A rapid propagation system via tissue culture for Rosa multiflora was established using the stem segments with buds of the current-year as the experimental material. The results showed that the best explants were stem segments with axillary buds. The best disinfection method was to soak the explants in 75% ethanol for 30 seconds, and then soak them in 10% sodium hypochlorite solution for 20 minutes. The survival rate can reach 96%. The optimal bud-induction medium was MS+1.0 mg∙L^-1 6-BA+0.01 mg∙L^-1 NAA+0.1 mg∙L^-1 GA₃. The budding rate can reach 98% after 30 days of cultivation. WPM was the best basal medium for the proliferation of sterile regenerated plantlets, and the proliferation coefficient was 2.87. The best medium for rooting was 1/2MS+1.0 mg∙L^-1 6-BA+0.1 mg∙L^-1 NAA, and the rooting rate can reach 93%. The transplanting survival rate of sterile regenerated plantlets was 98%. On this basis, the transient expression system of R. multiflora was established. The results showed that the optimal transformation conditions for transient expression were OD₆₀₀ of 0.8 for the bacterium culture medium, vacuum negative pressure of -0.10 MPa and vacuum suction twice for 15 minutes each time. The transient expression efficiency can reach 96%. The results of this study laid a foundation for the establishment of regeneration and genetic transformation system of R. multiflora, and also provided technical support for studying on the gene function of Rosa plants.

Due to the long flowering cycle, unpredictable flowering period and low seed setting rate of most bamboo plants, bamboo breeding has been a challenge in the research of bamboo plants. Polyploid breeding, as a common mean of plant breeding, is able to obtain progeny with excellent traits through artificial induction. In bamboo breeding, there are fewer studies on polyploid breeding. In this study, based on the existing regeneration system of Dendrocalamus asper, the embryonic calluses of D. asper were treated with colchicine using the liquid suspension method and the solid medium mixed culture method, respectively. The results showed that, based on the differentiation and browning rates of the calluses, the better results were obtained by treating the calluses with 50 mg∙L^-1 colchicine for 48-72 hours using the liquid suspension method. A total of 54 regenerated plants, including 7 control plants, and 16 chromosome doubled plants were successfully obtained from D. asper using flow cytometry to test all the regenerated plants. In terms of chromosomal doubling, treatment with 100 mg∙L^-1 colchicine for 48 h produced the highest number of chromosome-doubled plants with a polyploidy rate of 54.54%. Compared with 6-ploid plants, the 12-ploid plants presented larger and thicker leaves, and larger lower epidermal stomata, implying their superiority in stress tolerance physiology. This study provides an efficient polyploid breeding technique based on the in vitro indirect regeneration system of bamboo, and offers a new solution for breeding new polyploid germplasm of bamboo.

A large number of software applications for plant identification based on plant images have been developed in recent years. However, those applications are mostly used for identifying the common species countrywide, and thus cannot meet the needs of identifying region-specific vegetation types. In this study, we developed an artificial intelligence model for identifying the dominant plants in Hulunbeier and Xilinhot grassland in Inner Mongolia, based on the image datasets in the Plant Photo Bank of China. The Top5 accuracy of this model reaches 94.6% in the actual field identification tests. Our model provides a new method for the intelligent identification of the major plant species in a specific area.

Cereal starch endosperm is the main source of human staple food, but the methods for observing its mature cell structure are not well developed. Micro CT technology, a non-destructive three-dimensional imaging technique, is a powerful tool for studying plant morphology. However, due to the uniform density of cereal starch endosperm, whose cell structures cannot be distinguished clearly by conventional micro CT techniques. In this study, we used phosphotungstic acid to treat ten different kinds of cereal seeds of seven crops, and then dried them by CO₂ critical point dryer. We found that the cell structure of the treated endosperms can be clearly displayed by micro CT. Our method provides a new way for studying the structures and functions of cereal starch endosperm cells.

