Sėmeninių linų tyrimai somatinių ir generatyvinių audinių kultūrose ; Investigation of linseed flax in the cultures of somatic and generative tissues
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LITHUANIAN UNIVERSITY OF AGRICULTURE Aušra Blinstrubien ė INVESTIGATION OF LINSEED FLAX IN THE CULTURES OF SOMATIC AND GENERATIVE TISSUES Summary of doctoral dissertation Biomedical sciences, agronomy (06B) Akademija, 2005 This doctoral dissertation was prepared at the Laboratory of Genetics – Biotechnology, Department of Crop Science and Animal Husbandry of the Lithuanian University of Agriculture in 1999-2005. Part of the work was carried out at the Laboratory of Plant Physiology of the Lithuanian Institute of Horticulture. Scientific supervisor: Prof. dr. habil. Algirdas Sliesaravi čius (Lithuanian University of Agriculture, biomedical sciences, agronomy – 06B). Scientific consultant: Dr. Natalija Burbulis (Lithuanian University of Agriculture, 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. Vidmantas Stanys (Lithuanian Institute of Horticulture, biomedical sciences, agronomy – 06B). Members: Prof. dr. habil. Pavelas Duchovskis (Lithuanian Institute of Horticulture, biomedical sciences, agronomy – 06B). Prof. dr. habil. Rimantas Veli čka (Lithuanian University of Agriculture, biomedical sciences, agronomy – 06B). Ass. prof. dr. Liuda Žil ėnait ė (Lithuanian University of Agriculture, biomedical sciences, agronomy – 06B). Ass. prof. dr.
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| Publié par | lithuanian_university_of_agriculture |
| Publié le | 01 janvier 2006 |
| Nombre de lectures | 39 |
| Langue | English |
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LITHUANIAN UNIVERSITY OF AGRICULTURE
Aura Blinstrubien ė
INVESTIGATION OF LINSEED FLAX IN THE CULTURES OF SOMATIC AND GENERATIVE TISSUES Summary of doctoral dissertation Biomedical sciences, agronomy (06B)
Akademija, 2005
This doctoral dissertation was prepared at the Laboratory of Genetics Biotechnology, Department of Crop Science and Animal Husbandry of the Lithuanian University of Agriculture in 1999-2005. Part of the work was carried out at the Laboratory of Plant Physiology of the Lithuanian Institute of Horticulture. Scientific supervisor: Prof. dr. habil. Algirdas Sliesaravi č ius (Lithuanian University of Agriculture, biomedical sciences, agronomy 06B). Scientific consultant: Dr. Natalija Burbulis (Lithuanian University of Agriculture, 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. Vidmantas Stanys (Lithuanian Institute of Horticulture, biomedical sciences, agronomy 06B). Members: Prof. dr. habil. Pavelas Duchovskis (Lithuanian Institute of Horticulture, biomedical sciences, agronomy 06B). Prof. dr. habil. Rimantas Veli č ka (Lithuanian University of Agriculture, biomedical sciences, agronomy 06B). Ass. prof. dr. Liuda il ė nait ė (Lithuanian University of Agriculture, biomedical sciences, agronomy 06B). Ass. prof. dr. Sigut ė Kuusien ė (Lithuanian Institute of Forestry, biomedical sciences, biology 01B). Opponents: Prof. dr. habil. Eugenija Kup č inskien ė (Lithuanian University of Agriculture, biomedical sciences, biology 01B). Dr. Aura Brazaityt ė (Lithuanian Institute of Horticulture, biomedical sciences, agronomy 06B). Defense of the doctoral dissertation will take place during the public meeting of the Council of Agronomy Science on the 29 th of December 2005, at 11 a.m. in room No. 322, central building of the Lithuanian University of Agriculture. Address: Lithuanian University of Agriculture Studentu 11, LT-53361 Akademija, Kaunas district, 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 Ė S Ū KIO UNIVERSITETAS Aura Blinstrubien ė S Ė MENINI Ų LIN Ų TYRIMAI SOMATINI Ų IR GENERATYVINI Ų AUDINI Ų KULT Ū ROSE
Daktaro disertacijos santrauka Biomedicinos mokslai, agronomija (06B)
kAad meji,a 2005 
Disertacija rengta 1999-2005 metais Lietuvos em ė s ū kio universiteto Augalininkyst ė s ir gyvulininkyst ė s katedros Genetikos ir biotechnologijos laboratorijoje. Dalis tyrim ų atlikta Lietuvos sodininkyst ė s ir darininkyst ė s instituto Augal ų fiziologijos laboratorijoje. Mokslinis vadovas: Prof. habil. dr. Algirdas Sliesaravi č ius (Lietuvos em ė s ū kio universitetas, biomedicinos mokslai, agronomija 06B). Mokslinis konsultantas: Dr. Natalija Burbulis (Lietuvos em ė s ū kio universitetas, biomedicinos mokslai, agronomija 06B). Disertacija ginama Lietuvos em ė s ū kio universiteto Agronomijos mokslo krypties taryboje: Pirmininkas: Prof. habil. dr. Vidmantas Stanys (Lietuvos sodininkyst ė s ir darininkyst ė s institutas, biomedicinos mokslai, agronomija 06B). Nariai: Prof. habil. dr. Pavelas Duchovskis (Lietuvos sodininkyst ė s ir darininkyst ė s institutas, biomedicinos mokslai, agronomija 06B). Prof. habil. dr. Rimantas Veli č ka (Lietuvos em ė s ū kio universitetas, biomedicinos mokslai, agronomija 06B). Doc. dr. Liuda il ė nait ė (Lietuvos em ė s ū kio universitetas, biomedicinos mokslai, agronomija 06B). Doc. dr. Sigut ė Kuusien ė (Lietuvos mik ų