MD1055Z - Method for determining the sex of Actinidia arguta plants cultivated in vitro - Google Patents
Method for determining the sex of Actinidia arguta plants cultivated in vitro Download PDFInfo
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- MD1055Z MD1055Z MDS20160017A MDS20160017A MD1055Z MD 1055 Z MD1055 Z MD 1055Z MD S20160017 A MDS20160017 A MD S20160017A MD S20160017 A MDS20160017 A MD S20160017A MD 1055 Z MD1055 Z MD 1055Z
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- peroxidases
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000000338 in vitro Methods 0.000 title claims abstract description 13
- 244000298800 Actinidia arguta Species 0.000 title claims abstract description 11
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 23
- 108700020962 Peroxidase Proteins 0.000 claims abstract description 21
- 238000001228 spectrum Methods 0.000 claims abstract description 17
- 241000196324 Embryophyta Species 0.000 claims abstract description 15
- 239000000284 extract Substances 0.000 claims abstract description 13
- 239000007853 buffer solution Substances 0.000 claims abstract description 7
- 238000001962 electrophoresis Methods 0.000 claims abstract description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229920002401 polyacrylamide Polymers 0.000 claims abstract description 5
- 235000015097 nutrients Nutrition 0.000 claims abstract description 4
- 238000005119 centrifugation Methods 0.000 claims abstract description 3
- 238000011081 inoculation Methods 0.000 claims abstract description 3
- 238000002803 maceration Methods 0.000 claims abstract description 3
- 238000000926 separation method Methods 0.000 claims abstract description 3
- 238000005520 cutting process Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 2
- 230000020509 sex determination Effects 0.000 claims 1
- 108040007629 peroxidase activity proteins Proteins 0.000 abstract 2
- 230000006698 induction Effects 0.000 abstract 1
- 238000010186 staining Methods 0.000 abstract 1
- 241000219068 Actinidia Species 0.000 description 5
- 235000009434 Actinidia chinensis Nutrition 0.000 description 5
- 235000009436 Actinidia deliciosa Nutrition 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 235000016416 Actinidia arguta Nutrition 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 230000001133 acceleration Effects 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 230000005305 organ development Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000005337 ground glass Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
Invenţia se referă la biotehnologie, în special la un procedeu de determinare a sexului la plantele de Actinidia arguta cultivate in vitro. The invention relates to biotechnology, in particular to a process for determining the sex of Actinidia arguta plants cultivated in vitro.
În literatura de specialitate sexul plantelor la Actinidia arguta poate fi identificat numai după morfologia florilor, adică la momentul primului an de înflorire, care are loc la 4…6 ani. In the specialized literature, the sex of Actinidia arguta plants can only be identified by flower morphology, that is, at the time of the first year of flowering, which occurs at 4...6 years.
Este cunoscut procedeul de determinare a sexului în baza spectrului electroforetic al peroxidazelor (PO) în ţesuturile calusului, lăstarilor şi rădăcinilor de Actinidia deliciosa in vitro feminine şi masculine. Pentru obţinerea extractelor enzimatice s-a folosit soluţia tampon de 0,15 M Tris-HCl, suplimentată cu 6 mM acid ascorbic, 6 mM hipoclorit de cisteină şi 0,5 M zaharoză, pH=8,3. Extractele omogenizate au fost centrifugate timp de 5 min la 12 000 g şi temperatura de -4°C. Supernatantul a fost folosit în analizele enzimatice. Spectrele PO au fost obţinute prin efectuarea electroforezei în tuburi cu gel de poliacrilamidă 7%, pH=8,9. Vizualizarea benzilor corespunzătoare ale PO s-a efectuat cu utilizarea 0,1% benzidină în 0,1 M acetat de sodiu, pH=5,5, cu adaos de 0,1% de H2O2. Însă metoda menţionată, comparativ cu metoda propusă, are unele deosebiri şi neajunsuri. The procedure for determining sex based on the electrophoretic spectrum of peroxidases (PO) in the tissues of the callus, shoots and roots of Actinidia deliciosa in vitro, both female and male, is known. To obtain the enzymatic extracts, a 0.15 M Tris-HCl buffer solution was used, supplemented with 6 mM ascorbic acid, 6 mM cysteine hypochlorite and 0.5 M sucrose, pH=8.3. The homogenized extracts were centrifuged for 5 min at 12,000 g and a temperature of -4°C. The supernatant was used in the enzymatic analyses. PO spectra were obtained by performing electrophoresis in 7% polyacrylamide gel tubes, pH=8.9. Visualization of the corresponding PO bands was performed using 0.1% benzidine in 0.1 M sodium acetate, pH=5.5, with the addition of 0.1% H2O2. However, the mentioned method, compared to the proposed method, has some differences and shortcomings.
