Somatic Embryogenesis Induction and Genetic Stability Assessment of Plants Regenerated from Immature Seeds of Akebia trifoliate (Thunb.) Koidz

Abstract

Akebia trifoliata is a perennial woody plant with considerable potential in nutrition, food, and health, and the production of seedlings with high quality is critical for its economic utilization. Plant regeneration through somatic embryogenesis is a powerful alternative for propagating many plant species. In this study, a simple and practicable protocol was developed for plant regeneration from immature seeds of A. trifoliata via somatic embryogenesis, and the genetic stability of regenerated plants was also assessed. In the somatic embryo (SE) induction stage, the highest frequency of somatic embryogenesis (35.2%) was observed on the WPM medium containing 1.0 mg L−1 of thidiazuron (TDZ) and 1.0 mg L−1 of 6-benzyladenine (6-BA). The concentration of 6-BA was optimized at 1.0 mg L−1 for the proliferation and maturation of the induced SEs, and the combination of 2.0 mg L−1 of indole-3-butyric acid (IBA) and 0.5 mg L−1 of TDZ was the most responsive for root development and plant growth. The leaf morphological characteristics greatly varied among the established plants, and they could be grouped into three plant types, namely the normal type, Type Ι, and Type ΙΙ. Remarkable differences in the number, size, shape, and color of the leaflets were observed among the three types, while their ploidy level was the same via flow cytometry analysis. The Type ΙΙ and the Type Ι plants had the highest and the lowest net photosynthesis rate, transpiration rate, and stomatal conductance among the three groups, respectively, and both had a smaller size of stomatal guard cells than the normal type. Simple sequence repeat (SSR) analysis detected that 41 bands (43.62%) were different from those observed in the wild, indicating a high degree of polymorphism between the regenerants and their donor parent. The obtained plants might hold potential for future genetic improvement and breeding in A. trifoliata, and the established regeneration protocol might serve as a foundation for in vitro propagation and germplasm preservation of this crop.