Chimeric soybean plants with transgenic hairy roots is very important for soybean functional genomics. In this study, we used three soybean genotypes to compare their hairy root induction rate and plant survival rate under different co-cultivation conditions. Our results showed that co-culturing the explants infected by Agrobacterium rhizogenes for 1 d under dark conditions was an effective strategy to induce hairy roots. We also found that removing the adventitious roots (AR) at hypocotyl significantly increased number of hairy roots, enhanced their growth and subsequently improved the positive rate of transgenic hairy roots. Furthermore, we found that the inoculation with rhizobium at 14 d of induction was able to enhance the contact between the bacteria and the transgenic hairy roots at early growth stages, and thus improved the soybean’s nodulation efficiency. Taken together, we successfully established a simple and efficient method to generate chimeric soybean plants with transgenic hairy roots. This method can be widely used in soybean gene functional studies.

To solve the problem of breeding excellent seedlings of Erythropalum scandens, research has been conducted on the establishment and optimization of tissue culture and rapid propagation systems of E. scandens by taking apical bud-induced aseptic seedlings as the material. Explant sterilization, callus induction, callus differentiation, test-tube rooting and transplanting and domestication were studied. The results are as follows: the best ratio of sterilization was 60 seconds of 75% alcohol+10 minutes of 0.1% HgCl₂, and the success rate was 48.89%. The best formula for callus induction by aseptic seedling leaf was MS+0.5 mg·L^-1 6-BA+1.0 mg·L^-1 2,4-D+1.0 mg·L^-1 IBA, for 30 days, and the induction rate was 71.11%, with compact green and strong differentiation potential. The best formula for callus induction by aseptic seedling shoot was MS+1.0 mg·L^-1 6-BA+0.5 mg·L^-1 2,4-D+1.0 mg·L^-1 IBA, for 30 days, and the induction rate was 70.00%; The most suitable medium for induction of callus propagation and differentiation was MS+2.0 mg·L^-1 6-BA+0.5 mg·L^-1 TDZ+1.0 mg·L^-1 IBA, bud differentiation rate was 98.89%, and coefficient of propagation was 3.33. The most suitable medium for rootage was MS+1.5 mg·L^-1 6-BA+0.5 mg·L^-1 IBA, achieving a 100% rootage rate with 2.2 of the average number. Plantlets were transplanted to small particle peat soil, and 88.89% of rooted plants survived. The research has established the tissue culture and rapid propagation system of E. scandens, which can be applied in production and serve as a foundation for providing seedlings and factory production.

Rehmannia henryi is an important plant with great medicinal value, but no research of CRISPR/Cas9 has been done on this species. To establish the gene editing system of R. henryi, the gene encoding phytoene desaturase (PDS) in carotenoid biosynthesis was screened, and the CRISPR/Cas9 vector of RhPDS1 was constructed and transformed into R. henryi genome by Agrobacterium-mediated transformation method. The transcript of RhPDS1 with a 1 764 bp open reading frame (ORF) of RhPDS1 was obtained, the deduced amino acid sequence of RhPDS1 has the typical structural domains of phytoene desaturase. RhPDS1 showed higher expression levels in bud, flower and new leaf. Using CRISPR/Cas9 method, three regenerated shoots with albino phenotype were finally obtained, the differentiation rate of albino shoot was 3.7%. Sequencing analysis revealed that the three albino shoots belong to 2 editing events, in which deletion of 1 bp or (and) 5 bp occurred, respectively, which caused frame shift mutations. The contents of chlorophylls and carotenoids were significantly decreased in the albino mutants as compared to wild type, and the expression levels of RhPDS1 were also decreased in the albino mutants. Taken together, the RhPDS1 gene was cloned and knocked out by using CRISPR/Cas9 method, which laid down the foundations for functional genomics studies and de novo domestication of R. henryi.

Taxus contains the anti-cancer ingredient paclitaxel, is the top-protected endangered plant in China, and has great medicinal value but scarce resources. To solve the problem of paclitaxel source shortage, in this study, an in vitro culture of a taxane-rich Taxus system was established. The results showed that MS+2 mg·L^-1 6-BA+0.4 mg·L^-1 NAA+0.7 g·L^-1 Pro was the optimal treatment for absorption surface expansion, and the expansion rate reached 90%. The adventitious bud high-frequency synchronous growth induction was in the DCR+1 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+5 mg·L^-1 Glu+1 g·L^-1 AC. The best biomass amplification phase I was in MS+0.7 mg·L^-1 6-BA+0.07 mg·L^-1 NAA+0.1 mg·L^-1 Phe+50 mg·L^-1 PG, in which the biomass reached 542 mg. The optimized biomass amplification phase II was obtained in MS+0.5 mg·L^-1 6-BA+0.05 mg·L^-1 NAA+0.1 mg·L^-1 Phe+2 g·L^-1 AC, in which the biomass reached 1 612 mg. The content of taxanes in tissue culture materials is much higher than that of natural materials. The content of paclitaxel in tissue culture materials was 6.1 times that of natural materials. The contoent of baccatin III in tissue culture materials was 8.2 times that of natural materials. The content of 10-DAB in tissue culture materials was 68.1 times that of natural materials. This study established an in vitro culture system of taxane-rich Taxus, and solved the problem of paclitaxel drug shortage.

