SI20053A - Procedure of induction of direct in vitro organogenesis of onion - Google Patents
Procedure of induction of direct in vitro organogenesis of onion Download PDFInfo
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- SI20053A SI20053A SI9800247A SI9800247A SI20053A SI 20053 A SI20053 A SI 20053A SI 9800247 A SI9800247 A SI 9800247A SI 9800247 A SI9800247 A SI 9800247A SI 20053 A SI20053 A SI 20053A
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Univerza v Ljubljani, Biotehniška fakultetaUniversity of Ljubljana, Biotechnical Faculty
Postopek za indukcijo neposredne in vitro organogeneze pri čebuliA method for inducing direct in vitro organogenesis in onions
Področje izuma (Field of Invention)Field of Invention
Predmetni izum se nanaša na področje rastlinske biotehnologije, posebej na nov postopek za indukcijo neposredne in vitro organogeneze pri čebuli s specifično uporabo v mikropropagaciji ali genetskih transformacijah čebule.The present invention relates to the field of plant biotechnology, in particular to a novel method for inducing direct in vitro organogenesis in onions with specific use in micropropagation or onion genetic transformations.
Čebula (Allium cepa L.) spada med najbolj pomembne zelenjadnice, po obsegu pridelave je v svetovnem merilu na drugem mestu. Pridelovalno območje sega od arktičnih do tropskih predelov. Čebulo se da razmnoževati s semeni, čebulicami ali vegetativno. Načini, ki vključujejo in vitro razmnoževanje, spadajo med sodobnejše postopke vegetativnega razmnoževanja čebule. Pomen vegetativnega razmnoževanja je v hitri razmnožitvi genotipov pomembnih za žlahtnjenje rastlin ter za ohranjanje genetske strukture pomembnejših, tudi moško sterilnih linij. Postopki, ki omogočajo neposredno somatsko in vitro organogenezo, so poleg naštetega posebej pomembni kot pogoj za uspešno genetsko transformacijo čebule.Onion (Allium cepa L.) is one of the most important vegetables, it is second in the world in terms of production. The production area extends from the arctic to the tropics. Onions can be propagated by seeds, bulbs or vegetatively. Modes involving in vitro propagation are among the more modern methods of vegetative onion propagation. The importance of vegetative reproduction is in the rapid reproduction of genotypes important for plant breeding and for maintaining the genetic structure of more important, including male sterile lines. The procedures that allow direct somatic in vitro organogenesis are, in addition, particularly important as a condition for successful onion genetic transformation.
Stanje tehnike (Prior Art)Prior Art
Objavljenih je bilo več študij usmerjenih v proučevanje različnih in vitro odzivov pri čebuli, pretežno izzvani po poti aksilamega razraščanja.Several studies have been published focusing on the study of various in vitro responses in onions, mainly evoked by the axillary growth pathway.
Mikropropagacija je pri čebuli težavna, najprej je potrebno izbrati izhodiščno tkivo uporabljeno za vcepek. Del proučevanj je za izsečke uporabljal dele čebule ali čebulic, tudi nastalih in vitro. Najprimernejše tkivo so deli bazalne plošče s skrajšanimi osnovami listov. O tovrstnem pristopu poročajo:Micropropagation is difficult for the onion, and it is first necessary to select the starting tissue used for the inoculation. Part of the studies used portions of onions or bulbs, including those generated in vitro, for the extracts. The most appropriate tissues are parts of the basal plate with shortened leaf bases. This approach is reported by:
Hussey G (1978) In vitro propagation of the onion Allium cepa by axillary and adventitious shoot proliferation. Sci Hortic 9: 227-236;Hussey G (1978) In vitro propagation of the onion Allium cepa by axillary and adventitious shoot proliferation. Sci Hortic 9: 227-236;
Fujieda K, Matsuoka N, Fujita Y (1979) Vegetative multiplication of onion, Allium cepa L., through tissue culture. J Japan Soc Hort Sci 48: 186-194;Fujieda K, Matsuoka N, Fujita Y (1979) Vegetative multiplication of onion, Allium cepa L., through tissue culture. J Japan Soc Hort Sci 48: 186-194;
Hussey G, Falavigna A (1980) Origin and production of in vitro adventitious shoots in the onion, Allium cepa L. J Exp Bot 31: 1675-1686;Hussey G, Falavigna A (1980) Origin and production of in vitro adventitious shoots in the onion, Allium cepa L. J Exp Bot 31: 1675-1686;
Kahane R, Rancillac M, Teyssendier de la Serve B (1992) Long-term multiplication of onion {Allium cepa L.) by cyclic shoot regeneration in vitro. Plant Celi Tiss Org Cult 28: 281-288.Kahane R, Rancillac M, Teyssendier de la Serve B (1992) Long-term multiplication of onion {Allium cepa L.) by cyclic shoot regeneration in vitro. Plant Whole Tiss Org Cult 28: 281-288.
Drugi del proučevanj je za izsečke uporabljal dele nezrelih socvetij. Matsubara S, Hihara H (1978) Onion bulblet regeneration on receptacles in vivo and in situ. J Japan Soc Hort Sci 46: 479-486, poročata o uporabi baze nezrelega socvetja.The second part of the study used sections of immature inflorescences for excision. Matsubara S, Hihara H (1978) Onion bulblet regeneration on receptacles in vivo and in situ. J Japan Soc Hort Sci 46: 479-486, report the use of an immature inflorescence base.
Pike LM, Yoo KS (1990) A tissue culture technique for clonal propagation of onion using immature flower buds. Sci Hortic 45: 31-36 ter Mohamed-Yasseen Y, Splittstoesser WE, Litz RE (1993) In vitro bulb formation and plant recovery from onion inflorescences. HortScience 28: 1052 pa so za izsečke uporabili razrezane dele nezrelih socvetij ali posamične nezrele cvetne brste.Pike LM, Yoo KS (1990) A tissue culture technique for clonal propagation of onion using immature flower buds. Sci Hortic 45: 31-36 ter Mohamed-Yasseen Y, Splittstoesser WE, Litz RE (1993) In vitro bulb formation and plant recovery from onion inflorescences. HortScience 28: 1052 used cut sections of immature inflorescences or individual immature flower buds for excision.
