MD1090Z - Process for haploid induction in barley - Google Patents
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- MD1090Z MD1090Z MDS20160028A MDS20160028A MD1090Z MD 1090 Z MD1090 Z MD 1090Z MD S20160028 A MDS20160028 A MD S20160028A MD S20160028 A MDS20160028 A MD S20160028A MD 1090 Z MD1090 Z MD 1090Z
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- 230000006698 induction Effects 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title claims abstract description 13
- 235000007340 Hordeum vulgare Nutrition 0.000 title claims abstract description 12
- 240000005979 Hordeum vulgare Species 0.000 title claims abstract 3
- 235000015097 nutrients Nutrition 0.000 claims abstract description 17
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 6
- 239000001110 calcium chloride Substances 0.000 claims abstract description 6
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- 229920000936 Agarose Polymers 0.000 claims abstract description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 5
- 229930195725 Mannitol Natural products 0.000 claims abstract description 5
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- 241000983759 Linaria genistifolia Species 0.000 claims abstract description 3
- 229930182470 glycoside Natural products 0.000 claims description 18
- 150000002338 glycosides Chemical class 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 4
- 238000009835 boiling Methods 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- 238000013375 chromatographic separation Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 3
- 210000005069 ears Anatomy 0.000 abstract description 2
- 238000011081 inoculation Methods 0.000 abstract description 2
- 229930182489 iridoid glycoside Natural products 0.000 abstract description 2
- 150000008145 iridoid glycosides Chemical class 0.000 abstract description 2
- 229920001917 Ficoll Polymers 0.000 abstract 2
- 238000012423 maintenance Methods 0.000 abstract 2
- 238000011534 incubation Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 230000000408 embryogenic effect Effects 0.000 description 11
- 241000209219 Hordeum Species 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 5
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- 241000196324 Embryophyta Species 0.000 description 4
- 230000013020 embryo development Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000167853 Linaria Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 101710199392 TATA-box-binding protein 1 Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
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- 230000001548 androgenic effect Effects 0.000 description 1
- 239000012223 aqueous fraction Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
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- 235000013339 cereals Nutrition 0.000 description 1
- 229910052927 chalcanthite Inorganic materials 0.000 description 1
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- 230000002380 cytological effect Effects 0.000 description 1
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- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
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- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
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- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
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- 235000009529 zinc sulphate Nutrition 0.000 description 1
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Abstract
Description
Invenţia se referă la biotehnologie, şi anume la un procedeu de inducere a haploizilor la orz. The invention relates to biotechnology, namely to a process for inducing haploids in barley.
Una din cele mai aplicabile metode de inducere a haploizilor la orz este cultura in vitro a anterelor ca urmare a inducerii embriogenezei microsporale. Această cale este de perspectivă, fiind utilizată pe larg în crearea a numeroase linii de cereale, dar reuşita acestei tehnici este influenţată în primul rând de factorii genetici, care determină frecvenţa formării calusurilor şi embrionilor. Deosebit de important în obţinerea răspunsului pozitiv în cultura anterelor este pretratamentul spicelor/anterelor şi componenţa mediilor nutritive ce conduc microsporii spre formarea de structuri embriogene. One of the most applicable methods for inducing haploids in barley is the in vitro culture of anthers as a result of the induction of microsporal embryogenesis. This route is promising, being widely used in the creation of numerous cereal lines, but the success of this technique is influenced primarily by genetic factors, which determine the frequency of callus and embryo formation. Particularly important in obtaining a positive response in anther culture is the pretreatment of the spikes/anthers and the composition of the nutrient media that lead the microspores to the formation of embryogenic structures.
Este cunoscut procedeul bazat pe prelevarea spicelor la stadiul de microspori uninucleaţi (ce corespunde fazei de burduf), sterilizarea şi plasarea în cutii Petri cu puţină apă distilată sterilă pentru 3 zile în frigider la 4°C, excizarea anterelor, inocularea lor pe mediul nutritiv ce conţine manitol (0,7M), CaCl2 (40mM) şi agaroză (8g/l) şi incubarea la întuneric timp de 4 zile la 24°C, după care acestea sunt transferate în cutii Petri ce conţin mediul de inducere FHG şi plasate la întuneric la o temperatură de 24°C [1]. The procedure is known based on sampling the spikelets at the uninucleate microspores stage (corresponding to the bellows phase), sterilizing and placing them in Petri dishes with a little sterile distilled water for 3 days in a refrigerator at 4°C, excising the anthers, inoculating them on a nutrient medium containing mannitol (0.7M), CaCl2 (40mM) and agarose (8g/l) and incubating them in the dark for 4 days at 24°C, after which they are transferred to Petri dishes containing the FHG induction medium and placed in the dark at a temperature of 24°C [1].
