Callus Proliferation Research Articles

In vitro establishment of cell suspension culture of Ceropegia bulbosa for improved production of cerpegin content through elicitation of engineered carbon and ZnO nanoparticles.

The present investigations aimed to conserve C. bulbosa a threatened plant species and for production of cerpegin through cell culture technology using ENP elicitation. Leaf explants were aseptically cultured with normal MS medium-supplemented PGRs BA and NAA various concentrations, and the best callus induction response was recorded on 4.5 + 4.5 μM. The prospective special effects of the ENPs on plant cell cultures are the key part of our study and used to evaluate leaf callus culture proliferation with the reduction of browning, establishment, biomass, and metabolite formation. The CNP concentrations (0, 2, 4, 8, 12 mg/l) are used for the callus proliferation and browning reduction. The cell suspension cultures are also established, and they were elicited with EZnONPs (0, 25, 50, 100, 150 mg/l) for evaluation of biomass, antioxidant, non-antioxidant enzyme activation, toxicity, ROS defense activation, and metabolite development in cell cultures. The metabolite extraction, UV, and NMR characterization confirm that the toxic and nontoxic effect of ENPs on leaf cell cultures varies with high to low concentration.

The effect of MgNPs synthesized by using green method on the produc-tion and proliferation of Capisum annuum callus culture In Vitro

Nanotechnology, consider as one of the newest important methods towards improve the production of callus using plant tissue culture. The objective of the current work was to create a unique callus induction methodology. and proliferation from Capisum annum in vitro. Therefor different concentrations of Mg nanoparticles were used which synthesized using green method. For callus inducement, young foliage, and stem from the sterile germinated seedlings using Sodium hypochlorite (NaOCL) 6% for 10 min were used as explants. They were cultivated in MS media supported by several combinations of kinetin and 2, 4- Dichlorophenoxyacetic. The maximum percentage responsive reached 100% from leaf explants at 2, 4-D 1.5 mg/L and Kin 1.0 mg/L. While 2, 4-D 1.5 mg/L and Kin 0.5 mg/L achieved the highest fresh weight for callus proliferation. The best MgNPs concentration for callus inducement and proliferation of capsicum leaves and stem explants was MS media supported with 1.0 and 0.3 ml/L respectively. While the highest proliferation mean value for callus was recorded at the concentration of 6.0 ml/L MgNPs in MS medium reached 6.1 gm. AFM photographs prove that MgNPs were in nanometer size and had the spheres and the rods shape. The average grain size of the MgNPs was 23.8 to 38.24 nm.

Colchicine induced polyploidy from thallus segments of Kappaphycus alvarezii (Doty) for genetic improvement

Regeneration of polyploidy from young thallus segments of Kappaphycus alvarezii was optimized for genetic improvement. Kappaphycus thallus segment cultured on sterile sea water supplemented with various combinations of Indole acetic acid, Kinetin and Acardian Marine Plant Extract Powder revealed differential response on callus proliferation and development of new thallus. Presence of Acardian Marine Plant Extract Powder (3 mg/l) in combination with Indole acetic acid and Kinetin (0.01 mg/l each) had induced the longest emerging thallus. Exposure of thallus to colchicine at 0.01% with above combination was optimal to induce high frequency regeneration of polyploidy mostly from the meristematic cells. Anatomical study of colchicine induced polyploidy revealed larger cortical cells with irregular thickening of epidermal layer. Phase contrast and Scanning Electron Microscopic study revealed increase in cell size in cortical region with significantly larger number of spherical shaped carrageenan globules in colchicine induced polyploidy than normal thallus. Single cells isolated using enzymatic treatments from colchicine induced polyploidy, shown chromosome number with a ploidy status of 4n ≈ 40. Whereas in normal thallus, only half the number of chromosomes (2n ≈ 20) were observed. Polyploidy were successfully acclimatized gradually using raft method for further evaluation. This is the first report reveals the induction and regeneration of polyploidy in Kappaphycus. The possible application of this finding in genetic improvement of Kappaphycus is discussed.

Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem

BackgroundSomatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution.ResultsWe generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay.ConclusionsThis study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Fındıkta Dilciksiz Aşı Tekniğinde Kaynaşmanın Anatomik ve Histolojik Gelişimi

The anatomical and histological development of the graft union in splice grafting in hazelnut (Corylus avellana L.) was examined. One-year suckers were used as rootstock and scion materials. Grafting was done manually in the first week of December. The grafted plants were kept for 28 days at 26-28 °C and 70-80% relative humidity conditions. The cross and longitudinal sections with a thickness of 12-20 microns taken from the graft union area by a rotary microtome 14, 18, 21, 26, 32, 52 ve 140 days after grafting were examined microscopically. Early callus proliferation from rootstock and particularly scion two weeks after grafting was generally slow. The initial cambial differentiations in callus tissues were seen in sections 18 days after grafting. Cambial continuity between rootstock and scion was established 32 days after grafting. The sections in the following periods exhibited that the graft partners were in vascular relationship. The development of the graft union was successfull with all its stages. It was observed that the amount of callus tissue proliferated during the first two weeks after grafting directed the subsequent development of the union. In this respect, it is thought that developing methods that encourage callus formation in the early periods of the union will increase the success in related studies. The anatomical and histological examinations revealed that rootstock and scion thicknesses that are very close to each other and accordingly well matching of graft partners from cambial zones affect the development of union positively and prepare a suitable basis for early vascular differentiation.

Metabolite profiling and screening of callus browning-related genes in lotus (Nelumbo nucifera).

Callus browning is a major drawback to lotus callus proliferation and regeneration. However, the underlying mechanism of its formation remains largely unknown. Herein, we aimed to explore the metabolic and molecular basis of lotus callus browning by combining histological staining, high-throughput metabolomics, and transcriptomic assays for lotus callus at three browning stages. Histological stained brown callus cross sections displayed severe cell death symptoms, accompanied by an obvious accumulation of polyphenols and lignified materials. Widely targeted metabolomics revealed extensively decreased accumulation of most detected flavonoids and benzylisoquinoline alkaloids (BIAs), as well as a few phenolic acids, amino acids and their derivatives in callus with browning symptoms. Conversely, the contents of most detected tannins were significantly increased. Subsequent comparative transcriptomics identified a set of differentially expressed genes (DEGs) associated with the biosynthesis and regulation of flavonoids and BIAs in lotus. Notably, callus browning was coupled with significantly up-regulated expression of two polyphenol oxidase (PPO) and 17 peroxidase (POD) encoding genes, while the expression of ethylene associated genes remained at marginal levels. These results suggest that lotus callus browning is primarily controlled at the level of metabolism, wherein the oxidation of flavonoids and BIAs is crucially decisive.

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.).

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.

Colchicine-sensitivity Test in Cassava Leaf Lobes and its Effect on Callus and Somatic Embryo Formation

Background: Colchicine acts as a polyploidy inducer but at high concentrations, it causes high cell mortality. To improve the efficiency of colchicine polyploidization in cassava, leaf lobes colchicine-sensitivity tests were carried out and LD50 determined at 0.00, 0.05, 0.10, 0.20 and 0.25g/l colchicine concentrations in the varieties Ankrah, Dagati, Tomfa and Tuaka. Methods: Colchicine treated leaf lobes were regenerated into callus on either 8 mg/l 2, 4-D or Picloram. The calli were subsequently regenerated into somatic embryos by NAA. LD50 of 0.09, 0.11, 0.13 and 0.09 mg/L colchicine concentration were determined for Ankrah, Dagati, Tomfa and Tuaka respectively in 2, 4-D. Similarly, LD50 of 0.12, 0.10, 0.14 and 0.10 mg/L were respectively obtained in Picloram. Result: In 2, 4-D, Ankrah and Tuaka were more sensitive to colchicine than Dagati and Tomfa whereas in picloram, Dagati and Tuaka showed more sensitivity. Callus proliferation differed significantly among varieties and influenced by the concentration of colchicine.

A Stable and Efficient Genetic Transformation Method for Embryogenic Callus of Fraxinus mandshurica