Nastanek kalusnega tkiva je uspel iz številnih tkiv, kot na primer delov čebul, čebulic ali koreninic (Dunstan Dl, Short KC (1978) Shoot production ffom onion callus cultures. Sci Hortic 9: 99-110), delov mlajših listov, nezrelih spolnih embrijev, nezrelih neoplojenih semenskih zasnov, zrelih delov bazalnih plošč (Phillips CG, Luteyn KJ (1983) Effects of picloram and other auxins on onion tissue cultures. J Amer Soc Hort Sci 108: 948-953).Callus tissue formation has succeeded from many tissues, such as parts of onions, bulbs or roots (Dunstan Dl, Short KC (1978) Shoot production ffom onion callus cultures. Sci Hortic 9: 99-110), parts of younger leaves, immature sexual embryos , immature non-fertilized seed designs, mature parts of basal plates (Phillips CG, Luteyn KJ (1983) Effects of picloram and other auxins on onion tissue cultures. J Amer Soc Hort Sci 108: 948-953).
Najbolj pogosto uporabljani rastlinski rastni regulatorji (fitohormoni) za indukcijo poganjkov so bili naftalenocetna kislina (NAA), 6-benzilaminopurin (BAP), medtem ko je bil za indukcijo kalusa pikloram učinkovitejši od NAA ali 2,4-diklorofenoksiocetne kisline (2,4-D).The most commonly used plant growth regulators (phytohormones) for the induction of shoots were naphthalenacetic acid (NAA), 6-benzylaminopurine (BAP), while for the induction of callus, picloram was more effective than NAA or 2,4-dichlorophenoxyacetic acid (2,4- D).
Značilnost objavljenih protokolov je relativno nizka učinkovitost formiranja poganjkov, iz ene donorske rastline je z njihovo uporabo možno pridobiti relativno majhno število poganjkov. Protokoli osnovani na indukciji poganjkov iz delov čebul ali čebulic so dali do 10 poganjkov na izseček, medtem ko je število možnih izsečkov, pridobljenih iz ene čebule, omejeno (Fujieda et al. 1979). Protokoli osnovani na inokulaciji nezrelih cvetov ali delov nezrelih socvetij so bili različno uspešni. Pike in Yoo (1990) poročata o 10% indukciji cvetov, iz induciranih cvetov pa je nastalo okoli 5 poganjkov. Mohamed-Yassen et al. (1993), ki so inducirali poganjke iz na 4 dele razrezanih nezrelih socvetij so uspeli izzvati do 10.6 poganjkov na izseček, torej 42.4 na socvetje. V nobenem od naštetih del ni opisana neposredna organogeneza poganjkov preko nastanka organogenih skupkov, kar je predmet tega izuma.A feature of published protocols is the relatively low efficiency of shoot formation, and relatively small numbers of shoots can be obtained from a single donor plant. Protocols based on the induction of shoots from onions or bulbs gave up to 10 shoots per section, while the number of possible shoots derived from one onion is limited (Fujieda et al. 1979). Protocols based on the inoculation of immature flowers or parts of immature inflorescences have been variously successful. Pike and Yoo (1990) report 10% induction of flowers, and about 5 shoots emerged from the induced flowers. Mohamed-Yassen et al. (1993), who induced shoots from 4 sections of cut immature inflorescences were able to elicit up to 10.6 shoots per section, ie 42.4 per inflorescence. None of the above works describes the direct organogenesis of shoots via the formation of organogenic clusters, which is the object of the present invention.
In vitro gojene poganjke je možno razmnoževati tudi z in vitro odebelitvijo bazalih delov ter razrezom tako nastalih čebulic (Kahane at al. 1992), vendar je postopek zamuden, za en ciklus razmnožitve potrebujejo 3-4 mesece. Objavljen je bil tudi protokol za nastanek somatske embriogeneze (Phillips and Luteyn 1983). Avtoija sta inducirala nastanek kalusa iz meristemskih vršičkov sejancev, korenin sejancev, zrelih embrijev, nezrelih oplojenih ovul in iz zrelih bazalnih plošč.In vitro-grown shoots can also be propagated by in vitro thickening of the basal lobes and dissection of the bulbs thus formed (Kahane et al. 1992), but the process is time-consuming and requires 3-4 months for one propagation cycle. A protocol for the formation of somatic embryogenesis has also been published (Phillips and Luteyn 1983). The autoies induced callus formation from meristematic seedling tips, seedling roots, mature embryos, immature fertilized ovules, and from mature basal plates.
Opis izuma z izvedbenimi primeri (Description of Invention with Working Examples)Description of Invention with Working Examples
Iz navedenega pregleda del je razvidno, da do sedaj še ni bila razvita učinkovita metoda za neposredno somatsko regeneracijo, ki bi vodila preko nastanka organogenih skupkov ter kasnejšega razvoja velikega števila poganjkov, in bi temeljila na postopku, ki ne vključuje nastanka kalusa ter je učinkovit pri večini testiranih kultivarjev. Taka metoda je pomembna z dveh vidikov. Prvič, uporaba tovrstne regeneracije je mogoča za in vitro razmnožitev velikega Števila poganjkov nastalih iz ene same izhodiščne rastline. Drugič, nastanek organogenih skupkov preko neposredne somatske organogeneze je en od pogojev za uporabo metod prenosa genov z metodo genetske transformacije, tako osnovane na biolističnem pristopu kot pristopu po poti okužbe z bakterijo Agrobacterium in podobnih metodah.The aforementioned review of the works shows that an effective method for direct somatic regeneration, leading to the formation of organogenic clusters and subsequent development of a large number of shoots, has not yet been developed and is based on a process that does not involve callus formation and is effective in most cultivars tested. Such a method is important from two perspectives. First, the use of such regeneration is possible for the in vitro propagation of a large number of shoots derived from a single parent plant. Second, the formation of organogenic clusters via direct somatic organogenesis is one of the conditions for the use of gene transfer methods using the genetic transformation method, both based on the biolistic approach and the Agrobacterium infection pathway and similar methods.
