Callus Lines Research Articles

Clinical Study on the Treatment of Long Bone Fracture Non-Union with Extracorporeal Shock Wave Therapy Combined with Platelet-Rich Plasma

Objective: The objective of the study was to evaluate of the therapeutic efficacy of extracorporeal shock wave therapy (ESWT) combined with platelet-rich plasma (PRP) injection in patients with long bone fracture non-union. Material and Methods: A total of 36 patients identified with long bone fracture non-union treated from September 2020 to September 2023 were enrolled into this study. Employing a random number table method, they were randomly divided into three groups, with 12 cases in each group. Based on the treatment modality, the groups were categorized as the ESWT group, PRP group, and combination ESWT + PRP group. Routine radiographs and musculoskeletal ultrasound were obtained before treatment and at 3-, 6-, and 9-month post-treatment intervals to observe for bone callus formation and assess fracture line imaging scores with the aim to evaluate the treatment efficacy of each group. Results: With the extension of treatment time, the bone callus and fracture line imaging scores of the three groups gradually increased (P < 0.05). At 3-, 6-, and 9-month post-treatment, the scores of the ESWT combined with the PRP group were significantly better than those of the singular ESWT group and PRP group, and the differences were statistically significant (P < 0.05). Conclusion: Therapy with singular ESWT, singular PRP, and combination ESWT + PRP has demonstrated effective improvement in fracture healing for patients with long bone fracture non-union. The synergistic effects of combination therapy were more significant, surpassing the efficacy of singular ESWT or PRP applications. The combined use of ESWT and PRP represents a safe and promising alternative treatment for long-bone fracture non-union, making it a compelling choice in the context of fracture healing. Keywords: Extracorporeal shock wave therapy, Platelet-rich plasma, Non-union of fracture.

BIOMASS AND VALUABLE METABOLITES DYNAMIC ACCUMULATION IN STRAWBERRY CALLUS CULTURES

Strawberry is one of the most commonly cultivated and consumed plant, due to its organoleptic properties and nutritional values, exerting multiple beneficial health effects. In this study, two distinct strawberry callus lines obtained in different lighting conditions were characterized. The biotechnological potential of these cultures can be demonstrated by biomass and secondary metabolites accumulation. For this reason, growth rate, cellular morphology and viability were assessed. For metabolic potential evaluation, biochemical analyses of total phenolic and flavonoid content, anthocyanin pigments concentrations and antioxidant capacity were performed. The red callus line displayed higher growth rate, total phenolic content and antioxidant activity than the white line. Flavonoids and anthocyanins were present only in the red line. The biomass and these metabolites accumulation varied between subcultures, but were maintained at a high rate, being superior to values reported in planta. Our results show that both callus cultures lines were highly proliferative and the red one being a source of valuable bioactive secondary metabolites, with high antioxidant activity.

Cellular and metabolic characteristics of peach anther-derived callus

Callus is an important material for horticulture research, especially for biotechnological applications, and can be used for the production of high-value metabolites. In this paper, anthers of peach (Prunus persica L.) varieties ‘Zaojiubao’, ‘Yanhong’, and ‘Xueyulu’ were used to induce callus lines, and the tissue morphology, subcellular structure, and metabolite profiles were characterized. Peach anther-derived calli were successfully induced in MS+1.0 mg/l ZT+1.0 mg/l 2,4-D + 0.5 mg/l IAA medium, and were stably subcultured in MS+1.0 mg/l BA+0.2 mg/l IBA+0.1 mg/l GA3 medium. SSR analysis confirmed the callus lines originated from somatic cells. Furthermore, microscopic observation of callus cells revealed some interesting cytological features, such as abundant mitochondria, which may be associated with vigorous growth and strong resistance to senescence, and suggests their potential for protoplast-related experiments such as transient expression. The metabolites in anther-derived calli were comparatively analyzed. Naringenin-7-O-glucoside (prunin), which has medicinal value as an anti-inflammatory and antiviral compound, was highly enriched in the callus lines, and cyanidin-3-O-glucoside caused the red coloration of peach anther calli. Additionally, chlorogenic acid, and amino acid derivatives such as aspartic acid and leucine, as well as other organic acids, lipids, and alkaloids, were detected in the callus lines. This study showed a series of peach anther-derived callus lines have great potential for cell biology and transformation research, especially for the production of high value components, which can be used for medicinal purposes, health products, and food additives.

Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene.