In order to establish the regeneration system of Citrus australasica, the effects of different plant growth regulators combinations, medium types and dark culture time on callus induction and plant regeneration of C. australasica were studied using stem segments as explants. The results showed that the best medium for adventitious bud induction was 1/2MS+4.0 mg∙L^-1 ZT+30.0 g∙L^-1 sucrose. Dark culture for 14 days and then light culture had the best promoting effect. The induction rate of callus and adventitious bud was 100%, and the average number of adventitious bud regeneration per explants was 4.83. The optimal rooting medium was 1/2MS+0.5 mg∙L^-1 NAA, and the regenerated plants with the 94.43% rooting rate were obtained, and the average number of roots was 3.9. In the mixture of grass carbon:perlite: vermiculite=2:1:1 (v/v/v), tissue culture seedlings had the best growth, and the survival rate was more than 90%. This study established in vitro regeneration system of C. australasica, which laid the foundation for the genetic improvement and rapid propagation of C. australasica fine varieties.

In this study, a plant regeneration system via somatic embryogenesis was established in Alocasia genus. We obtained embryogenic cell suspension cultures of A. reginula through embryogenic calli induced from petioles, and achieved a high frequency of plant regeneration using embryogenic cell aggregates. Efficiency of embryogenic calli induced from petiole explants was highest (81.3%) on a Murashige and Skoog (MS) medium supplemented with 2.0 mg·L^-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg·L^-1 thidiazuron (TDZ). The embryogenic calli were crushed into cell aggregates and then transferred to liquid MS media supplemented with 2.0 mg·L^-1 2,4-D and 1.0 mg·L^-1 TDZ for suspension cultivation. By subculturing biweekly, lots of cell aggregates were gained from embryogenic suspension cultures after 12 weeks. The cell aggregates within 28 weeks of suspension culture were transferred to solid 1/2MS media without plant growth regulator for differentiation culture, with an average of 2.3-2.5 plantlets regenerated from each cell aggregate. The formation and germination of somatic embryos were observed by optical microscopy and scanning electron microscopy (SEM). After the regenerated plantlets were transplanted to a greenhouse for 4 months, the achieved survival ratio was 95.3%. Flow cytometry (FCM) demonstrated that there was no chromosome ploidy variation in randomly selected 50 surviving plants. In addition, the nuclear DNA content was estimated at 10.94 pg·(2C)^-1, and the genome size was 5 290.12 Mb·C^-1. There was no significant variation in their phenotypes from the time the plants were transplanted to the greenhouse until they bloomed spontaneously. These results provide good technical support for the commercial production of seedlings and biotechnological breeding of A. reginula.

In this study, we used the leaflets of marigold (Tagetes erecta) Milestone Yellow as explants to investigate the major factors impacting the transformation efficiency of Agrobacterium-mediated method. The factors included antibiotic concentration, strain type, bacterial concentration, infection time, co-culture time, acetosyringone concentration and anti-browning agent type. We found that the suitable concentrations of cefotaxim sodium salt and kanamycin sulfate were 100 mg·L^-1 and 10 mg·L^-1, respectively. We also found that the strain EHA105 had the highest transformation efficiency up to 4%. The best infection conditions for EHA105 were at bacterial concentration of 0.1 for OD₆₀₀, infected for 5 minutes, and co-cultured for 1 day. In addition, the bud germination rate could be improved by both applying 100 μmol·L^-1 acetosyringone during the infection process and adding 0.2 g·L^-1 polyvinyl pyrrolidone in the screening medium. This study laid a foundation for marigold gene function research and transgenic breeding.

Plasma membrane (PM) proteins are important components of cell membranes and play important roles in material transport, ion exchange, signal transduction, and metabolic process. Their movements in the PM are in response to the developmental cues and environmental stimuli. Studying the regulatory mechanism of PM protein movement is crucial for a better understanding of the plant development and adaptation to environment. In the recent years, the rapid development of microscopic technologies enables us to move one step closer to reveal the regulatory mechanism of PM protein dynamics. In this paper, we systematically summarized PM protein dynamics and the factors affecting it. We also provided an introduction to commonly-used microscopic imaging techniques applied on PM protein dynamics research. This review will serve as a useful reference for the further investigation of biological functions of PM proteins.

Tn5 is a bacterial transposon. The engineered Tn5 can efficiently tag DNA while adding the adapter sequences. Therefore, it has been widely used in the preparation of high-throughput sequencing libraries. Cleavage Under Target & Tagmentation (CUT&Tag) is an improved technology for studying the interaction between protein and DNA, which has the advantages of good repeatability, high signal-to-noise ratio, and easy operation. This technology uses Protein A (pA) or Protein G (pG) and Tn5 to form a fusion protein, which can locate specific antibodies (the antibody is used to identify the target protein) and break the DNA near the target site while introducing sequencing adapters. Then, DNA was extracted, followed by PCR amplification to obtain the sequencing library. However, different types of antibodies have different affinities for pA and pG, thus limiting the application of CUT&Tag for some antibodies. To overcome this limitation, the expression vector of pG-Tn5 was constructed by recombination, and pG-Tn5 recombinant protein was obtained by prokaryotic expression and affinity purification. We used RNA polymerase II (Pol II)-specific antibodies (Pol II Ser5P, mouse IgG1 and rabbit IgG) to compare the efficiency of pA-Tn5 and pG-Tn5 in library preparation of CUT&Tag in Arabidopsis. The results showed that the IgG1 antibody had higher affinity for pG-Tn5, and the quality of the constructed library was better when pG-Tn5 was used. The rabbit IgG antibody has comparable affinities to the two enzymes. A lower starting amount of plant material can be applicable in CUT&Tag. This study provides a reference for the selection of Tn5 fusion proteins against different antibodies in future CUT&Tag experiments.

