Research progress on tissue culture, rapid propagation and virus-free technology of strawberry

Strawberry viral diseases have been identified as a major constraint on the growth and sustainability of China's strawberry industry. This article provides a comprehensive overview of four major viral pathogens: Strawberry Crinkle Virus (SCV), Strawberry Mosaic Virus (SMoV), Strawberry Mild Yellow Edge Virus (SMYEV), and Strawberry Vein Banding Virus (SVBV). It details the symptoms associated with each virus, their transmission mechanisms—particularly through aphid vectors and contaminated propagation materials—and the compounded effects of mixed infections on plant health and yield. The synergistic impact of multiple viral infections is noted to significantly reduce productivity and plant vigor, underscoring the importance of effective virus management strategies. To address these challenges, the study summarizes three primary virus elimination techniques: meristematic tissue culture, meristematic tissue culture combined with thermotherapy, and ultra- low temperature treatment (cryotherapy). Each method is explained in terms of its underlying principles and efficacy. Meristematic culture exploits the virus-free nature of apical meristems, as these tiny, actively dividing tissues often remain uninfected due to their rapid cell division outpacing viral replication. Practically, 0.2-0.5 mm apical tips are excised and cultured, yielding virus- free plantlets in 60%- 70% of cases for Benihoppe strawberry viruses. Meristematic culture combined with thermotherapy enhances virus inactivation through controlled heat exposure—typically 38-40 ℃ for 4-6 weeks—weakening viral particles before meristem excision, boosting success rates to 66%-73%. Cryotherapy exploits the difference in frost resistance between viruses and plant meristems, utilizing ultra-low temperatures (-196 ℃ in liquid nitrogen) to disrupt viral integrity and replication; freezing induces ice crystal formation that ruptures virus- containing cells while preserving meristem vitality, achieving 90% + virus elimination for stubborn pathogens like strawberry mild yellow edge virus. This article conducts a comprehensive and in-depth analysis of the rapid propagation technology in strawberry tissue culture, with a special focus on pinpointing the key factors that have a significant impact on the efficiency and success of the entire process. The physiological state and developmental stage of explants are paramount, as they critically determine the ultimate outcomes of tissue culture. For instance, explants in the vigorous growth phase tend to have higher regeneration potential compared to those in the dormant stage. Hence, it is strongly recommended to select healthy and actively growing plant materials during the appropriate season, such as spring when plants are in a state of robust growth, to maximize survival rates and ensure the successful establishment of cultures. Furthermore, the disinfection duration should be flexibly and appropriately adjusted based on the sampling time and growth condition of the explants. Young and tender explants sampled in the growing season, which are more delicate, require shorter disinfection periods to prevent tissue damage. In contrast, older and tougher explants collected during the dormant period can tolerate a slightly longer disinfection duration to achieve thorough surface sterilization, thereby laying a solid foundation for subsequent tissue culture steps. Additionally, the article investigates the effects of plant growth regulators (PGR) on rooting culture indicators, including root length and rooting percentage. It outlines the essential technical considerations during the pre-transplant hardening and acclimatization phase, aiming to enhance the overall quality of tissue-cultured plants and improve transplant survival rates. Proper acclimatization protocols, including gradual exposure to ambient environmental conditions and appropriate substrate selection, are highlighted as critical steps for ensuring successful establishment of tissue-cultured plantlets in soil. The paper also discusses the optimal medium composition for inducing bud formation in explants, particularly focusing on the selection of plant growth regulator concentrations. It further explores the role of plant growth regulators during the subculture phase, especially their impact on the proliferation coefficient of tissue-cultured plantlets. The study elaborates on how auxins and cytokinins, such as 6-benzylaminopurine (6-BA), indole-3-butyric acid (IBA), and naphthaleneacetic acid (NAA), regulate bud proliferation. It emphasizes that the balance and concentration of these growth regulators are crucial for achieving optimal bud multiplication, and that these parameters should be tailored according to the specific strawberry variety being cultured. For example, in the subculture of Benihoppe strawberry, the optimal combination of plant growth regulatory substances is 1.0 mg · L-1 6-BA+0.2 mg·L-1 IBA, while the optimal growth regulatory substances in the proliferation medium of Ningyu strawberry is 0.1 mg ·L- 1 6-BA+0.1 mg ·L- 1 NAA. The article also addresses common challenges encountered during tissue culture, including incomplete virus elimination, explant browning due to phenolic oxidation, vitrification (glassy, water- soaked shoots), microbial contamination and mutation. The underlying causes of these problems are analyzed, and potential mitigation strategies are proposed, such as optimizing culture media formulations, adjusting environmental conditions, and employing antioxidants or antimicrobial agents. The study highlights the importance of selecting appropriate gelling agents, regulating light intensity and temperature, and maintaining aseptic conditions throughout the culture process. Finally, the author reflects on the future development of strawberry tissue culture technology, emphasizing the need to develop species- and variety-specific protocols that accommodate the genetic diversity of strawberry cultivars. The ultimate goal is to establish a robust, standardized, and efficient system for propagation and virus elimination, thereby supporting the large-scale production of healthy planting materials. This study aims to provide valuable insights and references for researchers and practitioners in the field of strawberry biotechnology, contributing to the advancement of sustainable strawberry cultivation practices.