Sedaj pa smo presenetljivo ugotovili, da so lahko zreli čebulni cvetovi ali ovariji inducirani za nastanek neposredne organogeneze, ki vodi do nastanka kompaktnih organogenih tvorb (organogenih skupkov), iz katerih se lahko kasneje izdolžijo Številni poganjki. Indukcijski postopki in sestavine gojišč so zelo podobni, kot so bili uporabljeni za povsem drug odziv, to je ginogenetsko regeneracijo haploidnih rastlin čebule (Bohanec B, Jakše M, Ihan A, Javornik B (1995) Studies of gynogenesis in onion (Allium cepa L.): induction procedures and genetic analysis of regenerants. Plant Science 104: 215-224; Jakše M, Bohanec B, and Ihan A (1996) Effect of media components on the gynogenic regeneration of onion (Allium cepa L.) cultivars and analysis of regenerants. Plant Celi Rep 15: 934-938). Glavne razlike med protokolom za indukcijo haploidov in tu opisanim postopkom 2 je uporaba BDS gojišča (Dunstan Dl, Short KC (1977) Improved growth of tissue cultures of onion, Allium cepa. Physiol Plant 41: 70-72) namesto B5 (Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root celiš. Exp Celi Res 50:151-158) indukcijskega gojišča, povišana vsebnost vitaminov in inozitola, uporaba gellan-guma namesto agarja in krajše indukcijsko tretiranje. Te relativno majhne spremembe povzročijo povsem drugačen odziv istih čebulnih organov ob gojenju in vitro, kakršen še ni bil nikjer opisan.We have now surprisingly found that mature onion flowers or ovaries can be induced to produce direct organogenesis, which leads to the formation of compact organogenic formations (organogenic assemblages) from which many shoots may subsequently lend. The induction procedures and the components of the culture medium are very similar to those used for a completely different response, that is, the gynogenetic regeneration of haploid onion plants (Bohanec B, Jakse M, Ihan A, Javornik B (1995) Studies of gynogenesis and onion (Allium cepa L. ): induction procedures and genetic analysis of regenerants Plant Science 104: 215-224; Jakse M, Bohanec B, and Ihan A (1996) Effect of media components on gynogenic regeneration of onion (Allium cepa L.) cultivars and analysis of regenerants.The Plant Whole Rep 15: 934-938). The main differences between the haploid induction protocol and method 2 described here is the use of BDS medium (Dunstan Dl, Short KC (1977) Improved growth of tissue cultures of onion, Allium cepa. Physiol Plant 41: 70-72) instead of B5 (Gamborg OL. Miller RA, Ojima K (1968) Nutrient requirements for suspension cultures of soybean root whole. Exp Whole Res 50: 151-158) induction medium, elevated vitamin and inositol content, use of gellan gum instead of agar and shorter induction treatment. These relatively small changes result in a completely different response from the same onion organs to in vitro cultivation than has been described before.
Predmetni izum je postopek za indukcijo neposredne in vitro organogeneze pri čebuli, ki obsega stopnje:The present invention is a method for inducing direct in vitro organogenesis in onions, comprising the steps of:
(i) Inokulacija cvetov tik pred odprtjem cvetnega odevala na indukcijsko gojišče, ki vsebuje zadostno koncentracijo rastlinskih rastnih regulatorjev in hranilne komponente ter strjevalce gojišča za iniciacijo nastanka neposredne somatske organogeneze. Omenjeni rastni regulatorji v tej fazi vsebujejo učinkovito mešanico avksinov in citokininov, gojišče pa vsebuje mikro in makro elemente, vitamine, inozitol, prolin, ogljikove hidrate in strjevalce gojišč.(i) Flower inoculation shortly before opening the flower dresser to an induction medium containing a sufficient concentration of plant growth regulators and nutrient components and culture media to initiate the formation of direct somatic organogenesis. The growth regulators mentioned at this stage contain an effective mixture of auxins and cytokinins, and the medium contains micro and macro elements, vitamins, inositol, proline, carbohydrates, and culture media.
(ii) Po ustreznem času indukcije prenos cvetov z indukcijskih na diferenciacijska gojišča, ki vsebujejo zadostno količino citokininov ter mikro in makro elemente, vitamine, inozitol, prolin, saharozo in strjevalce gojišč do nastanka neposredne organogeneze.(ii) After appropriate time of induction, the transfer of flowers from induction to differentiation media containing a sufficient amount of cytokinins and micro and macro elements, vitamins, inositol, proline, sucrose and clotting media for direct organogenesis.
(iii) Opcijsko odstranitev cvetnega odevala s cvetov in gojenje izoliranih ovarijev ob prenosu z indukcijskega na diferenciacij sko , gojišče, drugi postopki so enaki kot so opisani v točkah (i) in (ii) in vodijo do nastanka neposredne organogeneze.(iii) Optional removal of the flower dresser from the flowers and cultivation of isolated ovaries upon transfer from induction to differentiation, culture medium, other procedures are the same as described in (i) and (ii) and lead to the emergence of direct organogenesis.
(iv) Ločevanje v točkah (ii) ali (iii) nastalih skupkov somatskih poganjkov ter prenos večjih poganjkov na koreninjenje oziroma subkultiviranje preostalih kompaktnih organogenih struktur.(iv) Separation in (ii) or (iii) of the resulting clusters of somatic shoots and transfer of larger shoots to the rooting or subculturing of the remaining compact organogenic structures.
(v) Aklimatizacija ukoreninjenih prej omenjenih poganjkov.(v) Acclimatization of rooted shoots mentioned above.