Neajunsul procedeului cunoscut este rata redusă a anterelor ce prezintă răspuns pozitiv la condiţiile in vitro în vederea obţinerii plantelor haploide. The drawback of the known process is the low rate of anthers that respond positively to in vitro conditions in order to obtain haploid plants.
Problema pe care o rezolvă invenţia propusă constă în sporirea cotei de antere care formează structuri embriogene de natură microsporală. The problem solved by the proposed invention consists in increasing the proportion of anthers that form embryogenic structures of a microsporal nature.
Invenţia soluţionează problema prin aceea că se propune un procedeu de inducere a haploizilor la orz, care include colectarea spicelor la etapa de microsporogeneză, sterilizarea acestora cu clorură şi cu alcool etilic de 70%, plasarea în cutii Petri cu apă distilată sterilă, menţinerea la întuneric la o temperatură de 4°C în decurs de 3 zile, excizarea, sub un microscop binocular, a anterelor din spiculeţul central al părţii de mijloc a spicului, inocularea anterelor într-un mediu nutritiv ce conţine 0,7 M manitol, 40 mM CaCl2 şi 8 g/l agaroză în cutii Petri, izolarea cutiilor cu parafilm şi incubarea la întuneric la o temperatură de 24°C timp de 4 zile, transferarea anterelor pe mediul nutritiv de inducere FHG cu un conţinut de Ficol de 200 g/l şi suplimentat cu 0,0001% de glicozide iridoice, obţinute din plante de Linaria genistifolia, prin extragere cu soluţie hidrometanolică la fierbere şi separare cromatografică ulterioară, menţinerea la întuneric la o temperatură de 24°C în decurs de 12..14 zile, după care anterele cu răspuns pozitiv se transferă pe mediul FHG cu un conţinut de Ficol de 400 g/l. The invention solves the problem by proposing a process for inducing haploids in barley, which includes collecting the spikelets at the microsporogenesis stage, sterilizing them with chloride and 70% ethyl alcohol, placing them in Petri dishes with sterile distilled water, keeping them in the dark at a temperature of 4°C for 3 days, excising, under a binocular microscope, the anthers from the central spikelet of the middle part of the spikelet, inoculating the anthers in a nutrient medium containing 0.7 M mannitol, 40 mM CaCl2 and 8 g/l agarose in Petri dishes, isolating the dishes with parafilm and incubating them in the dark at a temperature of 24°C for 4 days, transferring the anthers onto the FHG induction nutrient medium with a Ficol content of 200 g/l and supplemented with 0.0001% iridoic glycosides, obtained from Linaria plants. genistifolia, by extraction with boiling hydromethanolic solution and subsequent chromatographic separation, keeping in the dark at a temperature of 24°C for 12..14 days, after which the anthers with a positive response are transferred to FHG medium with a Ficol content of 400 g/l.