The technique of identification and preparation of chromosome(s) are important tools in genetic research. Oligonucleotide fluorescence in situ hybridization (oligo-FISH) is an emerging chromosome identification technique in recent years. Flexible and efficient probes are the key factors in the process of oligo-FISH. The labelling process of traditional single-stranded oligo probes (ss-oligos) is complicated and the cost of obtaining individual probes is high. By improving the ss-oligo probes labelling process, we obtain the probes by PCR amplification with specific primers targeting the whole chromosome (fragment), which simplifies the probe labelling process, reduces the cost and improves the labelling efficiency. In this study, we describe in detail the synthesis and labelling of a modified double-stranded labelled oligo probes (ds-oligos) library in rice (Oryza sativa), the preparation of mitotic chromosomes and the hybridization process of ds-oligo probes. By designing gradient experiments, the optimal denaturation time and temperature of chromosome and oligo probe in rice were found to be 85°C for 3.5 min and 90°C for 6 min, respectively. This is the first study to establish a chromosomal double-stranded labelled oligo-FISH system in rice, which provides a powerful tool for the preparation and precise identification of chromosomes in a variety of plants.

Hordeum brevisubulatum is a perennial grass belonging to the genus Hordeum in the Poaceae with strong cold and salt-alkali tolerance, which is an excellent germplasm resource for mining stress resistance genes. However, there are few reports on the genetic transformation system in H. brevisubulatum. In this study, the mature embryos in wild barley (Mengnong 1 hybrid) seeds were employed as explants to induce high quality embryogenic calli. A highly efficient tissue culture system was established for Mengnong 1 hybrid wild barley with approximate 70% of differentiation rate and the propagation coefficient was 35. Furthermore, Agrobacterium tumefaciens strain EHA105 was used to infect the wild barley callus line YZ101, and the infection efficiency was approximate 30% with optimized infection conditions. This study will facilitate gene function deciphering and developing novel germplasm in H. brevisubulatum through molecular design breeding.

Asteraceae plants have high ornamental value due to their rich petal variation in ray florets. Chrysanthemum indicum is a wild species and closely related to C. × morifolium. Ray florets mutants of C. indicum in natural populations, such as flat, spoon and tubular types, are excellent material for studying petal shapes. However, there is currently a lack of research on its regeneration system. In this experiment, considerable ray floret mutants of C. indicum were found on Pingtan Island, Fujian province, including the plants with flat, spoon and tubular types of ray florets coexisting in the same capitulum, in which leaves and transverse thin cell layers (tTCLs) were used as explants to establish a regeneration system. The results showed that the best medium for callus induction and adventitious bud differentiation of tTCLs was MS+1.0 mg∙L^-1 6-BA+0.5 mg∙L^-1 NAA, and the induction rate was 100% on the 14th day. The average differentiation time was 25 days, and the differentiation rate reached 82% on the 40th day. The best rooting medium was 1/2MS+0.5 mg∙L^-1 NAA, and the rooting time was 10 days. All of the rooted and regenerated seedlings were survived after transplanting and kept the mix type in capitulum. An efficient regeneration system for C. indicum in Pingtan with various ray floret forms was established in this experiment. The results not only laid the foundation for the establishment of a transformation system but also provided technology for future research on the morphological variation in ray florets.

Flow cytometry requires preparation of suspensions of intact nuclei, which is crucial step for analysis. A newly formulated buffer, PVPK12-mGB₂ compared with two common buffers (LB01 and CyStain^® PI Absolute P), were used to isolate nuclei from fresh or silica gel desiccated leaf tissues of different species. PVPK12-mGB₂ exhibited the best performance on all fresh leaves tested of plant species, of which the interfering secondary metabolites highly accumulated, the quality of extracted nuclei were able to satisfy demands for flow cytometric analysis. Moreover, results of the present study substantiate the enhanced effectiveness of PVPK12-mGB₂, for silica gel desiccated plant tissue, compared to other buffers tested. The study established an optimal pretreatment of plants for DNA flow cytometry, which provided reference method for sampling and analysis in remote regions.

Switchgrass (Panicum virgatum) is an important C₄ perennial lignocellulosic bioenergy and forage crop. In order to rapidly develop novel germplasm of switchgrass with high cell wall conversion rate, we cloned the ferulic acid 5-hydroxylase gene (PvF5H) from the heterotetraploid switchgrass cultivar Alamo. According to the PvF5H sequence, the editing target sgRNA was designed and used to construct CRISPR/Cas9-PvF5H editing vector. Finally, 59 positive transgenic switchgrass lines were generated by Agrobacterium tumefaciens mediated transformation. Sequencing analysis showed that the PvF5H was edited in most of the transgenic lines (94.9%), and the homozygous editing efficiency was 55.4%. Thus, we have successfully established a highly efficient gene editing system for switchgrass. It will facilitate manipulating target genes involved in cell wall quality and can be employed to breed novel switchgrass cultivars for production of biofuels and fodders in future.