Značilni parametri predmetnega postopka so navedeni v nadaljevanju, čeprav obseg izuma ni omejen nanje, ampak obsega vse možne variacije, ki so za strokovnjaka očitne iz opisa. Uporabljeno indukcijsko in diferenciacij sko gojišče vsebuje kot strjevalce gojišča gellan-gum, mešanico agarja in gellan-guma ali samo agar.The characteristic parameters of the present process are set forth below, although the scope of the invention is not limited thereto, but comprises all possible variations that are apparent to the person skilled in the art from the description. The induction and differentiation medium used contains gellan-gum medium, agar-gellan-gum mixture, or agar alone as solidifying agents.
Gojenje na indukcijskem gojišču traja od 3 do 12 dni.Growing on an induction medium lasts from 3 to 12 days.
V indukcijskem in diferenciacijskem gojišču je vir ogljikovih hidratov saharoza, glukoza ali maltoza.In the induction and differentiation medium, the carbohydrate source is sucrose, glucose, or maltose.
Indukcijska in/ali diferenciacij ska gojišča vsebujejo od 25 do 100 g/1 saharoze.Induction and / or differentiation media contain from 25 to 100 g / l sucrose.
'6'6
Indukcijsko gojišče poleg citokinina vsebuje kot vir avksina 2,4-diklorfenoksiocetno kislino ali pikloram.In addition to cytokinin, the induction medium contains 2,4-dichlorophenoxyacetic acid or picloram as the auxin source.
Indukcijsko gojišče poleg avksina ne vsebuje citokinina ali vsebuje benzilaminopurin, tidiazuron ali isopentenyladenine (2ip).In addition to auxin, the induction medium contains no cytokinin or contains benzylaminopurine, thidiazuron or isopentenyladenine (2ip).
Diferenciacijsko gojišče vsebuje kot vir citokinina tidiazuron ali benzilaminopurin.The differentiation medium contains either thidiazuron or benzylaminopurine as a source of cytokinin.
Proces indukcije in diferenciacije poteka na svetlobi ali v temi.The process of induction and differentiation takes place in light or in the dark.
Sestava makro in mikro elementov v indukcijskih in diferenciacijskih gojiščih pripravljena po BDS (Dunstan and Short 1977), B5 (Gamborg et al. 1968) ali MS (Murashige in Skoog, 1962)*.Composition of macro and micro elements in induction and differentiation media prepared according to BDS (Dunstan and Short 1977), B5 (Gamborg et al. 1968) or MS (Murashige and Skoog, 1962) *.
*Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15: 473-497* Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497
Podroben opis postopkaA detailed description of the process
Preizkušan rastlinski material je izviral iz različnih, javnosti dostopnih virov, kultivarji so bili prejeti iz genskih bank ali kupljeni na drobno, inbridirane linije so izvirale iz ameriškega javnega žlahtniteljskega programa (Dr. M.J. Havey, USDA, Madison, Wisconsin, ZDA). Preizkušam so bili naslednji genotipi čebule:The plant material tested came from a variety of publicly available sources, cultivars were received from gene banks or purchased at retail, and inbred lines were sourced from the U.S. Public Breeder Program (Dr. M.J. Havey, USDA, Madison, Wisconsin, USA). The following onion genotypes were tested:
Belokranjka (Slovenija), Ptujska rdeča (Slovenija), Stuttgarter Riesen, Timor, Shenshu Yellow, Yamaguchi Koudaka, Texas Early Grano 502, eksperimentalni hibridi ΧΡΗ 3371 Fi (Asgrow) in križanca 70723 (B1717BxB2923B) in 70719 (B2371CxB2923B), inbridirane linije B2355B, B2923B, MSU2935B, MSU5718B, MSU8155B.Belokranjka (Slovenia), Ptuj Red (Slovenia), Stuttgarter Riesen, Timor, Shenshu Yellow, Yamaguchi Koudaka, Texas Early Grano 502, experimental hybrids ΧΡΗ 3371 Fi (Asgrow) and crossbreeds 70723 (B1717BxB2923B) and 70719 (B2371CxB2923B B25, inbreds) , B2923B, MSU2935B, MSU5718B, MSU8155B.
Cvetni brsti v zreli fazi tik pred odprtjem cveta so bili porezani s prej navedenih donorskih rastlin, ki so bile gojene v rastlinjaku. Površinska sterilizacija cvetov je bila dosežena z 10 minutno potopitvijo cvetov v dinatrijevo dikloroizocianumo kislino v koncentraciji 16.6 g/1 z dodatkom nekaj kapljic močila Tween 20. Po tretiranju so bili cvetovi 3 krat sprani v sterilizirani vodi.The flower buds in the mature stage just before the flower opened were cut from the aforementioned donor plants grown in the greenhouse. Surface sterilization of the flowers was achieved by submerging the flowers for 10 minutes in disodium dichloroisocyanic acid at a concentration of 16.6 g / l by adding a few drops of Tween 20. After the treatment, the flowers were washed 3 times in sterilized water.
Pri postopku 1 so bili cvetovi gojeni v petrijevkah premera 100 mm (30 cvetov na petrijevko), ki so vsebovale indukcijska gojišča, kot je opisano kasneje. Na indukcijskih gojiščih so bili cvetovi (kjer ni drugače rečeno) 6 dni. Po indukcijskem tretiranju so bili cvetovi preneseni na diferenciacijska gojišča, kot je opisano kasneje.In Procedure 1, the flowers were grown in 100 mm diameter petri dishes (30 flowers per petri dish) containing induction media as described later. On the induction media, the flowers (where not stated otherwise) were 6 days. After induction treatment, the flowers were transferred to differentiation media as described later.
Pri postopku 2 smo za razliko od postopka 1 pred prenosom cvetov z indukcijskega na diferenciacijsko gojišče izrezali ovarije, torej odstranili cvetno odevalo in prašnike.In process 2, unlike procedure 1, the ovaries were cut before the transfer of flowers from the induction medium to the differentiation medium, thus removing the floral garment and anthers.