Membrane microdomains, which are highly dynamic structures rich in sterols and sphingolipids on the plasma membrane, play crucial roles in various biological processes such as signal transduction, vesicle transport, endocytosis, and exocytosis. Consequently, the investigation of membrane microdomain dynamics is an important area of research in plant cell biology. Fluorescence probes combined with fluorescence microscopy are widely used to monitor the status of living plant cells. The PA probe (push-pull pyrene) is a novel, highly efficient and stable fluorescence probe based on pyrene: however, its application in imaging studies of living plant cells is limited. In this study, we used PA probes and laser scanning confocal microscopy, combined with image processing and the polar normalized value mapping method, to quantitatively analyze the order of the plasma membrane in Arabidopsis root cells. The results revealed that the emission spectrum of the liquid-ordered phase in the plasma membrane of Arabidopsis root cells labeled with the PA probe ranged from 500-550 nm, whereas the emission spectrum of the liquid-disordered phase ranged from 580-700 nm. Treatment of wild-type plants with the sterol extraction agent MβCD resulted in a decrease in plasma membrane order. In the smt2/smt3 double mutant lacking the key methyltransferase in sterol synthesis, the plasma membrane order was consistent with that of the wild-type plants after treatment with MβCD. In the smt2/smt3 mutant, the plasma membrane order of the root hair cells was lower than the plasma membrane order of the wild-type root hair cells, indicating that sterols, as key components of membrane microdomains, play an important role in regulating the order of the plasma membrane. This study provides a straightforward and rapid detection method for monitoring the dynamic characteristics of living plant cell membranes and changes in membrane microdomains.

In this study, the seed embryos of Pyrus calleryana cv. ‘Cleveland’ were used as materials to systematically study the sterilization of seed embryo explants, callus induction, adventitious bud proliferation and rooting medium. The results showed that the germination rate of young embryos stored at 4°C for 21 d was the highest (67.23%) The suitable sterilization treatment of seed embryo explants was 75% alcohol (30 s)+10% H₂O₂ (10 min)+0.1% HgCl₂ (14 min)_. The most suitable medium for seed embryo germination was 1/2MS+4.0 mg·L^-1 6-BA+0.5 mg·L^-1 NAA, and the germination rate was 89.67%. The optimum culture medium for callus induction was 1/2MS+1.0 mg·L^-1 IBA+1.0 mg·L^-1 6-BA, and the callus induction rate was 93.33%. The best differentiation medium was 1/2MS+0.2 mg·L^-1 IBA+2.0 mg·L^-1 6-BA, and the regeneration frequency was 87.44%. Best transgenerational proliferation medium was 1/2MS+1.5 mg·L^-1 6-BA+0.1 mg·L^-1 IBA, and the proliferation rate was 100%. The most suitable medium for root formation was 1/2MS+ 20 g·L^-1 sucrose+1.0 g·L^-1 active carbon+1.5 mg·L^-1 IBA+0.05 mg·L^-1 NAA, and the rooting rate was 82.63%. In summary, the seed embryo regeneration system of P. calleryang cv. ‘Cleveland’ was established, which provides scientific basis and guidan-ce for the efficient reproduction of high-quality pear germplasm resources.

To establish a tissue culture regeneration system for cultivated Chinese yam (Dioscorea polystachya), the effects of different concentrations of plant growth regulators, and stem segments on axillary bud induction and plant regeneration were investigated with different concentrations of plant growth regulators, media types, and shoot tips and stem segments at four different positions under the shoot tip as explants. The results revealed that MS+1.0 mg∙L^-1 6-BA+0.5 mg∙L^-1 KT hormone ratio and 12-20 cm stem segment under the stem tip were the best formulas for the induction of adventitious buds in D. polystachya, and the induction rate reached 90.0%. In the subculture, the optimal concentration was MS+0.5 mg∙L^-1 6-BA+0.05 mg∙L^-1 NAA+0.1 mg∙L^-1 KT, and the proliferation coefficient increased with increasing of 6-BA within a certain range when the low concentration of NAA was constant. With the same concentration of exogenous hormones, DKW medium was significantly better than the conventional 1/2MS medium, and the rooting rate is significantly improved, reaching 92.86%. Adding 1 mg PVP (polyvinylpyrrolidone) and increasing the number of transfers can significantly reduce browning. This study effectively solved the problem of optimizing the explant position for the stable of in vitro propagation of cultivated yam germplasm resources, laying a good foundation for the large-scale production of their high-quality virus-free seedlings.