Deosebirea dintre specii. În procedeul cunoscut s-a studiat Actinidia deliciosa, iar în procedeul propus s-a studiat Actinidia arguta. Speciile respective se deosebesc după mai multe caractere morfo-fiziologice. Difference between species. In the known procedure, Actinidia deliciosa was studied, and in the proposed procedure, Actinidia arguta was studied. The respective species differ in several morpho-physiological characters.
Fazele de dezvoltare a inoculilor cultivaţi in vitro, precum şi conţinutul mediilor de cultivare. Pentru activarea şi obţinerea ţesuturilor feminine şi masculine de Actinidia deliciosa s-a aplicat procedeul de cultivare constituit din două etape, cu o perioadă lungă de propagare, fiind folosite două tipuri de medii de cultivare. În procedeul propus, obţinerea minibutaşilor feminini şi masculini in vitro este posibilă într-o singură etapă, cu utilizarea unui singur mediu de cultivare (Brevet MD 605 Z 2013.10.31). Development phases of inoculums cultivated in vitro, as well as the content of the cultivation media. For the activation and obtaining of female and male tissues of Actinidia deliciosa, a two-stage cultivation process was applied, with a long propagation period, using two types of cultivation media. In the proposed process, obtaining female and male mini-cuttings in vitro is possible in a single stage, using a single cultivation medium (Patent MD 605 Z 2013.10.31).
Spre deosebire de spectrele electroforetice ale PO studiate la Actinidia deliciosa din celulele calusului nediferenţiat, lăstarilor (2,5…3 cm) şi rădăcinilor la etapele incipiente de dezvoltare, în procedeul propus, pentru obţinerea spectrelor electroforetice ale PO s-au folosit frunze de Actinidia arguta ale minibutaşilor feminini şi masculini, cultivaţi in vitro, cu toate organele vegetative dezvoltate. Fiecare minibutaş de Actinidia arguta avea înălţimea de aproximativ 10 cm şi 7…8 perechi de frunze, fiind la etapa finală de dezvoltare in vitro, cu transferul ulterior în condiţii ex vitro. Unlike the electrophoretic spectra of PO studied in Actinidia deliciosa from undifferentiated callus cells, shoots (2.5…3 cm) and roots at early stages of development, in the proposed procedure, to obtain the electrophoretic spectra of PO, Actinidia arguta leaves of female and male mini-cuttings, cultivated in vitro, with all vegetative organs developed, were used. Each mini-cutting of Actinidia arguta was approximately 10 cm tall and had 7…8 pairs of leaves, being at the final stage of in vitro development, with subsequent transfer to ex vitro conditions.
Spre deosebire de componenţa soluţiei tampon folosită în cea mai apropiată soluţie pentru obţinerea extractelor enzimatice, în procedeul propus a fost utilizată soluţia tampon de 0,05 M Tris HCl, fără adaosuri, cu pH=6,8. Acesta este un efort mai puţin costisitor faţă de cea mai apropiată soluţie. Unlike the composition of the buffer solution used in the closest solution for obtaining enzyme extracts, the proposed process used a 0.05 M Tris HCl buffer solution, without additives, with pH=6.8. This is a less expensive effort compared to the closest solution.
Neajunsurile procedeului cunoscut menţionat diminuează exactitatea şi productivitatea, sporesc eforturile financiare şi cele umane, depuse pentru obţinerea rezultatelor. The shortcomings of the known procedure mentioned diminish accuracy and productivity, and increase the financial and human efforts required to obtain results.