Tea plant is an important economic crop with high heterozygosity and variation. There are few reports on the in vitro regeneration system with high efficiency. In this study, the stem segment of a tea cultivar Shuchazao was used as the initial explants to identify the factors which could efficiently influence the regeneration of adventitious buds. The results showed that MS+2 mg∙L^-1 6-BA was the best formula for normal buds induction, and the induction rate was 84.44%. The expansion rate of the absorption chassis was 80%, which was beneficial to the subsequent induction of adventitious buds. MS+2 mg∙L^-1 6-BA+0.2 mg∙L^-1 NAA+0.1 mg∙L^-1 KT+1 mg∙L^-1 proline was the suitable formula for the induction of adventitious buds, and the induction rate was 88.89%. The average number of shoots was 7.8. 1/2MS+3 mg∙L^-1 IBA was the suitable formula for adventitious root induction, and the rooting rate was 85.56%. The regenerated plants were subjected to molecular verification by RAPD and ISSR techniques. The results showed that no significant variation was found in the two consecutive generations of regenerated plants in vitro.

Plant virus disease is an important factor restricting the safe production of crops. Virus detection can identify viruses and determine the types of viruses, which is the key to disease monitoring, early warning and prevention in crops production. In this study, a detection system based on Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 12a (RPA-CRISPR/Cas12a) was established for potato virus Y (PVY). The results showed that: (1) Cas12a and other components in the CRISPR/Cas12a detection system were necessary for the detection; (2) The target location of crRNA had a significant effect on Cas12a nuclease activity, and the reaction efficiency was the highest when the target of crRNA contained part of PAM site sequence; (3) The minimum detection limit of RPA-CRISPR/Cas12a was 3×10² copies∙μL^-1, which was higher than that of PCR and qPCR methods; (4) The combination of RPA-CRISPR/Cas12a system with crude extraction of nucleic acid and reverse transcription could detect PVY in a non-laboratory setting and the whole process took about 60 minutes. The RPA-CRISPR/Cas12a detection system of PVY established in this study provides an effective method for real-time and rapid visual detection of plant viruses under non-laboratory conditions.

Reactive oxygen species (ROS) are a ‘double-edged sword’ in plants. On the one hand, ROS, as a signal molecule, plays pivotal roles in many aspects of life activities; on the other hand, excessive accumulation of ROS can cause oxidative damage to biological macromolecules. Accurate detection of ROS is essential to assess its intracellular redox status. Due to the characteristics of short half-life and strong reactivity of ROS components, their qualitative and quantitative analysis are difficult. It is critical to select the appropriate detection method and improve the spatiotemporal accuracy of detection for research in plant sciences and in other fields. At present, fluorescent probe analysis has attracted the attention of researchers because of its advantages of high sensitivity, good selectivity, low detection limit and strong intuition. This article introduces the detailed operation protocol and attentions for ROS detection using 2′,7′-dichlorodi-hydrofluorescein diacetate (H2DCFDA) fluorescent probe based on flow cytometry and confocal microscope. These methods can be used to detect ROS levels and distribution in model plant tissues, including Oryza sativa, Arabidopsis thaliana, Zea mays and Glycine max.

Plant transformation is an important tool for genetic engineering. The key technology of genetic transformation is to introduce foreign genes into plants genomes quickly and efficiently and reduce the time to obtain transgenic offspring. Peanut (Arachis hypogaea) is an important oil and cash crop in China. The genetic transformation system is still not well established in peanuts. It seriously restricts the function research of peanut genes and the molecular breeding progress. Here, we established a rapid and stable genetic transformation system in peanuts. The Agrobacterium tumefaciens was injected into the second stem of peanut to produce transgenic plants. Then positive transgenic peanut was transplanted and tamped backfill to cover the injection point. Those seeds above the injection point were picked for further screening and analyzing. The results showed that over 40% of transgenic plants were obtained and displayed chimeric in T₀ generation. The T₀ seeds were harvested about 5 months after rapid-transformation. About 9% of T₁ peanuts were hybrids rather than chimeras. To solve the problem of few seed in some transgenic plants, the rapid-transformation system was combined with traditional tissue culture. This rapid-transformation system has potential value in garlic (Allium sativum), potato (Solanum tuberosum), and freesia (Freesia refracta). Altogether, this study establishes a rapid and stable genetic transformation system for peanuts, which sheds light on other plants’ genetic transformation.

Recent advances in the genomic sequencing of tea plants have laid the foundation for tea research at the molecular and gene levels. However, the transgenic technologies are immature and the life cycles are long for tea trees, it is still difficult to conduct functional analyses of tea genes. This study used young leaves of Camellia sinensis var. sinensis cv. ‘Tieguanyin’, established a useful formula by testing multiple concentration combinations of cellulase, pectinase, macerozyme and mannitol. By evaluating the quantity, viability and debris of resulted protoplast, we successfully established a highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in Tieguanyin seedling leaves, with a transformation rate reaching 56.25%. Using this system, the subcellular localization of two pivotal enzymes in the theanine metabolism pathway (the theanine synthetase (TSI) and the glutamine synthetase (GSII-1.1)) were explored. Results show that, these two enzymes are both localized in the cytosol of the Tieguanyin protoplasts. Together, the establishment of this tea mesophyll protoplast extraction and transient expression system would lay a technological foundation for studying the function of tea genome.

