LITHUANIAN UNIVERSITY OF AGRICULTURE LITHUANIAN INSTITUTE OF HORTICULTURE DanguolėJukevičienė GYNOGENESIS OF EDIBLE ONION (Allium cepaL.) AND CREATION OF HOMOZYGOUS LINES Summary of doctoral dissertation Biomedical Sciences, agronomy (06 B), plant growing, horticulture, plant protection and phytopathology branch (B 390) Akademija, 2006
2 Dissertation was worked out during 2001 2005 at the Lithuanian Institute of Horticulture Scientific supervisor: Prof. Dr. Habil. Vidmantas STANYS (Lithuanian Institute of Horticulture, Biomedical Sciences, Agronomy 06 B, plant growing, horticulture, plant protection and phytopathology branch B 390). Dissertation is defended at the Council of Defence for Biomedical Sciences, Agronomy: Chairman: Prof. Dr. Habil. Algirdas SLIESARAVIČIUS (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy 06 B, plant growing, horticulture, plant protection and phytopathology branch B 390). Members: Assoc. Prof. Dr. Bronislovas GELVONAUSKIS (Plant Gene Bank, Biomedical Sciences, Agronomy 06 B, plant growing, horticulture, plant protection and phytopathology branch B 390); Dr. Giedrė DABKEVIČIENĖ Institute of Agriculture, (Lithuanian Biomedical Sciences, Agronomy 06 B, plant genetics branch B 225); Dr. Tadeuas IKNIANAS (Lithuanian Institute of Horticulture, Biomedical Sciences, Agronomy 06 B, plant growing, horticulture, plant protection and phytopathology branch B 390); Dr. Rytis RUGIENIUS (Lithuanian Institute of Horticulture, Biomedical Sciences, Biology 01 B, vessel plants physiology branch B 310). Opponents: Prof. Dr. Habil. Pavelas DUCHOVSKIS (Lithuanian Institute of Horticulture, Biomedical Sciences, Agronomy 06 B, plant growing, horticulture, plant protection and phytopathology branch B 390); Dr. Laimutė BALČIŪNIENĖ (Vilnius University, Biomedical Sciences, Biology 01 B, plant growing, horticulture, plant protection and phytopathology branch B 390). Defence of dissertation will be held in the public meeting of the Council of Defence for the Agronomy Sciences on February 15th, 2006, at 11:00 a.m. in the room 322, Central building of the Lithuanian University of Agriculture. Address: Studentųstr.11, LT-53361, Akademija, Kaunas district, Lithuania Summary of doctoral dissertation was distributed on January 15th, 2006 Doctoral dissertation is available at the libraries of the Lithuanian Institute of Horticulture and the Lithuanian University of Agriculture.
3 LIETUVOS EMĖSŪKIO UNIVERSITETAS LIETUVOS SODININKYSTĖS IR DARININKYSTĖS INSTITUTAS DanguolėJukevičienė VALGOMOJO SVOGŪNO (Allium cepaL.) GINOGENEZĖIR HOMOZIGOTINIŲLINIJŲKŪRIMAS Daktaro disertacijos santrauka Biomedicinos mokslai, agronomija (06 B), augalininkystė, sodininkystė, augalųapsauga, fitopatologija B 390
4 Disertacija rengta 2001-2005 metais Lietuvos sodininkystės ir darininkystės institute. Mokslinis vadovas: prof. habil. dr. Vidmantas STANYS (Lietuvos sodininkystės ir darininkystėinstitutas, biomedicinos mokslai, agronomija 06 B,s augalininkystė, sodininkystė, augalųapsauga, fitopatologija B 390). Disertacija ginama Lietuvos emėsūkio universiteto Agronomijos mokslo krypties taryboje: Pirmininkas prof. habil. dr. Algirdas SLIESARAVIČIUS (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija 06 B, augalininkystė, sodininkystė, augalųapsauga, fitopatologija B 390). Nariai: doc. dr. Bronislovas GELVONAUSKIS (Augalų genų bankas, biomedicinos mokslai, agronomija 06 B, augalininkystė, sodininkystė, augalųapsauga, fitopatologija B 390); dr. Giedrė DABKEVIČIENĖ (Lietuvos emdirbystės institutas, biomedicinos mokslai, agronomija 06 B, augalųgenetika B 225); dr. Tadeuas IKNIANAS (Lietuvos sodininkystės ir darininkystėinstitutas, biomedicinos mokslai, agronomija 06 B,s augalininkystė, sodininkystė, augalųapsauga, fitopatologija B 390); dr. Rytis RUGIENIUS (Lietuvos sodininkystės ir darininkystės institutas, biomedicinos mokslai, biologija 01 B, induočiųaugalųfiziologija B 310). Oficialūs oponentai: prof. habil. dr. Pavelas DUCHOVSKIS (Lietuvos sodininkystės ir darininkystės institutas, biomedicinos mokslai, agronomija 06 B, augalininkystė, sodininkystė, augalųapsauga, fitopatologija B 390); dr. Laimutė BALČIŪNIENĖ (Vilniaus Universitetas, biomedicinos mokslai, biologija 01 B, augalininkystė, sodininkystė, augalų apsauga, fitopatologija B 390). Disertacija bus ginama vieame Agronomijos mokslo krypties tarybos posėdyje, kuris vyks 2006 m. vasario 15 d., 11 val. Lietuvos emėsūkio universiteto centriniųrūmų322 auditorijoje. Adresas: Studentųg. 11, LT-53361, Akademija, Kauno raj., Lietuva Disertacijos santrauka isiuntinėta 2006 m. sausio 15 d. Disertaciją peri galimaūrėti Lietuvos sodininkystės ir darininkystės instituto ir Lietuvos emėsūkio universiteto bibliotekose.
5 INTRODUCTION Edible onion is one among six vegetable species, which has the biggest economic importance in Lithuania. Growing area of edible onion is 2,6 Thou. ha (2003 2004 data). Edible onions have high nutritional and dietary properties and are used for preparation of different courses. Edible onions are valuable raw material in food processing industry (pickling, freezing, drying). Biochemical combinations of edible onion are distinguished for their therapeutically properties. They are valuable for prevention of catching a cold, heart and blood - vessels diseases and cancer. Edible onion is a diploidic species with the main chromosome number x=8 (HAVEY, 1993). Edible onions are biennial, cross-pollination plants. Breeding of new onion varieties is a long term complicated process. Breeding of varieties population is based on the individual selection. Variation of morphological and economical traits is characteristic for varieties created using the above mentioned method (DORUCHOWSKI, 1993; BREWSTER, 1994). While heterotic hybrids are distinguished according to uniformity of mentioned traits. Lines with cytoplasmatic male sterility are used for they creation. Breeding of heterotic hybrids is time and material expenditure consuming. Breeding of edible onion can be speeding up by using methods of haploids for creation of homozygous lines. The only gynogenesis is a successful for edible onion haploid obtaining. Optimal methodical suggestions, which would enable to grow high amounts of gynogenic embryos are still not available. There is no enough data about poliploidisation methods of edible onions haploid plants. Aim of investigation. investigate the peculiarities of edible onions To (Allium cepaL.) gynogenesis. To evaluate conditions for creation of dihaploid plants. To determine biological assumptions for preparation of methodical suggestions, that would enable improving of gynogenesis. Goals of investigation 1. To investigate the dependence of edible onion gynogenesis from plant donor genotype. 2. the influence of explant for formation ofTo evaluate morphostructures in unfertilized flower and ovary culture. 3. To determine the influence of plant donors and explant growing conditions on frequency of gynogenesis. 4. To evaluate the morphogenetic influence of exogenous growth regulators on isolated unfertilized flower culture. 5. To determine the ploidy level, homozygosity and morphobiological traits of regenerants. 6. To optimize the conditions of edible onion poliploidisation.