Petrijevke so bile zavite s Parafilmom™ (American National Can, Greenwich, CT, ZDA) za preprečitev izhlapevanja. Inokulirani cvetovi in ovariji so bili gojeni v rastnih komorah s 16/8 urno osvetlitvijo pri 21-23 °C in osvetlitvi približno 80 pmol m’2s'1.Petri dishes were wrapped with Parafilm ™ (American National Can, Greenwich, CT, USA) to prevent evaporation. Inoculated flowers and ovaries were grown in growth chambers with 16/8 hour illumination at 21-23 ° C and illumination of about 80 pmol m ' 2 s' 1 .
Gojišča so bila pripravljena v skladu z uveljavljeno prakso za delo s rastlinskimi tkivnimi kulturami, kot je na primer opisano v delu RLM Pierik (1987) In vitro culture of higher plants. Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster, pp. 344.The media were prepared in accordance with established practice for working with plant tissue cultures, as described, for example, in RLM Pierik (1987) In vitro culture of higher plants. Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster, pp. 344
Osnovno gojišče je vsebovalo BDS makro in mikro elemente in vitamine (kjer ni drugače rečeno) kot sta jih opisala Dunstan in Short 1977 (komercialno dostopne pri Duchefa Biochemie BV, Haarlem, Nizozemska) in 500 mg/1 inozitola, 200 mg/1 prolina, 100 g/l saharoze (kjer ni drugače rečeno); pH gojišča je bil uravnan na 6.0 pred avtoklaviranjem. Sestavine vseh gojišč so, za vsa v primerih navajana tretiranja, podrobneje navedene v Tabelah 11 A in 11 B.The basic medium contained BDS macro and micro elements and vitamins (unless stated otherwise) as described by Dunstan and Short 1977 (commercially available from Duchefa Biochemie BV, Haarlem, The Netherlands) and 500 mg / 1 inositol, 200 mg / 1 proline, 100 g / l sucrose (unless otherwise stated); The pH of the medium was adjusted to 6.0 before autoclaving. The constituents of all the media are listed in Tables 11 A and 11 B in detail for all the treatments cited.
Cvetovi, gojeni na indukcijskih gojiščih, so se v prvih dneh gojitve odprli, ovariji so se močno povečali. Po prestavitvi na diferenciacijska gojišča so se po treh tednih pojavile prve vidne tvorbe na bazi ovarijev. Formirane tvorbe so bile dobro vidne zlasti pri postopku 2, ko je bilo cvetno odevalo odstranjeno in so bile tvorbe vidne skozi gojišče. V začetni fazi so imele te tvorbe globularen embriogen videz in so bile popolnoma bele barve (priloga 1, risba 1).The flowers grown on the induction media opened in the first days of cultivation and the ovaries increased significantly. After switching to differentiation media, the first visible ovarian-based formations appeared after three weeks. Formations formed were clearly visible especially in process 2, when the floral reflector was removed and the formations were visible through the medium. In the initial phase, these formations had a globular embryogenic appearance and were completely white in color (Appendix 1, Figure 1).
Nadaljni razvoj je bil viden v 1 tednu. Globulame strukture so se povečale in vidni so bili posamični zametki poganjkov (priloga 1 risba 2). Del teh struktur se je v naslednjih dveh tednih izdolžil in formiral poganjke dolge približno 2 cm (priloga 1 risba 3). Tedaj je bilo poganjke možno oddvojiti in gojiti posamično ali v skupkih. Ostali del organogenih skupkov je ostal v osnovni kompaktni obliki in ob subkultiviranju na brezhormonsko gojišče je tvoril nove poganjke. Tvorba novih poganjkov je ostala na brezhormonskem gojišču ohranjena vsaj 3 subkulture. Najprimernejše gojišče za izdolževanje poganjkov je bilo osnovno gojišče (ali osnovno gojišče v polovični koncentraciji) brez dodanih fitohormonov ter z znižano koncentracijo saharoze ali glukoze (od 20 do 70 g/1, priporočljivo 30 g/1). Poganjki so postopoma postajali zeleni, izdolžili so se ter v tej ali naslednji subkulturi na istem gojišču tvorili korenine, enako kot z drugimi postopki pridobljeni mikropropagirani poganjki. Za pospešeno tvorbo korenin je bilo možno gojiščem tudi dodati avksine, na primer 0.5-1.0 mg/1 indolmaslene kisline (IBA).Further development was seen within 1 week. The globulam structures were enlarged and the individual embryos of the shoots were visible (appendix 1, drawing 2). Part of these structures lengthened over the next two weeks and formed shoots approximately 2 cm long (Annex 1, Figure 3). At that time, the shoots could be separated and grown individually or in groups. The rest of the organogenic assemblages remained in the basic compact form and formed new shoots when subcultured to the hormone-free medium. The formation of new shoots remained at least 3 subcultures on a non-hormonal medium. The most suitable medium for lentil shoots was a basic medium (or a basic medium in half concentration) with no added phytohormones and a reduced sucrose or glucose concentration (20 to 70 g / l, 30 g / l recommended). The shoots gradually turned green, elongated, and in this or the next subculture formed roots on the same medium, in the same way as micropropagated shoots obtained by other methods. For accelerated root formation, auxins, for example 0.5-1.0 mg / l of indole butyric acid (IBA), could also be added to the culture media.
Število posamičnih poganjkov, ki so se regenerirali, je bilo težko določiti, saj so poleg izdolženih poganjkov ostale prisotne še kompaktne organogene tvorbe. Povprečni nastal skupek je bil ob koncu gojenja na diferenciacijskem gojišču sestavljen iz 5-10 izdolženih poganjkov ter preostalega kompaktnega organogenega tkiva.The number of individual shoots that regenerated was difficult to determine, as compact organogens remained in addition to the elongated shoots. The average cluster formed at the end of cultivation on the differentiation medium consisted of 5-10 elongated shoots and the remaining compact organogenic tissue.
