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Hmelj (Humulus lupulus L.) je dvodomna zeljnata trajnica in ovijalka. Pridelovanje hmelja je danes razširjeno predvsem zaradi uporabe v pivovarski industriji. Hmelj je gostiteljska rastlina številnih škodljivih organizmov, vse pogosteje se pri pridelavi hmelja srečujemo z gospodarsko pomembnima boleznima, verticilijsko uvelostjo hmelja ter hudo viroidno zakrnelostjo hmelja. Žlahtniteljski cilji se osredotočajo na selekcijo in vzgojo tolerantnih genotipov na omenjeni bolezni. Za žlahtnjenje rastlin se vse bolj uporabljajo sodobnejši pristopi rastlinske biotehnologije. Za ta namen smo razvili protokol za izolacijo protoplastov iz listov hmelja. Protoplasti so rastlinske celice brez celične stene, pri katerih je vsebina celice obdana le s celično membrano. Pogosto jih uporabljamo za genske transformacije. V našem delu smo ugotovili, da je za rast hmelja v tkivni kulturi najprimernejše MS gojišče z vitamini in glukozo, brez dodatka hormonov. Največji izplen protoplastov smo dobili z enourno plazmolizo v 0,4 M manitolu in prekonočno inkubacijo 0,4 g rastlinskega tkiva v 8 ml encimske raztopine ER3 z 2,0 % koncentracijo celulaze Onozuka R-10 in 0,5 % koncentracijo macerocima R-10. Protoplaste smo izolirali iz listov različnih genotipov hmelja. Uporabili smo liste rastlin hmelja, gojenih v in vitro razmerah in v rastlinjaku ter divji hmelj, ki je uspeval na prostem. Najboljše rezultate smo dobili pri in vitro vzgojenih rastlin genotipa 'Celeia', kjer je bila živost 85,6 %. Divji hmelj, rastlinski material iz rastlinjaka ter in vitro vzgojene rastline genotipa 'Styrian Eureka' so se izkazali kot neprimeren rastlinski material za izolacijo protoplastov z uporabljenim postopkom. Hop (Humulus lupulus L.) is a dioecious perennial climbing plant, mainly known for the use in the brewing industry. Among several diseases that damage hop, the most devastating for hop production are severe hop stunt disease caused by citrus bark cracking viroid (CBCVd) and Verticillium wilt. Developing resistant cultivars are therefore the principal goals of hop breeding programmes. Today, almost all plant breeding programs involve use of new techniques based on advances in biotechnology. With that aim we developed a protocol for isolation of mesophyll protoplasts from hop leaves. Protoplasts are cells with removed cell wall, bounded only by the plasma membrane. They are often used for genetic transformations. In our work, we found that MS medium with vitamins and glucose, without the addition of hormones, is the most suitable for hop growth. The highest yield and viabilty of isolated protoplasts were obtained after one hour plasmolysis in 0.4 M mannitol from 0.4 g leaves digested overnight in 8 ml of 2.0 % cellulase Onozuka R-10 and 0.5% macerozyme R-10 enzymatic solution. Protoplasts were isolated from different hop genotypes and different types of plant material. We used leaves of wild hop, hop grown in greenhouse and under in vitro conditions. The highest number of viable protoplasts (85.6 % viability) were obtained from genotype ‘Celeia’. Wild hop, hop plants grown in greenhouse and genotype ‘Styrian Eureka’ grown under in vitro conditions appeared not suitable plant material for protoplast isolation with used procedure.