institutas, biomedicinos mokslai, biologija 01B). Oponentai: Prof. habil. dr. Eugenija Kup č inskien ė (Lietuvos em ė s ū kio universitetas, biomedicinos mokslai, biologija 01B). Dr. Aura Brazaityt ė (Lietuvos sodininkyst ė s ir darininkyst ė s institutas, biomedicinos mokslai, agronomija 06B). Disertacija bus ginama vieame Agronomijos mokslo krypties tarybos pos ė dyje 2005 m. gruodio m ė n. 29 d. 11 val. Lietuvos em ė s ū kio universiteto centrini ų r ū m ų 322 auditorijoje. Adresas: Lietuvos em ė s ū kio universitetas Student ų g. 11, LT-53361 Akademija, Kauno raj., Lietuva. Tel. (8-37) 752254, faks. (8-37) 397500. Disertacijos santrauka isiuntin ė ta 2005 m. lapkri č io m ė n. 28 d. Disertacij ą galima peri ū r ė ti Lietuvos em ė s ū kio universiteto ir Lietuvos emdirbyst ė s instituto bibliotekose.
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INTRODUCTION Flax ( Linum usitatissimum L.) is one of the oldest cultivated plants in the temperate regions (Kaul et al., 1994). Linseed flax cultures cover 3.5-4.0 million ha in the world. The largest areas it occupies in India, Canada, China, USA, Germany, Argentina, Great Britain. Linseed flax is an important oil plant. The seeds of linseed flax contain 25-45% of fat used in medicine, cosmetics, food, chemistry and technical industry, as well as up to 30% of proteins. Linseed cake and coarses, containing about 30-34% of proteins, 5-8% of fat, 9-12% of fibre as well as various mineral materials and microelements, ferments and vitamins, are used as cattle fodder. Linseed flax stalks, depending on the variety, contain about 12-18% of fibre which is used to produce packages, strings, paper (Kutuzova et al., 1999). The composition of fat acids in extracted seed oil, depending on the genetic properties of variety, growing and meteorological conditions, slightly fluctuates: stearic (C 18:0 ) 3-5%, palmitic (C 16:0 ) 5.0-7.2%, oleic (C 18:1 ) 3-4%, linoleic (C 18:2 ) 14-17%, linolenic (C 18:3 ) 50-60%. The main structural part of flax oil, i.e. linolenic acid is extremely important, because it cannot be synthesized in an organism, it must be obtained with food. Among all the fat acids in flax oil, linolenic acid is the most unstable, as it is biologically extremely active, which determines application possibilities of this oil. Oils containing much linolenic acid (more than 3-5%) get spoiled very fast and become bitter (Froment et al., 1998). Breeding of linseed flax is practiced in many countries of the world: India, Hungary, Uzbekia, Argentina, Russia, Canada, Romania, France, Finland, Chekia, Ukraine. Studies on the varieties of linseed flax were started in 1998 in Upyte Experimental Station of the Lithuanian Institute of Agriculture. Breeding of flax even nowadays is a long and complicated process, based on interspecific hybridization and selection of the best plants, therefore the development of genetically stable lines takes a very long time 10-12 years. Only advanced selection technologies can help to develop new varieties, valuable for the local industry, which could open a new market for this culture. Developing genetic diversity of the initial genetic material of plants, the role of biotechnology methods in plant selection is constantly increasing. Tissue culture technologies in flax selection may be used seeking to provide varieties with new and useful characteristics (resistance to diseases, improved oil quality and tolerance to herbicides) through somatic hybridization and somaclonal variation. One of the most perspective biotechnological methods, used in plant selection is the culture of anthers, which became possible only having revealed the phenomenon of androgenesis. The system of haploid development is used both in fundamental genetic studies and practical plant selection programs. The possibilities to use cell technologies in many objects remained unrealised due to the lack of fundamental knowledge inducing the cultures of isolated tissues and cells of individual plant varieties as well as regenerating plants (Stanys, 1997). It is important to study biological requirements for in vitro system of plants and to select conditions ensuring a sufficient output of plant regenerants of desirable genotypes. Study aim is to study endogenic and exogenic factors preconditioning morphogenesis of linseed flax in vitro. To achieve the aim, the following objectives were raised: • to study the influence of the genotype, explant and nutrient medium on the dedifferentiation in the culture of somatic tissues; to determine the influence of genotype, explant type and nutrient medium on organogenesis • in the culture of somatic tissues; • to investigate the influence of genetic origin and growth conditions of the donor plant on androgenesis process in vitro ; • to define the influence of nutrient medium on androgenesis process in vitro ; • to study the influence of the genotype and nutrient medium on gynogenesis in vitro . Novelty. For the first time, optimal conditions for the dedifferentiation of linseed flax stem segments cells and those of secondary differentiation in vitro were ascertained. Light technology of a solid body was used for investigation and an optimal combination of light parameters has been selected, promoting photomorphogenesis in vitro . Factors determining gynogenesis process in vitro were evaluated and potential gynogenic genotypes for the development of initial selection material were selected. For the first time in Lithuania, optimal conditions for the dedifferentiation of hypocotyls cells and those of secondary
