The effect of 2,4-dichlorophenoxyacetic acid, benzyl amino purin and cupric sulphate on in vitro propagation system from shoot apices of shoot tiller of hybrid Napier grass (Pennisetum purpureum Schum)

An efficient micropropagation method of hybrid Napier grass (Pennisetum purpureumn Schum) for in vitro plant production and material breeding was established from multiple-shoot clumps (MCS) regeneration system. This system was important for forage breeding system. Shoot apices from shoot-tillers produced MSC on Murashige-Skoog (MS) induction medium containing several combinations of BAP and 2,4-D in induction stage. The addition of 5 μM (v/v) and 50 μM (v/v) CuSO4 were added in best medium for inoculation to proliferate the clump in proliferation/multiplication stage. Plant regeneration was achieved by culturing on solid MS with several combination of medium containing NAA and BAP in regeneration stage. The best results for induction were Murashige-Skoog (MS) induction medium containing 2 mgL−1 BAP and 0.1 mgL−1 2,4-D. The proliferation stage on MS medium containing 5 μM CuSÜ4 effective for proliferation (50% multiple shoot formation). The regeneration stage using 0.1 mgL−1 NAA and 2.0 mgL−1 BAP (51.6% number of shoot can regenerate). All plantlets were successfully grown up in an acclimatization stage. Based on the results, the hybrid Napier grass regeneration via MSC was a stable tissue culture system (no albino plats), which could be applied either for further genetic transformation assay or for alternative supply of nursery plant in the future.

Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

Establishment of Tissue Culture Regeneration System for Medicago ruthenica L. cv. ‘Zhilixing’

Background: Medicago ruthenica L. ‘Zhilixing’ is a new variety with superior forage and seed yield compared to the wild type. The cold, drought and salt tolerance of Zhlixing are better than those of alfalfa, suggesting that this variety can serve as a high-quality genetic resource for improving the stress resistance of alfalfa. However, because of the lack of tissue culture regeneration system, it is difficult to perform genetic transformation studies on stress resistance genes. This study aimed to establish an efficient tissue culture regeneration system for Zhilixing variety. Methods: Three types of explants were selected and tested on four types of basal media supplemented with different combinations of auxin and cytokinin for callus induction and differentiation, based on orthogonal tests to select the combinations of auxin and cytokinin suitable for callus induction and differentiation. Two-factor combination method was used to formulate a suitable rooting medium. Result: The hypocotyledonary axis was found to be an excellent explant for callus induction on MS medium. The optimum callus induction medium contained thidiazuron (TDZ, 0.5 mg/L), 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/L) and naphthaleneacetic acid (NAA, 0.5 mg/L) where the callus induction rate was 93.33%. The differentiation medium was supplemented with TDZ (0.75 mg/L), 2,4-D (0.25 mg/L) and 6-benzyladenine (6-BA, 1.5 mg/L) where the differentiation rate was 63.33%. Thidiazuron played the key role in both processes of callus induction and differentiation. Half-strength MS containing 0.1 mg/L of NAA was the most efficient rooting medium.

MEK1/2 Inhibitor (GDC0623) Promotes Osteogenic Differentiation of Primary Osteoblasts Inhibited by IL-1β through the MEK-Erk1/2 and Jak/Stat3 Pathways

Objective. We evaluated the effects and mechanisms of GDC0623 on osteogenic differentiation of osteoblasts induced by IL-1β. Methodology. Osteoblasts were treated with 20 ng/ml IL-1β and 0.1 µM GDC0623. Cell proliferation levels were evaluated by the cell counting kit 8 (CCK8), EdU assay, and western blotting [proliferating cell nuclear antigen (PCNA) and Cyclin D1]. Osteoblasts were cultured in an osteogenic induction medium for 1–3 weeks after which their differentiations were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, calcium concentration, immunocytochemistry staining, real-time quantitative PCR (RT-qPCR), and immunofluorescence staining. The osteogenesis-associated mechanisms were further evaluated by western blotting using appropriate antibodies. Results. Relative to the control group, IL-1β induced the rapid proliferation of osteoblasts and suppressed their osteogenic differentiations by upregulating the activities of MEK-Erk1/2 as well as Jak-Stat3 pathways and by elevating MMP13 and MMP9 levels. However, blocking of the MEK-Erk1/2 signaling pathway by GDC0623 treatment reversed these effects. Conclusion. Inhibition of Jak-Stat3 pathway by C188-9 downregulated the expression levels of MMP9 and MMP13, activated MEK-Erk1/2 pathway, and inhibited osteogenic differentiation.

Effect of Light Conditions on In Vitro Adventitious Organogenesis of Cucumber Cultivars

The response on callus and shoot formation under different light incubation conditions was evaluated in cucumber (Cucumis sativus L.). Four-day-old cotyledon explants from the inbred line 'Wisconsin 2843' and the commercial cultivars 'Marketer' and 'Negrito' were employed. A four-week culture was conducted on MS-derived shoot induction medium containing 0.5 mg L-1 IAA and 2.5 mg L-1 BAP, under an 8-h dark/ 16-h light regime, or by a one- or two-week dark pre-incubation followed by the same photoperiod. Significant differences were obtained for the regeneration of shoots in all cultivars. The response in both frequency and number of shoots under continuous photoperiod was at least 3-6 fold higher than with dark pre-incubation. The highest genotypes response was obtained by 'Negrito' and 'Marketer' with identical values. All explants formed callus, and in two of the three cultivars, the response on callus extension was not significantly affected by incubation conditions. The results clearly show that shoot induction under continuous photoperiod regime was beneficial for adventitious shoot regeneration in cucumber.

