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Analysis of potential approaches for production of haploids of cannabis (Cannabis sativa L.)
Get PDFZaradi široke uporabe in vse večje prepoznavnosti konoplje (Cannabis sativa L.) se potreba po žlahtnjenju in iskanju novih sort povečuje. Konoplja je zaradi fizioloških in morfoloških značilnosti zelo zanimiva rastlina za pridelovanje. Te lastnosti pa vplivajo tudi na potencialne pristope za pridobivanje novih sort. Mnoge žlahtniteljske tehnike se pri konoplji že uporabljajo. Ena izmed tehnik je tudi induciranje haploidov, ki so rastline z gametnim številom kromosomov. Če bi uspeli pridobiti haploidno linijo in nato podvojene haploide, bi to olajšalo možnost za pridobitev F1 hibridnih linij, ki jih pri konoplji še ni. Hibridne linije so genetopisko in fenotipsko izenačene, na račun izražene heteroze pa imajo boljše lastnosti, kot starševske linije. Tehnik indukcije haploidov je več, npr. ginogeneza in situ in in vitro ter androgeneza . V nalogi smo te tehnike opisali in se naslonili na postopke, ki so jih razvili pri drugih rastlinskih vrstah, ter na raziskave na konoplji, ki bi lahko igrale vlogo v teh postopkih.Due to the wide use and increasing recognition of cannabis (Cannabis sativa L.), search for new varieties and the need for breeding is increasing. Cannabis is a very interesting plant for cultivation because of its physiological and morphological characteristics. These aspects also affect potential approaches to obtain new varieties. Many breeding techniques are already in use for cannabis. One of the techniques is to induce haploids, which are plants with gametic number of chromosomes. Successfully acquiring a haploid line and then doubled haploids, would make it easier to obtain F1 hybrid lines that currently do not exist in cannabis. Hybrid lines are genetically and phenotypically uniform. Expressed heterosis would induce better traits in hybrids than in parental lines. There are several techniques for haploid induction like gynogenesis in situ and in vitro and androgenesis. We described these techniques and leaned on processes developed on other plant species, as well as on researches on cannabis that could play a role in these processes
Izvrednotenje morfoloških lastnosti dihaploidnih linij zelja (Brassica oleracea var. capitata L.) in izbranih hibridov
Get PDFComparison of different reporter genes for expression of ß-glucuronidase for destructive and non-destructive histochemical gus-assay in transgenic tobacco
Get PDFEstablishment of different genotypes of miniature rose tissue culture and induction of flowering in vitro
Get PDFHaploid Embryogenesis and micropropagation of new donor cabbage genotypes (Brassica oleracea var. capitata L.)
Get PDFV okviru programa žlahtnjenja zelja (Brassica oleracea var. capitata L.) smo preverjali odzivnost posameznih genotipov na haploidno embriogenezo. Iz izhodiščnih rastlin smo porezali brste, iz njih izolirali mikrospore in jih gojili v NLN gojišču brez dodatka ali z dodatkom 0,02 % aktivnega oglja. Po 33 dneh smo prešteli formirane embrije. Oglje ni imelo statistično značilnega vpliva na indukcijo embrijev, le-ti pa so bili večji in bolj regenerativno sposobni. Pri treh genotipih je bilo povprečno število embrijev višje kot 50 embrijev/petrijevko, pri ostalih petih genotipih pa je bilo število embrijev nižje (11,4 – 39,0 embrijev/petrijevko). Embrije smo prestavili na B5 gojišče za regeneracijo ali pa smo jih predhodno tretirali še z abscizinsko kislino (ABA). Ob takojšnji prestavitvi na B5 gojišče, se je v rastline regeneriralo 23,8 - 46,0 % embrijev. Po tretiranju z ABA in izsušitvi smo dosegli višji odstotek regeneracije (37,0 - 73,3 %) in normalen razvoj rastlin. Ploidnost smo izmerili 671 regenerantom in od tega je bilo 275 podvojenih haploidov (41 %), ki smo jih uspešno aklimatizirali. Delež aklimatizacije je