Glicozidele iridoice sumare (GIS) din Linaria genistifolia s-au obţinut din material vegetal uscat (300 g partea aeriană). La materialul vegetal mărunţit s-au adăugat 1,5 l de soluţie apoasă de metanol 60%, apoi a fost supus refluxului prin fierbere timp de 6 ore, procedura fiind repetată de 3 ori. Extractele obţinute au fost combinate şi concentrate prin distilare în vid, după care reziduul apos a fost decantat cu cloroform. Fracţia apoasă a fost trecută prin coloană cu Sephadex LH-20. Coloana a fost spălată consecutiv cu apă, soluţie apoasă de metanol de 10% şi metanol pur. Ambele eluate metanolice au fost combinate şi evaporate prin distilare în vid până la uscare. Extractul uscat obţinut (4,6 g) reprezintă GIS (Mashcenco N., Gurev A., Lupascu G., Gorincioi E. Iridoid glycosides from Linaria genistifilia (L.) Mill. in biological control of soil-borne fungal pathogens of wheat and some structure consideration. Chemistry Journal of Moldova. General, Industrial and Ecological Chemistry. 2015, 10 (1), 57-63). The total iridoic glycosides (GIS) from Linaria genistifolia were obtained from dried plant material (300 g aerial part). 1.5 l of 60% aqueous methanol solution was added to the crushed plant material, then it was refluxed by boiling for 6 hours, the procedure being repeated 3 times. The extracts obtained were combined and concentrated by vacuum distillation, after which the aqueous residue was decanted with chloroform. The aqueous fraction was passed through a Sephadex LH-20 column. The column was washed consecutively with water, 10% aqueous methanol solution and pure methanol. Both methanol eluates were combined and evaporated by vacuum distillation to dryness. The obtained dry extract (4.6 g) represents GIS (Mashcenco N., Gurev A., Lupascu G., Gorincioi E. Iridoid glycosides from Linaria genistifilia (L.) Mill. in biological control of soil-borne fungal pathogens of wheat and some structure consideration. Chemistry Journal of Moldova. General, Industrial and Ecological Chemistry. 2015, 10 (1), 57-63).
Rezultatul invenţiei constă în sporirea cotei de antere care formează structuri embriogene de natură microsporală în crearea haploizilor la orz. The result of the invention consists in increasing the proportion of anthers that form embryogenic structures of a microsporal nature in the creation of haploids in barley.
Exemplu de realizare a invenţiei Example of embodiment of the invention
În cercetare au fost antrenate soiurile de orz Strălucitor, Ciuluc şi Galactic. Plantele au fost crescute în condiţii de câmp conform tehnicilor standard. Spicele au fost colectate la etapa de microsporogeneză, stadiul microsporilor uninucleaţi (ce corespunde fazei de burduf). Etapa a fost apreciată prin examinarea citologică a preparatelor temporare prin colorare cu carmină acetică. The barley varieties Strălucitor, Ciuluc and Galactic were involved in the research. The plants were grown in field conditions according to standard techniques. The ears were collected at the stage of microsporogenesis, the stage of uninucleate microspores (which corresponds to the bellows phase). The stage was assessed by cytological examination of temporary preparations by staining with acetic carmine.
Spicele identificate la stadiul de microspori uninucleaţi au fost colectate. După sterilizare cu clorură de sodiu, spicele au fost trecute prin alcool 70%, apoi plasate în cutii Petri cu puţină apă distilată sterilă pentru 3 zile în frigider la 4°C, întuneric. Pentru fiecare genotip au fost selectate câte cel puţin 5 spice. În incinta boxei cu flux laminar, sub un microscop binocular, din spiculeţul central al părţii de mijloc a spicului au fost excizate anterele şi inoculate pe mediul nutritiv ce conţine manitol (0,7M), CaCl2 (40mM) şi agaroză (8 g/l). Cutiile Petri în care au fost inoculate anterele au fost izolate cu parafilm şi incubate la 24°C, întuneric, pentru 4 zile. După 4 zile anterele au fost transferate în cutii Petri ce conţin mediul nutritiv de inducere FHG (tabelul 1) şi menţinute la 24°C, la întuneric. După 12…14 zile anterele cu răspuns pozitiv au fost transferate pe mediu FHG cu conţinut de Ficol 400g/l. Spikelets identified at the uninucleate microspores stage were collected. After sterilization with sodium chloride, the spikelets were passed through 70% alcohol, then placed in Petri dishes with a little sterile distilled water for 3 days in a refrigerator at 4°C, dark. At least 5 spikelets were selected for each genotype. In the laminar flow cabinet, under a binocular microscope, the anthers were excised from the central spikelet of the middle part of the spike and inoculated onto a nutrient medium containing mannitol (0.7M), CaCl2 (40mM) and agarose (8 g/l). The Petri dishes in which the anthers were inoculated were isolated with parafilm and incubated at 24°C, dark, for 4 days. After 4 days, the anthers were transferred to Petri dishes containing the FHG induction nutrient medium (table 1) and maintained at 24°C, dark. After 12…14 days, the anthers with a positive response were transferred to FHG medium containing 400g/l Ficol.