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differentiation in vitro were ascertained. Factors determining androgenesis process in vitro were evaluated and potential androgenic genotypes for the development of initial selection material were selected. Practical value. Cultivation of linseed flax cells and tissues as well as plant regeneration in vitro may be used as the source of genetic variation developing initial breeding material. Combinations of light parameters ensuring optimal activity of photomorphogenesis and allowing to manage photophysiological processes in vitro in order to increase the yield of regenerants has been selected. Approval of the research work. The main statements and findings of the research work were presented and discussed at the international scientific conferences: Evaluation of productivity, economic and agricultural value of fibre and oil flax cultivars grown in Europe (Poznane (Lenkija), 2003 m.); Sustainable agriculture and plant breeding in Baltic states (Kaunas, 2003m.); Plant tissue culture: from theory to practice (Salaspils (Latvia), 2004 m.); Growth and development of plants. Theoretical and practical problems (Babtai, 2004m.). The main research findings have been published in 6 Lithuanian and foreign reviewable periodicals scientific articles, one of them in a publication included into the list of the Institute of Scientific Information (ISI) and in 4 conferences reports. Volume and structure of the work. The dissertation is written in Lithuanian. It consists of six main parts: introduction, research overview, methodology of the work, results of the work, conclusions, references. The dissertation comprises 104 pages, including 12 tables, 34 figures and 331 literature references. MATERIALS AND METHODS The value of linseed flax for breeding has been estimated in the Experimental Station of Lithuanian University of Agriculture in 2000-2002. Linseed flax studies in vitro were carried out at the Laboratory of Genetic Biotechnological, Department of Crop Science and Animal Husbandry of Lithuanian University of Agriculture in 2002-2005. Linseed flax photomorphogenetic processes were investigated at the Laboratory of Plant Physiology of the Lithuanian Institute of Horticulture in 2004-2005. Morphogenesis investigation of linseed flax in the culture of somatic tissues Having ascertained the value of linseed flax for breeding, investigation of somatic tissues in vitro was carried out with three linseed varieties Lirina, Barbara, Szaphir . Donor plants were grown in the growth chamber at light intensity of at least 4000 lx with a 16/8h (day/night) photoperiod, temperature 22/18°C (day/night) and 75% humidity. Plants were sown at a depth of 1 cm, 30 plants per pot (about 5 kg of soil in pots). All plants were grown in a mixture of compost and peat with a 2:1 ratio, pH 5.8-6.5. The plants were watered and fertilized with 1.15 g/pot NH 4 NO 3 , 0.96 g/pot KH 2 PO 4 , 0.43 g/pot K 2 SO 4 , 0.37 g/pot KCl. No means against diseases and pests were used. To study linseed flax morphogenesis of somatic tissues leaves, stem segments and hypocotyls were used as explants. Explants collected from donor plants grown under controlled conditions were externally sterilized in 70% hydrous ethanol solution for 1min., 2% natrium hypochloride 5 min., afterwards 3 times washed with sterile distilled water. Sterilization of explants and transfer of the culture was carried out under aseptic conditions. Sterile somatic explants were cultivated in test-tubes, containing 4 ml of nutrient medium, supplemented with sucrose 30000 mg l -1 and Difco-Bacto agar 6000 mg l -1 . The media pH 5.7±0.1. Isolated tissues and cells in vitro were grown in the cultivation chamber with light intensity 5000 lx, photoperiod 16/8h (day/night), temperature 25 ± 2 o C. Selection of organogenic explant types. The investigation was carried out with three linseed flax explant types (leaves, stem segments and hypocotyls) on basal MS medium (Murashige, Skoog, 1962) differing in the levels of growth regulators: without growth regulators; 0.5 mg l -1 BAP (6-benzylaminopurine) + 3.0 mg l -1 2,4D (2.4-dichlorphenoxyacetic acid); 1.0 mg l -1 BAP + 0.05 mg l -1 NAA ( α - naphtylacetic acid); 5.0 mg l -1 BAP + 0.5 mg l -1 NAA. In the experiment, at 50 explants of each variant were grown, the study was done with 4 replications. Every four weeks explants were transferred on fresh nutrient medium of the same composition. Optimization of the nutrient medium composition. Estimation of basal media MS, B 5 and combined medium MSB 5 . Two basal media MS (Murashige, Skoog, 1962), B 5 (Gamborg, 1975) and combined medium MSB 5 (MS macro salts and B 5 micro salts with vitamins) with different levels of growth regulators: 1.0 mg l -1 kinetin (6-furfurylaminopurine) + 0.1 mg l -