znašal 98 %. Z namenom optimizacije postopka mikropropagacije in klonskega razmnoževanja že pridobljenih linij iz predhodnih poskusov žlahtnjenja smo devet linij inokulirali na tri MS gojišča z različnimi koncentracijami hormonov. Največ poganjkov smo dobili na gojišču z 2,0 mg/l IBA in 3,0 mg/l BAP. Po končani razrasti poganjkov smo jih koreninili na polovičnem brezhormonskem MS gojišču. Delež koreninjenja je bil med 50 % in 78 %. Zdrave in ukoreninjene poganjke smo prestavili v rastlinjak, kjer je bila aklimatizacija 97 % uspešna.In the scope of cabbage (Brassica oleracea var. capitata L.) breeding program the responsiveness of new donor genotypes to haploid embryogenesis was tested. Microspores were isolated from flower buds of donor plants, which were then cultivated on NLN medium, with and without the addition of 0. 02 % active charcoal. After 33 days, the embryos were counted. Charcoal had no statistically significant effect on embryo induction but embryos were more vigorous with higher germination efficiency. For three genotypes the embryo yield was higher than 50 embryos per petri dish, while the other five genotypes had lower number of embryos (11.4-39.0 per petri dish). The next step was either direct inoculation of embryos to the B5 regeneration medium or treatment of embryos with abscisic acid and desiccation before the inoculation to B5 medium. The percentage of regenerated embryos without ABA treatment was 23.8 – 46.0 %. The embryos with ABA treatment acheived higher germination percentage (37.0 % - 73.3 %) followed by normal plantlet development. From the total number of 671 regenerated plants analysed with flow cytometry, 275 plants (41 %) were dubled haploids. 98 % of dubled haploids were successfully acclimatized. In order to optimize the micropropagation and clone reproduction of already obtained lines from previous breeding experiments, nine inbred lines we inoculated to MS medium with different concentrations of hormones. The highest number of shoots were achieved on MS medium with 2 mg/l of IBA and 3 mg/l of BAP. The shoots were then transplanted to rooting medium. The percentage of rooting was between 50 % and 78 %. Rooting was induced on half-strength MS hormone free medium. The healthy and rooted regenerants were then planted in the green house, where the acclimatization rate was 97 %
Determination of selfincompatibility alleles in inbred lines of white cabbage (Brassica oleracea var. capitata L.) with molecular markers
Get PDFZelje predstavlja eno najpomembnejših zelenjadnic na svetu. Klasično žlahtnjenje in pridobivanje novih sort zelja je zelo zamudno, saj je zelje dvoletnica. Samoinkompatibilnost je lastnost nekaterih cvetočih rastlin, ki bi lahko znatno skrajšala postopek žlahtnjenja. V ta namen je bil zasnovan poskus, v katerem smo 114 čistim linijam belega zelja iz pomnoženih DNA fragmentov določili inkompatibilnostni razred na osnovi markerjev SRK (S-lokus receptor kinaza) gena, ki je ženska domena (se izrazi v pestiču). Na začetku smo uporabili 9 začetnih oligonukleotidov, ki pomnožujejo različne domene SRK gena, v nadaljevanju poskusa pa smo jih 6 izločili, ker niso delovali pravilno ali pa so bili kontaminirani. Ugotovili smo, da 33,3 % čistih linij pripada razredu I. S pomočjo sekvenčne reakcije smo določili zaporedje 66 čistim linijam in jih razvrstili v podrazrede. V razredu I smo dobili 4 različne podrazrede, v razredu II pa 2. Na 12 različnih lokacijah po Sloveniji smo v parih posadili po eno rastlino belega zelja in eno rastlino rdečega zelja za poskus križanja. Seme smo pridobili samo na dveh lokacijah. Iz semena smo vzgojili sadike in na nivoju fenotipa ter s pomočjo mikrosatelitnih markerjev preverili, ali je prišlo do samooprašitve ali križanja. Delež samooprašitve je bil v inkompatibilnostnem razredu I visok, kar pomeni, da se je kljub močni samoinkompatibilnosti oprašilo