The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis. Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail. Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.

Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh

The grapevine (Vitis spp.) undoubtedly represents one of the woody crops most widely grown in temperate climates, and a highly valuable agricultural commodity. As most of the vegetatively propagated crops, grapevines are exposed to the attacks of a variety of pests and pathogens among which infectious intracellular agents (viruses, viroids, phloem‑ and xylem‑limited prokaryotes) play a major role, causing heavy losses, shortening the productive life of vineyars, and endangering the survival itself of affected vines. The importance of the grapevine industry and the magnitude of the problems caused by these pathogens has generated wide interest which, in turn, has fostered intensive research which has been especially active at the international scale from the late 1950’s onwards.

Directory of virus and virus-like diseases of the grapevine and their agents

The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is a powerful tool for editing plant genomes. Efficient genome editing of grape (Vitis vinifera) suspension cells using the type II CRISPR/Cas9 system has been demonstrated; however, it has not been established whether this system can be applied to get biallelic mutations in the first generation of grape. In this current study, we designed four guide RNAs for the VvWRKY52 transcription factor gene for using with the CRISPR/Cas9 system, and obtained transgenic plants via Agrobacterium-mediated transformation, using somatic embryos of the Thompson Seedless cultivar. Analysis of the first-generation transgenic plants verified 22 mutant plants of the 72 T-DNA-inserted plants. Of these, 15 lines carried biallelic mutations and seven were heterozygous. A range of RNA-guided editing events, including large deletions, were found in the mutant plants, while smaller deletions comprised the majority of the detected mutations. Sequencing of potential off-target sites for all four targets revealed no off-target events. In addition, knockout of VvWRKY52 in grape increased the resistance to Botrytis cinerea. We conclude that the CRISPR/Cas9 system allows precise genome editing in the first generation of grape and represents a useful tool for gene functional analysis and grape molecular breeding.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12832

CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation

Secondary somatic embryogenesis is the phenomenon whereby new somatic embryos are initiated from somatic embryos. Such cultures have been described in at least 80 Gymnosperm and Angiosperm species. In the initial step (primary somatic embryogenesis) such cultures have to be started from plant explants. In general, primary somatic embryogenesis from vegetative plant explants is, indirect and mostly driven by auxin (AUX) or auxin and cytokinin (AUX/CYT) supplemented media, whereas, from zygotic embryos it is direct and driven, to a larger extent, by CYT or growth regulator free media. Primary somatic embryogenesis from floral plant explants is between these two extremes. Indirect and direct somatic embryogenesis should be seen as two extremes of one continuum: in indirect somatic embryogenesis the embryos develop up to the (pre)-globular stage and in direct somatic embryogenesis to mature stages before they are subjected to secondary embryogenesis. In general, secondary embryogenesis requires no growth regulators in species with CYT driven primary embryogenesis. Whereas, continuous exposure to growth regulators is needed in species with CYT/AUX or AUX driven primary embryogenesis.

Secondary somatic embryogenesis and applications in plant breeding.

Callus derived from cultured anthers of Gloryvine ( Vitis vinifera L. x V. rupestris Scheele) contained both haploid (n=19) and diploid (2n=38) cells, but all the plantlets produced in vitro were diploid. The presence of haploid cells in callus derived from anthers was further indicated by the occurrence of nuclei with the 1C-DNA level. Haploid cells were not found in callus from internode explants. Multinucleate pollen grains were observed, but there was strong anatomical evidence that the anther-callus originated primarily from somatic cells of the anthers wall and the connective. There was considerable variation among anther-derived plants in leaf morphology. Progeny of selfed anther-derived plants exhibited segregation for leaf characters indicating heterozygosity and somatic origin. Isoenzyme patterns for indophenol oxidase, leucine amino peptidase and catechol oxidase in anther-derived plants and in the original genotype from which the cultures were established were indistinguishable.

Embryos and plantlets from cultured anthers of hybrid grapevines

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl−1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl−1 indole-3-acetic acid or 0.1 mgl−1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl−1) with indole-3-butyric acid (0.1 mgl−1) was added to NN medium supplemented with casein hydrolysate (250 mgl−1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris.

A protocol for preserving grape embryogenic cultures indefinitely has been defined, and through recurrent cycles of secondary embryogenesis, Vitis rupestris Scheele cultures are still regenerating after 10 years. The morphogenic competence of a sample of 1,204 somatic embryos with such a long history has been evaluated. Within a 15-month-long culture, secondary embryogenesis regeneration reached an average efficiency of 23%, proving that morphogenic competence is retained during prolonged culture times. While the culture maintenance described here was one of the crucial aspects of our genetic transformation protocol, several V. rupestris plants with various exogenous genes have been regenerated during these last 10 years.

Morphogenic competence of Vitis rupestris S. secondary somatic embryos with a long culture history

A highly efficient, reproducible method for somatic embryogenesis induction, plant recovery and embryogenic culture preservation has been developed for cvs Chardonnay and Brachetto a grappolo lungo ( Vitis vinifera ), starting from immature anthers and ovaries. Embryogenic induction efficiency was 2 % and 17 % in anthers for Chardonnay and Brachetto g.l., respectively, and 14 % in ovaries for both cultivars. Embryogenic cultures of both genotypes are still propagating 3.5 years after the initial induction and are still morphogenic. Embryo conversion into plantlets occurred at suitable efficiencies during a 100 d culture for both Chardonnay (37 % and 15 %) and Brachetto g.l. (30 % and 29 %), in the two media tested. Organogenesis was also obtained from cotyledonary leaves of Chardonnay.

/pdf/high-efficiency-somatic-embryogenesis-and-plant-germination-ja1vgtsh2x.pdf

High efficiency somatic embryogenesis and plant germination in grapevine cultivars Chardonnay and Brachetto a grappolo lungo

T-DNA chimeric integration and unexpected transgene expression are relevant constraints affecting transgenic plants. This study aims to properly investigate the occurrence of these events and to what extent they may be related. The final goal is to develop an effective screening tool for earlier selection of proper transgenic lines. A strategy based on qPCR and Southern blot was adopted for evaluating gus and Egfp chimerism degree in transgenic Vitis vinifera cv ‘Chardonnay’. Of nine transgenic lines, one had a very high chimerism value, which was shown to be associated with minimal transgene expression. The evaluation of the gus gene over time and space on a line selected as a model showed that transgene’s chimerism was stable and uniform throughout plant tissues whilst its expression was highly variable. Transgene chimerism issue was investigated in detail and useful hints were given for selecting the most favorable transgenic plants and for proper planning of in vitro and ex vitro experiments.

V. Poletti

Papers

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris.

Morphogenic competence of Vitis rupestris S. secondary somatic embryos with a long culture history

High efficiency somatic embryogenesis and plant germination in grapevine cultivars Chardonnay and Brachetto a grappolo lungo

Development of analytical tools for evaluating the effect of T-DNA chimeric integration on transgene expression in vegetatively propagated plants

Genetic transformation of tobacco and grapevine for resistance to viruses related to the rugose wood disease complex