridges A-T with multiple bridges A-T with retarded chromosomes and chromosomal fragments Micronuclei 12 days 1006 1 0 0 2 0 0 2 months 697 2 0 0 2 0 0 SVGB 14376 12 months 858 4 1 1 1 2 0 12 days 726 2 0 0 1 0 1 2 months 684 3 0 0 0 0 1 SVGB 15079 12 months 772 3 0 0 2 0 0 12 days 991 1 1 0 1 0 1 2 months 673 3 0 0 2 0 0 SVGB 15102 12 months 736 3 0 0 1 1 2 12 days 834 1 0 1 0 0 1 2 months 744 1 1 0 1 0 0 SVGB 15140 12 months 760 2 0 0 1 0 0 12 days 905 2 1 0 1 0 1 2 months 661 3 0 0 0 0 0 SVGB 15446 12 months 762 3 1 1 0 0 0       A-T = ana-telophase    ASPECTS OF IN VITRO POTATO CYTOGENETICS   77   Figure 3 – Metaphase with retarded chromosomes (top) and interphase with micronucleus (left) to the genotype SVGB 15102, after 12 days of subculture (original) Figure 4 – Ana-telophase with double bridges to the genotype SVGB 15140, after 12 days of subculture (original)   Figure 5 – Ana-telophase with fragments and retarded chromosomes to the genotype SVGB 14376 after 12 months of subculture (original) Figure 6 Ana-telophase with retarded chromosomes to the genotype SVGB 15446, after 2 months of subculture (original)   Figure 7 – Ana-telophase with fragment and retarded chromosomes to the genotype SVGB 15102, after 12 months of subculture (original) Figure 8 – Metaphases with retarded chromosomes to the genotype SVGB 15079, after 12 months of subculture (original)  Iustina CIOBANU, Dana CONSTANTINOVICI, L. CREŢU   78  CONCLUSIONS  The two storage period influenced the mitotic activity in root meristems of the five varieties of Solanum tuberosum L. Cell division rate decreased from the control variant after both cultivation periods, but the most pronounced reduction of the mitotic index was found after 2 months of subculture. The results obtained showed the presence of chromosomal aberrations which ranged from 0.29 to 0.55% at 12 days, between 0.39 - 0.74% at 2 months and between 0.39 - 1.04% at 12 months of subculture. The extension of subculture up to 12 months increased the recorded values with 52.6%, compared to the control variant. The increasing of culture period on a micropropagation medium and in slightly restrictive conditions of storage room, act in many ways, affecting cell growth and the process of cell division.  REFERENCES  Ciobanu Iustina Branduşa, Creţu L., Constantinovici Dana, 2011 – Research on the dynamics of cell division in some local populations of Solanum tuberosum L. from in vitro collection of Suceava Genebank.  Analele Univ. “Al. I. Cuza” Iaşi, Biologie vegetală, Tomul LVII, fasc. 2.s. II-a, p.5 -11. Cîmpeanu M., Maniu, M,, Surugiu, I,, 2002 – Genetica – Metode de studiu (Genetics - study methods). Edit. Corson, Iaşi, p. 31-36. Denton, I.R., Westcott, R.J., Ford-Lloyd, B.V., 1977 – Phenotypic variation of Solanum tuberosum L., cv. Dr. McIntosh regenerated directly from shoot-tip culture. Potato Research, 20:131-136. Estrada R., Schilde Rentschler, L., Espinoza, N., 1983 – In vitro storage of potato germplasm. In: Hooker, W.J. (ed.): Research for the Potato for the Year 2000. (CIP) International Potato Centre, Lima, pp. 80–81.  Ghiorghiţă G., 1999 – Bazele geneticii (Genetic basis). Edit. „Alma Mater”, Univ. Bacău. Ghiorghiţă G. Nicuţă, Petrescu, D., 2005 – Biotehnologiile azi  (The Biotechnology today). Edit. Junimea, Iaşi.  Gopal J., Chamail Anjali, Sarkar Debabrata, 2005 – Use of microtubers for slow growth in vitro conservation of potato germplasm. Plant Genetic Resource Newsletter, 141: 56-60. Ishige T., 1995 – In vitro preservation of potato genetic resources in NIAR. In: Proceedings of MAFF International Workshop on Genetic Resources, 15–17 March, 1994, Japan, pp.93–97. Kotkas K., 2004 – Influence of culture medium composition on in vitro preservation of potato varieties by means of meristemplants.  Anale I.C.D.S.Z., Braşov, vol. XXXI, p. 97-108. Lopez-Delgado, H., Jimenez-Casas, M., Scoot, I.M., 1998 – Storage of potato microplants in vitro in the presence of acetylsalicylic acid. Plant Cell Tissue and Organ Culture 54:145–152. Murashige  T., Skoog,  F., 1962  - A  revised  medium  for rapid growth  and bioassays with tobacco tissue culture. Phisiol. Plant., 15, p. 473-497.  Siddiqui S.U., Chaudharay, M.F., Anwar, R., 1996 - In vitro preservation of potato (Solanum tuberosum L.) germplasm. Pakistan J. Bot. 28: 37-40. Westcott R.J., 1981a - Tissue culture storage of potato germplasm. 1. Minimal growth storage. Potato Research, 24:331-342. Westcott R. J., 1981b - Tissue culture storage of potato germplasm. 2. Use of growth retardants. Potato Research, 24:343-352.  

The dynamics of cell division in some local potato varieties cultivated in vitro on micropropagation medium

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The dynamics of cell division in some local potato varieties cultivated in vitro on micropropagation medium

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