V tabelah 1-10 podani rezultati predstavljajo število cvetov (postopek 1) ali ovarijev (postopek 2), ki so tvorili organogene skupke. Statistično značilne razlike testirane z analizo variance in Duncanovim testom (p=0.05) so prikazane s črkami ki sledijo odstotkom regeneracije, ločeno za vsak genotip, skupine označene z različnimi črkami se statistično značilno razlikujejo.In Tables 1-10, the results presented represent the number of flowers (process 1) or ovaries (process 2) that formed organogenic clusters. Statistically significant differences tested by analysis of variance and Duncan's test (p = 0.05) are shown in letters that follow the percentage of regeneration separately for each genotype, groups marked with different letters differ statistically significantly.
Primer 1Example 1
Efekt strjevalcev gojiščaThe effect of culture media
Indukcijska in diferenciacijska gojišča so vsebovala tri različne sestavine za strjevanje gojišč in sicer gellan-gum (Il/Dl), mešanica gellan-guma in agarja (Difco-Bacto™, Difco Laboratories, Detroit, MI, ZDA) (I2/D2) ter agar (Difco Bacto) (I3/D3). Učinek različnih strjevalcev gojišč je bil testiran z uporabo postopka 1 in postopka 2, cvetovi so bili prestavljeni z indukcijskega na diferenciacijsko gojišče po.6 dneh. Rezultati so prikazani v tabeli 1.Induction and differentiation media contained three different media for solidifying media, namely gellan-gum (Il / Dl), a mixture of gellan-gum and agar (Difco-Bacto ™, Difco Laboratories, Detroit, MI, USA) (I2 / D2), and agar (Difco Bacto) (I3 / D3). The effect of different culture hardeners was tested using procedure 1 and procedure 2, and the flowers were shifted from induction to differentiation medium after 6 days. The results are shown in Table 1.
Tabela 1Table 1
Najvišji odstotek regeneracije je bil dosežen na gojiščih strjenih z gellan-gumom in to pri obeh uporabljenih postopkih. Na gojišču strjenem z gellan-gumom je več novonastalih poganjkov kazalo znake nezaželene hiperhidracije, manj hiperhidriranih poganjkov pa se je tvorilo na gojiščih z agarjem ali mešanico obeh sredstev.The highest percentages of regeneration were obtained on gellan gum-cured media in both methods used. On the gellan gum-coated medium, several newly emerged shoots showed signs of undesirable hyperhydration, and fewer hyperhydrated shoots formed on agar media or a mixture of both agents.
Primer 2Example 2
Trajanje indukcijske fazeDuration of induction phase
Preizkušeno je bilo različno trajanje indukcijske faze pred prestavitvijo na diferenciacijsko gojišče. Uporabljen je bil postopek 2 ter gojišči Il/Dl. Rezultati so prikazani v tabeli 2.Different duration of the induction phase was tested before transferring to a differentiation medium. Procedure 2 and the Il / Dl media were used. The results are shown in Table 2.
Tabela 2Table 2
Trajanje indukcijske faze je imelo signifikanten učinek na regeneracijo. Najvišja regeneracija poganjkov je bila dosežena na ovarijih, ki so bili prestavljeni na diferenciacijsko gojišče po 6 dneh, krajše ali daljše indukcijsko tretiranje pa je rezultiralo z nižjo odzivnostjo; pri 6 dneh je bila tudi stopnja hiperhidriranosti najnižja.The duration of the induction phase had a significant effect on regeneration. Highest shoot regeneration was achieved on ovaries that were switched to differentiation medium after 6 days, and shorter or longer induction treatments resulted in lower responsiveness; at 6 days, the level of hyperhydration was also lowest.
Primer 3:Example 3:
Vpliv vira ogljikovih hidratovEffect of carbohydrate source
Proučevan je bil vpliv različnih ogljikovih hidratov v indukcijskih in diferenciacijskih gojiščih, in sicer 50 g/l saharoze (I4/D4) in ekvimolame koncentracije 26.3 g/l glukoze (I6/D6) in 52.6 g/l maltoze (I7/D7). Uporabljen je bil postopek 2, učinek ogljikovih hidratov pa je bil testiran na treh različnih genotipih čebule, kot je navedeno v tabeli 3. Rezultati so prikazani v tabeli 3,The effect of different carbohydrates in induction and differentiation media was studied, namely 50 g / l sucrose (I4 / D4) and equimolam concentrations of 26.3 g / l glucose (I6 / D6) and 52.6 g / l maltose (I7 / D7). Procedure 2 was used and the effect of carbohydrates was tested on three different onion genotypes as indicated in Table 3. The results are shown in Table 3,
Tabela 3Table 3
Organogeni skupki so nastajali na vseh preizkušanih gojiščih ter pri vseh genotipih, med gojišči ni bilo statistično značilnih razlik, tudi stopnja hiperhidracije ni bila bistveno različna.Organogenic clumps were formed on all tested media and in all genotypes; there were no statistically significant differences between the media and the degree of hyperhydration was not significantly different.
Primer 4:Example 4:
Vpliv koncentracije saharoze v gojiščihEffect of sucrose concentration in culture media
Proučevan je bil vpliv vsebnosti saharoze v indukcijskih in diferenciacij skih gojiščih, in sicer 100 g/1 (Il/Dl), 50 g/1 (I4/D4) in 25 g/1 (I5/D5). Uporabljen je bil postopek 2, učinek saharoze pa je bil testiran na treh različnih genotipih čebule, kot je navedeno v tabeli 4. Rezultati so prikazani v tabeli 4.The effect of sucrose content in induction and differentiation media was studied, namely 100 g / l (Il / Dl), 50 g / l (I4 / D4) and 25 g / l (I5 / D5). Procedure 2 was used, and the sucrose effect was tested for three different onion genotypes as indicated in Table 4. The results are shown in Table 4.
Tabela 4Table 4
Organogeni skupki so nastajali na vseh preizkušanih gojiščih ter pri vseh genotipih, na gojišču s 50 g/1 saharoze je statistično značilno večje število skupkov nastajalo le pri genotipu MSU5718B. Pri dveh drugih genotipih je nižja koncentracija saharoze vplivala na manjši pojav hiperhidracije.Organogenic clumps were formed on all tested media and in all genotypes, on a medium with 50 g / l sucrose, a statistically significant higher number of clumps occurred only on the MSU5718B genotype. For the other two genotypes, the lower sucrose concentration resulted in a lower incidence of hyperhydration.