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1 IAA (indole-3-acetic acid); 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; 2.0 mg l -1 2iP(6-(y-y-dimethylallylamine)purine); 1.0 mg l -1 kinetin were investigated using stem segments and hypocotyls. Estimation of macro and micro salts concentrations in the nutrient medium. A combined medium MSB 5 with different macro and micro salts concentrations (1.0MSB 5 ; 0.75MSB 5 ; 0.5MSB 5 ) and with different levels of growth regulators: 1.0 mg l -1 kinetin + 0.1 mg l -1 IAA; 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; 2.0 mg l -1 2iP; 1.0 mg l -1 kinetin was investigated using stem segments and hypocotyls. In the experiment, at 50 explants of each variant were grown, the study was done with 4 replications. Every four weeks explants were transferred on fresh nutrient medium of the same composition. Selection of light flow for morphogenesis in vitro. Under phytotron conditions the influence of light flow generated by four spectrum components of solid body on linseed flax morphogenesis in vitro was studied. Lighting spectrum was composed using powerful light diodes, radiating at 447, 638, 669 and 731 nm wavelengths (Table 1). Table 1. Density of photons flow, μ mol m -2 s -1 Flow of illum Light flow % 447 638 inators μ 6m6o9l m -2 s -1 of different 7w3a1v elength ( λ nm) total: 100 15 113.0 18.20 3.60 149.80 80 12 90.4 14.56 2.88 119.84 60 9 67.8 10.92 2.16 89.88 40 6 45.2 7.28 1.44 59.92 20 3 22.6 3.64 0.72 29.96 For four weeks stem explants of linseed flax Szaphir were grown in a combined medium MSB 5 with cytokinin and auxin combination 1.0 mg l -1 BAP + 0.05 mg l -1 NAA. In the experiment, at 15 explants of each variant were grown. Morphogenic potential of somatic tissue was estimated analyzing morphological parameters formed in the explant frequency of callus formation (%), frequency of the formation of adventitious shoots (%). In the cultivated linseed flax tissues, concentrations of endogenic phytohormones, i.e. gibberelin acid (GA 3 ), abscisic acid (ABA), IAA and zeatin (µg/g) were ascertained using liquid chromatography method (HPLC) with diode matrix detector. For phytohormone extraction, 1-2 g of the studied plants were taken. The samples were mashed using liquid nitrogen, extracted for a day with 100% izopropanol at 4 ° C temperature, centrifuged (2500 speed/min, 5min, 4 ° C), while the extract was vaporized using rotation evaporator (B UCHI 205). Phytohormones were depurated using NH 2 extraction cartridges, and evaporated again to water stage. For GA 3 , ABA, IAA and zeatin distribution, Adsorbosil C 18 , 125 x 4,6 mm, 5 μ m column with ante-column were used. Into the system at 20 μ L of the sample were injected, delution was carried out using gradient: up to 6 min. 39% methanol with 1% vinegar acid, from 6 to 12 min. methanol concentrations increased up to 50%. For GA 3 and ABA detection 254 nm, while for IAA and zeatin 275 nm wavelength was used. Selection of the nutrient medium for shoot rooting. To investigate the influence of nutrient media components on root formation the regenerated 1.5-3.0 cm long shoots were transferred onto MS medium, differing in the levels of macro and micro salts: 1.0 MS macro + 1.0 MS micro; 0.5 MS macro + 1.0 MS micro; 0.5 MS macro + 0.5 MS micro; 0.25 MS macro + 0.25 MS micro. To investigate the influence of exogenous auxins on rhizogenesis in vitro, the regenerated 1.5-3.0 cm long shoots were transferred onto 0.5MS (0.5 macro salts + 0.5 micro salts) medium supplemented with growth regulators: 0.02 mg 1 -1 2,4 D; 0.1 mg 1 -1 NAA; 0.2 mg 1 -1 NAA; without growth regulators. In the experiment, at 50 explants of each variant were grown, the study was done with 4 replications. In all experiments the morphogenic somatic tissue potency was evaluated by analyzing morphological parameters of the structures formed in the explant. The evaluation was based on the frequency of callus, callus mass (mg), shoots formation (%), frequency of shoots rooting (%), frequency of callus and adventitious shoots formation, on the shoots cultivated for rooting (%). Morphogenetic studies of linseed flax in the cultures of generative tissues For the studies of linseed flax generative organs in vitro, the following varieties were chosen: Lirina, Barbara, Mikael, Szaphir, Atalante as well as developed by the selectionist dr. K. Ba č elis in Upyt ė Experimental Station of the Lithuanian Institute of Agriculture F 1 hybrid plants: hybrid No.30 Barbara x Lirina; hybrid No.23 Lirina x Barbara; hybrid No.31 Barbara x Mikael; hybrid
