The special conditions during in vitro culture result in the formation of plantlets of abnormal morphology, anatomy and physiology. After ex vitro transfer, these plantlets might easily be impaired by sudden changes in environmental conditions, and so need a period of acclimatization to correct the abnormalities. This review is focused upon contemporary information on the changes in leaf structure, water relations and photosynthesis during acclimatization of plantlets to ex vitro conditions. It also describes some ways of improving plant survival and for the speeding up of acclimatization.

Acclimatization of Micropropagated Plants to Ex Vitro Conditions

Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apoptotic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies ('immunotoxins') or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application.

Ribosome-inactivating proteins: progress and problems.

https://flore.unifi.it/bitstream/2158/1073524/1/2016%20Pertot%20A%20critical%20review.pdf

A critical review of plant protection tools for reducing pesticide use on grapevine and new perspectives for the implementation of IPM in viticulture

The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in ‘Chardonnay’ suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.

/pdf/crispr-cas9-mediated-efficient-targeted-mutagenesis-in-4gkm00hgt4.pdf

CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

Downy mildew (DM), caused by pathogen Plasmopara viticola (PV) is the single most damaging disease of grapes (Vitis L.) worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease. Approximately 8.5 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF) library and 14,549 for the control (CON) library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs), which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism. This study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.

https://ufdcimages.uflib.ufl.edu/AA/00/01/39/50/00001/1471-2229-10-234.pdf

Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology.

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.

Transgenic plants of Vitis vinifera cv. Seyval blanc

New Horizons for Grapevine Breeding

Somatic embryos of Seyval blanc, Dornfelder, Re- gent, Riesling, Muller-Thurgau and Rupestris du Lot originating from leaf discs or anther culture were checked with regard to their ability to be cultivated in suspensions for an induction of secondary embryo- genesis Culture parameters like carbohydrates, phytohormones, pH, intervals of media change, the anti-oxidant dithioerythritol (DTE), and the size of embryos were tested to optimize the suspension cul- ture conditions In liquid culture, glucose was better utilized than sucrose or maltose The pH (58 - 50) and the embryo size mainly influenced the quality of em- bryogenic suspensions Medium change proved to be necessary at a pH 4 weeks Browning of media was avoided by addition of 3 or 6 mM DTE Homogeneous suspension cultures were maintained by fragmentation of embryos accord- ing to their developmental stage From suspensions containing embryos of globulary- and heart-stage (05-20 mm) 50 % new somatic embryos were ob- tained whereas further developed embryos (torpedo- stage, >20 mm) were not suitable for cultivation in suspension Best induction of secondary embryos was obtained from Seyval blanc, Dornfelder, Muller-Thurgau and Rupestris du Lot After transfer to a LS- medium the seedlings of Dornfelder and Muller-Thurgau regenerated to rooted in vitro plantlets

/pdf/establishment-of-embryo-suspension-cultures-of-grapevines-it7er7b0nz.pdf

Establishment of embryo suspension cultures of grapevines ( Vitis L.)

An efficient regeneration protocol was developed for leaf explants derived from in vitro plantlets of grapevine cv. Seyval blanc. High frequency somatic embryo induction could be obtained on a modified NN69 medium with 20 µM NOA in combination with the synthetic cytokinin TDZ (4 µM). The application of 2.5 mM phenylalanine promoted embryo induction dramatically by reducing the induction period, and by increasing the regeneration rate. Embryos could be induced on almost 80 % of the plated leaf discs, and the regeneration capacity could be maintained for at least two years. Explants of Vitis thunbergii and the interspecific hybrid Chancellor could now be regenerated by the application of the regeneration system elaborated for Seyval blanc.

/pdf/development-of-a-regeneration-protocol-for-high-frequency-1birmgtdge.pdf

Development of a regeneration protocol for high frequency somatic embryogenesis from explants of grapevines (Vitis spp.)

Somatic embryos originating from anther cultures of Vitis vinifera cvs Dornfelder, Muller-Thurgau and Riesling cultured on solid or in liquid medium were used for transformation via Agrobacterium tumefaciens strain LBA 4404. After two days of cocultivation the embryos proliferated in liquid NN69 medium containing kanamycin for selection and cefotaxim for removal of bacteria. Subsequent cultivation of larger embryos was carried out on solid NN69 medium supplemented with kanamycin. Highest conversion rates of selected germinated embryos to rooted plantlets were obtained from embryos of suspension cultures of Dornfelder and Riesling. Transgenic plants of both varieties were adapted to glasshouse and field conditions.

/pdf/influence-of-culture-technique-and-genotype-on-the-2zzia5mxgw.pdf

Influence of culture technique and genotype on the efficiency of Agrobacterium -mediated transformation of somatic embryos ( Vitis vinifera ) and their conversion to transgenic plants

Margit Harst

Papers

Transgenic plants of Vitis vinifera cv. Seyval blanc

New Horizons for Grapevine Breeding

Establishment of embryo suspension cultures of grapevines ( Vitis L.)

Development of a regeneration protocol for high frequency somatic embryogenesis from explants of grapevines (Vitis spp.)

Influence of culture technique and genotype on the efficiency of Agrobacterium -mediated transformation of somatic embryos ( Vitis vinifera ) and their conversion to transgenic plants