Primer 5:Example 5:
Vpliv sestave avksinov v indukcijskih gojiščihEffect of auxin composition in induction media
Proučevan je bil vpliv sestave avksinov v indukcijskih gojiščih. Uporabljen je bil postopek 2, učinek avksinov pa je bil testiran na štirih različnih genotipih čebule, kot je navedeno v tabeliThe effect of auxin composition in induction media was studied. Procedure 2 was used and the effect of auxins was tested on four different onion genotypes as indicated in the table
5. Indukcijska gojišča so vsebovala 2 mg/l 2,4-D (14), 1 mg/l. piklorama (18) in 2 mg/l piklorama (19) ali 5 mg/l NAA (110). Diferenciacijsko gojišče je bilo Dl ali D4.5. Induction media contained 2 mg / l 2,4-D (14), 1 mg / l. picloram (18) and 2 mg / l picloram (19) or 5 mg / l NAA (110). The differentiation medium was D1 or D4.
Rezultati so prikazani v tabeli 5.The results are shown in Table 5.
Tabela 5Table 5
Organogeni skupki so nastajali na treh od štirih preizkušanih gojišč, na gojišču I4/D4 jih je bilo pri obeh genotipih statistično značilno največ, tudi stopnja hiperhidriranosti je bila pri tej kombinaciji najmanjša.Organogenic clusters were formed on three of the four tested media, with the statistically significant highest in both genotypes I4 / D4, with the lowest level of hyperhydration in this combination.
Primer 6Example 6
Vpliv sestave citokininov v indukcijskih gojiščihImpact of cytokinin composition in induction media
Proučevan je bil vpliv sestave citokininov v indukcijskih gojiščih. Uporabljen je bil postopekThe effect of cytokinin composition in induction media was studied. The procedure was used
2, učinek citokininov pa je bil testiran na dveh različnih genotipih čebule, kot je navedeno v tabeli 6. Indukcijska gojišča so vsebovala 2 mg/1 BAP (14), nič citokinina (111), 1 mg/1 tidiazurona (112) ali 2 mg/12ip (113), diferenciacijsko gojišče je bilo D4.2, and the effect of cytokinins was tested on two different onion genotypes as indicated in Table 6. Induction media contained 2 mg / 1 BAP (14), zero cytokinin (111), 1 mg / 1 thidiazuron (112) or 2 mg / 12ip (113), the differentiation medium was D4.
Rezultati so prikazani v tabeli 6.The results are shown in Table 6.
Tabela 6Table 6
Statistično značilno najvišji odstotek indukcije je bil dosežen na gojišču 112 še posebej pri prvem genotipu, pri drugem pa je bil statistično neznačilno nižji od gojišča 14. Tudi stopnja hiperhidriranosti je bila na gojišču 112 zelo nizka. Odsotnost citokinina v indukcijskem gojišču (Il 1) je močno znižala odstotek regeneracije.A statistically significant highest induction percentage was achieved on the medium 112, especially in the first genotype, and in the second genotype it was statistically insignificantly lower than the medium 14. The hyperhydration rate was also very low on the medium 112. The absence of cytokinin in the induction medium (Il 1) greatly reduced the percentage of regeneration.
Primer 7Example 7
Vpliv sestave citokininov v diferenciacijskih gojiščihImpact of cytokinin composition in differentiation media
Proučevan je bil vpliv sestave citokininov v diferenciacij skih gojiščih. Uporabljen je bil postopek 2, učinek citokininov pa je bil testiran na štirih različnih genotipih čebule, kot je navedeno v tabeli 7 ter na gojiščih z dvema različnima vsebnostima saharoze. Diferenciacijska gojišča so vsebovala 2 mg/1 TDZ (Dl) ali 5 mg/1 BAP (D10 in Dl 1), indukcijsko gojišče je bilo II ali 14. Rezultati so prikazani v tabeli 7.The effect of cytokinin composition in differentiation media was studied. Procedure 2 was used, and the effect of cytokinins was tested on four different onion genotypes as indicated in Table 7 and on media with two different sucrose contents. Differentiation media contained 2 mg / 1 TDZ (Dl) or 5 mg / 1 BAP (D10 and Dl 1), the induction medium was II or 14. The results are shown in Table 7.
Tabela 7Table 7
Organogeni skupki so nastajali na vseh preizkušanih gojiščih ter pri vseh genotipih. Nižja vsebnost saharoze je pri vseh preizkušanih genotipih zvišala regeneracijo in znižala hiperhidriranost.Organogenic clumps were formed on all tested media and on all genotypes. Lower sucrose content increased regeneration and reduced hyperhydration in all genotypes tested.
Primer 8Example 8
Vpliv svetlobeThe influence of light
Proučevanje bil vpliv indukcije in diferenciacije organogenih skupkov na svetlobi ali v temi pri dveh genotipih. Uporabljen je bil postopek 2, gojišči sta bili I4/D4. Rezultati so podani v tabeli 8.The effect of the induction and differentiation of organogenic assemblages on light or in darkness in two genotypes was studied. Procedure 2 was used and the media were I4 / D4. The results are given in Table 8.
Tabela 8Table 8
Organogeni skupki so nastajali na obeh preizkušanih tretiranjih ter pri obeh genotipih, v obeh primerih je bila regeneracija organogenih skupkov boljša na svetlobi kot v temi.Organogenic clusters were produced on both treatments tested and on both genotypes, in both cases the regeneration of the organogenic clusters was better in light than in darkness.
Primer 9Example 9
Vpliv sestave makro in mikro elementovImpact of composition of macro and micro elements
Proučevan je bil vpliv indukcije in diferenciacije organogenih skupkov na gojiščih, ki so vsebovala različne sestavine makro in mikro elementov v indukcijskem in diferenciacijskem gojišču in sicer BDS (I4/D4), B5 (I14/D12) ali MS (I15/D13). Uporabljen je bil postopek 2, rezultati so podani v tabeli 9.The effect of induction and differentiation of organogenic assemblages on media containing different constituents of macro and micro elements in the induction and differentiation medium, namely BDS (I4 / D4), B5 (I14 / D12) or MS (I15 / D13), was studied. Procedure 2 was used and the results are given in Table 9.