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No.02 Mikael x Barbara. The plants were grown under the same conditions as for the studies of somatic tissues. For the studies of linseed flax morphogenesis in the culture of generative cells, anthers and ovaries were used. Buds taken from donor plants grown under controlled conditions were externally sterilized in 70% hydrous ethanol solution for 1min., 2% natrium hypochloride 10 min., afterwards 3 times washed with sterile distilled water. Sterilization of explants and transfer of the culture was carried out under aseptic conditions. Sterile explants were cultivated in plastic Petri dishes (at 10 pcs./dish of generative explants), containing 3 ml of medium, supplemented with 60000 mg l -1 sucrose and solidified with Difco-Bacto agar 6000 mg l -1 . The media pH 5.7± 0.1. Isolated tissues and cells in vitro are grown in the dark cultivation chamber for 28 days, then they are transferred into light and are cultivated under controlled conditions: lighting 5000 lx, photoperiod 16 h., environmental temperature 25 ± 2 o C. Evaluation of factors determining the efficiency of androgenesis in anthers culture Determination of the stage of microspores development. From each donor plant at 10 buds of varying size are taken, which then are kept for 24 hours in the fixer (ethyl alcohol:acetic acid (3:1)). Having measured the length of bud (mm), anthers are extracted with a pincette and staned with 1% acetocarmine (3 min.). The anther is then transferred onto a drop of 45% acetic acid, covered with a glass, dried with filtration paper and smashed, lightly tapping with a wooden pencil. For microscope analysis, 10x and 40x objective-lenses are used. In each anther, development stage of 50 microspores nuclei is identified. The anthers are isolated from buds in which most microspores are of the late mononuclear development stage. Selection of the combination of growth regulators in the induction medium. Four different combinations of auxins and cytokinins on a modified MS basal medium were tested using genotypes Lirina, Barbara , Mikael, Szaphir, Atalante: 2.0 mg l -1 BAP + 1.0 mg l -1 NAA; 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D; 1.0 mg l -1 BAP + 2.0 mg l -1 IAA; 1.0 mg l -1 BAP + 0.05 mg l -1 NAA. Evaluating the influence of the genotype, flax varieties Lirina, Barbara, Mikael and their reciprocal hybrids No.30, No.23, No.31, No.02 were studied. Anthers were cultivated on a modified MS basal medium with the following combination of cytokinins and auxins: 2.0 mg l -1 BAP + 1.0 mg l -1 NAA; 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D. Optimization of sucrose level in the induction medium . 6%, 9% and 12% levels of sucrose in the modified MS medium containing 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D were investigated using genotypes Lirina, Barbara, Mikael, Szaphir, Atalante. Evaluating the influence of the genotype, varieties Lirina, Barbara, Mikael and their reciprocal hybrids No.30, No.23, No.31, No.02 were studied. Anthers were cultivated on a modified MS basal medium with 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D, differing in the levels of sucrose (6%, 9% and 12%). Evaluation of the productivity of anthers according to temperature during the growth of donor plants. Linseed flax cultivars Lirina, Barbara and Mikael were grown in the greenhouse (22/18°C, day/night) and in the growth chambers (18/14°C, day/night). Subsequently, anthers harvested from these plants were cultured on modified MS induction medium with three different growth regulators combinations: 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; 2.0 mg l -1 BAP + 1.0 mg l -1 NAA; 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D. Regeneration of plants from anther induced callus. In the induction media formed callus were transferred to the regeneration medium, containing MS mineral salts and vitamins supplemented with 375 mg l -1 glutamine, 1.0 mg l -1 BAP, 30000 mg l -1 sucrose and 6000 mg l -1 agar. After organogenesis induction, buds were cut and transferred to a shoot elongation medium containing MS mineral salts and vitamins supplemented with 0.01 mg l -1 BAP + 0.001 mg l -1 NAA, 30000 mg l -1 sucrose and 6000 mg l -1 agar. The percentage of callus forming shoots was calculated after each subculture. Regenerated shoots were then transferred to the rooting medium containing MS mineral salts and vitamins supplemented with 0.001 mg l -1 -1 NAA, 30000 mg l -1 sucrose and 6000 mg l agar. A complete randomized design was used for all experiments. For each treatment 120 anthers were cultured (10 anthers/Petri dish; 12 replicates/treatment) and each experiment was done in triplicate. The number of anthers producing callus was scored 28 days after the initial inoculation. The percentage of anthers with callus was calculated as the number of anthers producing calli/100 inoculated anthers. The number of callus forming shoots was first recorded 28 days following transfer to the regeneration medium and then again every 28 days after each subculture transfer onto fresh regeneration medium. Regenerated plants were evaluated by inspection of the morphological characteristics.
