A New Perspective on Cryotherapy: Pathogen Elimination Using Plant Shoot Apical Meristem via Cryogenic Techniques.

Plant pathogens cause different diseases on crops and industrial plant species that result in economic losses. Pathogen-free plant material has usually been obtained by traditional procedures such as meristem culture, thermotherapy, and chemotherapy. However, there are many limitations of these procedures such as mechanical challenges of meristem excision and low regeneration rate, low resistance to high temperatures, phytotoxicity, and mutagenic effects of the chemicals used in the procedures. Cryotherapy is a newly developed biotechnological tool that has been very effective in virus elimination from economically important plant species. This tool has overcome the abovementioned limitations. This chapter aims to highlight the importance of the cryogenic procedures (vitrification, encapsulation-vitrification, droplet vitrification, two-step freezing, dehydration, encapsulation-dehydration) in order to generate virus-free germplasm.

Functional Analysis of Coilin in Virus Resistance and Stress Tolerance of Potato Solanum tuberosum using CRISPR-Cas9 Editing.

The role of the nuclear protein coilin in the mechanisms of resistance of potato Solanum tuberosum cultivar Chicago to biotic and abiotic stresses was studied using the CRISPR-Cas9 technology. For the coilin gene editing, a complex consisting of the Cas9 endonuclease and a short guide RNA was immobilized on gold or chitosan microparticles and delivered into apical meristem cells by bioballistics or vacuum infiltration methods, respectively. Editing at least one allele of the coilin gene considerably increased the resistance of the edited lines to infection with the potato virus Y and their tolerance to salt and osmotic stress.

Distribution of potato spindle tuber viroid in reproductive organs of petunia during its developmental stages.

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Thermotherapy, chemotherapy, and meristem culture in banana.

Bananas that provide a staple food to the millions of people are adversely affected by several viruses such as Banana bunchy Top Virus (BBTV), Banana Streak Virus (BSV), and Cucumber Mosaic Virus (CMV). These viruses are known to have a devastating effect on crop production and constraint to the international exchange and conservation of banana germplasm-a cornerstone for breeding new cultivars. The viruses are particularly problematic in vegetative propagated crops, like bananas, because of their transmission in the planting material. Different virus eradication techniques have been developed, such as thermotherapy, chemotherapy, and meristem culture for providing virus-free planting material. Meristem culture proved to be the most effective procedure to eradicate phloem-associated viruses. This method requires isolation of meristematic dome of plant under the aseptic conditions and culture in an appropriate nutrient medium to develop new virus-free plants. Thermotherapy is another widely used virus eradication technique, which is initially carried out on in vivo or in vitro plants and eventually combined with meristem culture technique. The plantlets are initially grown at 28°C day temperature and increase it by 2°C per day until reaches 40°C and the night temperature at 28°C; maintain plants at 40°C for 4 weeks; excise meristem and culture onto the regeneration medium. In chemotherapy technique, antiviral chemical compound Virazole(®) is applied on meristem culture. Combination of these techniques is also applied to improve the eradication rate.

Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine.

The plum pox virus (PPV) and prunus necrotic ringspot virus (PNRSV) cause serious disease problems in stone-fruit trees. In this work, the possibility of obtaining plant material free from these viruses through thermotherapy and meristem-tip culture from infected nectarine shoots (Prunus persica var. nectarina Max, cv. 'Arm King') was studied. In addition, the detection of these viruses in in vitro cultures and young acclimatized plantlets with double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was studied. Meristem-tip explants (0.8-1.3 mm) derived from sprouted buds of winter wood and spring shoots from field grown plants had a 2-5% regeneration response. However, application of thermotherapy to potted nectarine trees (3 weeks at a maximum temperature of 35 degrees C) facilitated excision of longer meristem tips (1.3-2.0 mm) that resulted in a significantly higher regeneration response (38%) in woody plant medium (WPM) without plant growth regulators. Such explants formed multiple shoots with the addition of 8 microM benzylaminopurine and 0.8 microM indoleacetic acid. When they were tested for the presence of PPV and PNRSV, 86% and 81% were found to be virus-free as detected by DAS-ELISA and multiplex RT-PCR, respectively. Individual shoots excised from virus-free cultures readily rooted in vitro (half-strength WPM plus 2 microM indolebutyric acid) and grew to plantlets. The combination of an efficient protocol for virus elimination and the establishment of highly sensitive diagnostics resulted in the production of nectarine plants free from PPV and PNRSV.

Cellular basis of potato spindle tuber viroid systemic movement.

Viroids are small, nontranslatable pathogenic RNAs that replicate autonomously and traffic systemically in their host plants. We have used in situ hybridization to analyze the trafficking pattern of Potato spindle tuber viroid (PSTVd) in tomato and Nicotiana benthamiana. When PSTVd was inoculated onto the stem of a plant, it replicated and trafficked to sink, but not source, leaves. PSTVd was absent from shoot apical meristems. In the flowers of infected plants, PSTVd was present in the sepals, but was absent in the petals, stamens, and ovary. The replicative form of PSTVd was detected in the phloem. Our data demonstrate that (i) PSTVd traffics long distance in the phloem and this trafficking is likely sustained by replication of the viroid in the phloem, and (ii) PSTVd trafficking is governed by plant developmental and cellular factors. The dependency of PSTVd and other viroids on cellular mechanisms for RNA trafficking makes them excellent tools to study such mechanisms.

The gradual reduction of viroid levels in hop mericlones following heat therapy: a possible role for a nuclease degrading dsRNA.

A high concentration of hop latent viroid (HLVd) was detected in infected mericlones of Osvald's hops grown in vitro. This concentration was about 8-fold higher than in leaves of young, field-grown plants, reaching about 30 pg/mg of fresh mass. Treatment of these in vitro-grown plants at high temperature (35 degrees C) for two weeks lead to a dramatic (about 70-90%) decrease of HLVd content. More detailed investigations performed with mericlone 6147 of Osvald 31 showed that HLVd levels decrease gradually during subsequent cycles of heat treatment. A nuclease activity capable of cleaving HLVd and fully double-stranded RNA was shown to increase significantly in hop tissues during thermotherapy cycles, or after the heat shock. The nuclease activity was found to have similar properties to those extracted earlier from tobacco anthers. This enzyme resembles a sugar-unspecific nuclease which has a maximum activity at pH 5.5. Analysis of the activity with viroid and dsRNA showed that both, endo- and exonucleolytic activities were attributable to the enzyme. A strong tissue-specific gradient of viroid (the lowest level in stem apex and the highest level in roots) was observed in young plants, showing a negative correlation with the dsRNAse activity. In senescent plants, the highest viroid concentration was observed in maturated cones and in upper stems. High nuclease activity in the upper stem tissue suggests that viroid RNA must be protected in this tissue against degradation.