Tabelul 1 Table 1
Compoziţia mediului nutritiv utilizat pentru inducerea embriogenezei la orz Composition of the nutrient medium used for the induction of embryogenesis in barley
Nr. FHG de inducere a embriogenezei Componentele mediului nutritiv Cantitatea (mg/l) Macroelemente 1 KNO3 1900 2 NH4NO3 165 3 CaCl2·2H2O 440 4 MgSO4·7H2O 370 5 KH2PO4 170 6 Fe Na2EDTA 37,5 Microelemente 7 MnSO4·4H2O 22,3 8 H3BO3 6,2 9 ZnSO4·7H2O 8,6 10 KI 0,83 11 Na2MoO4·2H2O 0,25 12 CuSO4·5H2O 0,025 13 CoCl2·6H2O 0,025 Vitamine 14 Mioinozitol 100 15 Tiamină HCl 0,4 Alţi factori 16 Glutamină 730 17 BAP 1 18 Maltoză 62000 19 Ficol 200000 pH 5,8No. FHG for embryogenesis induction Components of the nutrient medium Quantity (mg/l) Macroelements 1 KNO3 1900 2 NH4NO3 165 3 CaCl2·2H2O 440 4 MgSO4·7H2O 370 5 KH2PO4 170 6 Fe Na2EDTA 37.5 Microelements 7 MnSO4·4H2O 22.3 8 H3BO3 6.2 9 ZnSO4·7H2O 8.6 10 KI 0.83 11 Na2MoO4·2H2O 0.25 12 CuSO4·5H2O 0.025 13 CoCl2·6H2O 0.025 Vitamins 14 Myoinositol 100 15 Thiamine HCl 0.4 Other factors 16 Glutamine 730 17 BAP 1 18 Maltose 62000 19 Ficol 200000 pH 5.8
Procedeul, conform invenţiei, include suplimentarea mediului nutritiv de inducere FHG cu GIS (Nr.1). Pentru stabilirea concentraţiei optime au fost utilizate două variante: 0,001% GIS şi 0,0001% GIS. De asemenea, a fost aplicată schema de inoculare fără pretratament (Nr.2), conform căreia după sterilizare anterele au fost plasate pe cele trei variante ale mediului nutritiv de inducere (FHG, FHG+0,001% GIS şi FHG+0,0001% GIS). The method, according to the invention, includes supplementing the FHG induction nutrient medium with GIS (No. 1). To establish the optimal concentration, two variants were used: 0.001% GIS and 0.0001% GIS. Also, the inoculation scheme without pretreatment (No. 2) was applied, according to which after sterilization the anthers were placed on the three variants of the induction nutrient medium (FHG, FHG+0.001% GIS and FHG+0.0001% GIS).
Conform rezultatelor obţinute (tabelul 2) s-a constatat că GIS a prezentat efect stimulator în inducerea reacţiei pozitive a anterelor pe mediul nutritiv FHG doar în concentraţia de 0,0001% (varianta FHG+0,0001% GIS). În varianta fără pretratament GIS a avut un impact pozitiv doar la soiul Ciuluc. According to the results obtained (table 2), it was found that GIS had a stimulating effect in inducing the positive reaction of anthers on the FHG nutrient medium only in the concentration of 0.0001% (FHG + 0.0001% GIS variant). In the variant without GIS pretreatment, it had a positive impact only on the Ciuluc variety.
