scholarly journals Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Charleson Poovaiah ◽  
Lorelle Phillips ◽  
Barbara Geddes ◽  
Cathie Reeves ◽  
Mathias Sorieul ◽  
...  
Keyword(s):  
Genome Editing ◽  
Hybrid Approach ◽  
Single Copy ◽  
Embryogenic Tissue ◽  
Natural Forests ◽  
Planted Forests ◽  
Large Deletions ◽  
Regenerated Plants ◽  
Nucleotide Insertions ◽  
Increasing Demand

Abstract Background To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. Results We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. Conclusions We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.

scholarly journals Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

2020 ◽  
Author(s):  
Charleson Poovaiah ◽  
Lorelle Phillips ◽  
Barbara Geddes ◽  
Cathie Reeves ◽  
Mathias Sorieul ◽  
...  
Keyword(s):  
Genome Editing ◽  
Hybrid Approach ◽  
Single Copy ◽  
Embryogenic Tissue ◽  
Natural Forests ◽  
Planted Forests ◽  
Guide Rna ◽  
Large Deletions ◽  
U6 Snrna ◽  
Nucleotide Insertions

Abstract BackgroundTo meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies.ResultsWe evaluated U6 snRNA promoters from three different species that were able to drive expression of guide RNA (gRNA) for CRISPR/Cas9 genome editing in P. radiata. Using a single-copy cell wall gene GUX1 as a target, we have demonstrated genome editing using CRISPR/Cas9 in somatic embryogenic tissue and plantlets derived from the edited tissue. We generated biallelic INDELs with an efficiency of 15% using a single gRNA. Twelve percent of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We also assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33%, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic.Conclusion We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using DNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.

scholarly journals CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yoshihiko Nanasato ◽  
Masafumi Mikami ◽  
Norihiro Futamura ◽  
Masaki Endo ◽  
Mitsuru Nishiguchi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cryptomeria Japonica ◽  
Fluorescent Protein ◽  
Japanese Cedar ◽  
Targeted Mutagenesis ◽  
Embryogenic Tissue ◽  
Breeding Period ◽  
Single Mutation ◽  
Knock Out ◽  
Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

Rapid and low-cost strategy for detecting genome-editing induced deletion: A single-copy case

2018 ◽  
Vol 1019 ◽  
pp. 111-118 ◽  
Author(s):  
Nan Cheng ◽  
Qin Wang ◽  
Ying Shang ◽  
Yuancong Xu ◽  
Kunlun Huang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Low Cost ◽  
Single Copy ◽  
Cost Strategy ◽  
Low Cost Strategy

scholarly journals The C:N:P Stoichiometry of Planted and Natural Larix principis-rupprechtii Stands along Altitudinal Gradients on the Loess Plateau, China

Forests ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 363
Author(s):  
Fujing Bo ◽  
Yunxiang Zhang ◽  
Han Y. H. Chen ◽  
Pingan Wang ◽  
Xuming Ren ◽  
...  
Keyword(s):  
Nutrient Cycling ◽  
Natural Forests ◽  
Resorption Efficiency ◽  
The Loess Plateau ◽  
Planted Forests ◽  
C:N:P Stoichiometry ◽  
Soil C ◽  
Nitrogen Resorption ◽  
N:P Ratios ◽  
Nitrogen Resorption Efficiency

Carbon:nitrogen:phosphorus (C:N:P) stoichiometry plays a critical role in nutrient cycling, biodiversity, and ecosystem functionality. However, our understanding of the responses of C:N:P stoichiometry to elevation and forest management remains elusive. Here we sampled 18 Larix principis-rupprechtii sites along altitudinal gradients (1700-2300 m) on Guandishan Mountain in the Loess Plateau, China. We determined the leaf, litter, and soil C N P contents and C:N:P stoichiometric ratios, as well as nutrient resorption efficiency (NuRE), and diameter at breast height (DBH) increments in both planted and natural stands, and then tested the impacts of elevation and stand origin on these parameters’ management. We found different C:N:P stoichiometry between natural and planted forests. The results revealed that: soil C, N, and N:P ratios, litter C:P and N:P ratios, leaf C:N and N:P ratios increased significantly; however, soil C:N ratios, litter P, leaf N and P, nitrogen resorption efficiency (NRE), and DBH increments decreased significantly with elevation in the planted forests. Soil C,N and N:P ratios, litter C, as well as C:N and C:P ratios increased significantly with elevation in natural forests. The soil N, P and N:P ratios, litter C:P and N:P ratios, leaf C, C:P and N:P ratios, nitrogen resorption efficiency (NRE), phosphorus resorption efficiency (PRE), and DBH increments were, on average, higher in the planted, rather than natural forests. Our results indicated that there was an enhancing P-limitation in both the planted and natural forests, and the plantations were more restricted by P. Moreover, compared to natural forests, plantations converged toward a higher conservative N- and P-use strategy by enhancing resorption efficiencies of internal nutrient cycling and a higher annual growth rate.