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Evaluation of factors determining the efficiency of gynogenesis in unfertilized ovaries culture Selection of the combination of growth regulators in the induction medium . Three different combinations of auxins and cytokinins on a modified MS basal medium were tested using genotypes Lirina, Barbara, Mikael, Szaphir, Atalante: 2.0 mg l -1 BAP + 1.0 mg l -1 NAA; 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D; 1.0 mg l -1 BAP + 0.05 mg l -1 NAA. Evaluating the influence of the genotype, flax varieties Lirina, Barbara, Mikael and their reciprocal hybrids No.30, No.23, No.31, No.02 were studied. Ovaries were cultivated on a modified MS basal medium with the following combination of cytokinins and auxins: 2.0 mg l -1 BAP + 1.0 mg l -1 NAA; 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D. Optimization of sucrose level in the induction medium . 6%, 9% and 12% levels of sucrose in the modified MS medium containing 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D were investigated using genotypes Lirina, Barbara, Mikael, Szaphir, Atalante. Evaluating the influence of the genotype, varieties Lirina, Barbara, Mikael and their reciprocal hybrids No.30, No.23, No.31, No.02 were studied. Ovaries were cultivated on a modified MS basal medium with 1.0 mg l -1 BAP + 2.0 mg l -1 2,4D, differing in the levels of sucrose (6%, 9% and 12%). Regeneration of plants from callus induced by unfertilized ovaries . In the induction media formed callus were transferred to the regeneration medium, containing MS mineral salts and vitamins supplemented with 375 mg l -1 glutamine, 1.0 mg l -1 BAP, 30000 mg l -1 sucrose and 6000 mg l -1 agar. After organogenesis induction, buds were cut and transferred to a shoot elongation medium containing MS mineral salts and vitamins supplemented with 0.01 mg l -1 BAP + 0.001 mg l -1 NAA, 30000 mg l -1 sucrose and 6000 mg l -1 agar. The percentage of callus forming shoots was calculated after each subculture. Regenerated shoots were then transferred to the rooting medium containing MS mineral salts and vitamins supplemented with 0.001 mg l -1 NAA, 30000 mg l -1 sucrose and 6000 mg l -1 agar. A complete randomized design was used for all the experiments. For each treatment, 40 ovaries were cultured (10 ovaries/Petri dish; 4 replicates/treatment) and each experiment was done in triplicate. The number of ovaries producing callus was scored at 28 days after the initial inoculation. The percentage of ovaries with callus was calculated as the number of ovaries producing calli/100 inoculated ovaries. The number of callus forming shoots was first recorded 28 days following transfer to the regeneration medium and then again every 28 days after each subculture transfer onto fresh regeneration medium. Regenerated plants were evaluated by inspection of the morphological characteristics. The data of the investigations were calculated using the computer programme STAT 1.55 from SELEKCIJA (Tarakanovas, 1999) and ANOVA for EXEL, vers. 2.1. Significant differences were calculated using the disperse analysis and grouped by Duncans criteria P ≤ 0.05. Mean value and SE for each genotype were calculated based on the number of independent replication. RESULTS AND DISCUSSION Morphogenesis of linseed flax in the culture of somatic tissues The influence of explants on callus formation and organogenesis Regenerating plants in vitro , it is extremely important to induce an easily divisible organogenic callus. However, different plant species are characterized by peculiar callus induction and organogenesis processes (Dixon, 1985; Bhaskaran, Smith, 1990; Stanys, 1997) influenced by the explant type, its genetic origin and the ratio of auxins and cytokinins in the induction medium (Bjowani, Razdan, 1990; Hanzel et al., 1985; Kuusien ė , Sliesaravicius, 1996). Scientists studying the development of linseed flax somatic tissues in vitro used cotyledons, hypocotyls, meristems and stem segments as explants. However, only hypocotyl showed a successful and efficient plant regeneration (Friedt, 1989). In these dedifferentiation and organogenesis studies various explants of linseed flax cultivars Lirina, Barbara, Szaphir were used: leaf, stem segment and hypocotyl. Analysing study data it was found, that the callus formation and organogenesis of linseed flax depends on the explant type and genotype potential to induce callus, as well as on the ratio of growth regulators in the nutrient medium. Explants of linseed flax, used to induce callus, are fragments of the organ, the tissues of which are characterized by cells of varying differentiation. These cells of varying differentiation, affected by corresponding growth regulators in the in vitro environment, had to undergo a complex dedifferentiation process, had lost characteristic to them structures and reobtained the condition of divisible cells. Cell division induced in this way forms primary callus. Depending on the genotype of linseed flax and explant