Using the scape as explants, we successfully established the tissue culture and rapid propagation technology for the large-scale production of Hemerocallis fulva cv. ‘Fenmeiren’. Our results showed that the survival rate of explant was 95%, which explants were obtained in June and sterilized in the 15% (v/v) sodium hypochlorite solution for 8 minutes. The best proliferation medium was MS + 1.0 mg·L^-1 6-BA + 0.004 mg·L^-1 TDZ + 0.1 mg·L^-1 NAA. After 30 days of cultivation, the monthly proliferation coefficient reached 2.9. On the medium of MS + 0.1 mg·L^-1 6-BA + 0.1 mg·L^-1 IBA, the tissue culture seedlings did not differentiate and grew vigorously. The optimal rooting medium was 1/2MS + 0.4 mg·L^-1 IBA + 20 g·L^-1 sucrose, and the rooting rate was 95%. by use of perlite:peat=1:2 (v/v) as the transplanting matrix, the survival rate of transplanting was 85% and the qualified rate was 75%. At present, the large-scale production of this cultivar has been achieved and 2.0 × 10⁵ tissue culture seedlings were produced, which all performed well in the field.

In order to establish the tissue culture and regeneration system of wild Anemone silvestris, we investigated effects of different concentrations of plant growth regulators on callus induction, adventitious bud differentiation, proliferation and rooting of different explants including epicotyl, hypocotyl, leaf and petiole. The results showed that adventitious buds could be induced in all four explants, among them the epicotyl was the best. The best medium for hypocotyl induction was 1/2MS+2.0 mg∙L^-1 6-BA+0.1 mg∙L^-1 NAA with the highest induction rate of 86.67%; The optimal medium for proliferation was as follow 1/2MS+1.0 mg∙L^-1 6-BA+0.05 mg∙L^-1 NAA, and the proliferation coefficient was 3.67; The optimal rooting medium was 1/2MS+0.3 mg∙L^-1 IBA with the rooting rate of 100%; In the medium of peat:vermiculite = 2:1 (v/v), the survival rate of tissue culture seedling was at 98.33%. This study effectively solved the shortage of germplasm resources of A. silvestris in garden and medicinal production, and provided technical support for the realization of factory seedling cultivation.

To increase the biomass of suspension culture of Vaccinium dunalianum, we used callus induced from leaves as start material, and investigated the effects of sucrose concentration, pH value, culture medium volume, initial inoculation size, and shaking speed using single factor experiment method. We optimized the suspension culture conditions using Box-Behnken response surface methodology. Our results showed that the optimal conditions for cell suspension culture were: 40 g·L^-1 sucrose, pH5.2, culture medium volume 45 mL, callus inoculation size of 2.64 g, and shaking speed of 149 r·min^-1 based on the improved WPM medium. Under these optimal conditions, the dried cell biomass was 0.184 4 g which was close to the theoretical prediction of 0.184 5 g, and the growth curve of cells showed ‘S’ type. These results provide a basis for further study on the regulation of the secondary metabolites of cell suspension culture in V. dunalianum.

Natural deep eutectic solvent (NADES) was used to extract steviosides from Stevia rebaudiana as an efficient, green and environmentally friendly new method. Compared with traditional solvent water, the optimal NADES formula was selected to extract S. rebaudiana dry leaves by measuring the concentration of stevioside and rebaudioside A, two main components in steviosides. The extraction conditions of NADES extracting steviosides were optimized by Box-Behnken design from response surface methodology. The best NADES formula for steviosides extraction was selected: 1,2-propanediol:glycerol:water=8:1:1 (v/v/v), the extraction concentration of stevioside was 2.59 mg∙mL^-1 (16.40% higher than water), and the extraction concentration of rebaudioside A was 1.06 mg∙mL^-1 (12.62% higher than water). The NADES extraction conditions were optimized by response surface methodology: extraction time was 90 min, extraction temperature was 60°C, ultrasonic power was 80 J∙s^-1, the predicted extraction concentration of stevioside was 3.49 mg∙mL^-1 and the concentration of rebaudioside A was 1.43 mg∙mL^-1, which was close to the experimental verification value (the concentration of stevioside is 3.48 mg∙mL^-1, the concentration of rebaudioside A is 1.42 mg∙mL^-1). Under the best condition, the extraction of stevioside was 34.36% higher than the initial condition, and the extraction of rebaudioside A was 33.96% higher than the initial condition. NADES is environmentally friendly and has higher extraction efficiency than traditional solvents. It can be used for the green extraction of steviosides from S. rebaudiana. At the same time, it provides a promising prospect for the green extraction of natural products from other bulk economic plants.

The effects of different concentrations of hormone on Primula × pubescens tissue culture were studied through callus induction and differentiation, adventitious bud proliferation culture and rooting. The optimal medium at different stages was selected to optimize the regeneration tissue culture system of Primula × pubescens. Our results showed that the best medium for callus induction and differentiation was MS+0.2 mg∙L^-1 NAA+1.0 mg∙L^-1 6-BA, with 84% induction rate and 67% budding rate; the optimal medium for adventitious bud proliferation was MS+0.5 mg∙L^-1 NAA+0.2 mg∙L^-1 6-BA, and the proliferation rate was 67%; MS+0.2 mg∙L^-1 NAA medium was the best for rooting and elongation of tissue culture seedlings. The average number of roots per plant was 9, and the rooting rate was 70%. This study established a highly effect tissue culture and regeneration system of Primula × pubescens, which laid a foundation for genetic research and germplasm innovation of other Primula plants.

