Barley callus: a model system for bioengineering of starch in cereals

Carciofi, Massimiliano; Blennow, Andreas; Nielsen, Morten; Holm, Preben Bach; Hebelstrup, Kim H.

doi:10.1186/1746-4811-8-36

Cited by 31 publications

(32 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, progress in carrot genetics has been much slower than in many other crop species mainly due to biennial reproduction cycle, outcrossing, and high inbreeding depression effect (Rubatzky et al 1999 ). Callus tissue cultured in vitro may be considered as a valuable alternative to plant requiring long cultivation while implemented for basic research at cellular level, including genetic engineering (Carciofi et al 2012 ; Tran and Sanan-Mishra 2015 ). Carrot callus grows fast, which can serve as a convenient protoplast source or be easily stimulated for embryogenesis allowing massive clonal propagation.…”

Section: Introductionmentioning

confidence: 99%

Efficient CRISPR/Cas9-based genome editing in carrot cells

et al. 2018

View full text Add to dashboard Cite

Key messageThe first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system.AbstractClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot—a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116–119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop.Electronic supplementary materialThe online version of this article (10.1007/s00299-018-2252-2) contains supplementary material, which is available to authorized users.

show abstract

Section: Introductionmentioning

confidence: 99%

Efficient CRISPR/Cas9-based genome editing in carrot cells

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Types with one or two null alleles have 1.7–5.0% amylose content, and the type with all three null alleles has an amylose content of 0.6–0.7%121415. Previous research in barley ( Hordeum vulgare L.) indicated that GBSS I overexpression could increase the expression levels of SBE I, SBE IIa and SS I16. Efforts to generate high-amylose wheat varieties have focused on identifying alterations in a number of genes involved in the synthesis or branching of amylopectin.…”

mentioning

confidence: 99%

Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

Chen

Zhou

Liu

et al. 2016

Sci Rep

View full text Add to dashboard Cite

Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.

show abstract

“…Sequence-confirmed mutants of HvGBSSIa-eGFP were digested with Pac I (Fermentas, FastDigest) and recloned into the pUCE vector Ubi:USER:NOS ( Pac I digested and treated with calf intestine alkaline phosphatase) as described previously (Carciofi et al , 2012). …”

Section: Methodsmentioning

confidence: 99%