LITHUANIAN UNIVERSITY OF AGRICULTURE Nijol Petraityt PHENOTYPIC AND GENETIC DIVERSITY OF CARAWAY ( CARUM CARVI L.) POPULATION IN LITHUANIA Summary of doctoral dissertation Biomedical sciences, agronomy (06B) Akademija, 2005
This doctoral dissertation was prepared at the Department of Crop Science and Animal Husbandry of Lithuanian University of Agriculture in 1996-2004. Part of the work was carried out at the Laboratory of Plant Physiology of Lithuanian Institute of Horticulture. Scientific supervisor: Prof. Dr. habil. Algirdas Sliesaravi č ius (Lithuanian University of Agriculture, biomedical sciences, agronomy 06B). Scientific adviser: Prof. Dr. habil. Pavelas Duchovskis (Lithuanian Institute of Horticulture, biomedical sciences, agronomy 06B). The dissertation will be defended in the Council of Agronomy Science at the Lithuanian University of Agriculture: Chairman: Prof.Dr.habil. Pavelas Duchovskis (Lithuanian Institute of Horticulture, biomedical sciences, agronomy 06B) Members: Prof.Dr.habil. Eugenija Kup č inskien (Lithuanian University of Agriculture, biomedical sciences, biology 01B) Dr. Natalija Burbulis (Lithuanian University of Agriculture, biomedical sciences, agronomy 06B) Dr. Danguol Raklevi č ien (Institute of Botany, biomedical sciences, botany 04B) Dr. Bronislovas Gelvonauskis (Plant genepool, agronomy 06B) Opponents: Habil.Dr. Leonida Novickien (Institute of Botany, biomedical sciences, botany 04B) Ass.Prof.Dr. Honorata Danil č enko (Lithuanian University of Agriculture, biomedical sciences, agronomy 06B) Defence of doctoral dissertation will take place during the public meeting of the Council of Agronomy Science on the 28 th of December, 2005 at 11 a.m. in room No. 322, the central building of the Lithuanian University of Agriculture. Address: Lithuanian University of Agriculture Student g. 11, LT-53361 Akademija, Kauno raj., Lithuania. Phone: (370) 37 752254, Fax: (370) 37 397500 The summary of the doctoral dissertation was distributed on the 28 th of November, 2005. The doctoral dissertation is available in the libraries of the Lithuanian University of Agriculture and the Lithuanian Institute of Agriculture.
LIETUVOS EM S KIO UNIVERSITETAS
PAPRASTOJO KMYNO ( CARUM CARVI L.) LIETUVOS POPULIACIJOS FENOTIPIN IR GENETIN VAIROV Daktaro disertacijos santrauka Biomedicinos mokslai, agronomija (06B)
Disertacija rengta 1996-2004 metais Lietuvos em s kio universiteto Augalininkyst s ir gyvulininkyst s katedroje. Dalis tyrim atlikta Lietuvos sodinikyst s ir darininkyst s instituto Augal fiziologijos laboratorijoje. Mokslinis vadovas: Prof. habil. dr. Algirdas Sliesaravi č ius (Lietuvos em s kio universitetas, biomedicinos mokslai, agronomija 06B). Mokslinis konsultantas: Prof. habil.dr. Pavelas Duchovskis (Lietuvos sodininkyst s ir darininkyst s institutas, biomedicinos mokslai, agronomija 06B) Disertacija ginama Lietuvos em s kio universiteto Agronomijos mokslo krypties taryboje: Pirmininkas: Prof. habil. dr. Pavelas Duchovskis (Lietuvos sodininkyst s ir darininkyst s institutas, biomedicinos mokslai, agronomija 06B). Nariai: Habil. dr. Eugenija Kup č inskien (Lietuvos em s kio universitetas, biomedicinos mokslai, biologija 01B) Dr. Natalija Burbulis (Lietuvos em s kio universitetas, biomedicinos mokslai, agronomija 06B) Dr. Danguol Raklevi č ien (Botanikos institutas, Biomedicinos mokslai, botanika 04B) Dr. Bronislovas Gelvonauskis (Augal genetini itekli bankas, Agronomija 06B) Oponentai: Habil. dr. Leonida Novickien (Botanikos institutas, Biomedicinos mokslai, botanika 04B) Doc. dr. Honorata Danil č enko (Lietuvos em s kio universitetas, biomedicinos mokslai, agronomija 06B) Disertacija bus ginama vieame Agronomijos mokslo krypties tarybos pos dyje 2005 m. gruodio m n. 28 d. 11 val. Lietuvos em s kio universiteto centrini r m 322 auditorijoje. Adresas: Lietuvos em s kio universitetas Student g. 11, LT-53361 Akademija, Kauno raj., Lietuva. Tel. (8-37) 752254, faks. (8-37) 397500 Disertacijos santrauka isiuntin ta 2005 m. lapkri č io m n. 28 d.Disertacij ą galima peri r ti Lietuvos em s kio universiteto ir Lietuvos emdirbyst s instituto bibliotekose. INTRODUCTION All over the world there are known around 30 Carum genus specie, which differ in fruit shape and size, stalk color, leaf and leafstalk shape and other traits. Caraway ( Carum carvi L .) is a most widely spread and used plant owing to its ability to accumulate essential oils (Dastikait , 1997). It is the only herbaceous plant in the vast Apiaceae family in the Lithuanian spontaneous flora. Its fruits are used in pharmacy, perfumery and food industry. Furthermore, caraway is the most widely grown medicinal and aromatic plant in Lithuania, which fruits are exported to EU (Raduien , 2004). Long away caraway has been used to improve food taste and value. Presently in many American and European countries caraway ( fructis carvi ) is an official medicine (Skliarevskis, 1985). It has antiseptic, pain sedative, antispasmodic, depletive, antimicrobic and antioxidant properties (Nikol č iuk, igar, 1996; Vokk, Loomaegi, 1998). Caraway essential oil and its compound carvane hold high economic potential (Nemeth, Pluhar, 1996). Amongst monoterpenes, carvane as potato sprouts inhibitor is most important and holds forth a hope to use it in practice (Kleinkopf et al 2003, Carvalko, 2005). Recently more attention has been paid to investigations of terpenes, including carvane, in order to protect and cure human organism against oxidant stresses as