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type, differences in cell proliferation were ascertained. In most cases, the leaves of all studied genotypes were characterized by a higher potential to induce callus than isolated stem segments and hypocotyls, except isolated leaves of the genotype Barbara (in the medium with 1.0 mgl -1 BAP + 0.05mgl -1 NAA) and Lirina (in the medium without growth regulators) (Table 2). Table 2. Influence of the explant type on callus induction in MS medium Type of explant Growth regulators leaf stem segment hypocotyl g l -1 Liri- Barba- Szap- Liri- Barba- Szap- Liri- Barba- Szap-m na ra hir na ra hir na ra hir without hormones 12a 17a 20a 15a 12a 10a 10a 15a 14a 0.5 BAP+3.0 2,4D 60b 80c 100b 30b 40b 30b 25b 30b 28b 1.0 BAP+0.05 NAA 100d 60b 100b 100d 96d 98d 40d 45d 38d 5.0 BAP+0.5 NAA 75c 60b 100b 60c 50c 50c 30c 38c 34c Means are significantly different at P ≤ 0.05 (Duncans multiple range test). The obtained study results confirm the opinion of other scientists that the structure of induced callus depends on the explant type, its tissue potential to dedifferentiation (Bjowani, Razdan, 1983), as far as the callus of all genotypes induced from leaf tissues was greenish, grainy, that of hypocotyl was light green, solid, while that formed from stem segment was greenish, velvet and solid. The culture of tissues and cells is inseparably related to growth regulators. The intensity of induced callus growth of all genotypes depended on the ratio of auxins and cytokinins in the nutrient media. Applying 0.5 mg l -1 BAP and 3.0 mg l -1 2,4 D for callus induction, mass increment of the callus was characterized by a very slow rate. Having increased BAP concentration up to 1.0 mg l -1 and using auxin NAA 0.05 mg l -1 , the mass of callus grew intensively, however, further increase of BAP (up to 5.0 mg l -1 ) and NAA (up to 0.5 mg l -1 ) concentrations has slowed down the growth of callus. Analyzing study results it was found that the growth of callus was influenced not only by growth regulators, but also by endogenetic factors, depending on the type of explant. The callus of isolated explants of all genotypes during the first four cultivation weeks was growing more intensively than during the following cultivation period. Essential differences were ascertained between the mean initial callus mass and mean callus mass after the 1st subcultivation among genotypes and explant types used to induce callus. It was ascertained that the increment of callus mass during the 1st cultivation period depended on the initial mass of callus. Having transfered the callus of all studied genotypes into fresh nutrient medium of the same composition, the most intensive growth was observed on the callus induced by stem segments. Callus mass increment of all studied genotypes induced by stem segments was higher than that of hypocotyls by 1.1 times (genotype Szaphir), 1.2 times (genotype Lirina) and 1.3 times (genotype Barbara) respectively. Callus mass induced by leaf tissues was growing from 1.5 (Szaphir) to 2.3 times (Lirina) slower as compared to the growth of callus mass induced by stem segments. Depending on the genotype and explant type, organogenesis started on the 21-28 day after explant isolation. Organogenesis of all studied genotypes in vitro was taking place only from the callus of isolated stem segments and hypocotyls. D.R. Duncan with co-authors (1985) states that grainy callus is not characterized by regeneration capabilities, which is confirmed by our studies as well, i.e. grainy callus induced by the leaves of all studied linseed flax genotypes had no organogenic properties. Usually, a very low ratio of auxins/cytokinins is used for callus organogenesis (Cullis, Clearly, 1986; Marshall, Courduries, 1992). In these studies callus was forming adventitious shoots also under a very low ratio of auxins/cytokinins. In the nutrient medium with 0.5 mg l -1 BAP + 3.0 mg l -1 2,4 D organogenesis was not taking place, while in the medium without growth regulators only the callus induced by the hypocotyls of Barbara and Szaphir genotypes (respectively 2 and 6%) was characterized by organogenic properties. The most intensive organogenesis was in the medium with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA depending on the type of explant - inducing callus (Table 3). Table 3. Influence of the explant type on organogenesis in MS medium Type of explant Growth regulators stem segment hypocotyl mg l -1 Lirina Barbara Szaphir Lirina Barbara Szaphir 1.0 BAP+0.05 NAA 70b 95b 100b 40b 58b 65b 5.0 BAP+0.5 NAA 10a 20a 30a 8a 15a 28a Means are significantly different at P ≤ 0.05 (Duncans multiple range test). 10