Volatile Compounds of Healthy and Insect-Damaged Hippophae rhamnoides sinensis in Natural and Planted Forests

2012 ◽  
Vol 67 (5-6) ◽  
pp. 244-248 ◽  
Author(s):  
Shixiang Zong ◽  
Youqing Luo ◽  
Jiao Zhou ◽  
Shujing Liu
Keyword(s):  
Volatile Compounds ◽  
Hippophae Rhamnoides ◽  
Cold Trap ◽  
Natural Forests ◽  
Dynamic Headspace ◽  
Planted Forests ◽  
Hippophaë Rhamnoides ◽  
Naturally Occurring ◽  
Gas Chromatography Mass Spectroscopy ◽  
Holcocerus Hippophaecolus

Volatile compounds of healthy and insect-damaged stems of Hippophae rhamnoides sinensis were analysed using dynamic headspace and thermal-desorption cold-trap injector gas chromatography/mass spectroscopy (TCT-GC/MS). Sixteen compounds, belonging to alkanes, alcohols, aldehydes, esters, ketones, and ethers, were identified in the stems of healthy H. rhamnoides sinensis; the compounds in H. rhamnoides sinensis occurring naturally or cultivated in plantations were similar, but the relative contents were significantly different. In plants damaged by Holcocerus hippophaecolus, the nature and content of the volatile compounds were greatly changed. Butanedione and butyl glyoxylate were newly generated after damage by the pest, and the relative levels of pentanal, heptanal, eucalyptol, terpineol, and camphor were sharply increased in both naturally occurring and plantation-grown plants. n-Decane, trans-2-nonen-1-ol, and n-hexadecane levels increased in plants cultivated in the plantation and decreased in natural forests, whereas the levels of other types were reduced. Thus, both the nature and the content of volatile compounds of H. rhamnoides sinensis are affected by H. hippophaecolus damage, providing a theoretical basis to identify the mechanism of pest destruction

Inheritance and resistance to insects in CryIA(c) transgenic cabbage

2007 ◽  
Vol 4 (1) ◽  
pp. 57-61
Author(s):  
Li Han-Xia ◽  
Yin Ruo-He ◽  
Lu Ya-Chun ◽  
Zhang Yu-Yang ◽  
Zhang Jun-Hong
Keyword(s):  
Diamondback Moth ◽  
Dominant Gene ◽  
Single Copy ◽  
Mendelian Inheritance ◽  
Kanamycin Resistance ◽  
Pcr Analysis ◽  
Regenerated Plants ◽  
Resistance To Insects ◽  
Bt Gene ◽  
Polymerase Chain

AbstractUsing hypocotyl segments of aseptic seedlings of cabbage (Brassica oleracea var. capitata) as explants, regenerated plants with kanamycin resistance were obtained mediated by Agrobacterium tumefaciens (strain LBA4404). The transformed plants with the CryIA(c) (Bt) gene were confirmed by Southern blotting analysis, indicating the integration of the transgene into the cabbage genome. The majority of the transgenic plants had only a single copy of the inserted CryIA(c) gene. Leaf section bioassays showed that resistance against larvae of diamondback moth in CryIA(c) transgenic cabbage was significantly enhanced. The inheritance patterns of the transgene in T1 offspring of transgenic cabbage were investigated using polymerase chain reaction (PCR) analysis and a kanamycin resistance test on the leaves of young seedlings. The results showed that dominant gene loci, CryIA(c) or neomycin phosphotransferase gene (NPTII), followed Mendelian inheritance, with a ratio of 3:1 segregation in T1 populations.