well as cancerous and cardio diseases (Wagner, Elmadfa, 2003). In 2005 caraway was included into ten plants of the European priority research. In Lithuania together with the decline of natural meadows caraway habitats shrink, species biodiversity and genetic resources become sparse (Balevi č ien , 1998). Wild plant populations are still being formed by such evolution forces like natural selection, mutation, migration and hybridization. More stable survival potential is found in wild plant populations with more individuals because this provides a better chance to form genotype combinations adapted to changing environment . Therefore, species genetic diversity is a guarantee of its survival. In order to preserve species genetic fund it is important to accumulate and investigate intraspecific diversity, to assess and select the most valuable samples suitable for registering into national genefund registers and utilize them in breeding (Budvytyt , 2000). Since 1996 in Lithuania there have been carried the programs Investigation and preservation of plant and animal genetic resources in Lithuania (Genefund), later National state plant genetic resources program, in which Carum carvi was one of the objective species. Aims of work were to assess the ecologic and phytocenologic affection of Lithuanian caraway habitats and the diversity of main morphologic, productivity and biochemical traits of samples grown in natural cenopopulations and introduced collections as well as to select cenopopulations with valuable traits for breeding. Tasks of the work: ● to investigate phytocenological and ecological variation of Carum carvi habitats; ● to establish the diversity and stability of morphological and productivity traits in natural ( in situ ) Carum carvi cenopopulations; ● to assess variation and stability of phenotypic traits of samples growing in collections ( ex situ ). 6
● to assess Carum carvi genetic diversity among and inside cenopopulations by employing the random amplified polymorphic DNA (RAPD) method. Originality of the research work . For the first time in Lithuania geographic, ecological and phytocenological assessment of caraway ( Carum carvi L.) habitats was carried out, ecological and antropogenic factors limiting the spread of population were established. Adaptation potential ex situ was examined. Stability of morphological parameters and the content of essential oil and carvone as well as phenological and phenotypic diversity variation of habit and leaf shape, leaves photosynthetic pigments, variation of petal color were assessed. The impact of temperature and genotype was evaluated at the stage of flower differentiation. Caraway intercenopopulation genetic diversity was determined at the molecular level. Practical importance of the work. Performed investigations and observations in situ underlie the basis for caraway preservation in natural environment. The correlations of main biochemical and phenotypic traits, phenotypic traits and stress resistance were established. Biochemical and phenotypic traits were identified for selection of valuable samples for breeding. 57 samples are put in long term seed storage Species descriptors will be used at the International Plant Genetic Resources Institute (IPGRI) to compose common inventories of Carum carvi species. Dissertation content and volume. The dissertation consists of introduction, methods, research results and discussion, conclusions and references (247 references), the list of published works on the dissertation subject and annexes. The dissertation consists of 110 pages. MATERIALS AND METHODS Research object : Natural cenopopulations of caraway ( Carum carvi L.) in various geographic places of Lithuania. Research place and time . Investigation of caraway cenopopulation and sample collection were performed in July August of 1996-2004. In 1998-2004 caraway seed samples were sown and grown ( ex situ ) at the trial station of LUA. The analysis of material collected in situ and ex situ (biometric parameters) and distillation of essential oils were carried out at the Genetics and Biotechnology Laboratory of LUA. Collections were grown and field trials were established at the trial station of LUA. The vegetation trial was performed in the greenhouse and phytotrons of Plant Physiology Laboratory of LIH, genetic trials at Genetics Laboratory of LIH. Equipment and methods. Cenopopulations were marked by a collection number that consisted of description year, initials of the describer, and serial. Collected samples were given a collection number. Descriptions of geobotanic community were performed according to J. Broun Blanquet (1964) methodology. Community sintaxonomic dependence was established according to Lithuanian and international plant classification (Rothmaler et al., 1988; Балявичене , 1991; Raomavi č ius, 1991). Plants were characterized according to handbooks (Snarskis, 1954; Lekavi č ius, 1989). Geographic coordinates of habitats were established by apparatus Garmin GPS-12. The map was compiled according to the square system (Gudinskas, 1993). Investigations were carried out in meadows of different economic activities: meadows and pastures. Soils were evaluated according to J. Mavilas (Mavila, 1998) scale. For plant assessment there were used biometric traits - stalk height, branching height, caulicle diameter, stalk number, inflorescences number, 1000 seeds weight, seed yield of a plant, and biochemical parameters essential oil content in