Fraxinus mandshurica is a widely used greening and ornamental tree species. However, its genetic transformation system has been hampered by problems such as low transformation efficiency, among others, which can hinder research related to molecular breeding and the analysis of functional genes. Thus, in this study, a novel genetic transformation method for efficient transformation of the embryonic callus of Fraxinus mandshurica was investigated. The method was optimized in terms of factors such as antibiotics, infection solution concentrations, co-culture time, and somatic embryo maturation. The results indicated that the optimal antibiotic concentration was 10 mg·L−1 of hygromycin (Hyg). At this point, the callus proliferation multiple was only 0.12. The highest transformation efficiency was found to be 93.93% when the absorbance of the infection solution concentration at OD600 was 0.4. Interestingly, transformation efficiency was found to be highest (77.9%) at 48 h of co-culture, with a GUS staining rate of 88.23%. The medium for somatic embryo maturation of transformed callus was half-strength MS medium (MS 1/2) containing 60 g·L−1 polyethylene glycol, 1 mg·L−1 abscisic acid, 400 mg·L−1 casein enzymatic hydrolysate (CH), 20 g·L−1 sucrose, 1 g·L−1 activated charcoal, and 5 g·L−1 gellan gum. The medium for somatic embryo germination was MS ½, containing 0.2 mg·L−1 of N-(Phenylmethyl)-9H-purin-6-amine(6-BA) and 5.0 mg·L−1 of gibberellin (GA). These results are of significance for the verification of the gene function and future genetic improvement of Fraxinus mandshurica.

Establishment of Agrobacterium-mediated transformation system to Juglans sigillata Dode 'Qianhe-7'.

An efficient genetic transformation system is of great significance for verifying gene function and improving plant breeding efficiency by gene engineering. In this study, a stable Agrobacterium mediated genetic transformation system of Juglans sigillata Dode 'Qianhe-7' was investigated using callus and negative pressure-assisted and ultrasonic-assisted transformation selection. The results showed that the axillary shoot leaves were suitable to induce callus and the callus proliferation rate could reach 516.27% when induction calli were cultured on DKW medium containing 0.5mgL-1 indole-3-butyric acid, 1.2mgL-1 2,4-dichlorophenoxyacetic acid and 0.5mgL-1 kinetin for 18 d. In addition, negative pressure infection was the optimal infection method with the lowest browning rate (0.00%), high GFP conversion rate (16.67%), and better growth status. To further prove the feasibility of this genetic transformation system, the flavonol synthetase (JsFLS5) gene was successfully transformed into the into leaf-derived callus of 'Qianhe-7'. JsFLS5 expression and the content of total flavonoids in transformed callus were improved significantly compared with the untransformed callus, which proved that we had an efficient and reliable genetic transformation system using leaf-derived callus of Juglans sigillata.

Regeneration of Transgenic Ficus lyrata via Indirect Somatic Embryogenesis and Isolation of Variants for Development of New Cultivars

Ficus lyrata is a popular ornamental foliage plant with unique violin- or guitar-shaped green leaves. In our previous study, a grapevine gene VvMybA1 was introduced into F. lyrata via Agrobacterium-mediated transformation, which resulted in the availability of purple-leaved plants. Since VvMybA1 is a transcription factor, the regeneration of transgenic purple-leaved plants might potentially produce variants with multicolored leaves. The objective of this study was to establish a method for regenerating purple-leaved F. lyrata and determine if variants with different coloration or variegation could be isolated from regenerated populations. Leaf explants derived from a completely purple-leaved transgenic plant were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA). Callogenesis occurred in leaf explants, and a subculture of callus-borne explants on the same medium resulted in callus proliferation and the occurrence of somatic embryos. Somatic embryos were more effectively induced from callus pieces cultured on MS medium supplemented with 8.88 μM of BA and 0.27 μM of NAA. More than 30 embryos were induced per callus piece, and the embryos matured and converted to plantlets. MS medium supplemented with 4.92 μM of indolyl-3-butanoic acid (IBA) greatly improved root development. Plantlets were transplanted into soilless substrate and grown in a shaded greenhouse for morphological evaluation. Nine variants with different degrees of coloration and variegation were isolated from the regenerated populations. Our results suggest that the regeneration of transgenic plants that harbor a transcription factor, such as VvMybA1, could be an additional way of isolating novel variants for the development of new cultivars of ornamental plants.

Development of somatic embryogenesis of mangrove fan palm (Licuala spinosa Thunb.) from culturing immature zygotic embryo

AbstractMangrove fan palm is an endemic plant that grows well and has for a long period been related to traditional ceremonies in southern Thailand. It is an endangered plant with a slow growth and is difficult to propagate in mass production. Tissue culture technique can be used to produce a large number of plants within a short time and to preserve in vitro germplasm resulting in the plant not becoming extinct from natural habitats. Thus, the objectives of this research were to study effects of auxins and embryo preparation on somatic embryogenesis. The sterile immature zygotic embryo was cultured on MS medium with different types and concentrations of auxins. The results showed that 1 mg/l dicamba gave the best results in callus induction at 100% and callus diameter at 4.67 mm after 8 weeks of culture. For callus proliferation, callus chopped at 100 times and transferred to 0.1 mg/l dicamba containing oil palm culture medium gave the highest callus diameter at 10.33 mm after 4 weeks of culture and somatic embryos were also produced. Upon transferring somatic embryos to MS medium with 0.2 M sorbitol, a large number of various developmental stages of secondary somatic embryos were induced within 4 weeks. Therefore, the finding of this study could be highlighted that dicamba and chopping callus have an important role to induce callus and somatic embryo. This is the first report achieved on in vitro propagation of this plant and it will be beneficial for genetic conservation and biotechnological applications in the future.

Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.

Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4h per day for 3days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Reactive oxygen metabolism in the proliferation of Korean pine embryogenic callus cells promoted by exogenous GSH

Exogenous glutathione (GSH) promotes the proliferation of embryogenic callus (EC) cells in Korean pine in the course of somatic embryogenesis, and reactive oxygen species (ROS) may play an important role in regulating the proliferation of EC cells by exogenous GSH. However, the concrete metabolic response of ROS is unclear. In this study, two cell lines of Korean pine with high proliferative potential 001#-001 (F, Fast proliferative potential cell line is abbreviated as F) and low proliferative potential 001#-010 (S, Slow proliferative potential cell line is abbreviated as S) were used as test materials. The responses of ROS-related enzymes and substances to exogenous GSH and L-Buthionine-sulfoximine (BSO) were investigated in EC cells. The results showed that the exogenous addition of GSH increased the number of early somatic embryogenesis (SEs) in EC cells of both F and S cell lines, decreased the amount of cell death in both cell lines. Exogenous addition of GSH promoted cell division in both cell lines, increased intracellular superoxide dismutase (SOD) and catalase (CAT) activities, inhibited intracellular hydrogen peroxide (H2O2), malondialdehyde (MDA) and nitric oxide (NO) production, and increased NO/ROS ratio. In conclusion, the exogenous GSH promoting the proliferation of Korean pine EC cells, the activity of intracellular antioxidant enzymes was enhanced, the ROS level was reduced, and the resistance of cells to stress was enhanced.

EFFECT OF THIDIAZURON AND 2,4-DICHLORO PHENOXY ACETIC ACID ON CALLUS DEVELOPMENT IN TEA CLONE UPASI 9 TDZ AND 2,4-D ON TEA CALLUS INITIATION

A commonly cultivated tea clone, UPASI 9, is currently propagated through cuttings, an endeavor that has been proved to be environmentally hazardous (due to the use of large quantities of excavated soil) and unprofitable. As an alternative, this study highlights tissue culture as a viable option for the robust and easy regeneration strategy for the tea clone UPASI 9. A study was initiated to establish an in vitro culture protocol for Nilgiris tea (Camellia sinensis) by standardizing explant sources, disinfestation methods, and the constitution of culture media. Segments were dissected from greenwood stem (current year growth) internodes of field-growntea plants. Disinfestation was achieved by successive treatments of fungicide (bavistin), 0.1% mercuric chloride, and 7.5% CaCl2 . MS medium supplemented with sucrose (30 g/l), citric acid (100 mg/l), and ascorbic acid (100 mg/l) was used with combinations of the auxins (2,4- dichlorophenoxyacetic acid; 2,4-D) and cytokinin (Thidiazuron; TDZ). It was found that 1.1 mg of 2,4-D and 1.1 mg of TDZ induced the most callus proliferation. Though equal proportions of TDZ (1.1, 2.2, 3.3 mg) and 2,4-D (1.1, 2.2, 3.3 mg) were effective in inducing callus formation, it was noticed that 1.1 mg each of TDZ and 2,4-D made a major impact by reducing the time required for the callus initiation and proliferation

<i>In vitro</i> propagation, shoot regeneration, callus induction, and suspension from lamina explants of <i>Sorbus caloneura</i>

