| 1. |
- Abrahamsson, Malin, et al.
(författare)
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Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death
- 2012
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 109, s. 391-400
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Tidskriftsartikel (refereegranskat)abstract
- Somatic embryogenesis is a useful tool to propagate conifers vegetatively. However, a major limitation in many pine species is the low quality of cotyledonary somatic embryos. The aim of this study has been to elucidate the developmental pathway of somatic embryos in Scots pine (Pinus sylvestris), to identify deviations from the normal pathway and to identify processes that might disturb normal development. Initially we compared the developmental pathway of somatic embryogenesis in representative cell lines yielding cotyledonary embryos with normal and abnormal morphology. Early embryos carrying suspensor cells in excess of the normal number (supernumerary) were more frequent in cell lines giving rise to abnormal cotyledonary embryos. In this study we show that the frequency of early somatic embryos with supernumerary suspensor cells increased after treatment with the auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Furthermore, the yield of developing embryos increased significantly after treatment with the antiauxin 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB), but the morphology of the embryos was not affected. The number of cells undergoing PCD was analyzed using a TUNEL-assay. The frequency of TUNEL-positive cells was high both in proliferating cultures and during differentiation of early somatic embryos. However, the pattern of TUNEL-positive cells was similar in normal somatic embryos and in embryos with supernumerary suspensor cells. Together our results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.
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| 2. |
- Andersson, Mariette
(författare)
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Colour change in potato (Solanum tuberosum L.) tubers by disruption of the anthocyanin pathway via ribonucleoprotein complex delivery of the CRISPR/Cas9 system
- 2024
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Ingår i: Plant Cell, Tissue and Organ Culture. - 0167-6857 .- 1573-5044. ; 157
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Tidskriftsartikel (refereegranskat)abstract
- Potato is an important part of the traditional Norwegian diet, and the crop faces several challenges with respect to pests and diseases, as well as the increasingly challenging changes in climate. Genome editing may provide tools to improve the resilience of Norwegian potato cultivars to new climate challenges. We have altered the skin colour of two potato cultivars, 'Desiree' and 'Nansen' from red to yellow, as a proof-of-concept for the use of CRISPR/Cas9 in a Norwegian cultivar. Our method has involved the use of protoplasts and we have grown the regenerants for three successive clonal tuber generations to evaluate the stability of the edited plants over time and under varying temperature conditions in contained rooms in a greenhouse. We found that the protoplast method is well suited to achieving CRISPR/Cas9 applications. The results show that the yellow skin is consistent over the three generations of tuber propagation. We found some suspected somaclonal variation in the protoplast regenerants. Some of the variation which we observed under high temperatures (up to nearly 40oC) during the second growth cycle, disappeared when cultivated under lower temperatures in the third cultivation cycle.
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| 3. |
- Andersson, Mariette, et al.
(författare)
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Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
- 2021
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 145, s. 291-305
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Tidskriftsartikel (refereegranskat)abstract
- Key message We compared the StPPO2 gene editing outcomes and efficiencies through Agrobacterium-mediated transformation and protoplasts transfection with DNA or RNPs, and demonstrated that genome editing efficiency depends on the CRISPR/Cas9 delivery approach in potato.Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.
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| 4. |
- Andersson, Mariette, et al.
(författare)
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Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing
- 2021
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 144, s. 463-467
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Tidskriftsartikel (refereegranskat)abstract
- The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)-mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency.Key message RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts.
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| 5. |
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| 6. |
- Dobrowolska, Izabela, et al.
(författare)
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Histological analysis reveals the formation of shoots rather than embryos in regenerating cultures of Eucalyptus globulus
- 2017
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 128, s. 319-326
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Tidskriftsartikel (refereegranskat)abstract
- Eucalyptus globulus is an important species in international forestry in regions with Mediterranean climates and comprises 65 % of the plantation hardwood in Australia. Propagation by somatic embryogenesis would offer many advantages and its development has received much attention. Structures regenerating on explants from hypocotyls of mature zygotic embryos of E. globulus cultured on medium with NAA, reported previously to be effective for embryogenic regeneration, were analyzed morphologically and histologically to clarify their pathway of development. Analysis of series of sections revealed organogenic, rather than embryogenic, pathways of regeneration in this system. We show that protocols for propagation of E. globulus must be carefully evaluated by microscopic examination of adequate numbers of serial sections.
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| 7. |
- Helmersson, Andreas, et al.
(författare)
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Embryogenic cell lines of Juniperus communis, easy establishment and embryo maturation, limited germination
- 2009
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 96, s. 211-217
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Tidskriftsartikel (refereegranskat)abstract
- Several coniferous species belonging to the Pinaceae family can be propagated via somatic embryogenesis, while species belonging to the Cupressaceae family cannot. The aim of this study was to identify possibilities and limitations with somatic embryogenesis in Cupressaceae. Juniperus communis was chosen as model species. We show that a high initiation frequency of embryogenic cell lines can be established from intact megagametophytes at the time when intensive cleavage polyembryogeny takes place. The embryogenic cell lines proliferate fast on medium lacking plant growth regulators. Early somatic embryos develop after transfer to medium with decreased content of nitrogen and calcium. The early embryos mature after exposure to abscisic acid. Mature cotyledonary embryos germinate after partial desiccation. A high proportion, over 40%, of the germinating embryos retain the embryogenic potential in the basal part, resulting in development of new embryogenic tissue
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| 8. |
- Kianersi, Farzad, et al.