Razvili smo uspešno metodo za odpravo okužb semen navadne konoplje (Cannabis sativa L.) in hkrati uspešno transformacijo hipokotilov, s katero smo v celice vnesli selekcijski gen hptII in reporterski gen ZsGreen, ki preko proteina izraža zeleno fluorescenco. Genske transformacije so se izvajale s tri dnevno kokultivacijo hipokotilov s suspenzijo bakterije Agrobacterium tumefaciens. Adventivno regeneracijo hipokotilov smo dosegli z gojenjem izsečkov na MS gojišču z vitamini (Duchefa, M0222), 30 g/l saharoze, 8 g/l agarja, 0,2 mg/l hormona TDZ, 0,5 mg/l hormona NAA, 200µM acetosiringona in pH 5,8 (t.i. gojišče KON3). Uspešnost transformacije smo potrdili z uporabo epifluorescentne lupe, saj so regenerirani hipokotili svetili fluorescentno zeleno. V drugem poskusu, kjer smo narezane hipokotile inokulirali na KON3 gojišče, smo preverjali ali oddaljenost izsečka od koreninskega vratu hipkotilov vpliva na adventivno regeneracijo tkiva. Ugotovljeno je bilo, da razlik v regeneraciji ni bilo in, da je za uspeh poskusa ključna izbira nepoškodovanih semen, ki imajo značilno marmoriran vzorec in nerazpokano semensko ovojnico. V nasprotnem primeru pride do večjega števila bakterijskih in glivičnih okužb predvsem endogenega izvora, saj le-teh ni mogoče odpraviti s klasičnim površinskim razkuževanjem. We have developed a successful method for the elimination of cannabis seed infections (Cannabis sativa L.) and at the same time the successful transformation of hypocotyls, with which we introduced the hptII selection gene and the ZsGreen reporter gene into plant cells, which expresses green fluorescence via protein. Gene transformations were performed by three-day cocultivation of hypocotyls with Agrobacterium tumefaciens. Adventitious regeneration of callus was achieved by growing explants on MS medium supplemented with vitamins (Duchefa, M0222), 30 g/l sucrose, 8 g/l agar, 0.2 mg/l TDZ, 0.5 mg/l NAA, 200µM acetosiringone and pH 5.8 (the KON3 medium). The success of the transformation was confirmed using an epifluorescent macroscope, as the regenerated hypocotyls glowed fluorescent green. In another experiment, where sliced hypocotyls were placed on KON3 medium, we examined whether the position of explants on the hypocotyl influenced adventitious regeneration. It was found that there were no differences in regeneration. For the success of the experiment, we found that the key is to choose undamaged seeds with a characteristically marbled seed coat pattern. Otherwise, a larger number of bacterial and fungal infections, mainly of endogenous origin, occur, as these cannot be eliminated by classical sterilization.

Tarčno 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.

V nalogi smo potrdili, da sta mogoča prenos in stabilna transformacija genov Cas9, Hpt II in ZsGreen v rastlino Nicotiana benthamiana, ki je vse bolj pomembna kot modelni organizem. Gensko transformacijo smo izvedli s pomočjo bakterije Agrobacterium tumefaciens, s katero smo kokultivirali listne izsečke in katera je omogočila vnos izbranih genov v rastlino. Adventivno regeneracijo transgenih poganjkov smo izvedli na trdnem gojišču Murashige in Skoog, ki smo mu dodali 0,1 mg l-1 avksina 1-naftalenocetne kisline (NAA), 1 mg l-1 citokinina 6-benzilaminopurina (BAP), 30 g l-1 saharoze, 150 mg l-1 timentina, 25 mg l-1 higromicina in 8 g l-1 agarja. Uspešnost transformacije smo v T0 generaciji preverili s PCR pomnoževanjem in agarozno gelsko elektroforezo, ki je sicer pokazala uspešnost prenosa genov, a je poleg tega pokazala tudi na prisotnost ostankov bakterije Agrobacterium tumefaciens. Zato smo se odločili za aklimatizacijo vseh rastlin, pri katerih so bili prisotni želeni geni, kljub prisotnosti vir genov. Stabilno transformacijo smo potrdili v T1 generaciji, s pomočjo katere smo potrdili prisotnost želenih genov in njihovo dedovanje v naslednjo generacijo. Molekulsko analizo smo v tem primeru izvedli s pomočjo KAPA3G Plant PCR kit, ki nam je omogočil hitrejše in boljše rezultate, saj je močno skrajšal pot od rastlinskega vzorca do rezultata. The thesis has confirmed transmission and steady genetic transformation Cas9, Hpt II and ZsGreen into the plant Nicotiana benthamiana, which is gaining on the importance as a model organism. The gene transformation was carried out with the aid of a bacterium Agrobacterium tumefaciens which helped us to co-cultivate the leafy parts and which enabled import of the chosen genes into a plant. Adventitious regeneration of the transgenetic offshoot was carried out on a solid nutrient medium Murashige and Skoog with added 0.1 mg l-1 auxin 1-Naphthaleneacetic acid (NAA), 1 mg l-1 cytokinin 6- Benzylaminopurine (BAP), 30 g l-1 sucrose, 150 mg l-1 timentin, 25 mg l-1 higromicin and 8 g l-1 agar. The success of the transformation has been checked with PCR multiplication and agarose gel electrophoresis in T0 generation. It has proven the success of the tranmission of the genes, however, the presence of a bacterium Agrobacterium tumefaciens has also been found in traces. Therefore, the decision was made to acclimatize all the plants where the desired genes were present despite the source genes. A steady transformation has been confirmed in the T1 generation, which helped to confirm the presence of the desired genes and their inheritance in the next generation. Molecular analysis was performed with the help of the KAPA3G Plant PCR kit, which enabled faster and better results as it significantly shortened the way from a plant sample to the result.