fruit, carvone content in essential oil, leaf pigments - chlorophylls a and b and carotenoide content, anthocyanins content in petals. For evaluation of Carum carvi L. leaf shape were used descriptors for evaluation of Daucus carota leaves (Descriptors for wild and cultivated carrots ( Daucus carota L.), 1998). Phytochemical analyses essentials oils content in caraway fruits (distillation by water steam method) and essential oil composition (by gas chromotographer FISONC GC) were established at the Biochemistry Laboratory of KTU. Contents of chlorophylls carotenoids and anthocyanins were established by the colorimetric method in a 100 % extract of acetone according to Wetshtein ( Гавриленко ,1975) and by spectrophotometer Beckman DU-40 at "Tempus" laboratory of LUA. Organogenesis stages were determined according to development stage and methodology worked out by F. Kupermans school ( Купермаи и др . 1982). Vertical gel apparatus of Bio Rad company was employed for electrophoresis. Peroxidase and polyphenoloxidase isoforms in electrophoregrams were calculated according to V. Jaaskas methodology (Jaaska, 1972). Leaf area was calculated by employing Gis Arc/info and Rootage program packages. DNA was identified by plant DNA identification package Macherey Nagel according to the producers methodology (Genomic DNA from Plant, 2002). Data were processed mathematically and statistically according to G.N. Zajcev (1984) and V. Sakalauskas (1998), computing by Exel and package STATISTIKA . Meteorological conditions during research years. Thermal and humidity conditions at caraway development stages were characterized by Selianinovs hydrothermal coefficient the ratio of precipitation and the sum of above zero temperatures (Kudakas, Urbonas 1983). Spring hydrothermal coefficient (HTCsp) was calculated to show meteorological conditions from the beginning of caraway vegetation till the beginning of flowering stage (decades: March III, April I - May I-II). Summer hydrothermal coefficient (HTCsm) - flowering ripening stage (decades: May II-III - July II-III). When HTC is up to 0.3 years or stages are very dry, 0.4-0.5 - dry, 0.6-0,7 - arid, 0.8-1.0 insufficiently wet, 1-1.5 sufficiently wet, 1.5 and > - wet . Meteorological conditions in research years (1996-2004) were variable (Fig. 1). 1996 was the wettest year, 1998, 1997, 2001 and 2003 - sufficiently wet. 2000 was insufficiently wet. 2004 had draughty spring and the beginning of summer. 4,5 2 HTCsp HTCsm 4 3,5 1,4 1,3 1,2 1,3 3 2,5 2,2 1,1 1 2,1 1 2,1 1,5 ,9 2 1,6 1 5 0,8 , 1,51 1 0,9 0,7 0,5 0 1996 1997 1998 1999 2000 2001 2002 2003 2004 Figure 1. Spring and summer hidrotermal coefficients (1996-2004) RESULTS AND DISCUSSION 7
Ecological and phytocenological characterization of Carum carvi habitats Habitat locations. In 1996-2000 during expeditions were found and described 123 Carum carvi habitats. The highest number of caraway habitats was found in South and Southeast Lithuania (Fig. 2). In this region because of hilly and laky relief there are a lot of natural meadows while intensively cultivated ones are scarce. The number of habitats in Central and North Lithuania is limited because of intensive agriculture. Non-anthropogenic meadows are not numerous. In the southeast part of Var na district, and especially in alcininkai district in East Lithuania bordering with Belarus, as well in ilale district prevail rather acid sandy and sand loam soils (pH<6) unsuitable for caraway. In the western part habitats are limited by wetlands and heavy and rather acid soils.
Figure 2. Natural habitats of Carum carvi L. 1996-2000 Geobotanic assessment of habitats. Most of Carum carvi L. habitats belonged to the widely spread in all Lithuania confederation community Molinion-Arrhenatheretea elatioris R. TX. 1937 class Cynosurion cristati R.. Tx. 1947 - 48%, to Arrhenatherion elatioris (Br.-Bl. 1925) 36.6%, to Molinion caeruleae W. Kotch 1926 2.4%. To the confideration Trifolio-Geranietea sanguinei (Th. Müller 1961) class Trifolion medii belonged 6.5% of described communities. Trifolionm medii communities were found in the eastern, southeastern and southwestern (ilute district) part of Lithuania in meadows on the edges of woods and in woods. To relict Festuco-Brometea erecti Br.-Bl. Et R.Tx. 1943 class communities were ascribed 4.9% of habitats. All communities were found in the South Southeast Lithuania on slopes of rivers and hills, in places non-suitable for land cultivation. Two (2.6%) Carum carvi habitas were described in unkempt, grassy soils ascribed to Agropyretalia repentis Obert., Th. Müller et Görs Oberd et al. 1967. In these habitats grassland was not fully covered. 