6 7. prepare the method of edible onion diploid microvegetativeTo propagation and to determine its application for haploid propagation. Innovation of investigation results.Stimulation effect of using TDZ and NAR in media on the formation of edible onion embryogenic tissue has been revealed for the first time. Embryogenic cells in tissue subjected to their origin capable to form gynogenic embryos or plant during gemmagenesis. This enables increasing of haploid output significantly. Characteristic higher frequency of gynogenesis of unfertilized flower of edible onion isolated from the flower stems soaked in 2,4-D solution has been evaluated. By using experimental method it has been established that 3 day length gynogenesis induction period, using media containing 2,4-D and BA, is enough to induce gynogenesis of edible onion. High heterogeneity of edible onion variety population Lietuvos didieji has been demonstrated from the point of organogenesis in isolated unfertilized flower culture. Practical importance of investigation.It has been established that the efficiency of edible onion gynogenesis can be increase by decreasing plant donor growing temperature in the final phases of flower development and using of exogenous growth regulators TDZ and NAA in plant regeneration media as well as by flower stems soaking in 2,4-D solution for 14 days. Plants with characteristic high gynogenesis frequency have been determined according evaluation of edible onion variety population Lietuvos didieji organogenetic response. 10 homozygous lines of edible onion have been created. 2 lines with valuable agronomic traits have been determined. The lines will be used in breeding subsequent work. Approbation of the work.The main results of the dissertation work were presented at: international scientific conference Molecular biology in agriculturally important organisms, Kaunas, Lithuania 2000; international scientific conference Current problems and developments in doubled haploid production, Budapest, Hungary, 2002; international scientific conference Biotechnology Approaches for Exploitation and Preservation of Plant Resources, Yalta, Ukraine, 2002; international Baltic States scientific conference Plant tissue culture: from theory to practice, Salaspils, Latvia, 2004; international scientific conference Accelerating Technology Transfer in East European Onion Production, Skierniewice, Poland; international scientific conference Quality of vegetables. Theoretical and practical problems, Babtai, Lithuania, 2005.
7 Publications. The main results of the dissertation work are presented in 1 publication approved by the Information Scientific Institute (ISI), 2 publications, approved by the Lithuanian Scientific Publications List of the Department of Science and Higher Education, 3 foreign publications and 4 conference proceedings. Dissertation content and volume.The doctoral dissertation is written in Lithuanian. The volume of the dissertation is 100 pages. It consists of introduction, literature review, description of investigation conditions and methods, discussion on investigation results, summing-up, conclusions, list of scientific publications and references. 21 tables and 14 pictures are included in the dissertation. References include 132 sources. THE OBJECT, CONDITIONS AND METHODS OF INVESTIGATION Investigation of gynogenesis of edible onion (Allium cepaL.) and creation of homozygous lines were carried out at the laboratory of biotechnology, phytotron complex chamber and greenhouse, experimental vegetables field of the Lithuanian Institute of Horticulture (Babtai) during the period 2001 2005. Object of investigations.Gynogenesis of 10 edible onion varieties and 7 hybrids was investigated. 2431 unfertilized ovaries and 31873 unfertilized flower were isolated. Organogenesis response has been determined, by counting the following parameters: - total frequency of explants regeneration (%) = total amount of gynogenic embryos and plants regenerated during gemagenesis (unit) x 100 / amount of isolated flower (units); - frequency of explants regenerated gynogenic embryos (%) = amount of gynogenic embryos (unit) x 100 / amount of isolated flower (units); - frequency of explants regenerated plants during gemmagenesis (%) = amount of plants regenerated during gemmagenesis (unit) x 100 / amount of isolated flower (units); - frequency of callus formation (%) = amount of callus (unit) x 100 / amount of isolated flower (units). Influence of genotype on the gynogenesis frequency of edible onion. 11986 unfertilized flowers from 9 varieties and 4094 from 4 hybrids of edible onion were isolated. Growing conditions of plant donor, isolation of explants and induction of gynogenesis were conducted according to the standard procedure of gynogenesis induction (MUREN, 1989; MARTINEZ al., 1994; et BOHANEC, JAKE, 1999; PUDDEPHAT et al., 1999; MICHALIK al., 2000). et Flower buds were sterilized according to the following scheme: ethanol (70%)