A. Cunha and M.F. Ferreira (1996) suggest that stem segment is a sufficiently good source for callus induction, however, this callus was not characterized by regeneration capabilities. The results of our studies show contrary results, i.e. in the medium with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA, callus organogenesis of stem segments of all genotypes was 1.5 times (Szaphir), 1.6 (Barbara) and 1.8 (Lirina) times more intensive than that of hypocotyls. Most organogenic structures were formed from callus induced by explants of the genotype Szaphir. The most intensive callus formation and organogenesis of linseed flax callus were in the nutrient medium with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA. The current study indicates that the tissues of linseed flax hypocotyls and stem segments are competent for the organogenesis of adventitious shoots, thus, they may be used for the production of primary selection material. Influence of the nutrient medium on morphogenesis Murashige & Skoog (MS) (Murashige, Skoog, 1962) and Gamborg (B 5 ) (Gamborg, 1975) nutrient media are the most successfully and frequently used nutrient media in vitro . Depending on the plant species, nutrient media are often modified changing the composition of vitamins and growth regulators (Son, Bhojwani, 1999). Most often used growth regulators are cytokinins BAP, 2iP and kinetin (Bjowani, Razdan, 1990), auxins IAA and NAA. Influence of the nutrient medium on callus induction . Cell division is a critical factor in the regeneration process, therefore, it is differently controlled in various model systems. Primary cell division in the induction environment depending on growth regulators in the nutrient medium and on the selected explant type (Blervacq et al., 1995; Dedicova et al., 2000b). This is confirmed by the results of this study, as far as having analyzed the results it was found that to stimulate dedifferentiation and later differentiation (formation of structures from callus) of linseed flax explant, it is very important to use a proper nutrient medium (the ratio of macro, micro salts, carbohydrates, vitamins and especially growth regulator), genotype and explant type. Influence of the nutrient medium on organogenesis . Many scientists studying tissue culture point out that morphogenic processes are preconditioned by genetic and exogenic factors (Bhaskaran, Smith, 1990; Bjowani, Razdan, 1990). Problems arise with many plant species seeking to induce regeneration (Nhut et al., 2003). Our results show that nutrient medium influenced callus formation not only of linseed flax varieties Lirina, Barbara and Szaphir , but also the processes of organogenesis, depending on the genotype and explant type. The current study indicates different regeneration capabilities of linseed flax varieties. This confirms the statement (Yokoya et al., 1996) that although the out put of regenerated callus is influenced by many factors (temperature, explant, composition of nutrient medium and growth conditions), the most important role, however, is played by genetic factors. Seeking to regenerate shoots from the genotype Lirina callus induced by stem segments and hypocotyls, it is purposeful to use complex MSB 5 medium with 2.0 mg l -1 2iP. For the regeneration of callus shoots induced by the genotype Barbara stem segments, the following nutrient media may be used: MS or B 5 with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; MSB 5 with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; 2.0 mg l -1 2iP or with 1.0 mg l -1 kinetin. Seeking to obtain the regeneration of shoots from hypocotyls-derived callus, it is purposeful to use MS medium with 2.0 mg l -1 2iP; B 5 medium with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA or with 2.0 mg l -1 2iP; MSB 5 medium with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; 2.0 mg l -1 2iP or with 1.0 mg l -1 kinetin. Seeking to regenerate shoots from callus of the genotype Szaphir stem segments, it is purposeful to use MS medium with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; B 5 medium with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA or with 2.0 mg l -1 2iP; MSB 5 medium with 1.0 mg l -1 kinetin + 0.1 mg l -1 IAA; 2.0 mg l -1 2iP or with 1.0 mg l -1 kinetin. For shoot regeneration of this genotype from hypocotyls-derived callus, suitable are MS or B 5 media with 1.0 mg l -1 BAP + 0.05 mg l -1 NAA; 2.0 mg l -1 2iP; MSB 5 medium with 1.0 mg l -1 kinetin + 0.1 mg l -1 IAA; 2.0 mg l -1 2iP or with 1.0 mg l -1 kinetin. Stem segments were more intensively forming callus than hypocotyls, however, in most cases, hypocotyl-derived callus was characterized by better morphogenic capabilities than callus induced by stem segments. Regeneration of linseed flax shoots was from green, soft, smooth callus induced by hypocotyls and stem segments with and without transfer on fresh nutrient medium. Shoot regeneration from hypocotyl-derived callus, cultivated for four weeks in different media are presented in Table 4.
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