168 plant species were identified in habitats. Habitat assessment. Carum carvi investigation was carried out in meadows with different economic activity that conditionally were subdivided into meadows (dunkempt, hayfiel) and pastures (pasturable). To pastures were ascribed 61% of investigated habitats (mostly Cynosurion cristati communities), meadows - 39%. Soils of pastures were more fertile (Table 1). Granulometric composition of pastures varied more in comparison to meadows. Most prevailing soils in pastures were clay soils (27%), sandy loams (24%) and light loams (21%), in meadows sandy loams (28%) and clay loams of various heaviness (65%). T able 1 A grochemical characterization of pastures and meadows Habitat type Agrochemical indicators x min max V,% P 2 O 5 , mg/kg 184.0 59.0 580.0 65.8 Pasture K 2 O, mg/kg 199.7 81.0 332.0 34.6 Humus, % 2.5 1.1 5.3 47.3 pH KCL 7.0 5.3 7.8 6.7 P 2 O 5 , mg/kg 162,3 35,0 640.0 82.5 Meadow K 2 O, mg/kg 171,0 15,0 364.0 40.9 Humus, % 2,1 0,66 4.34 42.9 pH KCL 6,9 5,8 7.6 6.2 Caraway was most abundant in Cynosurion cristati confederation communities, especially when except pasturage no other activities were carried out. Cattle while grazing leave out some caraway providing for them good assimilation conditions and possibility to ripen seeds. Least of all caraway plants were found in Festuco-Brometea erecti class communities. Caraway is ascribed to well cropped grasses but only in such cases when it makes up not more than 5% of grass mass (Petkevi č ius, Stancevi č ius, 1982). Soil analyses of habitats show that most often caraway grew in neutral alkaline and close to neutral, light and medium light loam, fertile, humus, potassium and phosphorus rich soils. Meanwhile there were found habitats in cohesive sandy and peat soils (pH 5.3-6.5) with low levels of mobile potassium and phosphorus, and humus. Assessment of caraway growth parameters ( in situ ) Discussion on morphological parameters. Differences of Carum carvi morphological parameters were rather significant in habitats of different economic activities. Stalk height . The average height of caraway stalks in pasture phytocenoses was lower by 14.2% in comparison to those in meadows (Table 2). High variation of stem heights was established. The maximal height of caraway stalk in pasture cenopopulations was by 2.4 higher than a minimal one in meadows 2.1. Stalk height was directly affected by meteorological conditions dependence of stalk height on the ratio of precipitation and air temperature (hydrothermal coefficient (HTC)) was more distinctive in meadows in summer, in pastures in spring. Stalk branching height. The maximal branching height 8
of caraway growing in meadows was higher by 26.7% than of those growing in pastures. Both in meadows and pastures high correlation between stalk height and the beginning of branching height is distinct. Caulicle diameter. Caulicle diameter of caraway in meadow phytocenoses was smaller by 3.1% in comparison to those in pastures. Meanwhile the limit of caulicle variation between the minimum and the maximum was lower in meadow caraway (4.6 times) than in pasture caraway (5.6 times). Average negative correlation was established between caulicle diameter and branching height, average positive between caulicle diameter and inflorescence number. T able 2 Characteristics of Carum carvi L. parameters in situ and ex situ Indices In situ Ex situ x x min x max v% x x min x max v% Stalk P 63.2* 36.0 85.0 15.3 height, cm M 72.8* 48.0 100.0 13.3 Joint 68.0 36.0 100.0 15.8 76.5 53.3 101.0 14.6 Branching P 10.8* 1.0 24.21 47.5 height, cm M 15.4* 1.0 33.0 47.5 Joint 13.2 1.0 33.0 51.1 3.1 1.0 12.6 92.0 Caulicle P 0.6 0.3 1.9 26.9 diameter, M 0.7 0.4 1.8 32 cm Joint 0.63 0.3 1.8 30.2 1.2 7.0 3.8 40.1 Number P** 1.3 1.0 3.0 36.6 of stalks, M 1.3 1.0 2.8 44.8 pcm. Joint 1.3 1.0 3.0 40.1 5.4 1.0 25.0 101.8 Number P** 15.4 7.9 50.1 44.2 of M 14.8 7.0 51.8 39.8 recames Joint 1.3 7.0 51.8 40.1 68.6 12.5 253.8 74.6 1000 seed P** 2.02 1.7 2.5 8.1 weight, g M 1.99 1.5 2.6 10.2 Joint 2.01 1.5 2.6 9.1 2.1 1.7 2.9 11.2 Seed yield P** 2.0 1.2 2.8 22.2 of a lant, M 1.6 1.1 2.9 25.7 g Joint 1.8 1.2 2.8 17.8 3.7 1.7 19.1 86.6 Essential P** 5.4 2.8 7.7 20.3 oil content M 4.9 2.6 8.4 23.3 % Joint 5.1 2.6 8.4 22.1 4.8 3.2 7.0 16,6 Carvone P** 55.4 51.6 62.1 4.7 content, M 55.4 50.9 62.4 5.4 % Joint 55.4 50.9 62.4 4.9 52.9 49.6 60.7 5.0 * Means are significantly different at P ≤ 0.05 ; ** P-pasture, M-meadow Inflorescence number. In meadow and pasture caraway the inflorescence number of one plant differed insignificantly by 6.7%. In both types of habitats the minimal number of inflorescences was the same, while the maximal number was higher by 3.4% in meadows. Both in meadow and pasture caraway weak negative correlation was established between stalk branching and inflorescence number. Weight of 1000 fruits. The weight of 1000 caraway fruits from pastures was higher only by 1.5% (0.12g). However, higher variation was observed in meadows. In meadows the minimum of 1000 fruit weight differed 1.7 times, in pastures 1.4 times. Plant productivity . The average fruit yield of one plant was higher by 18% in pastures. Both in meadows and pastures the difference between the minimum and maximum parameters was 2.6 times. Average negative correlation was established between plant fruit yield and stalk height, positive correlation - between plant fruit yield and branching height in pastures and medium positive correlation between plant fruit yield and inflorescence number. Essential oil content in fruits . The essential oil content in biannual caraway fruits is 3.0-8.0% ( Полуденный , 1979; Baumaster, 1995). In research years the average and minimal essential oil content in caraway fruits from pastures was higher (respectively 9.6% and 6.5%) (Table 2). Meanwhile higher variation of this parameter was established in meadows. More favorable conditions for essential oil synthesis were in pastures which are more open to lighting and CO 2 assimilation, but in drier years in such habitats faster evaporation of moisture was a limiting factor (Fig. 3). Average negative correlation was established between caraway essential oil content and precipitation and air temperature (HTC).The most favorable year for essential oil synthesis