Cytogenetic Research Regarding Species with Ornamental Value Identified in the North-East Area of Romania

CYTOGENETIC RESEARCH REGARDING SPECIES WITH ORNAMENTAL VALUE

Cercetări Agronomice în Moldova Vol. XLIII , No. 4 (144) / 2010

CYTOGENETIC RESEARCH REGARDING SPECIES WITH ORNAMENTAL VALUE IDENTIFIED IN THE NORTH-EAST AREA OF ROMANIA

Lucia DRAGHIA*, Aliona MORARIU, Liliana CHELARIU

University of Agricultural Sciences and Veterinary Medicine of Iaşi

Received June 15, 2010

ABSTRACT - In the paper are presented existence of diploid and tetraploid data regarding the number of somatic populations, separated by geography and chromosome determine at three species of ecology. Population of Dianthus armeria, plants with ornamental value from identified in Ia i area (Bîrnova forest), is a spontaneous flora of Romania (Allium mixt population with x=15, formed by ursinum L., Centaurea phrygia L., Dianthus tetraploid individuals 4n=60, but in which armeria L.) and cultivated in the could be found also a minor diploid experimental field to evaluate the adapt cytotype with chromosome number 2n=30. capacity, aiming to use them in crops. For chromosome study was used a root tip Key words: Allium ursinum; Centaurea meristem obtained from plantlets aged 3-7 Phrygia; Dianthus armeria; Chromosome days. Chromosome was stained by Feulghen number. method, and the samples were examined at optic microscope Motic, relevant images REZUMAT - Cercetări citogenetice la being assumed by a video camera and specii cu valoare ornamentală, processed with its soft. The obtained results identificate în zona de nord-est a were compared with the ones already României. În lucrare sunt prezentate date cu existed in the literature, at populations from privire la numărul cromozomilor somatici, other ecologic and geographic areas. So at determinat la trei specii de plante cu valoare Allium ursinum, from Dobrovăţ forest (Iaşi ornamentală, provenite din flora spontană a area) all the analysed individuals had a României (Allium ursinum L., Centaurea chromosome number 2n=2x=14, specie phrygia L., Dianthus armeria L.) şi being characterized by a great karyological cultivate în câmpul experimental pentru stability. Studies made on Centaurea evaluarea capacităţii de adaptare, cu scopul phrygia, from Neamţ area, show the introducerii lor în cultură. Pentru studiul presence of tetraploid individuals, with cromozomilor s-a utilizat meristem 4n=44 chromosomes. The specie is known radicular, obţinut din plantule cu vârsta de as a diploid one, with chromosome number 3-7 zile. Colorarea cromozomilor s-a 2n =2x=22, but many authors confirm the realizat dupa metoda Feulghen, iar

* E-mail: [email protected]

51 Lucia DRAGHIA, Aliona MORARIU, Liliana CHELARIU preparatele au fost examinate la at others were caused numerous microscopul optic, imaginile relevante fiind chromosomial mitigations (polyploid) preluate cu camera video şi prelucrate cu by modification of chromosome soft-ul corespunzător acesteia. Rezultatele obţinute au fost comparate cu cele existente number from the ancestor gamet în literatura de specialitate, la populaţii din (Sărac, 2005). alte areale ecologice sau geografice. Astfel, Also very important in genetic la Allium ursinum, din pădurea Dobrovăţ studies at angiosperms was the (Iaşi), toţi indivizii analizaţi au avut diversity of genome size, correleted numărul de cromozomi 2n=2x=14, specia with different characters at nucleus fiind caracterizată printr-o stabilitate cariologică mare. Determinările efectuate la cellular level or at organism level Centaurea phrygia, provenită din zona (estimated time for mitotic and Neamţ, au scos în evidenţă prezenţa meiotic cell division, reflected in life indivizilor tetraploizi, cu 4n=44 cromozomi. cycle period; adapt capacity at low Specia este cunoscută ca diploidă, cu temperatures etc.) (Bennett and numărul de cromozomi 2n =2x=22, dar este Leitch, 1997; Ohri and Pistrick, 2001). confirmată, de mai mulţi autori, şi existenţa Studies regarding chromosome populaţiilor diploide şi tetraploide, separate atât geografic, cât şi ecologic. Populaţia de number, ploidy level, DNA content Dianthus armeria, identificată în zona Iaşi etc. were done at a great number of (pădurea Bîrnova), este mixtă, cu x=15, angiosperm species, inclusively at formată din indivizi tetraploizi 4n=60, dar în populations from different areas, cadrul careia se întâlneşte şi un citotip being know that the karyological minoritar diploid, cu număr de cromozomi differences are also induced by 2n=30. ecological conditions (Krahulcová,