veliko število rastlin, zato bi morali poskus ponoviti v bolj optimalnih pogojih.Cabbage is one of the most important vegetables in the world. It is a biennial plant. Therefore the classical breeding procedure for new cabbage varieties is very time-consuming. Selfincompatibility is a characteristic of some flowering plants that could significantly shorten the breeding process. For this reason, the incompatibility class of 114 inbred lines of white cabbage from amplified DNA fragments using SRK-specific primers (S-locus receptor kinase) was determined. SRK gene is a female determinant and it is expressed in the pistil. Initially, we used 9 SRK-specific primers that amplify different domains of the SRK gene, and in the further experiment, 6 of them were eliminated because they did not function properly or were contaminated. We found out that 33.3% of the inbred lines belonged to class I. Using the sequence analysis, we analyzed the sequence of 66 inbred lines and classified them into subclasses. In class I, 4 different subclasses, and in class II, 2 subclasses were identified. At different locations across Slovenia, we planted a white cabbage plant together with a red cabbage plant for test-crossing. Seeds were obtained in only two locations. Seedlings were tested for self-pollination on the phenotypic level and with molecular markers. The percentage of self-pollination was very high in incompatibility class I, meaning that despite the strong selfincompatibility, a large number of plants were self-pollinated. Further research under more optimal conditions would be appropriate
Use of plant-based platform for production of biopharmaceutical antibodies
Get PDFMonoklonska protitelesa, tako za terapevtske kot diagnostične namene, predstavljajo enega izmed najpomembnejših biofarmacevtskih izdelkov. Zaradi trenutnega in v prihodnje vedno večjega povpraševanja po njih je treba preiti na alternativne proizvodne platforme, ki bodo zmožne vsaj dopolniti že sedaj prekomerno preobremenjene tradicionalne proizvodne obrate. Ena od možnih alternativ je uporaba rastlinskih ekspresijskih sistemov. Ta tehnologija je že dokazala nekatere svoje prednosti v smislu varnosti, hitrosti, možnosti hitrega povečanja obsega, nižjih stroškov proizvodnje, visokih donosov, nadaljnje raziskave na področju glikoinženirstva pa posledično omogočajo celo izboljšanje biofarmacevtskih protiteles v smislu boljše stabilnosti in aktivnosti. Raznolikost rastlinskih sistemov (celične in tkivne kulture, uporaba vodnih rastlin, mahov, alg, celih rastlin) omogoča različno vrsto platform, ki lahko potencialno tekmujejo na različnih trgih, saj se prilagajajo konkretnim zahtevam. Ker je možnosti uporabe rastlin v ta namen veliko, sem se v diplomskem delu usmerila le na potencialno uporabo celih rastlin. Osredotočila sem se predvsem na načine transformacije (stabilna in prehodna), prednosti, pomanjkljivosti in težave, s katerimi se ta alternativa srečuje. Nekaj pozornosti sem namenila tudi pregledu trga in v kakšni meri bi lahko rastline doprinesle h globalnim potrebam po protitelesih. Na koncu sem podala še nekaj primerov, ki prikazujejo uspešno uporabo te tehnologije v večjem obsegu.Monoclonal antibodies, both for therapeutic and diagnostic use, represent one of the most important biopharmaceutical products. Due to current and future increasing demand, there is an urgent need for alternative production platforms that can at least act as a complement to the overexploited mammalian fermentation systems. One possible alternative for the large-scale production of biopharmaceuticals is the use of plant expression systems. This technology has already proven its advantages in terms of safety, speed, potential for rapid scale-up production, lower production costs, high yields and the possibility to engineer a tailored antibody glycosylation profile, which allows us to produce