Tabela 9Table 9
Organogeni skupki so nastajali na vseh preizkušanih gojiščih ter pri obeh genotipih. Najnižja stopnja hiperhidriranosti je bila na gojiščih I14/D12.Organogenic clumps were formed on all tested media and both genotypes. The lowest level of hyperhydration was found on I14 / D12 media.
Primer 10Example 10
Izvrednotenje efekta genotipaGenotype effect evaluation
Proučevan je bil vpliv genotipa na tvorbo organogenih skupkov nastalih po postopkih 1 in 2, gojišči sta bili II/Dl. Rezultati proučitve indukcije 12 genotipov čebule so podani v tabeli 10.The influence of the genotype on the formation of organogenic clusters formed by procedures 1 and 2 was studied, and the media were II / Dl. The results of studying the induction of 12 onion genotypes are given in Table 10.
Tabela 10 A: postopek 1Table 10 A: Procedure 1
B: postopek 2B: process 2
Organogeni skupki so nastajali na vseh preizkušanih gojiščih ter pri vseh genotipih. Bistvena razlika med postopkom 1 in 2 je bila v formiranju kalusa ob bazi cvetov. V nekaterih primerih je prišlo do adventivne (nedirektne) regeneracije poganjkov iz nastalega kalusa, kot je v tabeli posebej prikazano. V primeru uporabe postopka 2 (izrez ovarijev) do tvorbe kalusa ni prišlo, regeneracija je nastala izključno po poti direktne organogeneze. Razlike med posameznimi kultivarji so bile statistično signifikantne pri obeh postopkih. Najvišji dosežen odstotek regeneracije je bil 57.9% (postopek 2) oziroma 45.4% (postopek 1). Najnižji odstotek regeneracije je bil pri obeh postopkih dosežen pri liniji MSU8155B, vendar je potrebno omeniti, da je ta linija cvetela 3 tedne kasneje kot ostale, v tem času so bile v rastlinjaku temperature (ki lahko vplivajo na odzivnost) že precej višje.Organogenic clumps were formed on all tested media and on all genotypes. The essential difference between process 1 and 2 was the formation of callus at the base of the flowers. In some cases, adventitious (indirect) regeneration of shoots from the resulting callus occurred, as shown in the table separately. When process 2 (ovary excision) was used, callus formation did not occur, regeneration occurred exclusively by direct organogenesis. Differences between individual cultivars were statistically significant in both treatments. The highest percentages of regeneration achieved were 57.9% (process 2) and 45.4% (process 1). The lowest percentage of regeneration was achieved with MSU8155B in both processes, but it should be noted that this line bloomed 3 weeks later than the others, during which time the greenhouse temperatures (which may affect responsiveness) were already much higher.
Del genotipov seje odzval podobno pri obeh testiranih postopkih, del pa se je odzval bolje pri enem ali drugem postopku. Variiral je tudi odstotek hiperhidriranosti poganjkov, nasplošno je bil nekoliko nižji pri postopku 1 kot pri postopku 2.Part of the session genotypes responded similarly to the two procedures tested, and part responded better in one or the other procedure. It also varied the percentage of hyperhydrogenation of shoots, overall being slightly lower in process 1 than in process 2.
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Claims (13)
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SI9800247A SI20053A (en) | 1998-09-24 | 1998-09-24 | Procedure of induction of direct in vitro organogenesis of onion |
| AU57685/99A AU5768599A (en) | 1998-09-24 | 1999-09-22 | A process for the induction of direct (in vitro) organogenesis in onion |
| PCT/SI1999/000022 WO2000016610A1 (en) | 1998-09-24 | 1999-09-22 | A process for the induction of direct in vitro organogenesis in onion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SI9800247A SI20053A (en) | 1998-09-24 | 1998-09-24 | Procedure of induction of direct in vitro organogenesis of onion |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| SI20053A true SI20053A (en) | 2000-04-30 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SI9800247A SI20053A (en) | 1998-09-24 | 1998-09-24 | Procedure of induction of direct in vitro organogenesis of onion |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU5768599A (en) |
| SI (1) | SI20053A (en) |
| WO (1) | WO2000016610A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6995016B2 (en) | 2000-08-17 | 2006-02-07 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food | Process for inducing direct somatic embryogenesis in immature scutella cells of pooideae, and rapidly regenerating fertile plants |
| CN102870674B (en) * | 2012-09-05 | 2013-12-25 | 广州白云华南生物科技有限公司 | Method for quickly propagating red onion through tissue culture |
| EP2925739A4 (en) * | 2012-11-28 | 2016-07-27 | Stichting Dienst Landbouwkundi | Substituted dihydropyrtoines for somatic embryogenesis i plants |
| CN110226517B (en) * | 2019-06-26 | 2021-06-01 | 北京市农林科学院 | In-vitro regeneration method of onion and culture medium used by same |
| CN117441617B (en) * | 2023-12-22 | 2024-04-02 | 北京花乡花木集团有限公司 | Tissue culture method of north shallot |
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| ATE160174T1 (en) * | 1989-08-09 | 1997-11-15 | Dekalb Genetics Corp | METHODS AND COMPOSITIONS FOR THE PRODUCTION OF STABLE TRANSFORMED, FERTILIZABLE CORN PLANTS AND CELLS THEREOF |
-
1998
- 1998-09-24 SI SI9800247A patent/SI20053A/en unknown
-
1999
- 1999-09-22 WO PCT/SI1999/000022 patent/WO2000016610A1/en active Application Filing
- 1999-09-22 AU AU57685/99A patent/AU5768599A/en not_active Abandoned
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| Publication number | Publication date |
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| AU5768599A (en) | 2000-04-10 |
| WO2000016610A1 (en) | 2000-03-30 |
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