Cuvinte cheie: Allium ursinum; Centaurea 2003). There are many angiosperms phrygi;, Dianthus armeria; număr species at which karyological cromozomi. investigations are missing and at others the results are unsure due to a single population study or even a INTRODUCTION single individual (Bennett, 1998, cited by Baltisberger and Widmer, 2009). Chromosome characterization So, that the informations function of number, size, morphologic regarding karyotype of different taxa type helps to establish the karyotype, to be as complete as possible it is a basical component for species recommended that the studies to be identification, cytological data being done on more individuals from the very important in plants’ systematic same population or from populations (Stace, 2000, cited by Baltisberger with different ecological areas and and Widmer, 2009), for motivating also from zones of confluence areas of some taxonomic decisions, especially related species (Stace, 2000). in hybrids case. At some species, the The rich floral diversity of basic number of chromosomes didn’t Romania offers the possibility of change through evolution process but some ample karyological studies on

52 CYTOGENETIC RESEARCH REGARDING SPECIES WITH ORNAMENTAL VALUE plants. In literature could be found MATERIALS AND METHODS informations regarding chromosome number of some taxa from different Plant material. Karyological ecologic areas of Romania and which studies were made on some plant species belong to genus Achillea, identified and gathered from spontaneous Podospermum, Campanula, Dianthus, flora from North-East of Romania, Silene, Geranium, Nepeta, Stachys, species considered to have ornamental qualities: Allium ursinum L., Centaurea Teucrium, Nigritella, Aconitum, phrygia L., Dianthus armeria L. Consolida, Ranunculus, Geum, Allium ursinum L. (wild garlic), Potentilla, Veronica, Cerastium etc. Alliaceae family, geophytes perennial (Baltisberger, 2009; Băra et al., 2003; specie (with bulbs) originally from Boşcaiu et al., 1999). A special group temperate area of Europe, is frequently is formed by rare and endemic species found, in Romania, in oak and beech at which the cytogenetic studies are forest and present an ornamental interest not so numerous (Băra et al., 2003; through small and white flowers, grouped Tăcină, 1980). more together (20) in umbel type The continuation of karyological inflorescences. Flowering take place at investigations is necessary even at the the end of spring (may-june). Biological material (bulbs, seeds) was gathered from species with a know basic number of a population from Iaşi (Dobrovăţ forest). chromosomes to confirm the already Centaurea phrygia L. (C. austriaca existed data. Cytological Wild., C. phrygia subsp. phrygia), investigations on species from Asteraceae family, is a hemicryptophite spontaneous flora with ornamental perennial specie, originally from value to elucidate some aspects mountain areas of Europe (Oprea, 2005). regarding the number and morphology In Romania is frequently founded on of the chromosomes from these grasslands near the limits of hardwood species is one of the main aims of the and coniferous forests (from sessile level studies undertaken for introduction in up to firtrees). The height is around 1 m and decorates through inflorescences with crop and their usage as a germplasm violet flowers. Biological material (seeds) source in breeding works, for was gathered from a population from improving the assortment of Neamţ area. ornamental plants. Dianthus armeria L. (deptford The paper aims to investigate pink) it is an annual specie from and to check the existed informations Caryophyllaceae family, originally from regarding chromosome number at Europe. In Romania is sporadically found some studied species, the presented on grasslands and sub-shrubs, from steppe data being compared with the one area to beech level (Ciocârlan, 2000). existed in literature. Have thin stems, with a heigh of 30-50 cm and decorates by terminal inflorescences, consist of sesil flowers, with pink-red petals spotted with white and accompanied by bracts. Blossom from june till august. Biologic material is