We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, β-carotene, and two β-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and β-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the ε-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in β-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the β-carotene content of plants.

Variability of morphological features and nuclear DNA content in haploids and doubled haploids of androgenic callus lines of rice (<I>Oryza sativa</I> L.)

The work is relevant for understanding evolutionary processes in plant species. Twelve callus lines with multiple regeneration of haploids and doubled haploids were obtained in F1 hybrids of Oryza sativa L. through in vitro androgenesis. Intracallus variability of the morphological features of haploids was often accompanied by a decrease in the values of morphological features with an increase in the serial number (p < 0.05). The number of panicles on a plant and the number of flowers on a panicle on two callus lines in the second or third group were increased. No variability was detected in five callus lines, i.e., such a phenomenon was not a rule. The nuclear DNA content of doubled haploids in four groups of the same callus line was 1.03– 1.09 pg, and for haploids it was 0.53–0.58 pg. Intracallus variability of nuclear DNA content was detected between groups of haploids of the same line and among doubled haploids of the same line. Significant differences were found between the haploids of one callus line and the three other callus lines of the Sadko × Kuboyar hybrid towards an increase of nuclear DNA content (p < 0.0015). The theoretical possibility of the appearance of intraspecific variability among plants with a small number of chromosomes is considered. A scheme of genomic reorganization is proposed for such species: initial plant (2n) → aneuhaploid plants (n + 1) → megasporogenesis and microsporogenesis of the 0-n type, formation of fertile pollen (n + 1) → diploid plant (2n + 2). Aneuhaploid evolution explains the intraspecific variability of chromosome numbers among plant species with low ploidy. Aneuploid technologies can help in the artificial formation of new polyploid crops, and rice is given a primary role.

SOMATIC EMBRYOGENESIS AND PLANTLET FORMATION FROM LEAF EXPLANTS OF SPONGE GOURD (Luffa aegyptiaca (MILL.) A MEDICINALLY IMPORTANT PLANT

An effective technique for the stimulation of somatic embryogenesis and plantlet regeneration from Leaf explants was developed in the sponge gourd (Luffa aegyptiaca (Mill.), which is significant in the field of medicinal botany. Fast-growing, yellowish nodular callus lines containing somatic embryos were established on an initiation medium supplemented with 2.0 mg/L 2,4-D, 3.0 mg/L 2,4-5-T, and 2.0 mg/L NAA, respectively. Somatic embryos were induced directly from cotyledon explants on Murashige and Skoog's (MS) medium fortified with different concentrations of 2,4- Dichlorophenoxyacetic acid, 2,4,5-trich On MS medium that was supplemented with 2.0 mg/L2,4-D and 1.5 mg/L TDZ, as well as 3.0 mg/L2,4-D and 2.0 mg/L BAP, embryo growth and good maturation were successfully accomplished. On an MS medium containing 2.0 mg/L TDZ and 2.0 mg/L NAA (-Naphthalene acetic acid), the well-formed embryos germinated into full plantlets and became plantlets. NAA stands for -Naphthalene acetic acid. The plants that had been regenerated were first transplanted into plastic cups and then into pots for maturation. After that, they were moved to soil and developed to maturity with an 80% survival rate, and the tissue culture-reared plants produced viable seeds.

Using the Possibilities of in Vitro Culture for the Preparation of Dietary Supplements from Alfalfa Tissues

In vitro methods have environmental advantages of preparing bio additives as harmful chemicals such as fertilizers, herbicides, pesticides are used. In addition, obtaining biomass does not depend on seasonality and a long growing season. In this series of experiments, an accelerated method for obtaining a sterile alfalfa culture was developed by adding 1% potassium humate to the explant culture. From 4 varieties of alfalfa, 3 cell lines were selected, characterized by heterogeneity of callus tissues. 1 - line morphogenic structures; 2 - line - without meristematic foci and brown and dark brown in color, which were not further differentiated; 3- line forming polymerogenic tissues. In addition, it was possible to induce denser tissues from loose callus by passaging on the Risting medium in the Ray khan variety. Structured tissues with meristematic foci induced on Risting medium were lyophilized and the dried biomass was prepared for further biochemical analyses. Thus, we have optimized the conditions for obtaining biomass from alfalfa culture and carried out the selection of cell lines, and we assume that callus lines, upon receipt of positive biochemical analyzes, can be used as feed additives.

