Establishment of rapid propagation system for tissue culture of the fire blight-resistant Pyrus betulifolia rootstock JWKD01

【Objective】Fire blight, caused by Erwinia amylovora, is a host-specific bacterial disease that severely threatens Rosaceae plants. Screening resistant germplasm resources to cultivate resistant varieties is an important way to control this disease. The excellent Pyrus betulifolia rootstock JWKD01, which was screened by the Institute of Agricultural Science of the Second Division of the Xinjiang Production and Construction Corps, exhibits significant resistance to fire blight. However, current production relies primarily on seed propagation, which leads to significant progeny segregation and inconsistent traits. This results in uneven growth of trees, failing to meet the needs of large-scale production and standardized pear orchard establishment. Therefore, the aim of this study was to establish a stable and efficient tissue culture rapid propagation system for fire blight- resistant Pyrus betulifolia rootstocks, providing technical support for the large-scale propagation and application of disease-resistant rootstocks and molecular biotechnology breeding.【Methods】Newly sprouted shoots of the fire blight-resistant Pyrus betulifolia rootstock JWKD01 were collected from the rootstock germplasm nursery during the spring growth period as explants. Firstly, the leaves of the explants were removed, leaving approximately onethird of the petioles. The shoots were cut into about 1.5 cm stem segments with sterilized pruning shears, followed by rinsing under running water for 1-2 hours to remove surface impurities. Secondly, in the clean bench, disinfection treatments with different time gradients were performed using 75% ethanol and 1% NaClO to screen the optimal protocol. Following disinfection, the explants were rinsed three to five times with sterile water and blotted dry using sterile filter paper. They were then inoculated onto MS medium supplemented with 1.0 mg·L-1 6-BA and 0.3 mg·L-1 IBA for primary culture. Once the materials had proliferated to a sufficient quantity, a completely randomized experimental design was adopted for systematic research. The effects of different basic medium types and plant growth regulators (types and concentrations) on subculture proliferation and rooting culture were first investigated. Building on these results, rooting conditions were optimized by setting gradients of dark culture duration. Finally, the effects of different substrate types and ratios on survival rate and growth status were explored after transplantation.【Results】The explant disinfection experiment showed that the combined treatment of 75% ethanol for 60 s followed by 1% NaClO for 15 min produced the lowest total browning rate and contamination rate in Pyrus betulifolia JWKD01 explants, with the highest survival rate reaching 95.56%. The effects of different plant growth regulators on the proliferation were compared, the suitable proliferation medium for fire blight-resistant Pyrus betulifolia JWKD01 was DKW + 1.0 mg·L-1 6-BA + 0.5 mg·L-1 IBA + 0.02 mg ·L-1 NAA. Under this condition, the average plant height of tissue culture seedlings was 2.87 cm, and the proliferation coefficient was 6.19. The effects of basic medium and plant growth regulators were compared on the rooting of tissue culture seedlings, and the results indicated that the suitable rooting medium for fire blight-resistant Pyrus betulifolia JWKD01 was 1/2 MS + 0.5 mg·L-1 IBA + 0.8 mg·L-1 NAA, with a rooting rate of 90.00%, an average number of roots of 2.39, and an average root length of 4.45 cm. Furthermore, analysis of dark culture duration gradients showed that short-term dark pretreatment promoted thicker root formation; prolonged exposure induced finer root morphology while significantly increasing both rooting rate and root number. However, excessive dark duration resulted in underdeveloped roots characterized by excessive fineness, absence of lateral roots, and thereby suppressed overall root system development. A 7-day dark culture treatment exhibited the best comprehensive performance in terms of rooting rate and average root number, with vigorous root activity and statistically significant differences from other treatments. Thus, the optimal rooting condition for tissue culture seedlings of fire blight- resistant Pyrus betulifolia JWKD01 was dark culture for 7 d followed by transfer to regular culture, as this regime effectively promoted rooting of tissue culture seedlings. In addition, studies on the effects of different substrate types on the transplant survival rate and growth status of tissue culture seedlings found that single substrates (pure vermiculite or pure matrix soil) resulted in similar low survival rates (54.23% and 54.17%, respectively), with no significant difference between them but both significantly lower than composite substrates. Seedlings grown in single substrates displayed weak vigor, including yellowing leaves and delayed development of new roots. In contrast, composite substrates significantly improved transplant efficiency. After transplanting in the substrate with a ratio of matrix soil∶vermiculite∶perlite = 7∶2∶1, the survival rate of tissue culture seedlings was 82.22%. Moreover, plants cultivated in this substrate mix grew robustly, characterized by dark green leaves and welldeveloped root systems.【Conclusion】A tissue culture rapid propagation technology system for fire blight-resistant Pyrus betulifolia rootstocks has been established. Suitable explant disinfection methods, subculture proliferation medium, rooting medium and rooting conditions suitable for fire blight-resistant Pyrus betulifolia rootstocks have been screened out. The acclimatization and transplantation of fire blight-resistant Pyrus betulifolia rootstock rooting seedlings have been successfully carried out.