53 Lucia DRAGHIA, Aliona MORARIU, Liliana CHELARIU gathered from populations identified in (sometimes also x=9), at which are Bîrnova forest (Iaşi county). added the polyploid froms. Ploidy Chromosome counting. For level at the species from this genus chromosome study was used a root tip could be between 2x till 16x (Bennett meristems obtained from seeds et al. 2000). germinated for 3-7 days (moment when the roots reached the lenght of 1-1.5 cm). Kawano and Nagai (2005) show Chromosome staining was realized in that Asian species are reported as according with Feulghen method, the tetraploid with 4n=32, and the ones samples being examinated at optic from Europe are diploid with 2n=16. microscope Motic B1, with zoom 100x, At some Allium species were and relevant images were assumed by identified a variable number of Motic video camera and processed with chromosomes (Baranyi et al., 1999; its soft. Fritsch et al., 2002). Allium ursinum is an exception RESULTS AND DISCUSSIONS characterised by a great karyological stability. All the data from references Allium ursinum L. Place: Iaşi present this specie as being diploid county (Dobrovăţ forest) with 2n=14 chromosomes, without Genus Allium present a great recording variation of ploidy level on diversity of species distinct both by it (Baranyi et al., 1999; Karpaviciene, morphologic features and ecologic 2007). habitats and also by cytogenetic Our investigations confirm these characters (basic number of dates, the determined chromosome chromosomes, ploidy level and number being of 2n=14 (Fig. 1). genome type) (Ricroch et al., 2005). The modern ranking accept over 750 species, many of them with recognize a economic importance (Fritsch et al., 2002; Yousaf et al., 2004; Stearn, 1992). In Romania can be found around 30 species from which 26 are wild species (two of them could be found in crops) (Ciocârlan, 2000; Oprea, 2005). Ecologic, taxonomic, systematic, morphologic, anatomic, karyologic and other studies realised by various Figure 1 - Somatic metaphase of Allium ursinum specie (2n=14) authors gave the opportunity to elucidate aspects regarding Allium Centaurea phrygia L. Place: genus. Neamţ county From kariological point of view, In Central Europe (Germany, prevail the basic number of Czech Republic, Slovakia, Poland, chromosomes x = 7 and x=8

54 CYTOGENETIC RESEARCH REGARDING SPECIES WITH ORNAMENTAL VALUE

Austria, Hungary and West of (2007), diploid populations were Ukraine) were identified more of recorded in North Bohemia, North seven taxons of Centaurea phrygia Moravia, Eastern Carpathians and group, both species and sub-species only one population in Western (Koutecky, 2007). Dostal (1976), in Carpathians. Tetraploid populations Flora Europaea 4 (cited by Koutecky, were identify in Western Carpathians, 2007) identify at Centaurea phrygia in Czech Repubilc and Slovenia by species a number of nine sub-species. Majovsky et al. (1987) and Dostal From those ones in Romania were (1989), cited by Koutecky (2007). identified six (Oprea, 2005): subsp. In conclusion, the existence of phrygia, subsp. melanocalathia diploid and tetraploid populations (Borbas) Dostal, subsp. carpatica from this specie is confirmed by many (Porcius) Dostal, subsp. authors, but these cytotypes are pseudophrygia (C.A. Mey.) Gugler, separated by geography and ecology. subsp. ratezatensis (Prodan) Dostal, Data regarding the ploidy level subsp. rarauensis (Prodan) Dostal. of the population from these species In literature the basic number of on Romania territory are not clear chromosomes at the species of enough. In “Flora of Russia: the Centaurea genus varies from x = 7 to european part and bordering x = 16 being identified also numerous regions”, vol VII, by Fedorov and diploid and tetraploid species. Tzvelev (2003) Centaurea phrygia, Centaurea phrygia is a diploid subsp. carpatica, spreaded in western specie with a chromosome number 2n Carpathians on Romania territory, is = 2x = 22 (Dydak et al., 2009). diploid with cu 2n=22 cromosomes. Diploid number of chromosomes was In our research specie Centaurea reported at subspecies Centaurea phrygia is tetraploid with 2n=4x=44 phrygia subsp. pseudophrygia C.A. (Fig. 2). Mey., Centaurea phrygia subsp. stenolepis şi Centaurea phrygia subsp. phrygia (Dostal 1976, Wagenitz 1987 cited by Koutecky P., 2007). In 1989, Dostal identify at Centaurea phrygia subsp. phrygia diploid forms respectively tetraploids forms (Koutecky P., 2007). The literature shows a geographical separation of different cytotypes of Centaurea, some authors considering only diploid cytotype for Figure 2 - Somatic metaphase of Centaurea phrygia specie, in Central Centaurea phrygia (2n=44) Europe. In according with some ample studies realised by Koutecky

