INTRODUCTION: Plant tissue culture technology is characterized by growth that is not restricted by seasons, high efficiency in plant propagation, and high survival rates. It can effectively ensure the quality and quantity of new and superior varieties of Xanthoceras sorbifolium, and is beneficial for the conservation and rapid dissemination of germplasm resources, thus promoting the development of the Xanthoceras industry. Therefore, it is now urgent to solve the browning problem of explants during the tissue culture process of Xanthoceras, in order to lay the foundation for establishing a stable and efficient regeneration system for this species.
RATIONALE: Currently, the growth cycle of somatic embryogenesis culture in X. sorbifolium is relatively long, and the acquisition of embryogenic callus tissue is quite challenging, resulting in poor reproducibility of the regeneration system established through somatic embryogenesis. Some studies have directly induced adventitious buds via organogenesis in X. sorbifolium and found that when cotyledons and stem segments are used as explants to induce adventitious buds, browning of the explants is common, leading to a low proliferation rate of adventitious buds and affecting the growth of the explants. Therefore, this study focuses on analyzing the difficulties in inducing adventitious buds during the tissue culture process of X. sorbifolium, explores the most suitable growth conditions for inducing adventitious buds, and discusses the impact of factors such as the type of cytokinin, temperature, light intensity, and the tenderness of the explants on the browning of X. sorbifolium tissue culture, in order to provide a basis for establishing an efficient and stable regeneration system for X. sorbifolium.
RESULTS: The browning issue severely affects the growth condition of X. sorbifolium explants and the efficiency of adventitious bud induction. After identifying the optimal disinfection method and the best medium for adventitious bud induction, we investigated the factors affecting the browning of X. sorbifolium cotyledons one by one. We found that the highest induction rate of adventitious buds was achieved when 2.5 mg∙L^-1 6-BA was added to the medium, and thus we selected it as the suitable cytokinin for inducing adventitious buds. The cotyledons of X. sorbifolium were cultured under the best conditions of a light intensity of 19.5 µmol∙m^-2∙s^-1 and a temperature of 26°C. Although culturing in the dark can inhibit browning, it cannot induce adventitious buds. The tenderness of the explants has an important impact on browning; the more tender the explants are, the lower the degree of browning. When the cotyledons of X. sorbifolium harvested from July 1^st to July 8^th were used for tissue culture, almost no browning occurred. At this time, the fruit coat is not cracked, the cotyledons are plump and firm, the seed coat is white, and it is easy to remove. These cotyledons are the best experimental materials for the tissue culture of X. sorbifolium.
CONCLUSION: Using the cotyledons of X. sorbifolium as the experimental material, the optimal medium for inducing adventitious buds and the factors influencing explant browning were investigated. The results showed that the best disinfection method for cotyledons was to treat them with 75% ethanol for 30 seconds, followed by 0.1% effective chlorine for 10 minutes, resulting in a contamination rate of 29.33% and a mortality rate of 12%. The optimal medium formulation for inducing adventitious buds was MS+2.5 mg∙L^-1 6-BA+1.0 mg∙L^-1 NAA+30 g∙L^-1 sucrose+6.8 g∙L^-1 agar+0.1 g∙L^-1 myo-inositol, with an induction rate of 72.22%. Among different types of cytokinins, the addition of 6-BA to the medium resulted in the lowest explant mortality rate (12.50%) and the highest induction rate (73.61%). The optimal culture conditions were 19.5 µmol∙m^-2∙s^-1 light intensity at 26°C, with an induction rate of 72.22%. The cotyledons from VI stage seeds collected between July 1^st and July 8^th were plump and firm, with white seed coats that were easy to remove, making them the best material for inhibiting browning in Xanthoceras tissue culture, with the highest adventitious bud induction rate (97.22%).

Adventitious bud induction (A) and browning inhibition (B) of Xanthoceras sorbifolium. When establishing a regeneration system for X. sorbifolium through tissue culture methods, severe browning poses a significant challenge. Therefore, through the process of inducing adventitious buds, explant materials that can alleviate the browning of cotyledon tissue culture have been identified. (A) Bar=5 mm; (B) Bar=2 cm