Callus Culture of Scorzonera radiata as a New, Highly Productive and Stable Source of Caffeoylquinic Acids.

During our ongoing efforts to investigate biotechnological sources of caffeoylquinic acid (CQA) metabolites, we discovered the plant Scorzonera radiata Fisch. (Asteraceae), which is able to produce callus cultures with high yield and extremely high stability. An actively growing callus line, designated as Sr-L1, retained the ability to produce 11 CQAs during long-term cultivation (more than 20 years). A total of 29 polyphenolic compounds were identified in the leaves and Sr-L1 callus culture of S. radiata, including CQAs, lignol derivatives, flavonoids, and dihydrostilbenes. The composition of CQAs in the Sr-L1 culture was identical to that in the S. radiata leaves. Sr-L1 calli did not produce flavonoids and dihydrostilbenes, but produced lignol derivatives, which were absent in leaves. The HPLC-UV-HRMS determination showed the presence of monoacyl derivatives of CQAs such as 5-CQA, 4-CQA, cis-5-CQA, and 5-O-p-coumaroylquinic acid in the Sr-L1 culture. Among diacyl derivatives, 3,4-diCQA, 3,5-diCQA, cis-3,5-diCQA, 4,5-diCQA, 3-O-p-coumaroyl-5-O-CQA, and 3-O-caffeoyl-5-O-p-coumaroylquinic acid were found. The content of 5-CQA reached 7.54 mg/g dry weight and the content of 3,5-diCQA was as high as 18.52 mg/g dry weight. 3,5-diCQA has been reported to be of high nutritional and pharmacological value, as it alleviates inflammatory pain, reverses memory impairment by preventing neuronal apoptosis, and counteracts excessive adipose tissue expansion, serving as an attractive treatment option for obesity. The high content of 3,5-diCQA and the exceptional stability of biosynthesis make callus cultures of S. radiata a promising source for the development of drugs and nutraceuticals.

Gardenia carotenoid cleavage dioxygenase 4a is an efficient tool for biotechnological production of crocins in green and non-green plant tissues.

Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the colour and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the carotenoid cleavage dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison with CsCCD2L, in citrus callus engineered by Agrobacterium-mediated supertransformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. We demonstrate that co-expression of GjCCD4a with phytoene synthase and β-carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved β-carotene, in planta, and produced crocetin dialdehyde via C30 β-apocarotenoid intermediate. GjCCD4a also cleaved C27 β-apocarotenoids, providing a new route for C17 -dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast-like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis-9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non-green crop tissues/organs.

Drought Stress in Sugarcane: In-vitro Mutagenesis and Selection of Polyethylene Glycol (PEG) Tolerant Callus Lines

Drought is one of the key abiotic factors causing a loss in sugarcane output, which is one of the world's thirstiest crops. A mutant sugarcane callus line was used to create an in vitro selection system for drought resistance. The objective of this study was to obtain putative sugarcane mutant callus lines that had been chemically altered with sodium azide at various concentrations (0, 0.5, 1.0, and 1.5%). These lines were subsequently screened using high molecular weight PEG 6000 as a selection agent at various concentrations (0, 5, 10, 15, 20, 25, and 30%). The experiment demonstrated a decline in the proportion of live callus and sensitivity index with increasing mutation concentration and PEG dose, with little to no live callus at the maximum mutation and PEG concentration. Biochemical investigation of the survived callus for proline accumulation and salicylic acid content revealed an elevated level of up to 20 percent PEG and 1.0 percent mutagen, followed by a quick drop as PEG and mutagen concentrations increased. The findings can be used to create drought-tolerant sugarcane lines and putative mutants, as well as to screen them in vitro.

Antioxidant and Anticancer Potential of Bioactive Compounds from Rhinacanthus nasutus Cell Suspension Culture

The potential benefits of natural plant extracts have received attention in recent years, encouraging the development of natural products that effectively treat various diseases. This is the first report on establishing callus and cell suspension cultures of Rhinacanthus nasutus (L.) Kurz. A yellow friable callus was successfully induced from in vitro leaf explants on Murashige and Skoog medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid and 1 mg/L 1-naphthalene acetic acid. A selected friable callus line was used to establish the cell suspension culture with the same medium. The antioxidant assays showed that the leaf- and ethanolic-suspension-cultured cell (SCC) extracts exhibited high antioxidant potential. In addition, the in vitro cytotoxicity revealed by the MTT assay demonstrated potent antiproliferative effects against the oral cancer cell lines ORL-48 and ORL-136 in a dose-dependent manner. Several groups of compounds, including terpenoids, phenolics, flavonoids, quinones, and stilbenes, were identified by UHPLC–QToF–MS, with the same compounds detected in leaf and SCC extracts, including austroinulin, lucidenic acid, esculetin, embelin, and quercetin 3-(2″-p-hydroxybenzoyl-4″-p-coumarylrhamnoside). The present study suggests the value of further investigations for phytochemical production using R. nasutus cell suspension culture.