was 1999 and most unfavorable 1996. Essential oil dependence on environmental conditions acknowledge other authors as well (Salamon, 1994; Хотин , Шульчина , 1963). % Pasture Meadow HTCsm 8 5,8 6,1 6,16,9 6,1 ,7 2,5 5,3 5,3 5 5 2 64 4,94,1 5 4,8 5,1 3,6 3,8 3,7 3,5 1,5 1 2 0,5 0 0 1996 1997 1998 1999 2000 2001 2002 2003 2004 Figure 3. Dynamics of essential oil content in Carum carvi fruits (1996-2004) C arvone amount in essential oil . In assessments of caraway cenopopulations the main criterion is the amount of carvone, the main component of essential oil. The amount of carvone in essential oil of wild caraway can reach 48.3-70.0% (Kuusi, 1981; Galambosi, 1996). In research years 1999 and 2000-2004 carvone amount in essential oil in in situ grown caraway ranged from 50.9% till 62.4% (Table 2). In pastures carvone amount was slightly higher than in meadows. Differently from essential oil synthesis, carvone formation was positively affected by cooler and wetter weather. The lowest level of carvone was in 1999 (Fig. 4), when the summer was particularly dry and hot (HTCsm-0.9), the highest in 2004, when HTCsm was highest 1.8. Strong positive linear correlation was established between HTCsm of research years and carvone content means (r=0.8864). In dry and hot summers (1999, 9
2001, 2003) in caraway essential oil more carvone was found in meadows than in pastures where moisture evaporated faster. Negative medium strong dependence was established between caraway essential oil content and carvone content (r=0.6472). Pasture Meadow HTCsm 57 56,3 2 55,1 55556453,85454,153,254,354,855,411,5 53 53 52 0,5 51 0 1999 2001 2002 2003 2004 Figure 4. Dynamics of carvone in essential oil content in Carum carvi fruits (1999; 2001-2004) Assessment of characteristics similarities of caraway cenopopulations. Investigation results show high variation of Carum carvi cenopopultions in morphological, productivity and biochemical parameters. After hierarchic cluster analysis of cenopopultions there were identified four clusters, which substantially reflected growth conditions of cenopopulations. Cluster I (9.8% of investigated habitats) cenopopulations grew in more or less shaded areas, which directly affected their parameters. For competence with other plants and light deficiency caraway stalks were highest (93.8 cm), and essential oil content was lowest (4.9%) in comparison to other clusters (Fig. 5). Predominantly pastures in fertile soils belonged to cluster II (30.1%). Means of stalk and branching height are rather low, inflorescence number the highest (18.3), essential oil content - one of the highest. This is a group of cenopopulations with the most stable microclimate and the lowest variation of traits during the investigation years. Predominantly habitats of fertile hayfield and unkempt meadows (no economic activities) belonged to cluster III. The means of cenopopulation height and branching height were among the highest in this cluster. In these cenopopulations caraway grew in the second layer of sward, where lighting and assimilation conditions were worse. Fluctuation limits (2.6-7.2%) and variation (22.2%) of fruit essential oil content was highest that reveals variation of lighting conditions in the cenopopulations. The habitat of a single-stalker concise caraway was formed in many cenopopulations of such lengthy grasses. Most significant were cluster IV cenopopulations, which made up 26.8% of all cenopopulations. Here prevailing were Cynosurion cristati (65.7%) communities and other confederation communities in less fertile soils, mostly used as pastures. Systematic grazing provided more favorable philosphera conditions for less cropped caraway. In this cluster was found the shortest caraway, the mean of stalk height was nearly twice lesser than in cluster I, and the mean of branching height was smallest. In such type habitats caraway grew in the first layer of sward. For this reason CO 2 assimilation and lighting conditions were extremely favorable. They affect directly seed mass growth during ripening (Bouwmeester, 1998) and essential oil synthesis (Bouwmeester, Kuijpers, 1993). Caraway from this cluster had the highest average content of essential oil (5.5%). This is the only cluster which cenopopulations had partial geographic preference. Most of them (57%) were described in East and Southeast Lithuania. Due to increase of philosphere space in some habitats formed a specific, procumbent, multistalk or pasture caraway habitat. BStraalnkc hhienigg hhteight1000 seed weightEssential oil content Number of inflorescence 2,2 8 120 20 2,1 7 6 180001525 1,9 4 % 460010183 , 20 5 1,7 2 1 0 0 1,6 0 I II III IV A I II III IV B Figure 5. Characteristics of different clusters of caraway cenopopulations in situ. A-Morphological traits, B- Productivity traits Natural cenopopulations form is affected by genetic and environmental factors. The structure of plant organs and habitat are inherited. These parameters can vary in changing environment conditions. The amplitude of trait variability allows prognosing species survival possibility ex situ. Variation of adaptive phenotypic morphological parameters of caraway, especially stalk habitat form, shows its high liability and capacity to survive under changing environment conditions (Gill, Randnava, 1999). Assessment of caraway parameters e x situ. Stability of phenotypic traits, essential oil and its composition were investigated when plants were transferred from natural