Cassava vein mosaic virus (CsVMV) is a pararetrovirus that infects cassava plants in Brazil. A promoter fragment isolated from CsVMV, comprising nucleotides -443 to +72, was previously shown to direct strong constitutive gene expression in transgenic plants. Here we report the functional architecture of the CsVMV promoter fragment. A series of promoter deletion mutants were fused to the coding sequence of uidA reporter gene and the chimeric genes were introduced into transgenic tobacco plants. Promoter activity was monitored by histochemical and quantitative assays of β-glucuronidase activity (GUS). We found that the promoter fragment is made up of different regions that confer distinct tissue-specific expression of the gene. The region encompassing nucleotides -222 to -173 contains cis elements that control promoter expression in green tissues and root tips. Our results indicate that a consensus as1 element and a GATA motif located within this region are essential for promoter expression in those tissues. Expression from the CsVMV promoter in vascular elements is directed by the region encompassing nucleotides -178 to -63. Elements located between nucleotides -149 and -63 are also required to activate promoter expression in green tissues suggesting a combinatorial mode of regulation. Within the latter region, a 43 bp fragment extending from nucleotide -141 to -99 was shown to interact with a protein factor extracted from nuclei of tobacco seedlings. This fragment showed no sequence homology with other pararetrovirus promoters and hence may contain CsVMV-specific regulatory cis elements.

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Functional organization of the cassava vein mosaic virus (CsVMV) promoter.

In recent years, the rapid development of somatic cell genetics has made possible the transfer of alien genes over wide taxonomic distances by somatic hybridization. In this review, the potential of somatic hybridization in the breeding of crops within the Brassicaceae, Fabaceae, Poaceae and Solanaceae is discussed. It is evident from these studies that many hybrids, either symmetric or asymmetric, which are fertile have the potential to be used as a bridge between the alien species and the crop. Progeny analysis of some hybrid combinations also reveals intergenomic translocations which may lead to the introgression of the alien genes. Furthermore, fusion techniques enable the resynthesis of allopolyploid crops to increase their genetic variability and to restore ploidy level and heterozygosity after breeding at reduced ploidy level in polyploid crops.

The potential of somatic hybridization in crop breeding

A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. ‘Bright Yellow-2’ (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

Factors affecting the genetic engineering of plants by microprojectile bombardment

Genotype and auxin influence direct somatic embryogenesis from protoplasts derived from embryogenic cell suspensions of Asparagus officinalis L.

Protoplast technology is important in, on the one hand, the application of various cellular methods in plant breeding (i.e. somatic hybridization, cybridization, direct DNA transfer via polyethylene glycol (PEG), electroporation, micro-injection), and various fundamental studies (e.g. on membrane transport, cell compartmentation, the cytoskeleton in relation to the cell cycle and cell division) on the other hand. For practical applications, plant regeneration from protoplasts is a prerequisite. In 1989, regeneration from protoplasts was listed (Roest & Gilissen 1989) for 214 higher plant species (Spermatophyta), representing 97 genera and 31 families. Since then, regeneration procedures for more than 100 higher plant species have been reported. These include many economically important agricultural and horticultural crops, as well as woody plant species. In this paper these new species are listed, supplemented with specific information on the donor tissue used, the culture technique applied, and the type of development of the regenerants. In addition, recent achievements in fundamental aspects of protoplast research, which gradually provide further insight into the genetical, physiological and ultrastructural background of the phenomenon of totipotency of plant cells, will be briefly reviewed.

https://natuurtijdschriften.nl/pub/540899/ABN1993042001001.pdf

L. J. W. Gilissen

Papers

Regeneration from protoplasts-a supplementary literature review