Problema pe care o rezolvă invenţia constă în determinarea sexului la plante de Actinidia arguta la etapa de dezvoltare completă a organogenezei in vitro, ce permite de a identifica plantele feminine cu 4…5 ani înaintea înfloririi lor, ceea ce asigură accelerarea procesului de selecţie la această specie cu cel puţin 4 ani. The problem solved by the invention consists in determining the sex of Actinidia arguta plants at the stage of complete development of organogenesis in vitro, which allows identifying female plants 4...5 years before their flowering, which ensures the acceleration of the selection process in this species by at least 4 years.
Invenţia soluţionează problema prin aceea că se propune un procedeu de determinare a sexului la plantele de Actinidia arguta cultivate in vitro, care constă în determinarea spectrului electroforetic al peroxidazelor în frunzele plantei, obţinute la inocularea mugurilor laterali ai minibutaşilor şi cultivarea lăstarilor obţinuţi cu inducerea rizogenezei pe un mediu nutritiv, prin maceraţia frunzelor plantei în soluţie tampon 0,05 M Tris-HCl, în raport de 1:2 respectiv, la un pH=6,8, agitarea ulterioară în decurs de 20 min şi separarea extractului prin centrifugare la 15000 g timp de 15 min la temperatura de -4°C, disc-electroforeza extractului în tuburi cu gel de poliacrilamidă de 7,5%, în decurs de 3 ore la 2,5 mA/tub şi 300 V cu obţinerea spectrului electroforetic al peroxidazelor, developarea benzilor cu soluţie de 0,1% de benzidină în 0,2 M acetat de sodiu, la un pH=4,8, suplimentată cu 0,1% H2O2, totodată dacă spectrul obţinut conţine 8 peroxidaze, planta este de gen feminin, iar dacă conţine 4 peroxidaze - de gen masculin. The invention solves the problem by proposing a method for determining the sex of Actinidia arguta plants cultivated in vitro, which consists in determining the electrophoretic spectrum of peroxidases in the leaves of the plant, obtained upon inoculation of the lateral buds of the mini-cuttings and cultivating the shoots obtained by inducing rhizogenesis on a nutrient medium, by maceration of the plant leaves in 0.05 M Tris-HCl buffer solution, in a ratio of 1:2 respectively, at a pH=6.8, subsequent agitation within 20 min and separation of the extract by centrifugation at 15000 g for 15 min at a temperature of -4°C, disc-electrophoresis of the extract in tubes with 7.5% polyacrylamide gel, within 3 hours at 2.5 mA/tube and 300 V with obtaining the electrophoretic spectrum of peroxidases, developing the bands with a solution of 0.1% benzidine in 0.2 M sodium acetate, at a pH=4.8, supplemented with 0.1% H2O2, also if the spectrum obtained contains 8 peroxidases, the plant is female, and if it contains 4 peroxidases - male.
Rezultatul invenţiei constă în posibilitatea de a determina sexul minibutaşilor de Actinidia arguta la etapa completă de dezvoltare a organogenezei in vitro, cu transferul ulterior în condiţii ex vitro, identificarea plantelor feminine cu 4…5 ani înaintea înfloririi lor, ceea ce asigură accelerarea procesului de selecţie la această specie cu cel puţin 4 ani. The result of the invention consists in the possibility of determining the sex of Actinidia arguta mini-cuttings at the complete stage of organogenesis development in vitro, with subsequent transfer to ex vitro conditions, identifying female plants 4...5 years before their flowering, which ensures the acceleration of the selection process in this species by at least 4 years.
Exemplu de realizare a invenţiei Example of embodiment of the invention
Pentru obţinerea extractelor enzimatice s-au utilizat frunze de plantule masculine şi feminine, obţinute la inocularea mugurilor laterali ai minibutaşilor şi cultivarea lăstarilor obţinuţi cu inducerea rizogenezei pe un mediu nutritiv. Materialul vegetal a fost macerat cu sticlă pisată în prezenţa soluţiei tampon 0,05 M Tris HCl, pH=6,8, în raport 1:2. Apoi extractele omogenizate au fost agitate 20 min şi centrifugate 15 min la 15000 g, la temperatura de -4°C. Supernatantul a servit drept extract enzimatic pentru etapele ulterioare ale experienţelor. To obtain the enzymatic extracts, leaves of male and female seedlings were used, obtained by inoculating the lateral buds of mini-cuttings and cultivating the shoots obtained by inducing rhizogenesis on a nutrient medium. The plant material was macerated with ground glass in the presence of 0.05 M Tris HCl buffer solution, pH=6.8, in a ratio of 1:2. Then the homogenized extracts were stirred for 20 min and centrifuged for 15 min at 15000 g, at a temperature of -4°C. The supernatant served as the enzymatic extract for the subsequent stages of the experiments.