even more stable and efficient antibodies. The diversity of plant systems (cell or tissue cultures, use of aquatic plants, mosses, algae, whole plants) allows adaptation to specific requirements of different platforms. Since the possibilities of use are broad I only focused on the potential use of whole plants. I described different methods of plant transformation (stable and transient expression), advantages, shortcomings, difficulties of this alternative platform, and the potential contribution of plant made antibodies to the global market. Finally, I have presented a few examples that have shown the successful use of plant-platforms on a larger scale
Genome editing of centromere protein CENH3 in cabbage (Brassica oleracea var. capitata L.) using CRISPR/Cas9 technology
Get PDFTarčno preurejanje genoma z uporabo tehnologije CRISPR/Cas9 je vedno pogosteje uporabljen pristop za induciranje tarčnih mutacij tudi pri rastlinah. V naših poskusih smo uporabili deset različnih sgRNA za ciljanje štirih različnih mest v genomu zelja (Brassica oleracea var. capitata L.), med njimi tudi 6 sgRNA za spremembe centromernega proteina CENH3, ki je vključen v ločevanje kromosomov. Rastline z mutiranimi oblikami proteina CENH3 se lahko uporabijo kot opraševalske linije za indukcijo haploidov v procesu pridobivanja hibridov z visoko agronomsko vrednostjo. Za dostavo pripravljenih CRISPR/Cas9 vektorjev v celice rastlin zelja smo uporabili tri različne pristope: transformacijo protoplastov, agroinfiltracijo in stabilno transformacijo z uporabo bakterije Agrobacterium tumefaciens (A. t.). Za detekcijo tarčnih mutacij smo uporabili test z endonukleazo T7E1, sekvenciranje po Sangerju in sekvenciranje naslednje generacije. Slednje je pokazalo uspešno indukcijo tarčnih mutacij za vse testirane sgRNA pri vzorcih poskusov transformacije protoplastov in agroinfiltracije. Odstotki induciranih indel (insercije-delecije) mutacij so bili do 11,95 % po transformaciji protoplastov in do 14,42 % po agroinfiltraciji. Za regeneracijo rastlin z želenimi mutacijami smo optimizirali protokole za regeneracijo protoplastov z uporabo gojenja v alginatnih diskih in protokol za stabilno transformacijo z A. t. Protokoli, izdelani v okviru te doktorske disertacije, bodo pripomogli k uporabi tarčne mutageneze pri žlahtnjenju zelja.Genome editing using CRISPR/Cas9 technology is a prevailing approach for the induction of target mutations also in plants. In our work, we used ten different sgRNAs for genome editing of four different sites in cabbage (Brassica oleracea var. capitata L.), including 6 sgRNAs for modification of centromere protein CENH3 involved in chromosome segregation. Plants carrying mutated versions of CENH3 have the potential to be used as haploid inducers in the process of production of hybrids with high agronomic value. We used three different approaches for delivery of prepared CRISPR/Cas9 vectors into cabbage cells: protoplast transfection, agroinfiltration and stable transformation using Agrobacterium tumefaciens (A. t.). To detect target mutations we used T7E1 assay, Sanger sequencing, and next-generation sequencing. The latter showed successful induction of target mutations for all tested sgRNAs in protoplast transfection and agroinfiltration experiments. Mutations were induced at frequencies up to 11,95 % for protoplast transfection and up to 14,42 % for agroinfiltration. For regeneration of plants carrying desired mutations, we optimized protocols for the regeneration of protoplast using cultivation in alginate layers and protocol for stable transformation with A. t. Protocols developed through this doctoral dissertation will help to further use targeted mutagenesis in cabbage breeding
Optimization of induction of organogenic structures using flower buds and ovaries of onion (Allium cepa L.)
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