Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre
Lepidium campestre is a wild oil species with a number of traits that are beneficial from an agricultural point of view. CRISPR/Cas9 could be used transiently in protoplasts to accelerate domestication of L. campestre. In order for plants in Sweden to be classified as non-GMO they need to be modifie...
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| Format: | H2 |
| Language: | Inglés Swedish |
| Published: |
SLU/Dept. of Plant Breeding (from 130101)
2017
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| Subjects: |
| _version_ | 1855571915080466432 |
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| author | Selga, Louise |
| author_browse | Selga, Louise |
| author_facet | Selga, Louise |
| author_sort | Selga, Louise |
| collection | Epsilon Archive for Student Projects |
| description | Lepidium campestre is a wild oil species with a number of traits that are beneficial from an agricultural point of view. CRISPR/Cas9 could be used transiently in protoplasts to accelerate domestication of L. campestre. In order for plants in Sweden to be classified as non-GMO they need to be modified without the addition of foreign DNA, therefore transient Cas9 expression is used. Since there are no protoplast methods optimized for L. campestre the aim of this project was to develop efficient methods for protoplast isolation, transfection and regeneration suitable for this species. Multiple isolation parameters were compared. When using higher enzyme concentrations significantly more protoplasts were obtained. No significant difference was found between gently shaking the leaves or keeping them stationary during the enzyme digestion. Cutting leaves with razorblades or scissors showed no significant difference in number of protoplasts isolated, and differences in regeneration capacity could not be evaluated due to infections. No significant difference was found when increasing the enzyme incubation time from 15 h to 18 h. Transfection was performed using the plasmid pEAQ-HT-GFP and the PEG incubation time was tested. Transfection was performed successfully using 25 % PEG4000 with incubation times 5 min and 10 min. Two regeneration methods were performed and differences in infection frequency and microcalli production were observed. Method 3.B suffered more infections, possibly due to a higher sensitivity or a contaminated solution. Microcalli were obtained from one plate, which was regenerated according to method 3.A. This shows that regeneration of the protoplasts is possible, and supports further optimization of method 3.A. |
| format | H2 |
| id | RepoSLU11663 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés swe |
| publishDate | 2017 |
| publishDateSort | 2017 |
| publisher | SLU/Dept. of Plant Breeding (from 130101) |
| publisherStr | SLU/Dept. of Plant Breeding (from 130101) |
| record_format | eprints |
| spelling | RepoSLU116632017-10-11T05:59:43Z Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre Optimering av protoplastmetoder avsedda för transient CRISPR/Cas9-uttryck i Lepidium campestre Selga, Louise protoplasts lepidium campestre CRISPR/Cas9 Lepidium campestre is a wild oil species with a number of traits that are beneficial from an agricultural point of view. CRISPR/Cas9 could be used transiently in protoplasts to accelerate domestication of L. campestre. In order for plants in Sweden to be classified as non-GMO they need to be modified without the addition of foreign DNA, therefore transient Cas9 expression is used. Since there are no protoplast methods optimized for L. campestre the aim of this project was to develop efficient methods for protoplast isolation, transfection and regeneration suitable for this species. Multiple isolation parameters were compared. When using higher enzyme concentrations significantly more protoplasts were obtained. No significant difference was found between gently shaking the leaves or keeping them stationary during the enzyme digestion. Cutting leaves with razorblades or scissors showed no significant difference in number of protoplasts isolated, and differences in regeneration capacity could not be evaluated due to infections. No significant difference was found when increasing the enzyme incubation time from 15 h to 18 h. Transfection was performed using the plasmid pEAQ-HT-GFP and the PEG incubation time was tested. Transfection was performed successfully using 25 % PEG4000 with incubation times 5 min and 10 min. Two regeneration methods were performed and differences in infection frequency and microcalli production were observed. Method 3.B suffered more infections, possibly due to a higher sensitivity or a contaminated solution. Microcalli were obtained from one plate, which was regenerated according to method 3.A. This shows that regeneration of the protoplasts is possible, and supports further optimization of method 3.A. Lepidium campestre är en vild oljeväxt med ett antal egenskaper som är fördelaktiga inom jordbruk. Domesticering av denna växt kan delvis utföras genom transient uttryck av CRISPR/Cas9 i protoplaster. För att växter i Sverige inte ska bli GMO-klassificerade krävs det att de blir modifierade utan att främmande DNA tillförs, så därför används transient Cas9-uttryck. Eftersom det inte finns protoplastmetoder anpassade för L. campestre så är syftet för detta projekt att utveckla metoder för isolering, transfektion och regenerering av protoplaster lämpliga för denna art. Ett flertal parametrar för protoplastisolering jämfördes och modifierades. När enzymkoncentrationen höjdes isolerades signifikant fler protoplaster. Ingen signifikant skillnad upptäcktes mellan att försiktigt skaka bladen eller att hålla dem stilla under enzymbehandlingen. Att skära bladen med rakblad eller sax gav ingen signifikant skillnad i antal protoplaster isolerade, och eventuella skillnader i regenereringsförmåga kunde ej undersökas på grund av infektioner. Ingen signifikant skillnad hittades mellan antalet protoplaster isolerade efter 15 respektive 18 h enzyminkubering. Transfektionen genomfördes med plasmiden pEAQ-HT-GFP och inkubationstiden i PEG testades. Lyckade transfektioner genomfördes med inkubationstider 5 och 10 min i 25 % PEG4000. Två regenereringsmetoder jämfördes och skillnader i infektionsfrekvens och mikrocalliproduktion noterades. Metod 3.B fick fler infektioner, vilket kan bero på en högre infektionsrisk eller användning av en kontaminerad lösning. Mikrocalli producerades på en platta, som regenererades med metod 3.A. Detta visar att regenerering av protoplasterna är möjlig, och stödjer användning av metod 3.A. SLU/Dept. of Plant Breeding (from 130101) 2017 H2 eng swe https://stud.epsilon.slu.se/11663/ |
| spellingShingle | protoplasts lepidium campestre CRISPR/Cas9 Selga, Louise Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre |
| title | Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre |
| title_full | Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre |
| title_fullStr | Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre |
| title_full_unstemmed | Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre |
| title_short | Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre |
| title_sort | optimization of protoplast methods suitable for transient crispr/cas9 expression in lepidium campestre |
| topic | protoplasts lepidium campestre CRISPR/Cas9 |