Establishing a Virus-Free Rapid Propagation System for Strawberry ‘Miaoxiang 7’ Through Anther Culture

Shoot tip culture is currently the most widely used method for strawberry virus elimination, yet its efficiency has approached the theoretical limit of 80–85%. While anther culture offers a higher virus-free rate, it faces the technical bottleneck of low callus differentiation rates. To address this issue, this study used ‘Miaoxiang 7’ strawberry anthers as explants and systematically optimized key culture parameters. Different combinations of cytokinins and auxins were tested across various culture stages—including callus induction, adventitious bud differentiation from callus, proliferation, and rooting—to determine the most efficient plant growth regulator (PGR) formulations. This approach enhanced both the callus induction rate and differentiation efficiency. The regenerated plants obtained in this study achieved a virus-free rate of 98.39%. Flow cytometric ploidy analysis revealed that octoploids constituted the highest proportion, reaching 73.64%, among the regenerated plants. SSR molecular marker analysis indicated a genetic similarity coefficient of 0.9778–1.0000 between the regenerated plants and the maternal parent. Virus-free treatment holds potential for enhancing physiological growth indicators and fruit quality, demonstrating advantages in certain key metrics such as leaf area and soluble solids content. This technological system provides a viable approach for obtaining virus-free plants through anther culture, overcoming the technical limitation of low callus differentiation rates in anther culture. It offers reliable technical support for the sustainable development of the strawberry industry.