Establishment of an Efficient Tissue Culture Regeneration System from Cotyledons and Hypocotyls of Elaeagnus moorcroftii

INTRODUCTION: Elaeagnus moorcroftii belongs to the genus Elaeagnus of the family Elaeagnaceae, and it is an important native tree species integrating economic and ecological benefits in Northwest China. At present, studies on its tissue culture and in vitro regeneration are still relatively insufficient; only one study carried out direct cluster bud induction using tender stem segments as explants. However, this type of explant is limited in collection and low in regeneration efficiency, which is difficult to meet the needs of large-scale propagation and genetic transformation. Moreover, this rapid propagation method does not involve the dedifferentiation of explants to form callus. Establishing a tissue culture regeneration system through the callus pathway can not only break through the limitations of explant collection mentioned above and realize efficient large-scale rapid propagation, but also take callus as an ideal receptor for subsequent genetic transformation, laying a foundation for target gene manipulation, variety improvement, stress-resistant breeding and other related researches.Taking seeds of E. moorcroftii as materials, this study optimized the disinfection system for sterileseedlings, explored the effects of culture media, explant parts and seedling ages on callus induction, screened the optimal medium for embryogenic callus induction by combining morphological and histocytological identification, established the technical procedures of adventitious bud differentiation, rooting culture, acclimatization and transplantation, and constructed a stable and efficient in vitro regeneration system. This study provides theoretical and technical support for its large-scale seedling raising and subsequent molecular breeding research. RATIONALE: To fill the gap that there is a lack of an efficient tissue culture regeneration system established based on the callus pathway for E. moorcroftii, this study used cotyledons and hypocotyls from sterile seedlings as explants. Through systematic optimization of tissue culture conditions, embryogenic callus was successfully induced. Furthermore, a dual-regeneration system integrating both somatic embryogenesis and adventitious shoot organogenesis was established. Ultimately, a complete technical protocol was achieved, encompassing the entire process from callus induction to the successful acclimatization and transplantation of regenerated plantlets. RESULTS: The results showed that the best disinfection method for E. moorcroftii seeds was 75% alcohol for 1 minute and 0.1% mercuric chloride for 10 minutes, the survival rate reaches 86.77%, and the pollution rate is 2.5%. In the callus induction stage, when using 10-day-old cotyledons as explants and medium A3 (MS+2.0 mg•L–1 6-BA+0.5 mg•L–1 NAA+30 g•L–1 sucrose+6 g•L–1 agar), a relatively high callus induction rate (98.55%) can be obtained, and all the calluses are embryogenic calluses. The most suitable medium for adventitious bud induction was E1 (MS+0.6 mg•L–1 6-BA+0.1 mg•L–1 NAA+30 g•L–1 sucrose+6 g•L–1 agar). The adventitious bud induction rate reaches 63.35%, and the average number of buds is 4.08. The most suitable medium for rooting was S5 (1/2MS+1.0 mg•L–1 IBA+15 g•L–1 sucrose+6 g•L–1 agar+0.5 g•L–1 activated carbon). The rooting rate reaches 68.58%, the average number of roots is 3.79, and the average length of roots is 3.53 cm. The survival rate of rooting seedlings transplanted was above 65%. This study provides certain technical references for the rapid propagation and large-scale production of E. moorcroftii seedlings. CONCLUSION: This study established an efficient callus-based tissue culture regeneration system using cotyledons and hypocotyls from E. moorcroftii sterile seedlings as explants. By optimizing plant growth regulator combinations for callus induction and adventitious bud differentiation, we achieved an embryogenic callus induction rate of 98.55% and an adventitious bud induction rate of 63.35%. These results significantly improve the efficiency of in vitro regeneration, providing a theoretical basis and technical support for the large-scale seedling production of this species. Furthermore, this system lays a solid foundation for the subsequent construction of genetic transformation systems and transgenic breeding research.