(författare)
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Biosynthesis of rutin changes in Capparis spinosa due to altered expression of its pathway genes under elicitors' supplementation
- 2020
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Ingår i: Plant Cell Tissue and Organ Culture. - : Springer Nature. - 0167-6857 .- 1573-5044. ; 141:3, s. 619-631
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Tidskriftsartikel (refereegranskat)abstract
- Caper plant is (Capparis spinosa L.) a good source of rutin which plays a key role in the human diet. In this study, the effect of different concentrations of salicylic acid (SA) and methyl jasmonate (MeJA) on the weight of anther-derived calli and their rutin contents were assessed in caper plants. Also, we investigated the rutin content and expression pattern of some rutin related genes in leaves of caper plants at vegetative and fresh fruiting growth stages under SA and MeJA treatments. In the first experiment, the highest rutin contents were observed in anther-derived calli treated with 10 mu M MeJA and 100 mg L-1 SA after 2 weeks from initial treatments, which were 2.44 and 2.22-fold higher than control. Also, the treatment of caper plants with150 mu M MeJA and 100 mg L-1 SA resulted in a higher increase in the rutin content of leaves at the fresh fruiting stage (61.46 and 9.99 mg g(-1) DW, respectively), in the second experiment. Among the studied genes, the FLS gene showed the highest expression in the leaves of the MeJA- and SA-treated plants at vegetative growth stage, while in the fresh fruiting stage the highest expression was related to the RT gene. Use of 150 mu M MeJA and 100 mg L-1 SA enhanced the expression levels of the RT gene up to 7.36 and 2.89 times of the control, respectively. These results suggest that rutin content and the expression patterns of rutin biosynthesis genes in caper can be significantly enhanced by the SA and MeJA treatments in a growth stage-dependent manner. Key message Methyl jasmonate and salicylic acid treatments enhance the rutin contents of Capparis spinosa in vitro and in vivo and up-regulate the rutin biosynthetic related genes at two different growth stages.
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| 9. |
- Le, Kim-Cuong
(författare)
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Culture condition optimization and FT-IR analysis of Polygonum multiflorum Thunb. adventitious root cultures grown in an air-lift bioreactor system
- 2021
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 144, s. 371-381
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Tidskriftsartikel (refereegranskat)abstract
- Key message The optimum condition for bioreactor culture of Polygonum multiflorum is full-strength MS medium supplemented with 2 mg center dot L-1 IBA and 5% sucrose after 4 weeks of culture. FT-IR analysis can rapidly discriminate among root samples based on total metabolite equivalence.Bioreactor cultures have been used for biomass production and bioactive compounds accumulation in adventitious root cultures of medicinal plants. In this study, we evaluated the effects of different auxin types [indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA)] and concentrations (0.5, 1.0, 2.0 and 4.0 mg center dot L-1), Murashige and Skoog (MS) medium salt strength (0.25, 0.5, 0.75, 1.0, 1.5 and 2X), and sucrose concentrations (0, 1.5, 3, 5, 7 and 10%) on Polygonum multiflorum Thunb. adventitious root cultures in a 3-L balloon-type bubble bioreactor (BTBB). IBA (1, 2, and 4 mg center dot L-1) was more effective than NAA in promoting root growth. Additionally, low MS salt strength (0.25 and 0.5X MS) increased the accumulation of total phenolics and flavonoids but reduced biomass accumulation. Four weeks of culture in full-strength MS medium supplemented with 2 mg center dot L-1 IBA and 5% sucrose resulted in the highest root biomass [98.46 g center dot L-1 fresh weight (FW); 13.46 g center dot L-1 dry weight (DW)] and bioactive compounds accumulation (total phenolics compounds, 53.08 mg center dot g(-1) DW; total flavonoids, 25.10 mg center dot g(-1) DW). To determine whether metabolic fingerprinting of whole-cell extracts could be used to compare metabolic equivalence of P. multiflorum root samples, we treated adventitious roots with different culture conditions, and analyzed the treated adventitious roots and natural roots by Fourier transform infrared (FT-IR) spectroscopy. The results showed that the metabolic pattern of adventitious root samples was similar under different culture conditions; however, these samples could be discriminated from each other in pilot-scale bioreactors. Overall, our study provides useful information for industrial-scale cultivation of P. multiflorum adventitious roots.
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| 10. |
- Li, Xue-Yuan, et al.
(författare)
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Genetic transformation of the oilseed crop Crambe abyssinica
- 2010
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 100, s. 149-156
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Tidskriftsartikel (refereegranskat)abstract
- Engineering oilseed crops for industrial purposes requires a suitable crop that does not outcross to any food oilseed crop, thus eliminating problems of gene flow. Crambe abyssinica is such a dedicated crop as it does not hybridize with any existing food oilseed crops. However, lack of regeneration and transformation protocols has limited the use of C. abyssinica in genetic manipulation studies. In this study, efficient regeneration and transformation protocols for Crambe have been developed. Hypocotyls of C. abyssinica cv. Galactica were incubated on a Murashige and Skoog medium supplemented with various plant growth regulators (PGRs). Among the different PGR combinations tested, 10 mu M thidiazuron and 2.7 mu M alpha-naphthaleneacetic acid promoted highest frequency of regeneration, up to 60%. Among six Agrobacterium stains evaluated, each harbouring the cloning vector containing the neomycin phosphotransferase (nptII) and beta-glucuronidase (gus) genes. EHA101 and AGL-1 yielded the highest transformation frequencies of 1.3 and 2.1%, respectively. Putative transgenic lines were recovered, and confirmed as transgenic by Southern blot analysis. Subsequently, Agrobacterium-mediated transformation of hypocotyls of cv. Galactica with constructs harbouring the wax synthase and fatty acid reductase genes have also successfully recovered confirmed transgenic plants carrying these transgenes.
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