The mechanism of bud dehyperhydricity by the method of 'starvation drying combined with AgNO3' in Lycium ruthenicum.

Micropropagation is very important for rapid clonal propagation and scientific research of woody plants. However, the micropropagated materials usually show hyperhydricity, which seriously hinders application of the micropropagation. Lycium ruthenicum is an important species of eco-economic forests. Herein, treatment of 'starvation and drying combined with 30 µM AgNO3' (SDCAg+) removed serious hyperhydricity of L. ruthenicum buds regenerated from its green-inflorescence-explants, and then gene expression, metabolites of various phytohormones, chloroplasts, chlorophyll (Chl) and total soluble proteins of the hyperhydric and dehyperhydric leaves were compared and analyzed. The results suggested that the SDCAg+ treatment might remove hyperhydricity of L. ruthenicum through: reducing water uptake; increasing water loss; up-regulating the expression of chloroplast-ribosomal-protein genes from nuclear genome; down-regulating the expression of cytoplasmic-ribosomal-protein genes; up-regulating the synthesis of the total soluble proteins; restoring the lamellar structure of chloroplast grana and matrix; improving Chl synthesis and reducing Chl metabolism; increasing expression of light-harvesting Chl protein complex genes and content of Chla and b; up-regulating both photosynthesis and starch and sucrose metabolism KEGG pathways; up-regulating abscisic acid, salicylic acid and their signaling; down-regulating cytokinin, jasmonic acid, jasmonoyl-l-isoleucine and their signaling. Also, the above events interact to form a regulatory network of dehyperhydricity by SDCAg+ treatment. Overall, the study indicated key genes/pathways and physiological/subcellular changes involved in dehyperhydricity and then established a dehyperhydric mechanism model of L. ruthenicum. This not only proposed clues for preventing or removing hyperhydricity but also laid foundations for molecular breeding of L. ruthenicum and other species.

A novel micropropagation of Lycium ruthenicum and epigenetic fidelity assessment of three types of micropropagated plants in vitro and ex vitro.

Lycium ruthenicum is an excellent eco-economic shrub. Numerous researches have been conducted for the function of its fruits but scarcely focused on the somaclonal variation and DNA methylation. An efficient micropropagation protocol from leaves and stems of L. ruthenicum was developed in this study, in which not only the leaf explants but also the stem explants of L. ruthenicum were dedifferentiated and produced adventitious buds/multiple shoots on one type of medium. Notably, the efficient indirect organogenesis of stem explants was independent of exogenous auxin, which is contrary to the common conclusion that induction and proliferation of calli is dependent on exogenous auxin. We proposed that sucrose supply might be the crucial regulator of stem callus induction and proliferation of L. ruthenicum. Furthermore, results of methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation somaclonal variation (MSV) of CNG decreased but that of CG increased after acclimatization. Three types of micropropagated plants (from leaf calli, stem calli and axillary buds) were epigenetically diverged more from each other after acclimatization and the ex vitro micropropagated plants should be selected to determine the fidelity. In summary, plants micropropagated from axillary buds and leaves of L. ruthenicum was more fidelity and might be suitable for preservation and propagation of elite germplasm. Also, leaf explants should be used in transformation. Meanwhile, plants from stem calli showed the highest MSV and might be used in somaclonal variation breeding. Moreover, one MSV hotspot was found based on biological replicates. The study not only provided foundations for molecular breeding, somaclonal variation breeding, preservation and propagation of elite germplasm, but also offered clues for further revealing novel mechanisms of both stem-explant dedifferentiation and MSV of L. ruthenicum.