Tacrolimus-Induced Neurotrophic Differentiation of Adipose-Derived Stem Cells as Novel Therapeutic Method for Peripheral Nerve Injury

Peripheral nerve injuries (PNIs) are frequent traumatic injuries across the globe. Severe PNIs result in irreversible loss of axons and myelin sheaths and disability of motor and sensory function. Schwann cells can secrete neurotrophic factors and myelinate the injured axons to repair PNIs. However, Schwann cells are hard to harvest and expand in vitro, which limit their clinical use. Adipose-derived stem cells (ADSCs) are easily accessible and have the potential to acquire neurotrophic phenotype under the induction of an established protocol. It has been noticed that Tacrolimus/FK506 promotes peripheral nerve regeneration, despite the mechanism of its pro-neurogenic capacity remains undefined. Herein, we investigated the neurotrophic capacity of ADSCs under the stimulation of tacrolimus. ADSCs were cultured in the induction medium for 18 days to differentiate along the glial lineage and were subjected to FK506 stimulation for the last 3 days. We discovered that FK506 greatly enhanced the neurotrophic phenotype of ADSCs which potentiated the nerve regeneration in a crush injury model. This work explored the novel application of FK506 synergized with ADSCs and thus shed promising light on the treatment of severe PNIs.

The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Research on in vitro haploidization of heterozygotic breeding lines of tomato (Solanum lycopersicum L.)

Experiments were made in order to examin the influence of various factors on the induction of androgenesis in heterozygous breeding material of tomato. The factors like: length of buds the manner of sterilization, type and the composition of induction media, genotype and thermal shock were included in conducted experiments. Most of all sterile cultures with the highest number of anther-derived callus were obtain by applying 2,5% calcium hypochlorite for 5 minutes. Anther-derived calli was obtained in 9 breeding lines from 18 used genotypes. The induction medium with the composition based on B5 medium with the addition of 750 mg L-1 calcium chloride and 100 g L-1 sucrose proved to be the best for inducing androgenesis. The addition of thidiazuron and NAA to this medium in the following season and silver nitrate in the other experiminet improved the efficiency of this process, which was depended on the genotype. Despite the lack of statistically significant differences, the highest number of anther-derived calli was obtained when anthers were cooled for 2 days in +4oC in the refrigerating chamber.

A Reliable Regeneration Method in Genome-Editable Bell Pepper ‘Dempsey’

A reliable regeneration technique is critical for the improvement of pepper traits in the genome editing era. Recently, we reported that peppers were successfully and specifically edited using CRISPR tools, CRISPR/Cas9 and CRISPR/Cas12a (LbCpf1). Although genome-editing tools can be applied to modify peppers at the cellular level, feasible pepper regeneration techniques have not been developed. Therefore, we studied a pepper regeneration protocol for Capsicum annuum L. ‘Dempsey’, a bell pepper species that has been proven to be genome-editable. Three explant types were used in this study, including the first leaves, cotyledons and hypocotyls of pepper seedlings. The shoot buds of the tested explants were produced using 8 mg/L 6-benzylaminopurine (BAP)- and 6 mg/L indole-3-acetic acid (IAA)-containing shoot induction medium (SIM). The first leaves of the ‘Dempsey’ seedlings showed an average shooting rate of 69.8%, whereas the hypocotyls and cotyledons had approximately 25.5% and 19.5% shooting rates, respectively. The regenerated ‘Dempsey’ plants exhibited no alterations in fruit and fertile seed phenotypes. Furthermore, the parent ‘Dempsey’ and progenies of the regenerants were cytogenetically stable with the same chromosome numbers (2n = 24). Therefore, this regeneration protocol enables the precise molecular breeding of ‘Dempsey’ peppers when coupled with CRISPR tools.

EFFECT OF 2,4-D AND NAA ON SOMATIC EMBRYOGENESIS FROM APICAL PORTION OF GROUNDNUT (ARACHIS HYPOGAEA L.)

Somatic embryogenesis was carried out epicotyl portion of the mature embryo/apical portion. The somatic embryo induction medium containing 2,4-D or NAA (10.0 to 50.0 mg/l). Of the two concentrations tested 2,4-D (30.0mg/l) recorded the highest percentage of response followed by NAA (30.0mg/l). But the highest number of somatic embryo were recorded in 30.0mg/l of 2,4-D followed by NAA. The apical portion of the mature embryo formed direct embryos without any intervention of callus. The maximum percentage of embryogenic cultures were noticed in 30.0mg/l of 2,4-D followed by NAA at 30.0mg/l. for the differentiation of somatic embryos, the embryogenic masses were transferred to medium without any growth regulator. The maximum number of somatic embryos per culture was recorded in 30 mg/l of 2,4-D followed by 30.0 mg/l of NAA. Keywords: Arachis hypogaea L.,Somatic Embryogenesis, 2,4-D and NAA