<italic>Sorbus caloneura</italic> has high ornamental and medicinal value but is endangered, and significant effort is required to preserve this natural resource. In this study, the stem and sterilized leaves of <italic>S</italic>.<italic> caloneura</italic> were used to explore the effects of different plant hormones basic medium type, initial callus quality, initial liquid volume, and ratio of old liquid culture medium to new on stem proliferation, regeneration and callus suspension culture. Naphthylacetic acid (NAA) and 6-benzyladenine (6-BA) significantly affected the proliferation of stem tissue. Murashige and Skoog (MS) basal medium supplemented with 1.75 mg/L 6-BA, 0.25 mg/L NAA, and 0.25 mg/L indole butyric acid (IBA) yielded a proliferation rate of 100%, with an average number of adventitious shoots per stem of 4.9. The best callus induction was observed with MS basal medium containing 0.5 mg/L 6-BA, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/L thidiazuron (TDZ). The adventitious shoots were directly induced by MS basal medium containing 5.0 mg/L 6-BA, 0.5 mg/L NAA, and 1.0 mg/L kinetin (KT) and indirectly induced by medium containing 3.0 mg/L 6-BA, 0.3 mg/L NAA, and 0.1 mg/L TDZ. NAA significantly affected rooting rate, with ideal conditions found to be medium supplemented with 0.2 mg/L NAA and 1.5 mg/L IBA. MS basal medium with 4.5 g of calli and 120 mL of liquid culture medium without retention of the original culture medium yielded the best suspension effect for callus proliferation. Taken together, the results of this study lay a foundation for the breeding, preservation of germplasm resources, and genetic transformation of<italic> S</italic>.<italic> caloneura.</italic>

In vitro Induction and Proliferation of Callus in Piper longum L. through Leaf Culture

Piper longum L. (Family Piperaceae) is a well- known health promoter used to treat cough, chronic bronchitis, asthma, and diabetes mellitus. The study is aimed to develop a protocol for callus induction and proliferation in P. longum. The leaf explants from mature plants were cultured on an MS basal medium supplemented with various concentrations of plant growth hormones, viz. 2,4-Dichlorophenoxyacetic acid (2, 4-D), Kinetin (KN), 6-Benzylaminopurine (BAP), and α-Naphthalene acetic acid (NAA), as well as 10% coconut water. In primary culture, the callus was compact and light white. The best callus induction and growth were observed in the MS basal medium containing1.0 mg/L 2,4-D+ 2.0 mg/L KN at 12 weeks of primary culture. At eight weeks of secondary culture, the MS medium containing 2.0 mg/L BAP alone and 0.5 mg/L NAA + 2.0 mg/L BAP and 10% coconut water had the best callus proliferation. Compared to 2,4-D and KN alone, BAP alone supported rapid callus growth in the MS medium. In P. longum, large-scale callus formation from leaf explants could be exploited to produce, isolate, and increase bioactive secondary metabolites for therapeutic purposes.

Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation.

Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L-1 NAA, 0.5 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was optimal for callus induction and MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L-1 and 200 mg·L-1, respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD600 and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora.

Production of podophyllotoxin, kaempferol and quercetin in callogenesis of Dysosma versipellis — an endangered medicinal plant

• Dysosma versipellis is an endangered medicinal herb found in southwest regions of China. • Callus initiation was achieved in B5 salts with 2 mg/L 2,4-D, and 0.2 mg/L kinetin under complete darkness. • The maximum callus biomass proliferation was obtained on B5 basal salts supplemented with 2 mg/L 2,4-D. • Leaf callus showed maximum production of podophyllotoxin, kaempferol and quercetin than that of control. • UPLC analysis revealed that roots and rhizomes were the primary source of secondary compounds accumulation. Dysosma versipellis is an endangered medicinal herb found in southwest regions of China. It has long been used in Traditional Chinese Medicine for general remedies in China. This herb is commonly used as an alternative to Podophyllum species. The large-scale removal of D. versipellis rhizomes from natural habitat continues at higher rates than natural regeneration. This study reports the callus induction from D. versipellis explants, callus proliferation and production of tumor inhibitory compounds such as podophyllotoxin, kaempferol and quercetin. The results revealed that tender leaves of D. versipellis exhibited the highest callus induction efficiency of 86% than other explants tested. The best callus initiation was obtained in Gamborg's medium (B5) augmented with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg/L kinetin, 3% sucrose, 0.1% activated charcoal and 0.7% agar and incubated under complete darkness. The maximum callus biomass proliferation was obtained on B5 basal salts supplemented with 2 mg/L 2, 4-D. The UPLC analysis of leaf callus showed maximum production of podophyllotoxin, kaempferol and quercetin than that of control callus. Furthermore, analysis of whole plant parts for their secondary compounds found roots and rhizomes were the primary source of secondary compounds accumulation. Therefore, this bioactive-enriched callus biomass protocol may be used as a possible technique for sustainable production of podophyllotoxin, kaempferol and quercetin at commercial scale.