Salinity is recognized as one of the abiotic stresses negetively affecting crop productivity worldwide, which is mainly introduced by the consequence of Na + toxicity. Great advances have been made in screening methodologies for salt tolerant soybean [Glycine max (L.) Merr.] in recent years. But few studies have focused on the evaluation of the relationship of soybean salt tolerance with Na + content in different organs. The objective of our study was to develop a steady, measurable and effective method for salt tolerance evaluation of soybean germplasm based on measuring Na + content in soybean. Twenty nine cultivars were grown in 1/2 Haogland nutrient solution, in which 100 mmol L -1 NaCl was added when the second pair of simple primary leaves fully expanded. The visual foliar symptom was used to evaluate the scale of the salt tolerance. Roots, stems, leaves, and cotyledons were sampled at eight days after salt treatment. Different parts of the plant were measured by the atomic absorption spectrophotometer. Na + content was extremly correlated with the scale of salt tolerance content in stem, leaf and cotyledon but not in root. Clustering for salt tolerant (including scale of 1 and 2) and salt sensitive (including scale of 3, 4, and 5) soybean cultivars at seedling stage based on Na + contents in stem, leaf and cotyledon. The average Na + contents of leaf and cotyledon from the tolerant cultivars were significantly lower than those from the sensitive culitvars. There was significant difference of Na + contents in roots but there was not in stem between tolerant and sensitive soybean. The results indicated that Na + content in leaf and cotyledon can be used for evaluation of salt tolerance in cultivated soybean at the seedling stage. Therefore, the method of evaluating salt tolerant soybean cultivars at the seedling stage by Na + content of leaf and cotyledon in hydroponics, will be used in germplasm identification, gene cloning and cultivar development of salt tolerance in soybean.

De novo shoot regeneration from a callus plays a crucial role in both plant biotechnology and the fundamental research of plant cell totipotency. Recent studies have revealed many regulatory factors involved in this developmental process. However. our knowledge of the cell heterogeneity and cell fate transition during de novo shoot regeneration is still limited. Here, we performed time-series single-cell transcriptome experiments to reveal the cell heterogeneity and redifferentiation trajectories during the early stage of de novo shoot regeneration. Based on the single-cell transcriptome data of 35,669 cells at five-time points, we successfully determined seven major cell populations in this developmental process and reconstructed the redifferentiation trajectories. We found that all cell populations resembled root identities and undergone gradual cell-fate transitions. In detail, the totipotent callus cells differentiated into pluripotent QC-like cells and then gradually developed into less differentiated cells that have multiple root-like cell identities, such as pericycle-like cells. According to the reconstructed redifferentiation trajectories, we discovered that the canonical regeneration-related genes were dynamically expressed at certain stages of the redifferentiation process. Moreover, we also explored potential transcription factors and regulatory networks that might be involved in this process. The transcription factors detected at the initial stage, QC-like cells, and the end stage provided a valuable resource for future functional verifications. Overall, this dataset offers a unique glimpse into the early stages of de novo shoot regeneration, providing a foundation for a comprehensive analysis of the mechanism of de novo shoot regeneration.

Phalaenopsis orchids are popular ornamental plants worldwide. The application of the efficient multiplex genome editing tools in Phalaenopsis , will greatly accelerate the development of orchid gene function and breeding research. In this study, we establish a fast and convenient Phalaenopsis protoplast platform for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9 (PTG, polycistronic tRNA gRNA) system and PTGm-Cas9 (PTG-Cas9 system with modified sgRNA structure) system are designed to edit PDS gene of commercial Phalaenopsis ST166 at four target sites. We find that both PTG-Cas9 and PTGm-Cas9 system are functional in Phalaenopsis , and the PTGm-Cas9 system with modified sgRNA has a higher editing efficiency than PTG-Cas9 system. Further, we design another multiplex genome editing tool, termed as DPⅡ-Cpf1 system (dual Pol II promoter to drive the expression of Cpf1 endonuclease and crRNA), to edit PDS gene of Phalaenopsis at four target sites likewise. All the four targets are efficiently edited by DPⅡ-Cpf1 system, and the total mutation rate is about 3 times higher than that of PTGm-Cas9 system. Taken together, using the Phalaenopsis protoplast platform, we successfully establish two efficient multiplex genome editing tools for Phalaenopsis research, PTGm-Cas9 and DPⅡ-Cpf1. The multiplex genome editing tools established in this study have great application potentials in efficiently constructing large-scale knockout mutant libraries of orchid and speeding up orchid precise breeding.