populations to field collection (1997-2004). Stalk height. In research years the average caraway height e x situ was smaller by 12.5%. Fluctuation limits were wider by 24% than in situ , meanwhile, the variation was very similar (Table 2). Higher height ex situ was stipulated by better granulometric composition, nutrients level and pH of soil at the Experimental station. Stalk number . The average stalk number ex situ was 4.3 time bigger (4.1 stalks). Difference between the minimal and maximal values was 8.3 times higher (22 stalks). Variation was twice higher than in situ. Correlation between branching height and stalk number was more distinctive than in situ. Inflorescences number. The average inflorescences number ex situ was 4.6 times higher (43). Difference between the minimal and maximal values was 4.8 times bigger (202) than in situ. In literature it is mentioned that due to stress the number of side stalks, subsequently the number of inflorescences as well increases ( Батыгин ). This gives reasons for abundant number of caraway stalks and inflorescences under ex situ conditions . Weight of 1000 fruits. The average weight of 1000 caraway fruits grown e x situ had low variation -5%, though difference between the minimal and maximal values was similar . Strong negative linear correlation exists between this parameter and inflorescences number. Plant productivity. In ex situ conditions on average caraway gave twice higher yield, and in favorable years some populations gave 5-fold higher yield (19.1 g) in comparison to the average yield of research years. Variation of this parameter was among the highest ones. Essential oil content in fruits. In research years the average essential oil content in caraway fruits ex situ was by 7% lower than in situ (Table 2). Difference between the minimal and maximal values was lower by 18%. Variation was lower too. Other morphological and productivity parameters had stronger impact on fruit essential oil content than in situ . Medium weak negative correlations were established 10
between essential oil content in fruits and the weight of 1000 fruits, and between essential oil content and one plant yield . Carvone content in caraway fruits. The average carvone content in caraway fruits was slightly lower, by 4.6% than in situ. Difference between the minimal and maximal values was almost the same and variation was similar. Weak linear correlations were established between carvone amount in essential oil and one plant yield, carvone amount and inflorescences number. The main resources for which plants compete are light, heat, water and nutrients. These conditions for plants in situ and ex situ are fundamentally different. Growing of local flora plants under the same conditions as cultivated plants does not mean their transference from one place to another. Naturally these plants are phytocenoses components, while in the collection they are monoculture. Phytocenotic and ecologic environment lighting, weather and soil regime, nutrition conditions, alelopatic impact, rysosphere, etc. is changed for them. Variability of adaptive phenotypic morphological traits reveals high liability of Carum carvi . The most liable parameters are branching height and the number of stalks and inflorescences, the most stable ones the weight of 1000 fruits and biochemical composition of essential oil. Biochemical and morphological assessment of Carum carvi leaf and petal diversity Biochemical leaf assessment in different cenopopulations. Individual cenopopulations in meadow phytocenoses of the same species tolerate differently light deficiency, differ in leaf shape, pigments amount and their composition (Protczuk et al., 2002). Leaf color, pigments amount and composition show biochemical diversity within plant genus or species as well as adaptivity in intensive lighting and shade tolerance (Murchie, Horton, 1997; Thayer, Bjorkman, 1990; Rosevear, 2001; Artemyeva, Solovyeva, 2003). In May 2002 cenopopulations rosette leaf pigments (chlorophylls a and b, carotinoid content) of biannual caraway were analyzed and after hierarchic cluster analysis of research data three clusters were identified. In caraway leaves of cluster I cenopopulations the lowest ratio of chlorophylls a and b was found compared to other clusters, but the highest content of total chlorophylls and carotenoids (Fig. 6). To this cluster belonged 20.1% of cenopopulations investigated in the collection. Caraway leaves of cluster II cenopopulations had the lowest content of chlorophylls and carotenoids. The ratio of chlorophylls a and b occupied the intermediate position between clusters I and II. In cluster III total chlorophyll content was by 20%, and carotenoids -22% higher in comparison to cluster II. Meanwhile the total chlorophyll content was by 35%, and carotenoids by 25% lower in comparison to cluster I. In cenopopulations of cluster III the highest ratio of chlorophylls a and b was found in comparison to other clusters. To cluster III belonged 36.4% of investigated cenopopulations. 