scholarly journals Soil Physical Attributes Under Eucalyptus stands With Non-living and Living Plants

2019 ◽  
Vol 11 (3) ◽  
pp. 197
Author(s):  
Nayana Alves Pereira ◽  
João Carlos Medeiros ◽  
Julian Júnio de Jesus Lacerda ◽  
Jaqueline Dalla Rosa ◽  
Bruna Anair Souto Dias ◽  
...  
Keyword(s):  
Hydraulic Conductivity ◽  
Soil Water ◽  
Tropical Climate ◽  
Saturated Hydraulic Conductivity ◽  
Water Retention Curve ◽  
Soil Water Storage ◽  
Natural Forests ◽  
Soil Water Retention Curve ◽  
Planted Forests ◽  
Physical Attributes

The conservation of ecosystems has benefited from planted forests which provide reforested wood reducing the pressure on deforestation of natural forests. Soil physical attributes determine soil water storage capacity; therefore, they play an important role on plant roots’ development which may compromise plant’s survival. The study tested the influence of soil physical and water attributes on the survival of Eucalyptus spp. clones under dry tropical climate. Two areas were selected, including one with living plants and a second with non-living plants of Eucalyptus spp. clones. Moreover, five soil profiles were studied in each area and the parameters estimated were soil bulk density, total porosity, saturated hydraulic conductivity, soil water retention curve, pores size distribution, available water capacity, and S index. Soil physical and hydric attributes did not differ between the area with living plants and the one with non-living plants. The saturated hydraulic conductivity in the area surface layer was high for both the living plants and non-living palnts; 331 mm h-1 and 294 mm h-1, respectively. The S index (to give the value) indicated that the structure was suitable for the development of Eucalyptus trees. Furthermore, it was possible to affirm that soil physical and water attributes of the studied areas were promising for the cultivation of Eucalyptus spp. in the dry tropical climate.

scholarly journals Prime editing primarily induces undesired outcomes in mice

2020 ◽  
Author(s):  
Tomomi Aida ◽  
Jonathan J. Wilde ◽  
Lixin Yang ◽  
Yuanyuan Hou ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Frequency ◽  
Mouse Embryos ◽  
Biomedical Science ◽  
New Approach ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Large Deletions

SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

scholarly journals The relative contributions of forest growth and areal expansion to forest biomass carbon sinks in China

2015 ◽  
Vol 12 (12) ◽  
pp. 9587-9612 ◽  
Author(s):  
P. Li ◽  
J. Zhu ◽  
H. Hu ◽  
Z. Guo ◽  
Y. Pan ◽  
...  
Keyword(s):  
Sustainable Forest Management ◽  
Forest Biomass ◽  
C Sequestration ◽  
Forest Growth ◽  
Forest Area ◽  
Natural Forests ◽  
Biomass Density ◽  
Planted Forests ◽  
Leading Role ◽  
Forest C

Abstract. Forests play a leading role in regional and global terrestrial carbon (C) cycles. Changes in C sequestration within forests can be attributed to areal expansion (increase in forest area) and forest growth (increase in biomass density). Detailed assessment of the relative contributions of areal expansion and forest growth to C sinks is crucial to reveal the mechanisms that control forest C sinks and is helpful for developing sustainable forest management policies in the face of climate change. Using the Forest Identity concept and forest inventory data, this study quantified the spatial and temporal changes in the relative contributions of forest areal expansion and increased biomass growth to China's forest C sinks from 1977 to 2008. Over the last 30 years, the areal expansion of forests was a larger contributor to C sinks than forest growth for all forests and planted forests in China (74.6 vs. 25.4 % for all forests, and 62.4 vs. 37.8 % for plantations). However, for natural forests, forest growth made a larger contribution than areal expansion (60.4 vs. 39.6 %). The relative contribution of forest growth of planted forests showed an increasing trend from an initial 25.3 to 61.0 % in the later period of 1998 to 2003, but for natural forests, the relative contributions were variable without clear trends owing to the drastic changes in forest area and biomass density over the last 30 years. Our findings suggest that afforestation can continue to increase the C sink of China's forests in the future subject to persistently-increasing forest growth after establishment of plantation.

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