Tabelul 2 Table 2
Cota anterelor de orz cu răspuns pozitiv la condiţiile in vitro de cultivare în dependenţă de pretratamentul aplicat (%) Share of barley anthers with positive response to in vitro cultivation conditions depending on the pretreatment applied (%)
Genotip Nr.1 Pretratament Nr.2 Fără pretratament Antere inoculate (nr.) FHG FHG+0,001% GIS FHG+0,0001% GIS FHG FHG+0,001% GIS FHG+0,0001% GIS Strălucitor 0,7 0 1,39 0 0 0 144 Ciuluc 0,36 0 0,72 1,07 0 1,61 562 Galactic 0,58 0 1,72 0,58 0 0 175Genotype No.1 Pretreatment No.2 No pretreatment Inoculated anthers (no.) FHG FHG+0.001% GIS FHG+0.0001% GIS FHG FHG+0.001% GIS FHG+0.0001% GIS Brilliant 0.7 0 1.39 0 0 0 144 Ciuluc 0.36 0 0.72 1.07 0 1.61 562 Galactic 0.58 0 1.72 0.58 0 0 175
În varianta cu pretratament (Nr.1) GIS a condus la o creştere a răspunsului pozitiv al anterelor de cca 2 ori la soiurile Ciuluc şi Strălucitor, iar la Galactic de 2,97 ori. In the pretreatment variant (No. 1), GIS led to an increase in the positive response of the anthers of about 2 times in the Ciuluc and Strălucitor varieties, and 2.97 times in Galactic.
După una-două săptămâni de recultivare in vitro a anterelor cu răspuns pozitiv a fost atestată inducerea embriogenezei. Capacitatea embriogenă a microsporilor a fost influenţată de genotip, precum şi de mediul nutritiv (tabelul 3). After one to two weeks of in vitro recultivation of the anthers with a positive response, the induction of embryogenesis was attested. The embryogenic capacity of the microspores was influenced by the genotype, as well as by the nutritional environment (table 3).
Tabelul 3 Table 3
Capacitatea embriogenă a microsporilor de orz cultivaţi in vitro conform variantei cu pretratament Embryogenic capacity of barley microspores cultivated in vitro according to the pretreatment variant
Genotip FHG FHG+0,0001% GIS Cota anterelor cu răspuns pozitiv Structuri embriogene Cota anterelor cu răspuns pozitiv Structuri embriogene Strălucitor 0,7 1 1,39 1,67 Ciuluc 0,36 2,44 0,72 3,15 Galactic 0,58 1,5 1,72 2,83Genotype FHG FHG+0.0001% GIS Share of anthers with positive response Embryogenic structures Share of anthers with positive response Embryogenic structures Brilliant 0.7 1 1.39 1.67 Ciuluc 0.36 2.44 0.72 3.15 Galactic 0.58 1.5 1.72 2.83
Aplicarea schemei cunoscute de inducere a androgenezei pe mediul FHG a generat formarea de la 1 până la 2,4 structuri embriogene per anteră în dependenţă de genotipul analizat. The application of the known scheme for inducing androgenesis on the FHG medium generated the formation of 1 to 2.4 embryogenic structures per anther depending on the genotype analyzed.
Suplimentarea mediului nutritiv FHG cu GIS a favorizat sporirea cotei de structuri embriogene la toate trei genotipuri incluse în studiu. Conform procedeului propus capacitatea embriogenă a sporit la soiul Ciuluc de cca 1,3, la soiul Strălucitor de 1,67, iar la soiul Galactic de 1,89 ori. Creşterea numărului de structuri embiogene formate ca rezultat al diferenţierii microsporilor contribuie la eficientizarea căii de obţinere a haploizilor la soiurile de orz cu capacitate androgenă redusă. Supplementing the FHG nutrient medium with GIS favored the increase in the proportion of embryogenic structures in all three genotypes included in the study. According to the proposed procedure, the embryogenic capacity increased in the Ciuluc variety by about 1.3 times, in the Strălucitor variety by 1.67 times, and in the Galactic variety by 1.89 times. The increase in the number of embryogenic structures formed as a result of microspore differentiation contributes to the efficiency of the path to obtaining haploids in barley varieties with reduced androgenic capacity.
1. Cistue L., Valles M. P., Echavarri B., Sanz J.M., Castillo A. Barley anther culure. In: Maluszynski M., Kasha K. J., Forster B.P., Szarejko I. Doubled haploid production in crop plants. A manual. Kluwer Academic Publishers, Dordrecht/Boston/London, 2003, p. 29-34 1. Cistue L., Valles M.P., Echavarri B., Sanz J.M., Castillo A. Barley anther culture. In: Maluszynski M., Kasha K. J., Forster B.P., Szarejko I. Doubled haploid production in crop plants. A manual. Kluwer Academic Publishers, Dordrecht/Boston/London, 2003, p. 29-34
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