8 (rinsed)→calcium hypochlorite (10%) (7 min.)→sterile water (rinsed 3 times). Explants were planted on induction B5 media, supplemented with 2mg.l-12,4-D and 2mg.l-1BA and 100g.l-130 days later flowers were transferred on the. regeneration media, that includes MS media, supplemented with 1mg.l-1NAA, 2mg.l-12iP and 100g.l-1sucrose. Regenerated plants were multiplied on hormone free BDS media, supplemented with 100g.l-1 (D sucroseUNSTAN, SHORT, 1977). Explants and regenerants were growing in the plant growing room at the +23oC temperature, 16h photoperiod and illumination of 50μmolm-2 s-1. Influence of explants.2431 unfertilized ovaries and 1681 flowers were planted on B5 media. Growing conditions of plant donor, isolation of explants, plant regeneration and multiplication were conducted according to the standard procedure of gynogenesis induction. Influence of plant donor growing conditions.4 different temperature regimes were investigated: plants donors were grown in greenhouse (at the +22+5oC temperature, 14 - 16 h photoperiod (1); plants donors were grown according to the standard procedure of gynogenesis induction (2); plants donors were grown in the open field under uncontrolled conditions (3) and plants donors were grown in the open field+phytotron complex chamber with +14oC temperature (4). Influence of explants growing conditions.Influence of illumination (light and dark) and temperature (+22oC, +25oC and +27oC) on gynogenesis induction and plant regeneration in unfertilized flower culture were investigated. Influence of growth regulators. Effect of TDZ and NAA concentration. Gynogenesis induction and plant regeneration were conducted according to the scheme that included 20 treatments. Gynogenesis induction - growing of explants in B5 media, containing 2,4-D and BA, for 3, 6, 12 and 30 days. Later explants were transferred on the regeneration media containing MS media, supplemented with 5 mg.l-1TDZ and 0, 0, 0,05, 0,5 and 5,0 mg.l-1NAA and 100 g.l-1sucrose. After growing for 3, 6, 12 and 30 days on B5 induction media explants were planted on MS media, supplemented with 1 mg.l-1NAA, 2 mg.l-1 2iP and 100 g.l-1sucrose in the control treatment. Effect of treatment with 2,4-D solution. 4 different treatments with 2,4-D solution flower stems and flowers of edible onions were investigated. The standard procedure of gynogenesis induction was applied as control (1). Flower stems were cut down to 50 cm and soaked into 2,4-D solution (50 mg.l-1) for 14 days (2). Flower buds were soaked into 2,4-D solution (50 mg.l-1) for 10 s. (3). Flower stems were cut down to 50 cm and soaked into 2,4-D (50 mg.l-1)+AgNO3(100 mg.l-1) solution for 14 days (4). Regenerants ploidy level determination methods.Chromosome number was determined using Ploidy Analyzer Partec CA (Germany)
9 (BOHANEC, JAKE, 1999) and by cytological evaluation of root tip cells using carmine staining method (STANIENĖ, 1996). Determination of optimal poliploidisation method. gationtiIvnse included 2 experiments. 4 different concentration (0, 5,0, 10,0 15,0μM) of oryzalin were used for poliploidisation of edible onion diploid regenerants in the first experiment. Explants were treated with oryzalin solution for 48 hours and later stored at plant growing room at +23oC temperature, 16 h photoperiod and illumination of 50μmolm-2 s-1 . In the second experiment the survival and poliploidisation ability of edible onion haploids were investigated. 41 explants of haploids were treated with oryzalin at concentration of 10,0μM. Evaluation of morphological traits of edible onion bulbs. Morphology of bulbs was evaluated during harvesting. The diameter and height of 43 plant donor and 261 diploid regenerants were measured and weight of each determined. DNA isolation and RAPD analysis.DNA was isolated from young leaf tissue of edible onion according to Doyle and Doyle (1990). RAPD analysis ware carried out according to Bohanec and Jake (1999). The molecular sizes of the amplification products were estimated using synthesized mass primers (MBI Fermentas, Lithuania). Optimization of methods for edible onion microvegetative propagation. 4 different types of explants: stem dome; basal plate of sheaths without stem dome, basal plate of sheaths with stem dome and therminal part of sheaths were planted on MS media, supplemented with 1 mg.l-1NAA (in all treatments), different amounts of BA (0,9; 4,4; 8,9 and 13,1μM) and kinetin (1,1; 5,3; 10,6 and 15,8μM) and 30 g.l-1sucrose. Explants were grown in plant growing room at +23oC temperature, 16 h photoperiod and illumination of m -1 50μmol-2 s. Explants of edible onion haploid were planted on MS media, supplemented with 1 mg.l-1NAA, 10,6μM kinetin and 30 g.l-1sucrose. Statistical assessment of experimental data.Reliability of results was evaluated according to dispersal analysis method and data was grouped according Duncans multiple range test. Experimental data was evaluated by computer programmeANOVAforEXCELvers.2.1. andSTAT_ENGforEXCEL vers.1.55 (TARAKANOVAS, 1996). Scatter plot of organogenesis of edible onion variety Lietuvos didieji individual plant was determined according principled coordinate analysis method and computer programme SPSS. INVESTIGATION RESULTS Influence of genotype on gynogenesis frequency of edible onion. Investigation was carried out with 13 long day edible onion varieties and hybrids, originated from Lithuania, Czech, Netherlands and Poland (Table 1).
