measurements of chromosome aberration at tomato plants

Measurements of chromosome aberration at tomato plants(Lycopersicon esculentum mill.) regenerated from “in vitro”tissue cultureCristea Tina Oana* 1 , Ambăruş Silvica 1 , Călin Maria 1 , Prisecaru Maria 2 , Avasiloaiei D. I. 11 Vegetable Research and Development Station Bacău ; 2 University Vasile Alecsandri Bacău, Faculty ofBiology*Corresponding author. Email: tinaoana@yahoo.comAbstract Tissue culture “in vitro” have a key role for the development ofbreeding activities at tomatoes plants. Currently, the modern breedingtechniques based on genetic engineering, employ in a certain stage of newvarietal creation, the utilization of tissue culture for the regeneration of plants.But, during the “in vitro” culture, due to the composition of medium culture,alteration of chromosomes morphology may occur. Depending on the finalobjective of tissue culture, the abnormalities of chromosomes that mayappear at “in vitro” regenerated plants have to be avoided (cultures designedfor clonal multiplication) or to be amplified (cultures designed for somaclonalvariability). According with the literature, chromosome aberrations have beenused as a measure of reproductive success in plants for many years andhave been correlated with morphological changes, fertility-sterilityrelationships, mutations, etc.Subsequent studies at different plant species have shown that plantchromosomes exhibit many different types of aberration, as a result ofdifferent types of chemicals used for the preparation of “in vitro” culturemedium.The main objective of the present paper is screening of some aspectsregarding the type and frequency of chromosomes aberrations that appearedat tomatoes plants regenerated from “in vitro” culture are discussed. The maintypes of aberrations identified at regenerated plants are: chromosomeclumping, contraction, stickiness, paling, fragmentation, dissolution,chromosome and chromatid bridges, C-mitosis and endoploidy.The early detection of the chromosomal aberrations allows theselection of the growth conditions that better suit the final goal ofinvestigations (clone multiplication or by contrary the induction of somaclonalvariability).Key wordsgenetic,stickiness,tomatoeschromosomes,anaphase,One of the principal objectives of using chromosomesas a monitoring system is to determine whether or notthe „in vitro” tissue culture conditions are clastogen(meaning capable of breaking chromosomes). If thehormones or other chemicals used for the mediapreparation are clastogen, then this would permitexchanges that would be translated through cytologicalor genetic changes at regenerated plants level.The cultivation of different explants onnutritive media “in vitro” is often related with anincrease in the frequency of structural chromosomalalterations as well as an increase in the frequency ofgene mutations. How these factors are related to oneanother and how they cause changes in thechromosome and gene mutation rates are not wellunderstood. However, the fact that all these externalagents cause similar changes and indicate a broadfundamental process may be a primary cause ofmutations.It is highly important to determine thefrequency of aberration that occurs during “in vitro”regeneration of plants as it reflects directly in thephenotype of plants. In the “in vitro” multiplication ofvaluable plants utilized in classical breeding this typeof genetic variability must be avoid. The identificationof hormonal formuli that causes the minimumfrequency of chromosomal alteration is thus ofmaximum importance for the development of futuremultiplication technologies.274

Material and MethodsThe biological material is represented from 4genotypes of tomatoes (Lycopersicon esculentum Mill.)from Vegetable Research and Development StationBacau, Romania. The seeds were utilized for the “invitro” multiplication of these valuable genotypes andthe meristematic root tips were excised from the “invitro” plantlets regenerated on D1-D3 variants,characterized through the presence of BAP and Kinetinalone or in association with IAA – table 1.Experimental variants utilized in the cytogenetic studies at Lycopersicon esculentum Mill.Components D0 D1 D2 D3Macro elementsMS, 1962Microelements MS, 1962Vitamins B 5BAP seeds germinated “ex 2,0 mg/l - 1,5 mg/lKinetin vitro”- 2 mg/l -IAA - - 0,5 mg/lSucrose 3% 3% 3%Agar 8 ‰ 8 ‰ 8 ‰Table 1The control variant is represented by plantsgerminated “ex vitro” in Petri dishes.The cytogenetic studies were accomplished inmeristematic root cells, stained in Carnoy fixingsolution for 24 hours at 4 0 C then hydrolyzed with HClfor 7 minutes and colored with the basic coloringsolution Carr. The root meristems were displayed usingsquash technique and for each genotype and variant6000 cells were counted.Results and DiscussionsThe objective of the present study was todetermine whether or not the plants regenerated fromin vitro culture presents alteration in their geneticstructure. Thus, we tested the influence of mediumculture composition over the chromosomes structures.The cytogenetical studies accomplished in the presentstudy demonstrate that the cultivation of tomatoesshoot tips on nutritive medium modified with Kinetinand BAP allows the regeneration of new plants with astable genetic material that shows little geneticvariability. This variability manifested at cellular levelthrough the different types of chromosomalabnormalities does not exceed the natural variabilitypresent also on plants germinated in natural conditions.The results obtained are presented in tables 2, 3, 4 andfigures 1, 2, 3.Table 2Types and frequency of chromosomal aberrations observed in root meristematic cells - genotype MonoromVariant Total noof cells% Prophases withanomalies%Metaphaseswith anomalies%A+T withanomaliesOther typesD0 5745 0,65 0,83 0,88 1,19D1 6078 0,23 0,24 0,54 0,32D2 5974 0,12 0,32 0,41 0,28D3 6298 0,21 0,49 0,68 0,59275