The spectral techniques have been widely used in the field of photosynthesis research, such as the light absorption signals P515 and P700 redox kinetics, and chlorophyll fluorescence, which can detect the photosynthetic activities of plants quickly and accurately. P515 signal is widely present in higher plants and algae, which is caused by the shift of absorption spectrum of pigments on thylakoid membrane. We can detect the ratio of PSI to PSII reaction center, the proton conductivity of chloroplast ATP synthase, the cyclic electron flow rate around PSI, the proton motive force (pmf) and its components by the P515 fast and slow kinetics, and study the photoprotective mechanism by simultaneous detection of P515 signal and chlorophyll fluorescence. In this paper, we summarize the main measurement methods of P515, expound its principles, and the applications. The aim is to provide technical supports for further study on the mechanism of photosynthesis.

Using fresh embryos isolated from developing lotus seeds of 18 days after pollination as explants, a rapid propagation system of lotus was established through primary culture, subculture plantlets hardening and transplantation. Results showed that the primary culture medium of MS+0.5 mg·L^-1 6-BA+0.5 mg·L^-1 NAA+30 g·L^-1 sucrose+0.5 g·L^-1 activated carbon+0.8 g·L^-1agar was the optimum for the explant growth. The explant induction rate was up to 85% after cultured for 60 days, Qiuhongyang had the highest amount of stem nodes. In the subculture, the optimal concentration of sucrose in the medium was 80 g·L^-1. When dividing aseptic seedling for subculture, cutting with two stem nodes showed the highest multiplication coefficient. The multiplication coefficient of different varieties was ranged from 4.0 to 6.7, and Qiuhongyang was the highest (6.7). Aseptic seedlings were suggested to be subcultured every 50 days, with high multiplication rates for up to 6 rounds of subcultures. The rooted plantlets were transplanted to pots containing medium of peat:pond soil=1:2 (v/v) during May to July, and the survival rate of transplanted plants was higher than 83.9%. Based on these results, using this in vitro propagation system, it is estimated that 1 465 seedlings be developed from a single lotus seed within one year. This study has established a rapid in vitro propagation system for lotus, which provides the basis for the factory scale production of lotus plantlets.

The anthers of Paeonia lactiflora cv. ‘Fenyunu’ were used as explants to study the effects of different concentrations of 2,4-D on callus induction, somatic embryogenesis and plant regeneration. The cell composition of callus and the development process of somatic embryos were observed with cytohistological method, and the ploidy of regenerated plants was identified using root tip squash method. The results showed that the suitable medium for callus induction of P. lactiflora anther was MS+1 mg·L^-12,4-D+1 mg·L^-1NAA+0.1 mg·L^-1KT+30 g·L^-1sucrose+6.5 g·L^-1agar, and the callus induction rate was 14.7%. The callus was transferred to somatic embryo induction medium and underwent stages of spherical embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo, and the somatic embryo induction rate was 52.1%. Genuine leaves germinated in seedling medium and complete plants were obtained, and the seedling rate was 47.1%. Haploid and diploid plants were identified using root tip squash method. The study preliminarily established a culture system to implement plant regeneration through somatic embryogenesis, which also provided reference protocol for anther culture of other varieties of Paeonia. Regenerated plants are important materials for genetic research and haploid breeding of P. lactiflora.

In order to establish the regeneration system of Cerasus serrulata var. lannesiana cv. ‘Grandiflora’, the effects of different explants and plant hormone combinations on callus induction, adventitious bud differentiation, proliferation and rooting were studied using perennial mother plant leaflets, annual grafted seedling leaflets, axillary bud induction leaflets and proliferative first-generation leaflets as explants. The results showed that callus could be induced from all four explants, and adventitious buds could be derived from all explants except the leaflets of perennial mother plants. The higher the degree of explants’ juvenility, the greater the success of subsequent culture, with the best explants were the proliferative first generation leaflets. The best medium for callus induction was MS+0.5 mg·L^-1 6-BA+1.0 mg·L^-1 2,4-D, and the induction rate was 96.22%. The optimal medium for differentiation was MS+1.0 mg·L^-1 6-BA+0.1 mg·L^-1 2,4-D+0.1 mg·L^-1 TDZ, and the differentiation rate was 78.14%. The optimal medium for proliferation was MS+1.0 mg·L^-1 6-BA, and the proliferation coefficient reached 7.85. The optimal medium for rooting was 1/2MS without any hormone, and the regenerated plants with 100% rooting rate were obtained. The regenerated plants from different explants grew very differently, and those induced from first generation of proliferative leaflets had the best growth.

Establishment of a simple, quick, non-destructive, and continuous sampling procedure, can save planting cost and accelerate gene functional analysis and breeding procedures. In this study, we created a rapid, non-destructive, and continuous sampling technique using micro electric driller and air pump. We also optimized the high throughput DNA extraction method for the 384 deep-well plate and genotyping method. Furthermore, this technique could be applied in rice, maize and other crops for seed sampling and high throughput genotyping.