Atomic force microscopy (AFM) is a powerful tool for studying the ultrastructure and mechanical properties of plant cell walls, while good sample preparation is essential for AFM data acquisition. As micron-scale plant samples, pollen tubes are typical experimental materials for studying the structure and function of cell walls. Due to the difficulty in sample preparation, it is hard to obtain in situ AFM data in physiological state of pollen tubes as pollen tubes are hard to attach to the substrate firmly in fluid. In this study, the AFM preparation and detection methods are optimized using Nicotiana tabacum pollen tubes as the experimental materials. Thin solid medium is used as the adhesive, thereby pollen tubes are attached to the glass slide during germination and elongation in fluidic environment. Compared with the conventional drying-rehydration method, the optimized liquid-attaching method allows observation directly in fluid without drying treatment, which avoids the shrinkage and denaturation of pollen tubes caused by drying-rehydration process. Accordingly, liquid-attaching method can be applied for pollen tubes of different species and sizes, providing support to obtain high-resolution in situ AFM data of pollen tube cell walls in native physiological state under aqueous conditions.

Agropyron mongolicum is a perennial shrub grass in the Triticeae tribe of Poaceae family. It has high forage quality and tolerance to cold, drought, salt and sandstorm. Therefore, it is an excellent grass species for mining stress tolerant genes and restoring deteriorated grasslands. However, a highly efficient transformation system has yet to be established in A. mongolicum. It restricts the identification of genetic resources and the application of genetic improvement of this species. Here, we report an embryogenic callus line (ECL) #89 that was induced from the seeds of A. mongolicum cultivar Mengnong No. 1. Our studies showed the ECL #89 had high regeneration and Agrobacterium-infection efficiencies. An Agrobacterium-mediated transformation system with a transformation efficiency up to 30% was established by infecting the ECL #89 with EHA105. Additionally, we found that subcultures could decrease severely the regeneration capacity of ECL #89, which could be partially restored by adding (1 mg·L^-1) ABA or high concentration (45 g·L^-1) sucrose in the regeneration medium, from 5% to 35% and 42%, respectively. Our work will facilitate the developing of genome editing system, gene function characterization, and molecular breeding in A. mongolicum.

Dracocephalum rupestre is an important medicinal plant. However, its leaf explant regeneration system has yet to be established. Here we investigated the impact of plant growth regulators on callus induction, differentiation, adventitious bud proliferation, and rooting using field planted leaves and tissue-cultured seedling leaves of D. rupestre as explants. We found that the optimal medium for inducing callus from field planted leaves was MS+1.0 mg·L⁻¹ 6-BA+ 0.1 mg·L⁻¹ 2,4-D+1.0 mg·L⁻¹ IAA, achieving an induction rate of 84.51%. For adventitious bud differentiation, the preferred medium comprised MS+3.0 mg·L⁻¹ 6-BA+0.5 mg·L⁻¹ TDZ+0.5 mg·L⁻¹ IAA, resulting in a differentiation rate of 66.37%. Similarly, for inducing callus from tissue-cultured seedling leaves, the optimal medium included MS+2.0 mg·L⁻¹ 6-BA+0.1 mg·L⁻¹ 2,4-D+0.5 mg·L⁻¹ IAA, with an induction rate of 86.73%. The medium conductive to adventitious bud differentiation consisted of MS+2.0 mg·L⁻¹ 6-BA+2.0 mg·L⁻¹ TDZ+0.05 mg·L⁻¹ IAA, yielding a differentiation rate of 53.48%. Furthermore, the appropriate medium for adventitious bud proliferation was MS+2.0 mg·L⁻¹ 6-BA+0.05 mg·L⁻¹ NAA, achieving a proliferation rate of 83.57%. The rooting medium was 1/2MS+0.1 mg·L⁻¹ NAA+0.1 mg·L⁻¹ IBA, resulting in a rooting rate of 86.97%. Tissue-cultured seedlings exhibited optimal growth in a mixed substrate of peat and vermiculite at a ratio of 1:1 (v/v). This study successfully established a leaf explant regeneration system for D. rupestre, providing valuable technical support for the conservation and rapid propagation of its germplasm resources.

