Location: Horticultural Crops Production and Genetic Improvement Research Unit
Project Number: 2072-30500-002-013-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2026
End Date: May 31, 2030
Objective:
Efficient plant virus elimination protocols are critical for accelerating the deployment of new, elite plant cultivars from breeding programs into large-scale commercial production. This is especially vital for perennial small fruit crops (e.g., blueberry, strawberry, raspberry, blackberry) which, being outcrossing and genetically heterozygous, depend on in vitro clonal propagation. Release of new berry cultivars for nursery and commercial planting demands extensive virus testing, often requiring virus elimination when infections are detected. In Pacific Northwest (PNW) growing environments, insect-, nematode-, and pollen-vectored viruses are prevalent, frequently infecting breeding lines before sufficient data can be collected for release decisions. The perennial nature of these crops further exacerbates this challenge, as they undergo years of outdoor evaluation for health and performance.
The National Clean Plant Network (NCPN) laboratory at the USDA-ARS Horticultural Crops Production and Genetic Improvement Research Unit (HCPGIRU; Corvallis, OR) provides essential virus testing and elimination services to the PNW berry crop industry, supporting public and private breeders and distributing certified disease-free tissue culture plants for wholesale nursery production. Currently, the NCPN laboratory primarily employs heat treatment of plant meristems for virus elimination. However, studies in other clonally propagated crops (e.g., apple, banana, grapevine, potato, sweet potato) indicate that cryotherapy, or a combination of heat treatment and cryotherapy, offers greater efficiency and higher success rates for virus elimination. Adopting improved virus elimination protocols could significantly increase the efficiency of transferring elite plant material from breeding programs to nurseries and enhance NCPN laboratory sustainability by reducing costs and plant maintenance periods.
Specific objectives include:
1. Evaluate the efficacy of meristem cryotherapy protocols (proven successful in other clonally propagated crops) for eliminating common PNW viruses, Raspberry Bushy Dwarf Virus (RBDV) and Strawberry Necrotic Shock Virus (SNSV), in infected raspberry plants, comparing results against current heat treatment methods.
2. Assess the efficacy of meristem cryotherapy protocols (proven successful in other clonally propagated crops) for eliminating common PNW viruses, Blueberry Latent Virus (BIVL) and Blueberry Scorch Virus (BlScV), in infected blueberry plants, comparing results against current heat treatment methods.
3. Optimize cryotherapy protocols to maximize the recovery of healthy, virus-free meristems from both blueberry and caneberry (raspberry/blackberry) plants.
Approach:
Virus elimination protocols will be tested on infected blueberry plants (harboring BIVL and BlScV) and raspberry plants (harboring RBDV and SNSV). These specific viruses were selected due to their prevalence in Pacific Northwest growing environments, significant economic impact on growers, and high infection rates within USDA breeding populations. Infected plant sources are maintained by the NCPN laboratory at the USDA Horticultural Crops Research Laboratory in Corvallis, OR. Shoots from these infected plants will be propagated in vitro to provide standardized source material for shoot tip extraction and protocol testing.
Three distinct virus elimination approaches will be evaluated using in vitro plant cultures:
1. Thermotherapy only (heat treatment): 2 mm shoot tips will be extracted from in vitro plantlets, transferred to fresh tissue culture (EM) media, and incubated for two weeks. Incubation conditions will be 16 hours of light at 39 degrees C and 8 hours of dark at 24 degrees C.
2. Cryotherapy only: <1 mm in vitro shoot tips will undergo a sucrose PCM solution pre-treatment. These pre-treated shoot tips will then be placed in vials containing plant vitrification solution and directly immersed in liquid nitrogen for one hour. Following immersion, shoot tips will be transferred to a recovery solution, held at room temperature for 20 minutes, and then transferred to plates containing sterile regeneration media. They will be dark-treated for one week before returning to light conditions on EM media.
3. Thermotherapy followed by cryotherapy: This approach will combine the thermotherapy protocol (as described in approach 1) with the subsequent cryotherapy protocol (as described in approach 2).
For each approach, shoot tips will be evaluated after two months for survival (indicated by shoot growth or callus production) and their ability to regenerate and produce new growth under standard tissue culture maintenance conditions.
Successfully regenerated plantlets will be transferred to potted media, acclimated, and grown under greenhouse (GH) conditions following standard procedures. The virus infection status of these plants will be assessed using high-throughput sequencing or RT-PCR. Testing will occur four times over two years, including during two dormancy-breaking periods, to meet the minimum testing requirements currently implemented by the NCPN laboratory. Based on these testing results, the rates of recovery for virus-free clones will be determined.