1,210,80,60,4D0D1D2D30,20% Prophaseswith anomalies%Metaphaseswith anomalies%A+T withanomaliesOther typesFig. 1: Types and frequency of chromosomal aberrations observedin root meristematic cells – genotype MonoromTable 3Types and frequency of chromosomal aberrations observed in root meristematic cells – genotype L24SVariant Total no ofcells% Prophases withanomalies%Metaphaseswith anomalies%A+T withanomaliesOther typesD0 6012 0,21 0,88 1,89 1,16D1 6241 0,11 0,85 1,13 0,59D2 5320 0,12 0,72 0,9 0,89D3 6000 0,09 0,63 1,12 1,0921,81,61,41,210,80,6D0D1D2D30,40,20% Prophaseswith anomalies%Metaphaseswith anomalies%A+T withanomaliesOther typesFig. 2: Types and frequency of chromosomal aberrations observedin root meristematic cells - genotype L24STable 4Types and frequency of chromosomal aberrations observed in root meristematic cells – genotype L27SVariant Total noof cells% Prophaseswith anomalies%Metaphaseswith anomalies%A+T withanomaliesOther typesofanomaliesD0 5891 1,32 0,25 1,72 1,74D1 5942 1,20 0,24 1,21 0,97D2 6001 0,12 0,18 1,18 0,78D3 5012 0,29 0,12 0,87 0,96276

1,81,61,41,210,80,6D0D1D2D30,40,20% Prophases %Metaphaseswith anomalies with anomalies%A+T withanomaliesOther types ofanomaliesFig. 3: Types and frequency of chromosomal aberrations observedin root meristematic cells - genotype L27STable 5Types and frequency of chromosomal aberrations observed in root meristematic cells – genotype FRANCESCAVariant Total noof cells% Prophaseswith anomalies%Metaphaseswith anomalies%A+T withanomaliesOther typesofanomaliesD0 6021 0,52 0,41 1,96 1,11D1 5478 0,24 0,26 1,32 1,00D2 5965 0,16 0,12 1,02 0,89D3 5418 0,28 0,22 1,35 1,2921,81,61,41,210,80,6D0D1D2D30,40,20% Prophaseswith anomalies%Metaphaseswith anomalies%A+T withanomaliesOther types ofanomaliesFig. 3: Types and frequency of chromosomal aberrations observedin root meristematic cells - genotype FrancescaThe main types of aberrations identified atregenerated plants are: chromosome clumping,contraction, stickiness, paling, fragmentation,dissolution, chromosome and chromatid bridges, C-mitosis and endoploidy.For all the genotypes tested in thepresent study the highest incidence of aberrations wasobserved in ana-telophases. The most commonabnormalities were ana-telophases with simple ormultiple bridges, expelled or late chromosomes andmultipolar ana-telophases – figure 4. We also detectedabnormalities is metaphases that were abnormallyorganized, with ring chromosomes, minutes, expelledchromosomes, fragment, etc – figure 5.In a smaller number we detected prophases thatpresented different types of chromosomal aberrationslike late prophases, with ring chromosomes, expelledchromosomes etc – figure 6.277

Fig. 4 – Ana-telophases with bridge (left) and late chromosomes (right)Fig. 5 – Prophase with ring chromosomes (left) and binucleate cells (right) (genotype L24S)Fig. 6 – Sticky metaphases (left) and with expelled chromosomes (right) at L27S genotypeAll four genotypes had the same cytogenetic behavior,the plants regenerated from “in vitro” culturepresenting abnormalities in similar percentages as thecontrol. For example at the genotype L24S from theentire number of identified metaphases a rangebetween 0,63-0,85% of them were abnormal at variant278

D1-D3, while the control registered 0,88%, at L27Sgenotype the value were between 0,12-0,24% for D-D3and 0,25% for control.Regarding the percentage of cells with anatelophaseswith abnormalities the values are higherthan the one registered for metaphases. At genotypeFrancesca, for example, at plants regenerated from invitro cultures the percentage of cells with abnormalitiesin ana-telophases ranged between 1,02-1,35 %, whilethe control registered 1,96%. The same genotype had0,26% from cells in metaphases with abnormalitieswhile the control had only 0,41%.ConclusionsThe results obtained in our experiment provedthat in the “in vitro” conditions tested, the types andfrequency of chromosomal aberration are similar withthe control. No other genetic abnormality of the tissueculturedplants was observed suggesting that geneticfidelity of tissue cultured plants can be maintained ifappropriate plant growth regulators are used with lessnumber of subcultures in the multiplication stage.The cultivation of tomatoes shoot tips onnutritive medium modified with Kinetin and BAPallows the regeneration of new plants with a stablegenetic material that shows little genetic variability.This variability manifested at cellular level through thedifferent types of chromosomal abnormalities does notexceed the natural variability present also on plantsgerminated in “ex vitro” conditions.The main types of abnormalities in the rootcells of tomatoes are ana-telophases with bridges,metaphases with lagging chromosomes, expelledchromosomes or ring chromosomes, multipolar anatelophases,as well as binucleate cells and interphaseswith micro-nucleuses.References1. Li M., M. Zhang, 1991 - Technology for plantchromosome research. Northwest Forest Univ. Press,Shenyiang, China. p. 31–39.2. Budiman, M.A., L. Mao, T. Wood and R.A.Wing. 2000. A Deep-Coverage Tomato BAC Libraryand Prospects Toward Development of an STCFramework for Genome Sequencing. GenomeResearch 10:129-136.3. Mao, L., D. Begum, S.A. Goff, R.A. Wing.2001. Sequence and Analysis of the TomatoJOINTLESS Locus. Plant Physiology: 126:1331-1340.279