55 Lucia DRAGHIA, Aliona MORARIU, Liliana CHELARIU

Dianthus armeria L. Place: Our studies on Dianthus armeria Bîrnova forest (Iaşi county) from Bîrnova forest (Iaşi county) Dianthus genus content diploid, show the existence of some diploid tetraploid and hexaploid species, with individuals with 2n=30 chromosomes a number of chromosomes 2n =2x = (Fig. 3) and also of some tetraploid 30, 2n = 4x = 60 and 2n = 6x = 90 (x individuals with 4n=60 chromosomes = 15). More this genus include (Fig. 4), diploid frequency being less. diploid, tetraploid and hexaploid cytotypes in more species (Weiss et al., 2002). From 91 species of Dianthus genus studied by Carolin (1957) it was ascertain that the majority of species are diploid (67%), 18.7% tetraploid, 6.6% hexaploid and 7.7 % present various cytotypes (Balao et al., 2009). Chromosome number 2n =30 confirm also other previous studies realised on populations from different Figure 4 - Somatic metaphase of areas (Fedorov, 1969; Goldblatt and Dianthus armeria 2n=60 Johnson 1991, 1994, 1998, 2000; Petrova 1975, 1995; van Loon and Usually natural populations van Setten, 1982, cited by Pavlova, contain only one cytotype but could 2008). Chromosomes of Dianthus be found mixt populations in which armeria are small, and the pair of together with a preponderent cytotype chromosomes have a globular aspect could appear a minoritar cytotype. (Pavlova, 2008). After Petit et al. (1999) these minority cytotypes appear based on the existance of a previous one asnd could co-habit a period together with the one from wihch he derived. In the case Dianthus specie genus, at which the phenomena of polyploidy appear frequently, being an important factor of specie, the appearance in a mainly diploid population of some tetraploid cytotypes could be a possible phenomenon. Hybridization of those cytotypes lead to the appearance of some tetraploids individuals, sterils, Figure 3 - Somatic metaphase of so the formed cytotypes could co- Dianthus armeria 2n=30 habit in population for a long period

of time being reproductive separated.

56 CYTOGENETIC RESEARCH REGARDING SPECIES WITH ORNAMENTAL VALUE

Checking this hypothesis could at species’ level which could be be done in the future through artificial elucidated by new mollecular studies hybridizations of individuals with at this specie. different polidy levels, but only the In Table 1 are presented simple appearance of polyploid synthetically the results of cytogenetic cytotypes in diploid populations of studies realised on Centaurea Dianthus armeria suggest the phrygia, Dianthus armeria, Allium existence of some evolutiv processes ursinum.

Table 1 - Data regarding the number of mitotic chromosomes in somatic cells of some species from spontaneous flora

Chromosome number Chromosome Specie References (data from (own results) literature) Baranyi et al, 1999; 2n=14 Karpaviciene, 2007, 2n=14 Allium ursinum x=7 Ricroch et al, 2005, x=7 Bennett et al. 2000 Dostal, 1976, 1989; Wagenitz 1987; Majovsky et al. 1987; Pulkina, 1988; Lavrenco et al.; Centaurea 2n=22,44 4n=44 1990 Hellwig 2004 – cited by phrygia x=11 x=11 Koutecky, 2007 Dydak et al., 2009 Koutecky, 2007 2n=30 and 2n=30 Dianthus armeria Pavlova, 2008 4n=60 x=15 x=15

CONCLUSIONS Neamţ area, is tetraploid, with 4n=44 chromosomes. Allium ursinum is a specie Population of Dianthus armeria, characterized by a great karyological identified in Bârnova forest (Iaşi stability, all analyzed individuals area), is a mixt population with x=15, having chromosome number 2n=2x= formed by tetraploid individuals 14. 4n=60 but in which could be found a Centaurea phrygia is a diploid minoritar diploid cytotype with 2n=30 specie with chromosome number 2n = chromosome number. 2x = 22, but at which is confirmed by many authors the existence of diploid Acknowledgments. This work and tetraploid populations separated was supported by NCPM, project PN geographical and ecologic. The Partnership II-code-52-174/2008. studied population, gathered from

57 Lucia DRAGHIA, Aliona MORARIU, Liliana CHELARIU

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