Raspberry, blackberry and shallot are more and more used in our daily food. However, how can we grow good quality of these plants and produce them economically. Raspberry, blackberry and shallot, all are vegetatively propagated. If the mother plants are infected with viruses, then all the next generation plants used for production will also be infected with the same viruses, and the production and quality of the plants will be reduced dramatically. Therefore, we have to find good methods to detect the viruses, and get rid of viruses from the infected mother plants. Once the mother plants are clean, we should keep them healthy, and use them for future production.
Develop efficient virus detection methods. Newly developed methods based on nucleic acid detection of plant viruses will be implemented in the diagnosis of relevant viruses. The establishment of six PCR methods for aphid-transmitted viruses has made it possible to make a survey for these viruses. Field samples for earlier years and from 2019 have shown that Black raspberry necrosis virus is the virus most often associated with mosaic in raspberry leaves.
Eradicate viruses from mother plants. We will eradicate plant viruses by using a therapy (chemical or high-temperature treatment) combined with cutting meristems (small shoot tips)
Preserve clean mother plants with cryopreservation technique. Cryopreservation, storage of living biological samples in liquid nitrogen (LN, -196°C). In this condition, all cellular activities cease, and theoretically, plant materials can be stored without any changes for an indefinite period. In addition, we keep the plant materials away from any disease or pest, preserving the clean mother stock for future purpose. This project will establish and preserve healthy nuclear stock plant material as a basis for innovation.
PCR methods for six raspberry viruses have been established. Establishment of raspberry virus positive controls in tissue culture is ongoing, but virus-infected plants grow bad in tissue culture until now. A better strategy should be considered in order to establish virus-infected materials in tissue culture. NGS has been used for identifying potential virus infections in raspberry surveys. We have established the hardware for bioinformatics analysis, and we are working on the evaluation of potential software for virus detection.
Tissue culture is essential for cleansing plant material for viruses and to establish cryopreservation of the selected cultivars. Tissue culture of different raspberry cultivars has been established (Ninni, Glen Ample, Preussen and Norfolk Giant) from the healthy mother stock at Sagaplant. Experiments with cryopreservation of these materials have been successful. After cryopreservation about 60% of regeneration rate can be achieved. Tissue culture of three black berry cultivars have been established and have been tested for cryopreservation.
In vitro cultures of three shallot genotypes Kverve, 10603, Lunteviga and 10309 have been established. A droplet vitrification cryopreservation protocol was optimized using 10603 in vitro shoot tips. High regrowth rate of shallot 10603 after cryopreservation was obtained (between 50 % to 60 % ). For the two other cultivars the regrowth rates of 45% (Kverve) and 70% (Lunteviga) were obtained. The cryopreserved 10603 was compared with the in vitro grown counterparts with regards to the vegetative growth in vitro and in the greenhouse regarding yield, genetic integrity and the main nutrients. No significant difference has been found regarding to the vegetative growth and bulb production.
Onion yellow dwarf virus (OYDV) and shallot latent virus (SLV) have been detected by ELISA and RT-PCR and was reconfirmed by immunohistochemical staining using OYDV and SLV antiserum. Kverve and 10603 are co infected by both virus while Lunteviga is single-infected by SLV. Both thermotherapy combining meristem culture and thermotherapy combining cryotherapy were implemented to get virus-free shallot clones. Until now, 5 clones of 10603 and 4 clones of Kverve were confirmed as virus-free of both OYDV and SLV using RT-PCR. Those double-tested clones will be transferred to the greenhouse for the final assessment. Regenerated plants of Lunteviga and 10309 have been tested and are under propagation at NIBIO. In May 2019 virus free shallot bulbs of Kverve and 10603 were sent to the collection of genetic resources at NIBIO Landvik, and to Datsja for growing in the mountain areas.
We have had an active cooperation with Norwegian and international project partners.
The project web page at NIBIO: https://www.nibio.no/en/projects/diagnosis-virus-cleaning-and-cryopreservation-of-raspberry-blackberry-and-shallot-rub-al
Dette prosjektet har styrket NIBIO og samarbeidspartnerne sin kompetanse på virus, avansert diagnostikk, vevskultur og kryopreservering. Dette har allerede gjennom prosjektet styrket norsk plantehelse for bringebær og sjalottløk. Prosjektet har økt Sagaplant sin evne til å produsere friskt plantemateriale. Gjennom samarbeidet mellom NIBIO og Sagaplant kan Sagaplant produsere sertifisert bringebærmateriale av beste kvalitet. Gjennom kryopreservering kan både sortsmaterialet og plantehelsestatus bevares og sikres. Et slikt, norskprodusert, friskt plantemateriale gir Gartnerhallen sine dyrkere det beste plantemateriale som utgangspunkt for sin produksjon. Viktige sorter i Norsk Genressurssenter sin samling av norske sorter av sjalottløk er nå sikret. Metodikken som har blitt etablert i dette prosjektet kan nå brukes på resten av samlingen. Prosjektet har blitt gjennomført i samarbeid med internasjonale forskere som ligger i forskningsfronten på sine felt.
In Norway, raspberry is an economically important crop; blackberry and shallot are potential crops for increased production and consumption. All of them are vegetatively propagated, and are often infected with several viruses that cause significant economic losses. Prerequisites for long-term development and production of these crops are virus diagnosis, virus elimination, and preservation of healthy mother stock and important cultivars. NIBIO, in cooperation with Sagaplant, NORGO, Gartnerhallen and The Norwegian genetic resources centre, apply for this knowledge-building project for industry Rub&Al - Diagnosis, virus cleaning and cryopreservation of raspberry, blackberry and shallot.
The purpose of this project is to develop diagnostic tools (PCR and Next Generation Sequencing, NGS) for viruses in Rubus and Allium, to eradicate relevant shallot viruses by meristem culture combined with thermotherapy or chemotherapy, and to cryopreserve healthy nuclear stocks. Establishment of NGS and PCR methods will give us the ability to verify the phytosanitary status of the mother plants. Cryopreservation can keep plants away from field disease and natural disasters, and, most importantly, cost-efficiently secure the healthy status of healthy nuclear stock and plant genetic resources.
This project includes four work packages: 1) Diagnostic tools for viruses; 2) Tissue Culture to eradicate shallot viruses; 3) Cryopreservation; and 4) Survey of virus occurrence and influence.
This project will benefit NIBIO in gaining knowledge and developing diagnostic methods to certify health status of Norwegian production, aid Sagaplant and Gartnerhallen producing certified plant materials and securing nuclear stocks, and ensure Norwegian genetic resource centre acquire a collection and future applications of healthy Norwegian shallots cultivars. This project will establish and preserve healthy nuclear stock plant material as a basis for innovation.