3 2,3 3 2,5 1 1,94 1,5 2,2 2,5 2 ,87 2 2 1,5 1, 1,5 1 0,44 1 0,5 0,26 0,32 0,5 0 0 I II II a+b c a/b Figure 6. Different clusters of caraway cenopopulations by leaf pigments. (a+b -chlorophylls a and b content; c-carotinoids content; a/b - ratio of chlorophylls a and b) Cenopopulations of cluster I according to the ratio of chlorophylls a and b relatively can be ascribed to shade tolerating ecotypes. Most of these cenopopulations in situ grew in shaded habitats (orchards, parks, deserted homesteads) and communities of fertile high growing Arrhenatherion elatioris meadows. The higher content of carotenoids in leaves of this cluster cenopopulations than in other clusters cenopopulations could be a protective function while acclimatizing in more intensive (than in situ conditions) lighting (Rasevear et al., 2001; Radunz, Schmid, 2002). Cluster II cenopopulations amounting nearly to a half (45.5%) of investigated cenopopulations according to research data occupied the intermediate position. Soils of habitats belonging to this cluster were rather rich, favorable for caraway growth because of granulometric compostion and pH. Cenopopulations of custer III according to the ratio of chlorophylls a and b relatively could be ascribed to ecotypes tolerating intensive lighting. Soils of this cluster are among the poorest and most unfavorable because of soil pH (6.3-7.8) and granulometric composition (coherent sands, clays). It includes a part of habitats of C ynosurion cristati pasture type and most Festuco-Brometea erecti communities, (which can be ascribed to thermophilic meadows (Raomavi č ius, 1986)), where caraway grew in the first layer of sward. Meanwhile in such habitats microclimate changes caused by heat, cold or draught are more obvious (Stoutjiesdijk, Barkman, 1987). Some cenopopulations distinguished by a particularly high content of chlorophylls and carotenoids and a higher ratio of chlorophylls a and b than others. According to literature data such ecotypes in comparison to others have higher cold resistantce (Haldimann, 1998). According to data of other authors high chlorophyll content directly correlates with high plant productivity (Kabanova, Chaika, 2001). Morphological assessment of leaf diversity in different conopopulations. According to dissection degree and shape there were identified three morphological types of caraway leaves: normal to which belonged 93% of all investigated cenopopulations, dill 2.8 % and parsley 0.9 % . Leaf area calculation data showed that the largest average leaf surface area was of parsley type caraway leaves - 13.5 c 2 th smallest of normal type leaves 12.2 cm 2 . The average area of dill type leaves was 12.4 m , e cm 2 . The characteristic leaf color in the investigated cenopopulations was dark green (83.2 % of investigated cenopopulations), green (11.2 % ), light green (2.8 % ) and greenish grey (2.8 % ). For leaves of parsley type cenopopulations the characteristic color was dark green, for dill type light green and green. Impact of low temperatures on coloration of caraway petals and leaves. Various plant specie are differently adapted to low above zero temperatures and frost stresses (Kratsch, Wisl, 2000). Such stress arises various gene expression changes (Guy, 1990), such as increase of leaf hairiness (Roy et al., 1999), content of anthocianins (Larcher, 1995) and carotinoids (Haldimann, 1998). Temperature is the main climatic factor that allows revealing the diversity of leaf colors (Zhang et al., 2004). It is supposed that different coloration of the same species petals is related to resistance to low temperatures (Warren, Mackenzie, 2001).
In research years visual estimations of caraway petals coloration in the collection revealed that petals coloration of the same cenopopulations vary in different years. Caraway with pink flowers were more abundant in the years with changeable weather in spring, where cold spells interchanged with warm spells and late frost were more frequent. Particularly strong caraway petals coloration was found in 2001, when in April, after a rather warm period (in the first decade the temperature at the soil surface in certain days reached 28.3 o C), followed a cold spell and for nearly a week each night frosts occurred (in the second decade, up to -3.4 o C at the soil surface). In 2001, 90% of cenopopulations had rose or pink color of petals. The diversity of leaf colors was more observable too (Fig. 7). 9,% 1 1,9% % 7,4% 1,9% 3,7% Figure 7. Petals (A) and leaves (B) color of different Carum carvi cenopopulations 7,3% 24% (2001) (A - 1-Fuchsia; 2 - Rose - pink, 3 30,8 Pink>70%-white; 4 - Pink>50%-white; 5 -% Pink>30%-white; 6 White. B - 1- Blue green, 63% 2 Grey green, 3 Dark green, 4 - Green; 5-38,2 Light green) 1 2 3 4 5 6 1 2 3 4 5 B A White, pink and rose flower forms were identified according to petal color intensity. Table 3 Different forms of Carum carvi 2001 Leaf Petals -1 Carotinoids, anthocianins, Form/variety a Cbh lorophyllsa , +mb g g a/b mg g -1 -1 mg g Gintaras 0.84* 0.94* 1.81* 1 0.60 0 White flower 1.14* 1.16 2.3* 0.98 0.57 0 Pink flower 1.34* 1.29* 2.63* 1.04 0.61 16.46* Rose flower 1.64* 1.54* 3.19* 1.08 0.83* 39.13* *Means are significantly different at P ≤ 0.05 Cenopopulations with colored petals had higher levels of leaf pigments. Cenopopulations with rose petals had not only higher (58%) anthocianins content in petals but the highest leaf pigment amount as well (Table 3). Cultured caraway Gintaras and 9.1% of wild cenopopulations with white flowers had the lowest amount of leaf pigments. Individuals tolerance to low temperatures, capacity to react to changing environmental conditions reveal their ecological plasticity that is genetically determined (Larcher, 1995). Different caraway genotypes with different levels of leaf carotinoids, chlorophyll content and ratio, anthocianins amount in petals, different reaction to cold and lighting stress reveal