Abstract:

Objective: The tissue culture seedlings of Populus alba × P. glandulosa (84K poplar) that have been preserved through long-term subculture are prone to bacterial infection, leading to problems such as slow growth and low genetic transformation efficiency. However, there has been no report on rapid bacteria elimination technology. This study aims to isolate and identify the bacteria in the tissue culture seedlings of 84K poplar that are infected with bacteria, and to explore an efficient and convenient technology for eliminating bacteria from the tissue culture seedlings, so as to provide technolical reference for the multiplication of tissue culture seedlings of woody plants, their long-term subculture preservation, stress resistance, growth vitality, and the maintenance of an efficient and stable transgenic transformation system. Method: In this study, the tissue culture seedlings of 84K poplar infected with bacteria were used as materials. The strains were isolated and further identified by using 16S rDNA sequencing combined with NCBI-BLAST search. Treatments such as 0.1% mercury chloride, aseptic hydroponics, darkness, and darkness with variable temperature were compared, and in combination with the method of shoot tip culture, the tissue culture seedlings of 84K poplar infected with bacteria were subjected to bacteria elimination treatment. Furthermore, the experiment of callus induction to buds before and after bacteria elimination was compared to evaluate the effect of bacteria elimination. Result: There were three kinds of bacteria (84K-01, 84K-02, 84K-03) in the tissue-cultured seedlings of 84K poplar that were infected by bacteria. Among them, the similarity between 84K-01 and a species of the genus Curtobacterium (CP066341.1) was as high as 99.79%, the similarity between 84K-02 and a species of the genus Williamsia (JQ660098.1) was as high as 99.93%, and the similarity between 84K-03 and a species of the genus Luteibacter (CP077072.1) was as high as 99.86%. The treatments with 0.1% mercury chloride, darkness for 21 days, and darkness at variable temperatures for 21 days, combined with the method of shoot tip culture all had significant effects on eliminating the three types of bacteria in the tissue culture seedlings of 84K poplar (P<0.05). The treatment of darkness at variable temperatures for 21 days combined with the method of shoot tip culture had the best comprehensive effect, with the sterile rate of 51.85%. Further analysis of the callus induction and bud formation ability of the bacteria-eliminated 84K poplar showed that the callus induced from the leaves of the bacteria-eliminated seedlings grew faster and was able to form buds. Conclusion: Three types of bacteria have been isolated and identified from the tissue culture seedlings of 84K poplar infected with bacteria. The treatment of darkness at variable temperatures for 21 days, combined with the method of shoot tip culture has the best elimination effect. With the advantages of simplicity, rapidity, high stability, and no toxicity, this set of methods can be popularized and applied in the elimination of bacteria from the tissue culture seedlings of woody plants infected with bacteria.

Key words: Populus alba × P. glandulosa, isolation and identification, sterilization, dark treatment with variable temperature, shoot tip culture