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.