Elymus sibiricus cv. ‘Chuancao No.2’ is the main cultivated grass species for desertification control and construction of high-yield and high-quality pasture in northwest Sichuan Plateau. In this study, we tested five explants of E. sibiricus cv. ‘Chuancao No.2’ for callus induction, and found that only inflorescence calli were able to differentiate and regenerate. The calli of inflorescence with dense and hard structure cultured for 25 d and 35 d were used for Agrobacterium and biolistic mediated transformation respectively. The results showed that only biolistic-mediated transformation could produce positive transgenic calli of ‘Chuancao No.2’. In the process of biolistic-mediated transformation, the calli was pretreated in two ways: hyperosmotic culture and filter paper drying. The results revealed that the transformation efficiency of filter paper drying was higher than that of hyperosmotic treatment. For the inflorescence callus after 25 d induction, the transformation efficiency under the condition of 2 h drying of filter paper was highest which reached about 40%. In short, we applied the biolistic technology in ‘Chuancao No.2’ for the first time and successfully obtained the positive transgenic inflorescence calli. This work will lead to establishment of the robust transformation system for E. sibiricus in future.

In this study, a simple and stable genetic transformation system of foxtail millet (Seteria italica) was established and optimized, in which shoot tips were used as the explant. We transformed Yugu 1, an elite millet cultivar, by Agrobacterium-mediated transformation, and tested different treatments to boost transformation efficiency. We used a PCR-based assay to screen transformants in third-leaf stage seedlings. We determined an optimal lethal concentration of glufosinate (Basta) when sprayed to millet seedlings, and tested the different PCR-based genotyping methods with or without Basta spary. Using the newly established pipeline, we further optimized various crucial factors that affect genetic transformation efficiency. We found that an optimal concentration of bacterial culture was OD₆₀₀=1.4, an optimal concentration of acetolsyringone was 800 μmol∙L ^-1. We also obtained high transformation efficiency with an infecting pressure at 0.05 MPa, and an infecting time of 40 min. We used the above-mentioned transformation method to transform a Seteria italica calcineurin B-like protein 4 (SiCBL4) overexpression construct. Genetic stability analysis on T₂ generation transformed plants was performed by the combination assay of Basta resistance and real-time quantitative fluorescence RT-PCR, which can save the time of genotyping. Altogether, this study establishes a shoot tip-based stable genetic transformation system for foxtail millets, and also develops a robust pipeline to detect transgenic offsprings.

A regeneration system for organogenesis and somatic embryogenesis in vitro was established by using leaves of Agapanthus praecox as explants, and different cultivation media for transplanting were selected for the best effect. The results showed that picloram (PIC) was effective in callus induction of leaves, and the optimal medium was MS+2.0 mg·L ^-1 PIC. The callus induction rate was determined by the meristematic activity of leaf segments. The callus induction rate of the basal tissues on the 1 ^st-2 ^nd euphyll was 85.71%, and the callus induction rate was 66.48% in meristematic zone of 0-0.5 cm of the same leaf. The results also showed that the callus induction efficiency was higher in the middle of leaf transection compared with that at the edge. The optimal medium for adventitious bud induction was MS+1.5 mg·L ^-1 PIC+0.3 mg·L ^-1 6-BA, and the induction rate was 80.27%. The basic MS medium was suitable for somatic embryo induction, but the induction rate would be significantly increased if 0.05 mg·L ^-1 paclobutrazol and 1.0 mg·L ^-1abscisic acid were added. Plantlets proliferation was promoted by 1.0 mg·L ^-1 6-BA, and the proliferation coefficients of organogenesis and somatic embryogenesis pathway were 2.23 and 2.93, respectively. The combination of peat:perlite: vermiculite=1:1:1 (v/v/v) was proved the suitable substrate for transplanting and acclimatization of plantlets, with a survival rate of 100%. This regeneration system provides a rapid and efficient propagation technology for A. praecox, and also provides a reference for the regeneration of monocotyledon explants in vitro.

Whole mount clearing is a routine method in morphological study, which allows observation of plant internal structure without section. Using high refractive index materials as medium, clearing techniques reduce light scattering, acquire enhancive light quantity and increase depth of field and vertical planes in a particular focal plane, to facilitate the samples transparency for observation. Nevertheless, clearing materials may disturb the osmosis and pH of sample medium, which is adverse to cells morphology. So far, the effective clearing techniques have been widely used in several studies with ovule and leaf. However, the current protocol is not reliable enough for root tip clearing, because the thin cell wall is vulnerable under the treatment of clearing solutions, resulting in abnormal root tips and cells plasmolysis. To achieve a stable and optimized clearing method for root tip, we established a standard protocol via evaluation of root tip morphology, plasmolysis and cells clarity in Arabidopsis thaliana. With these improved clearing methods, we developed an optimized clearing observation system (including clearing time, pH and composition) for root tip, which could provide a reliable technique for vulnerable tissues clearing.