Using two different genotypes of Pinguicula cyclosecta and P. ‘Sethos’ as explants, an efficient system for adventitious bud regeneration of Pinguicula was established by studying the factors affecting regeneration. The results showed that the two kinds of Pinguicula had the same requirements for the disinfection scheme and basic medium, but the regeneration ability differed. The best medium for P. cyclosecta leaf regeneration was MS+1.0 mg∙L^-1 6-BA+0.2 mg∙L^-1 NAA, with a regeneration rate of 92.22% and a regeneration coefficient was 4.84. On MS+0.6 mg∙L^-1 6-BA+0.1 mg∙L^-1 NAA media, the proliferation coefficient of adventitious buds reached 4.98. The best medium for the leaf regeneration of P. ‘Sethos’ was MS+2.0 mg∙L^-1 6-BA+0.2 mg∙L^-1 NAA, with a regeneration rate of 77.78% and a regeneration coefficient of 6.12. On the MS+0.3 mg∙L^-1 6-BA+0.1 mg∙L^-1 NAA media, the proliferation coefficient reached 4.84. After root culture (1/2MS+0.1 mg∙L^-1 IBA), the root state of the two kinds of Pinguicula was the best. This study solved the problems of a low reproduction coefficient and difficulty in factory breeding of Pinguicula and provided technical support for its large-scale production and breeding improvement.

In order to establish an in vitro regeneration system for Lunaria annua, its true leaves were used as explants to study the effects of sterilization conditions, combinations and concentrations of plant growth regulator on the induction of callus and on the differentiation of adventitious buds and roots; The effect of rooting methods on the growth of seedlings and young plants was further explored. The results showed that the best disinfection treatment for leaf explants was a combination of 75% alcohol for 45 seconds and 0.1% HgCl₂ solution for 6 minutes. The most suitable medium for induction of callus and differentiation of adventitious buds was MS+0.5 mg∙L^-1 6-BA+2.0 mg∙L^-1 2,4-D. The induction rate of callus reached 93.37% and the differentiation rate of adventitious buds reached 84.08%. The best rooting medium was MS+0.1 mg∙L^-1 NAA, and the time from inoculating leaf explants to obtaining regenerated plants was about 90 days. In this study, a stable regeneration system was established, which laid a foundation for the development and utilization of L. annua resources and digging for functional genes.

An in vitro rapid propagation system using scale buds of Itoh hybrids ‘He Xie’ can overcome the shortcomings of the slow traditional breeding methods and promote the adoption of the excellent Itoh hybrid varieties. In this study, we used the buds of ‘He Xie’ as explants to investigate the effects of various factors including disinfection time, plant growth regulators (PGRs) concentration and root induction time, and different rooted seedling grades on the initiation, proliferation, rooting and domestication of ‘He Xie’ with one-way experimental design. Our results showed: the optimal disinfection time of buds by 2% sodium hypochlorite solution was 12 min, with a contamination rate of 9.09%; the optimal initial culture medium was MS+1.5 mg∙L^-1 6-BA+0.2 mg∙L^-1 GA₃+0.5 mg∙L^-1 AgNO₃; the optimal proliferation culture medium was MS+450 mg∙L^-1 CaCl₂+0.5 mg∙L^-1 6-BA+0.2 mg∙L^-1 IBA+0.2 mg∙L^-1 GA₃+0.5 mg∙L^-1 AgNO₃ with a proliferation rate of 3.3. Rootless seedlings were cultured on root induction medium 1/2MS+1.0 mg∙L^-1 putrescine+2.0 mg∙L^-1 IBA for 8 d at 4°C in dark and then 30 d at room temperature under light, and finally transferred to root formation medium 1/2MS+1.0 g∙L^-1 AC for 20 d, a rooting rate of 66.7% was observed. The rooted seedlings were transplanted on a growing matrix of perlite:vermiculite:charcoal soil=1:1:1 (v/v/v) for 60 d, with the highest transplant survival rate of 52.0% being observed for first-grade rooted seedlings, but most of the second- and third-grade seedlings being dead, suggesting that the quality of rooting is critical for transplant survival.

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.