MiR171 modulates induction of somatic embryogenesis in citrus callus.

Overexpression of miR171 restored SE competence in the recalcitrant citrus callus, and inhibition of miR171 function weakened SE competence in the strongly embryogenic citrus callus. Somatic embryogenesis (SE) is an important way of in vitro regeneration for plants. For perennial woody crops such as citrus, embryogenic callus is usually induced from unfertilized aborted ovules and widely used in biotechnology aided breeding. However, SE capacity always declines in callus during subculture, which makes regeneration difficult and hinders the application of biotechnology. We previously found that miR171 may be a regulator of SE in citrus, based on the abundant expression of csi-miR171c in the embryogenic callus and during SE of citrus. Here, we report that miR171 promotes SE and is required for SE in citrus. Overexpression of miR171 restored SE competence in the recalcitrant callus of 'Guoqing No.1' Satsuma mandarin (G1), whereas inhibition of miR171 function by Short Tandem Target Mimic (STTM) weakened SE competence in the strongly embryogenic callus of 'Valencia' sweet orange (V). The comparative transcriptomic analysis in miR171 overexpressed callus line (OE) and the wild type callus (WT) indicated that overexpression of miR171 decreased the expression level of its SCARECROW-LIKE (CsSCL) targets, and activated stress response related biological processes and metabolic processes that are required for cell differentiation. However, CsSCLs were up-regulated in the OE callus during SE induction process, which activated the cell division and developmental processes that are required for embryogenesis progress. Our results validate the function of miR171 in regulation of SE and reveal the biological responses provoked by miR171 in citrus that may promote SE.

Metabolic Engineering of Rice Cells with Vanillin Synthase Gene (VpVAN) to Produce Vanillin.

Vanillin production by metabolic engineering of proprietary microbial strains has gained impetus due to increasing consumer demand for naturally derived products. Here, we demonstrate the use of rice cell cultures metabolically engineered with vanillin synthase gene (VpVAN) as a plant-based alternative to microbial vanillin production systems. VpVAN catalyzes the signature step to convert ferulic acid into vanillin in Vanilla planifolia. As ferulic acid is a phenylpropanoid pathway intermediate in plant cells, rice calli cells are ideal platform for in vivo vanillin synthesis due tothe availability of its precursor. In this study, rice calli derived from embryonic rice cells were metabolically engineered with a codon-optimized VpVAN gene using Agrobacterium-mediated transformation. The putative transformants were selected based on their proliferation on herbicide-supplemented N6D medium. Expression of the transgenes wereconfirmed through a β-glucuronidase (GUS) reporter assay and polymerase chain reaction (PCR) analysis provided evidence of genetic transformation. The semiquantitative RT-PCR and real-time (RT)-qPCR revealed expression of VpVAN in six transgenic calli lines. High-performance liquid chromatography identified the biosynthesis of vanillin in transgenic calli lines, with the highest yielding line producing 544.72 (± 102.50) μg of vanillin-g fresh calli. This work serves as a proof-of-concept to produce vanillin using metabolically engineered rice cell cultures.

Variation burst during dedifferentiation and increased CHH-type DNA methylation after 30years of in vitro culture of sweet orange.

Somaclonal variation arising from tissue culture may provide a valuable resource for the selection of new germplasm, but may not preserve true-to-type characteristics, which is a major concern for germplasm conservation or genome editing. The genomic changes associated with dedifferentiation and somaclonal variation during long-term in vitro culture are largely unknown. Sweet orange was one of the earliest plant species to be cultured in vitro and induced via somatic embryogenesis. We compared four sweet orange callus lines after 30years of constant tissue culture with newly induced calli by comprehensively determining the single-nucleotide polymorphisms, copy number variations, transposable element insertions, methylomic and transcriptomic changes. We identified a burst of variation during early dedifferentiation, including a retrotransposon outbreak, followed by a variation purge during long-term in vitro culture. Notably, CHH methylation showed a dynamic pattern, initially disappearing during dedifferentiation and then more than recovering after 30years of in vitro culture. We also analyzed the effects of somaclonal variation on transcriptional reprogramming, and indicated subgenome dominance was evident in the tetraploid callus. We identified a retrotransposon insertion and DNA modification alternations in the potential regeneration-related gene CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16. This study provides the foundation to harness in vitro variation and offers a deeper understanding of the variation introduced by tissue culture during germplasm conservation, somatic embryogenesis, gene editing, and breeding programs.

Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger & Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

Agronomical, Physiological and Biochemical Characterization of In Vitro Selected Eggplant Somaclonal Variants under NaCl Stress.

Previously, an efficient regeneration protocol was established and applied to regenerate plants from calli lines that could grow on eggplant leaf explants after a stepwise in vitro selection for tolerance to salt stress. Plants were regenerated from calli lines that could tolerate up to 120 mM NaCl. For further in vitro and in vivo evaluation, four plants with a higher number of leaves and longer roots were selected from the 32 plants tested in vitro. The aim of this study was to confirm the stability of salt tolerance in the progeny of these four mutants (‘R18’, ‘R19’, ‘R23’ and ‘R30’). After three years of in vivo culture, we evaluated the impact of NaCl stress on agronomic, physiological and biochemical parameters compared to the parental control (‘P’). The regenerated and control plants were assessed under in vitro and in vivo conditions and were subjected to 0, 40, 80 and 160 mM of NaCl. Our results show significant variation in salinity tolerance among regenerated and control plants, indicating the superiority of four regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) when compared to the parental line (‘P’). In vitro germination kinetics and young seedling growth divided the lines into a sensitive and a tolerant group. ‘P’ tolerate only moderate salt stress, up to 40 mM NaCl, while the tolerance level of ‘R18’, ‘R19’, ‘R23’ and ‘R30’ was up to 80 mM NaCl. The quantum yield of PSII (ΦPSII) declined significantly in ‘P’ under salt stress. The photochemical quenching was reduced while nonphotochemical quenching rose in ‘P’ under salt stress. Interestingly, the regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) exhibited high apparent salt tolerance by maintaining quite stable Chl fluorescence parameters. Rising NaCl concentration led to a substantial increase in foliar proline, malondialdehyde and soluble carbohydrates accumulation in ‘P’. On the contrary, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ exhibited a decline in soluble carbohydrates and a significant enhancement in starch under salinity conditions. The water status reflected by midday leaf water potential (ψl) and leaf osmotic potential (ψπ) was significantly affected in ‘P’ and was maintained a stable level in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ under salt stress. The increase in foliar Na+ and Cl− content was more accentuated in parental plants than in regenerated plants. The leaf K+, Ca2+ and Mg2+ content reduction was more aggravated under salt stress in ‘P’. Under increased salt concentration, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ associate lower foliar Na+ content with a higher plant tolerance index (PTI), thus maintaining a normal growth, while foliar Na+ accumulation was more pronounced in ‘P’, revealing their failure in maintaining normal growth under salinity stress. ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an obvious salt tolerance by maintaining significantly high chlorophyll content. In ‘R18’, ‘R19’, ‘R23’ and ‘R30’, the enzyme scavenging machinery was more performant in the roots compared to the leaves. Salt stress led to a significant augmentation of catalase, ascorbate peroxidase and guaiacol peroxidase activities in the roots of ‘R18’, ‘R19’, ‘R23’ and ‘R30’. In contrast, enzyme activities were less enhanced in ‘P’, indicating lower efficiency to cope with oxidative stress than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’. ACC deaminase activity was significantly higher in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ than in ‘P’. The present study suggests that regenerated plants ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an evident stability in tolerating salinity, which shows their potential to be adopted as interesting selected mutants, providing the desired salt tolerance trait in eggplant.

Obtaining Salt Stress-Tolerant Eggplant Somaclonal Variants from In Vitro Selection.