Studiul spectrului electroforetic al PO s-a realizat prin utilizarea disc-electroforezei în tuburi cu gel de poliacrilamidă de 7,5%, în condiţii native, în conformitate cu metoda (Davis B.J.: Disc electrophoresis. II. Method and application to human serum proteins. - Ann. New York Aead. Sei., 1964, vol. 121, p. 404-427). În fiecare tub s-au introdus câte 0,1 ml extract enzimatic. Electroforeza a fost efectuată în frigider la temperatura +6°C pe parcursul a 3 ore, la 300 V şi 2,5 mA/tub. Developarea benzilor s-a realizat cu soluţie de 0,1% benzidină în 0,2 M acetat de sodiu, pH= 4,8, cu adaos de 0,1% H2O2. Fotografierea s-a realizat cu utilizarea sistemului de documentare - Vilber Lourmat. Rezultatele obţinute sunt prezentate în figură. Pe spectrul electroforetic al PO din figură sunt identificate nouă peroxidaze (PO1, PO2, PO3, PO4, PO5, PO6, PO7, PO8, PO9), dintre care cinci sunt specifice numai plantelor feminine, una este specifică plantelor masculine, iar trei sunt comune, ce ne permite să tragem concluzia că dacă spectrul obţinut conţine 8 peroxidaze, planta este de gen feminin, iar dacă conţine 4 peroxidaze - de gen masculin. The study of the electrophoretic spectrum of PO was carried out using disc electrophoresis in tubes with 7.5% polyacrylamide gel, under native conditions, according to the method (Davis B.J.: Disc electrophoresis. II. Method and application to human serum proteins. - Ann. New York Aead. Sei., 1964, vol. 121, p. 404-427). 0.1 ml of enzymatic extract was introduced into each tube. Electrophoresis was carried out in a refrigerator at +6°C for 3 hours, at 300 V and 2.5 mA/tube. The bands were developed with a solution of 0.1% benzidine in 0.2 M sodium acetate, pH= 4.8, with the addition of 0.1% H2O2. Photography was carried out using the documentation system - Vilber Lourmat. The results obtained are presented in the figure. On the electrophoretic spectrum of PO in the figure, nine peroxidases are identified (PO1, PO2, PO3, PO4, PO5, PO6, PO7, PO8, PO9), of which five are specific only to female plants, one is specific to male plants, and three are common, which allows us to conclude that if the obtained spectrum contains 8 peroxidases, the plant is female, and if it contains 4 peroxidases - male.
1. Auxtova O., Samaj J., Cholvadova B., Khandlova E. Isoperoxidase and isopolyphenol oxidase spectra in male and female tissues of Actinidia deliciosa in vitro. Biologia Plantarum, 1994, vol. 94(4), p. 535-541 1. Auxtova O., Samaj J., Cholvadova B., Khandlova E. Isoperoxidase and isopolyphenol oxidase spectra in male and female tissues of Actinidia deliciosa in vitro. Biologia Plantarum, 1994, vol. 94(4), p. 535-541
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| MD605Y (en) * | 2012-07-09 | 2013-03-31 | Inst De Genetica Si Fiziol A Plantelor Al Academiei De Stiinte A Moldovei | Process for microclonal propagation of Actinidia arguta plants in vitro |
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| MD605Y (en) * | 2012-07-09 | 2013-03-31 | Inst De Genetica Si Fiziol A Plantelor Al Academiei De Stiinte A Moldovei | Process for microclonal propagation of Actinidia arguta plants in vitro |
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| Title |
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| Auxtova O., Samaj J., Cholvadova B., Khandlova E. Isoperoxidase and isopolyphenol oxidase spectra in male and female tissues of Actinidia deliciosa in vitro. Biologia Plantarum, 1994, vol. 94(4), p. 535-541 * |
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