Tri-parental protoplast fusion of<i>Brassica</i>species to produce somatic hybrids with high genetic and phenotypic variability

Lian, Yuji; Lin, Gan; Zheng, Qin

doi:10.5958/0975-6906.2015.00079.6

Cited by 5 publications

(6 citation statements)

References 32 publications

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“…Efficient protoplast regeneration protocols exists for Brassica species ( Glimelius, 1999 ; Sheng et al, 2011 ; Lian et al, 2012 ; Kiełkowska and Adamus, 2017 ). Until now they have been used in somatic hybridization experiments aimed at overcoming barriers in sexual crosses or to modify cytoplasmic traits by altering organelle populations ( Lian et al, 2015 ; Kang et al, 2017 ) and in experiments of genetic transformation by direct DNA uptake ( Nugent et al, 2006 ). Combined with our protocol for DNA-free genome editing of Brassica , they will enable the development of plants with edited phenotypes without the use of transgenesis.…”

Section: Discussionmentioning

confidence: 99%

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes

et al. 2018

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The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro-transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus Brassica (B. oleracea, B. napus, and B. rapa) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the FRI and PDS genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 μg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into B. oleracea and B. rapa protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated Brassica plants without the use of transgenesis.

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Section: Discussionmentioning

confidence: 99%

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes

et al. 2018

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“…In B. oleracea L. var. capitata, plant regeneration from protoplast-derived cells and somatic fusion has also been reported [6][7][8][9]. Although much effort has been devoted to the elaboration of efficient protocols for protoplast culture, plant regeneration is the main problem hindering the utilization of protoplast technology in practice.…”

Section: Introductionmentioning

confidence: 99%

“…Although much effort has been devoted to the elaboration of efficient protocols for protoplast culture, plant regeneration is the main problem hindering the utilization of protoplast technology in practice. The ability of a single cell to regenerate into an organ (organogenesis) or embryo (embryogenesis) is a very complex process that depends on many factors, among which, the genotype of the protoplast donor plays a crucial role [4,6,[9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Exogenously Applied Polyamines Reduce Reactive Oxygen Species, Enhancing Cell Division and the Shoot Regeneration from Brassica oleracea L. var. capitata Protoplasts

Kiełkowska

Adamus

2021

Agronomy

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Polyamines (PAs) are organic molecules that are found in plants and animals. In plants, they are involved in the regulation of cellular growth, apoptosis, rooting, flower development, and stress responses. The effect of exogenously applied polyamines on the development of Brassica oleracea L. var. capitata protoplast cultures was studied. Protoplasts were isolated from hypocotyls of 2-week-old seedlings of three accessions and they were cultured in liquid media supplemented with putrescine (Put), spermidine (Spd), and spermine (Spm) at concentrations of 0 (control), 10, 20, and 40 µM. In the very early culture (24 and 48 h), cellular reactive oxygen species levels (ROS) in live cells were monitored using a fluorescent probe. The Put- and Spd-treated protoplasts exhibited lower fluorescence intensities, which corresponded to lower ROS accumulation as compared to the PA-free control. The protoplast viability was affected by the type of polyamine applied rather than its concentration. Put and Spd had a beneficial effect on the mitotic activity of the cultured cells, which was observed in all tested accessions. The highest frequency of shoot organogenesis (21%) was obtained from microcalli derived from the protoplasts cultured on the medium supplemented with 10 µM Put. Analysis of the ploidy level of the regenerants showed that the vast majority were diploids. Our results demonstrated that exogenously applied PAs maintained the viability of B. oleracea L. var. capitata protoplasts by alleviating oxidative stress and stimulating mitotic activity, which further affected the plant regeneration process.

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“…Brassica plants in the Brassicaceae Family are not only a rich reservoir of genes that are valuable for the improvement of cultivated species but also potential medicinal resources as well as beloved table vegetables for containing lots of nutrients such as carotene, dietary fiber and various trace elements [98,99]. Somatic hybridization via protoplast culture of many Brassica plants such as Chinese cabbage, cabbage, cabbage rape, cauliflower and turnip has been reported hitherto concerning germplasm resource, and resistant improvement and multi-parent fusion so on [100][101][102][103]. Broad phenotypic variations were obtained from the asymmetric somatic hybridization of cauliflower "Korso" (Brassica oleracea var.…”

Section: Somatic Hybridization In Department Of Vegetables Cropsmentioning

confidence: 99%

Advance in Cell Cultures, Cell Fusion and Cell Splitting of Medicinal and Edible Plant

Shi,

Wang

2023

Preprint

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With worsening environmental degradation and increasing international demand for human health, plant cell based technology will be more important. The aim of this review is to give an overview regarding key points of current progress and development trend of nature medical and edible plants from the framework of whole plant cell engineering including cell cultures, cell fusion and cell splitting derived biotechnology. Here we sum up three main application trends of medical plants cell cultures covering industrial output, technology upgrading and potential resource development. We also provide classification and typification insights to summarize the development of somatic hybridization in cereal, vegetables crops and other medicinal and edible plants as well as application of artificial polyploidy induction in major medicinal herbs. Development medicinal and edible homologous plants based on modern plant cell engineering will bring us a promising future.

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Tri-parental protoplast fusion ofBrassicaspecies to produce somatic hybrids with high genetic and phenotypic variability

Cited by 5 publications

References 32 publications

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes

Exogenously Applied Polyamines Reduce Reactive Oxygen Species, Enhancing Cell Division and the Shoot Regeneration from Brassica oleracea L. var. capitata Protoplasts

Advance in Cell Cultures, Cell Fusion and Cell Splitting of Medicinal and Edible Plant

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