10 Visible organogenetic structures subjected to variety were formed after 80 167 days of explants isolation. Two types of organogenesis response were determined gynogenic embryos and plant during gemmagenesis regeneration. Hybrids dispersed less according to the ability to form gynogenic embryos (0,1 0,7%) and (3,0 4,0%) than varieties (0,1 1,8%). Variety Olina showed characteristic significantly highest (1,8%) gynogenesis capability in the group of varieties. Explants of the only SummitF1 significantly higher showed gynogenetic response in the group of hybrids. The output of gynogenic embryos reached 0,7%. Table 1. Induced gynogenesis frequency of European edible onion varieties and hybrids in the isolated unfertilized flower culture Total Frequency of Frequency of Number of frequency of explants, explants, Variety / hybrids explants explants regenerated regenerated plants investigated regeneration, gynogenic during %* embryos, %* gemmagenesis, %* Lietuvos didieji 3927 4,8a 0,6c 4,2a Olina 231 4,3b 1,8a 2,5d Karmen 296 1,0f 0,4cd 0,6g tutgarten Riesen 863 2,0e 0,4cd 1,6f Red Baron 1010 2,2e 0,1fg 2,1e Kristine 2277 0,7fg 0,2ef 0,5g Fiesta 419 0h 0gh 0h Zytawska 222 0h 0gh 0h Wolska 203 2,5d 0,4cd 2,1e Renate F13,2c 0,2ef 3,0c 660 Hyton F1 760 4,3b 0,3de 4,0a Spirit F1 1466 0h 0,1fg 0,1h Summit F1 3,7b 4,4b 0,7b 1258 *Means are significantly different at p≤0.05 (Duncans multiple range test), means followed by the same letter do not differ significantly The frequency of plants regenerated during gemmagenesis, the same as during gynogenesis process, dispersed to a lower degree in the group of hybrids (3,0 4,0%) compare to varieties (0,5 4,2%). The highest output of plants regenerated during gemmagenesis was obtained from the variety Lietuvos didieji and hybrid HytonF1. The ratio between frequency of explants gynogenic embryos and plants during gemmagenesis regeneration describes the adequate part of gynogenesis in a total organogenesis. Mentioned ratio was 1:8 in the group of investigated varieties and 1:4 in the group of hybrids. The above mentioned results showed that gynogenesis is a rare occurrence and the frequency of gynogenesis depends on the plant donor genotype. Therefore the necessity arises for investigation of factors influencing
11 gynogenesis and for searching of methods increasing the output of gynogenic embryos. The frequency of gynogenesis of varieties Kristine and Zytawska did not differ from the results presented by Polish reseachers (MICHALIK al., et 2000). Frequency of gynogenic embryos formation of variety Wolska reached 0,4% in our investigations. Explants of the mentioned variety did not form gynogenic embryos according to the data of Polish researchers. The contrary process was determined in the variety Fiesta. This confirms our earlier assumption, that plant growing conditions have influence on the frequency of gynogenesis. These results encouraged investigate the peculiarities among individuals in a population. Distribution of different plants of variety Lietuvos didieji showed that plants divide into two groups according to the frequency and pathway of organogenesis. The frequency of gynogenic embryos formation was typical from 0,4% to 2,5% in the first group (Fig.1). 2 1,5.L21.DD.L.4 1L.D.1 L.D.9 4 0,507D..6.DL.1.DL0.LD.19L.D.15.L.DL8D.1. L.D.3 L. L.D17 -0,5L.D.5 L.D.13 -1L.D.2 -1,5.L.D81L.D.16 -2 -1,5 -1 -0,5 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 Principal coordinate axis 1 (PC1) Fig.1. Scatter plot of different plants of edible onion variety Lietuvos didieji according results of principal coordinate (PC) analysis on organogenesis frequency Plants with explants regenerated during gemmagenesis at the frequency 0,4 1,0% and individuals without regeneration ability were distributed in the second group. Plants L.D.1 and L.D.4 were distributed in PC1 scatter plot with high positive value, separately from the main groups. They showed characteristic highest gynogenesis frequency, respectively 3,6% and 5,2%. Analogically plant L.D.11 was distributed in PC2 scatter plot. The frequency of explants regenerated plants during gemmagenesis reached 8,6%. Plants were distributed separately from the main groups showing that individuals can be selected with high frequency of gynogenesis and gemmagenesis in the isolated unfertilized flower culture. This enables selection of the most valuable genotypes for scientific and practical purposes in populations of edible onion.