In this study, immature zygotic embryos of Larix olgensis were used as explants to induce embryogenic callus and optimize the regeneration system from somatic embryogenesis. The rejuvenation and preservation of the embryogenic potential of embryogenic callus, the somatic embryogenesis and plant regeneration were investigated through adjusting the nutrition and plant growth regulator. The results showed that the generation rates of embryogenic callus were significantly different among different lines. Under the conditions of S+0.2 mg·L ^-1NAA+0.5 mg·L ^-1BA+0.5 mg·L ^-1KT+0.5 g·L ^-1glutamine+0.5 g·L ^-1hydrolyzed casein+30 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel, the embryogenic potential of embryonic callus could be recovered and maintained for a long time. Somatic embryogenesis were induced from embryonic callus cultured in S+20 mg·L ^-1ABA+60 g·L ^-1PEG₄₀₀₀+60 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel for 6 weeks, and the generation rate of somatic embryo reached 100%. The normal somatic embryos were first cultured for 2 weeks under the conditions of WPM+6 mg·L ^-1phloglucinol+1.0 g·L ^-1active carbon+3.0 mg·L ^-1VB₁+20 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel, and then transferred to B₅+0.4 mg·L ^-1NAA+1.0 mg·L ^-1IBA+0.5 mg·L ^-1GA₃+2.0 mg·L ^-1VB₁+1.0 g·L ^-1active carbon+20 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel. After 2 weeks, somatic embryo plantlets with cotyledon stretch, hypocotyl elongation and normal root system were observed. This study established a method for the recovery and maintenance of embryogenic callus from larch, and further optimized the somatic embryogenic pathway, which will lay a foundation for the rapid breeding and genetic improvement of L. olgensis.

Leymus chinensis is a heterotetraploid grass. It is hard to use genetic transformation method for improving breeding since the efficiency of regeneration using callus induced from mature embryo is very low. In this study, we optimized the protocol of culturing mature embryo with L. chinensis as explants by screening optimum plant hormone concentrations, light conditions and culturing temperatures at different culturing stages including callus induction, differentiation, rooting and transplanting. Our results showed at the callus induction stage, the induction rate could reach up to 74.1% under 2.0 mg·L ^-12,4-D, variable temperature and dark culture. The differentiation rate could reach up to 57.1% with optimum condition of 1.0 mg·L ^-16-BA and 1.0 mg·L ^-1NAA. In the rooting stage, the survival rate was 100% when cultured with 0.25 mg·L ^-1NAA.

Transient gene expression is a favorite tool used for functional analysis of target genes in plant. Of three techniques applied for genetic transformation in the model plant Arabidopsis thaliana, biolistic delivery system was less used than protoplast- or Agrobacterium-mediated transformation. This is mainly due to the smaller leaf size of Arabidopsis, the complicated procedure of bombardment and the limited efficiency and consistency of gene expression. Here, we report applications of an optimized double-barreled particle bombardment system for transient transformation in Arabidopsis, which displayed high expression level of GFP and GUS reporter genes in leaf epidermal cells. By introducing the parallel control in the same shoot by co-bombardment, gene expression efficiency and consistency were dramatically improved, which allows quantitative analysis of target genes with several replicates. Furthermore, cell death inducers BAX, Avh238 or ATR13/Rpp13, were co-expressed with GUS in Arabidopsis rosette leaves and led to strong necrosis phenotypes visualized by significant reduction of number of GUS spots. On the contrary, Avrblb1/RB gene pair triggered strong cell death in Nicotiana benthaminana, but not in A. thaliana. Therefore, this time-saving protocol is an alternative to quantitatively evaluate biological functions of the gene of interest and high-throughput screening of immune suppressors in Arabidopsis and its mutants.

To optimize the suspension culture conditions of Achyranthes bidentata cells, effect of different factors like inoculum concentrations, subculture cycles, pH, light and Cu ²⁺ on cell growth and polysaccharides contents was studied. The results showed that, cells grew well with a good ability for polysaccharides synthesis under 50 g·L ^-1 inoculum concentration, 14 d subculture cycle, pH5-6 and light. In the presence of 50 μmol·L ^-1 Cu ²⁺, the cell dry weight could be the maximum with 44.63 g·L ^-1, and polysaccharides contents reached the peak at 4.02 mg·g ^-1.

SUMOylation, a post-translational modification, is essential for plant normal growth and development. To date, thousands of possible SUMO substrates have been identified, but due to the relatively low SUMOylation level, biological significance of the SUMOylation remains largely unknown. Here, we summarized the SUMOylation detection methods, including in vitro and in vivo SUMOylation assays, which help to understand the functions of SUMOylation in plants.

Protein phosphorylation is one of the important protein posttranslational modifications that is involved in the regulation of most cellular processes in plants. Protein kinases catalyze the phosphorylation by transferring the phosphate group in ATP to the substrate proteins. The phosphate is usually covalently linked to the hydroxyl group of specific amino acid residues in the substrates by an ester bond. The mostly studied phosphorylation sites are serine, threonine, and tyrosine residues. Here, we present protocols and related tips for the in vitro and in vivo protein phosphorylation assays.

Protein-Protein interactions play important roles in various eukaryotic biological processes. Compared to other techniques measuring protein-protein interactions in plants, the Luciferase Complementation Assay (LCA), based on Agrobacterium-mediated transient expression in Nicotiana benthamiana, is a simple, sensitive, reliable, highly quantitative and low background method that can be easily scaled up for high-throughput interactome studies. Here, we describe a protocol that includes two alternative data collection methods to qualitative and quantitative analyse luminescence or luminous intensity to detect protein-protein interactions in plant cells.