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Vzpostavitev metode transformacije kalusa pri hmelju (Humulus lupulus L.)
ID Jurič, Živa (Author), ID Štajner, Nataša (Mentor) More about this mentor... This link opens in a new window, ID Stajič, Ester (Comentor)

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Abstract
Magistrsko delo je osredotočeno na poskus, ki omogoča odbor najprimernejšega gojišča za rast kalusa hmelja in njegovo transformacijo z bakterijo Agrobacterium tumefaciens. Od 15 genotipov hmelja smo jih izbrali 5, ki so tvorili najbolj primeren kalus. Iz sterilnih rastlin hmelja smo na gojišča inokulirali liste, listne peclje in internodije. Internodiji so tvorili različno količino nastalega kalusa znotraj petrijevk, zato smo jih za nadaljnji potek izločili. Od 4 različno sestavljenih gojišč smo za nadaljnje postopke transformacije izbrali najbolj ustrezna dva. Transformacijo smo izvedli z uporabo binarnih plazmidov pCAMBIA1302-ZsGreen, pGFPGUSplus ter pCAMBIA1301-GUS. Uporabili smo sev bakterije LBA4044. Delež transformiranega kalusa smo v prvi fazi preverjali z uporabo epifluorescenčnega mikroskopa ter z GUS testom. Prisotnost genov smo preverili s PCR analizo ter agarozno elektroforezo. Prisotnost transgena ZsGreen smo potrdili pri vseh analiziranih vzorcih. Celoten genski konstrukt skupaj z hptII je bil pri plazmidu pCAMBIA1302-ZsGreen prisoten pri 81 % transformiranega kalusa. Pri uporabi plazmida pGFPGUSPlus, ki je vseboval transgene EGFP, gusA, in hptII smo fragmente potrdili pri 71 % vzorcih. Gena EGFP in gusA nista bila prisotna pri 5 od 75 vzorcev. Ugotovili smo, da na uspešnost transformacije kalusa pri hmelju vpliva različna rast kalusa na gojiščih, ki je močno odvisno od genotipa, ter izbrani plazmid. S pomočjo uporabljenega protokola smo dobili visok odstotek uspešnosti transformacije s plazmidom pCAMBIA1302-ZsGreen. Potrdimo lahko, da je kalus hmelja primeren za uporabo v postopkih transformacije.

Language:Slovenian
Keywords:hmelj, kalus, genske transformacije, Agrobacterium tumefaciens, fluorescenca, GUS test, plazmidi, ZsGreen, EGFP, gusPlus, gusA hptII
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2023
PID:20.500.12556/RUL-146959 This link opens in a new window
COBISS.SI-ID:156861699 This link opens in a new window
Publication date in RUL:17.06.2023
Views:575
Downloads:104
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Secondary language

Language:English
Title:Establishment of callus transformation method in hop (Humulus lupulus L.)
Abstract:
The master's thesis is focused on experiment that enables the selection of the most suitable culture medium for hop callus growth and transformation with bacteria Agrobacterium tumefaciens. From 15 hop genotypes, we selected 5 that had the highest rate of callus formation with high quality. We inoculated leaves, internodes and petioles from sterile hop plants on induction media. From 4 medium that had different compositions of hormones, we chose the most suitable one for growing callus. Transformation was performed using the plasmids pCAMBIA1302-ZsGreen, pCAMBIA1301-GUS and pGFPGUSplus. The LBA4044 bacteria strain was used. Transformed callus was first checked using epifluorescence microscope and a histochemical GUS test. In addition, the presence of the desired genes in the hop genome was verified by PCR analysis and agarose electrophoresis. The presence of ZsGreen transgene was confirmed in all analyzed samples. The entire gene construct together with hptII was present in 81 % of the calli transformed with plasmid pCAMBIA1302-ZsGreen. When using the plasmid pGFPGUSPlus, which contained EGFP, gusA, and hptII transgenes, the fragments were confirmed in 71 % of the samples. EGFP and gusA genes were absent in 5 samples. We found that the success of callus transformation in hops is mainly influenced by suitable callus induction, which strongly depends on the genotype, and the selected plasmid. With this protocol, we obtained a high transformation efficiency, especially with the plasmid pCAMBIA1302-ZsGreen. We can confirm that hop callus is suitable for transformation procedures.

Keywords:hop plant, callus, genetic transformations, Agrobacterium tumefaciens, GUS test, fluorescence, plasmids, ZsGreen, EGFP, gusPlus, gusA hptII

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