specific adaptive plasticity of caraway. Impact of temperature and genotype on caraway morphogenesis at the phase of flower differentiation. In 2003 a vegetation trial was performed in climate chambers in order to find out the impact of temperature and genotype on caraway morphogenesis at the stage of flower differentiation. Trials included three wild caraway genotypes of different ripening seasons (No. 341 - very early, No. 337 - early, No. 351 medium late) and control variety Gintaras '(late). At organogenesis stages II-IV plants were placed in climatic chambers where they underwent thermo induction phase (+4 0 , 60 days), and after that were transferred to three climatic chambers for 30 days at different temperature regimes (Table 4). Table 4 Different temperature treatments in climate chambers Climate chamber C 0 (10 days*) C 0 (10 days*) C 0 (10 days*) I 21/15 21/15 21/15 II 15/10 21/15 21/15 III 21/15 15/10 21/15 *Day period-16h Assessment of plant state during the trial revealed that plants were most sensitive to low above zero temperature at organogenesis stages Vb-Vc and in the third chamber with the most unfavorable regime reached only stage X. The investigated wild caraway genotypes regarding temperature were more tolerant than cultivated variety Gintaras and their morphogenesis phases proceeded shorter (Fig 8). 100%100% 80%80% 60%60% 40% 40% 20% 20% 0% 0% V-VI VII VIII-IX X XI XII V-VI VII VIII-IX X XI XII I II III Organoge nes is s tages Organogenesis stages A Nr. 341 Nr. 351 Nr. 337 Gintaras B Figure 8. Influence of different temperature treatments on Carum carvi organogenesis. A- stages obtained by all genotypes; B- stages obtained by different genotypes 12
Duration of various ontogenesis stages is not related to genotype ripening season. Variety earliness is determined by the combination of different stages (iknianien , 2002) Phenological observations and morphological measurements allow concluding that the speed of different ontogenesis stages and character are similar, genetically determined and under the same temperature conditions proceed similarly, while genotype earliness is determined by the duration combination of different ontogenesis stages. Assessment of Carum carvi L. genetic polymorphism. Morphological and biochemical analyses are performed by classical methods (utar-Voli č , 2002), but for assessment of plant genetic diversity these analyses are not sufficiently informative because phenotypic expression is highly variable depending on environment conditions and different gene expression occurs (Javornik, 2002). Molecular investigation reveals individual s diversity at DNA level, which is independent from growth conditions (Javornik, 2002). After establishment of wide diversity of caraway characteristics, for molecular analyses were selected 6 geographically remote cenopopulations varying in phenotypic traits and Lithuanian variety Gintaras . OPC02 primer was employed in PRC reaction to obtain DNA fingerprints from different caraway cenopopulations. For some cenopopulations were obtained DNR rofiles, recurrin within ceno o ulations of different ori in Fi . 9).
Figure 9 . DNA fingerprints of different caraway cenopopulations By molecular analyses it was established that for most individuals of investigated cenopopulations irrespective of their place of origin 2250; 1200, 950, 740, 550 bp DNA fragment was characteristic. Only one individual had 1500,1000, 875 and 375 bp DNA fragment and only one had 500 bp DNA. Agrobiological assessment of Carum carvi cenopopulations. In summary of research data of ex situ caraway, hierarchic -cluster analysis was performed according to phenological as well as petal and leaf color research data. The investigated cenopopulations were grouped into four clusters, which varied in morphological and biochemical parameters. Earliness of the investigated cenopopulations had impact on their morphological and productivity parameters. To early cluster I belonged cenopopulations with most intensive petal color and the darkest leaf color (Fig. 10). Cenopopulations of this cluster were behind cenopopulations of other clusters in all morphological and productivity parameters, when in fruit essential oil content and carvone content in essential oil they exceeded the others. Caraway of medium early cenopopulations of cluster II and medium late cenopopulations of cluster III had medium morphological and productivity parameters in comparison to clusters I and IV. Caraway of late cenopopulations of cluster IV had the highest plant and branching height, the greatest number of flowers, weight of 1000 fruit and one fruit yield, though it had the highest content of essential oils and carvone. The most important productivity parameters of caraway are essential oil content and carvone content in essential oil. According to these two parameters the most productive were medium late and late cenopopulations of clusters III and IV. r of petals Essential oil 3,5 3,3* 3,3* CCoolloor of leaf 5,5 % 56 Carvone Fowardness 3 3,2* 55,,3454,8*55 2,5 2,1* 5,2 5,4 54 2 1,7* 5,1 53,3 52 1,51 1,2* 1,5* 1 1 * ,8* 1,1* 0715,*6* 4,95 5,2 ,652,1*5523 0,5 , 44,,87 5,14 51 ,9 4,6 50 0 I II III IV Klasteris I II III IV Klasteris A B Essential oil yield Carvone yield 196* 150 146* 176 194 100 80* 92 102,1 102,1* Figure 1 0. Characteristics of different clusters of caraway cenopopulations ex situ. 50 A- Earliness and phenotypic traits; B-C- Productivity traits. 0 Klasteris I II III IV C