An efficient regeneration protocol was applied to regenerate shoots on salt stress-tolerant calli lines of aubergine (Solanum melongena). These NaCl-tolerant cell lines were obtained by two different methods. On the one hand, the developed callus tissue was transferred to a medium with a continuous salt content of 40, 80, 120, or 160 mM NaCl. On the other hand, the callus tissue was subjected to a stepwise increasing salinity to 160 mM NaCl every 30 days. With the second method, calli which could be selected were characterized by compact growth, a greenish color, and absence of necrotic zones. When grown on salt-free medium again, NaCl-tolerant calli showed a decline in relative growth rate and water content in comparison to the control line. This was more obvious in the 120 mM NaCl-tolerant callus. Lipid peroxidase activity increased in 40 and 80 mM NaCl-tolerant calli; yet did not increase further in 120 mM-tolerant callus. An increase in ascorbic acid content was observed in 80 and 120 mM NaCl-tolerant calli compared to the 40 mM NaCl-tolerant lines, in which ascorbic acid content was twice that of the control. All NaCl-tolerant lines showed significantly higher superoxide dismutase (SOD) (208–305–370 µmol min−1 mg−1 FW) and catalase (CAT) (136–211–238 µmol min−1 mg−1 FW) activities compared to control plants (231 and 126 µmol min−1 mg−1 FW). Plants were regenerated on the calli lines that could tolerate up to 120 mM NaCl. From the 32 plants tested in vitro, ten plants with a higher number of leaves and root length could be selected for further evaluation in the field. Their high salt tolerance was evident by their more elevated fresh and dry weight, their more increased relative water content, and a higher number and weight of fruits compared to the wild-type parental control. The presented work shows that somaclonal variation can be efficiently used to develop salt-tolerant mutants.

Transformation of European Ash (Fraxinus excelsior L.) Callus as a Starting Point for Understanding the Molecular Basis of Ash Dieback.

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity.

Linnaea borealis L. var. borealis-In Vitro Cultures and Phytochemical Screening as a Dual Strategy for Its Ex Situ Conservation and a Source of Bioactive Compounds of the Rare Species.

Linnaea borealis L. (Twinflower)—a dwarf shrub in the Linnaeeae tribe of Caprifoliaceae family—is distributed across the Northern Hemisphere. By means of this study, a reliable protocol for efficient micropropagation of uniform L. borealis L. var. borealis plantlets has been provided for the first time; callus culture was also established. Different initial explants, types of cultures, media systems, and plant growth regulators in Murashige and Skoog (MS) media were tested. Agitated shoot cultures in the liquid media turned out to be the best system for the production of sustainable plant biomass. After stabilization of the callus lines, the highest growth index (c.a. 526%) was gained for callus maintained on MS enriched with picloram. TLC and UHPLC-HESI-HRMS analysis confirmed the presence of phenolic acids and flavonoids, and for the first time, the presence of iridoids and triterpenoid saponins in this species. Multiplication of L. borealis shoot culture provides renewable raw material, allowing for the assessment of the phytochemical profile, and, in the future, for the quantitative analyses and the studies of the biological activity of extracts, fractions, or isolated compounds. This is the first report on in vitro cultures of traditionally used L. borealis rare taxon and its biosynthetic potential.

Callus induction and chemical characterization of cell suspension cultures of jojoba (Simmondsia chinensis L.)

Jojoba (Simmondsia chinensis L.) oil is also known as liquid wax or fixed oil. It is an important metabolite of jojoba having commercial importance in cosmetics as well as a potential biofuel source. We presented an efficient system for in vitro establishment of cell suspension cultures (CSC) from proliferating friable calluses. For this purpose, cotyledon, internode, and leaf explants were cultured on MS medium + 1, 2, 4, 6, 8 or 10 µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D), α-Naphthalene acetic acid (NAA) alone or in combination with 1 or 2 µM N6-benzylaminopurine (BAP) or Kinetin. Results demonstrated that 100% healthy, friable and variegated calluses were obtained on 8 µM, 10 µM 2, 4-D or 2, 4-D 10 µM + 2 µM BAP and represented as callus lines (CL) CL-1, CL-2 or CL-3, respectively, after 38 days. One-gram callus tissue per CL was then immersed in the respective liquid medium and agitated on an orbital shaker at 60-70 rpm under the growth room conditions (25 ± 2 °C, 16 h light period) for the preparation of CSC. After 15 days, CSC was sieved and large clumps were removed. Growth measurement of CSC was determined by cell counting, packed cell volume (PCV) and cell viability. The highest number of viable cells was obtained at 2.57 OD with CL-3, where PCV was highest (0.35 ml) on CL-1 of 38 days old calluses. 2,3,5-Triphenyltetrazolium chloride was a reliable approach for the determination of cell viability of CSC.