Shoot Cultures Research Articles

Cryopreservation of aromatic ginger Kaempferia galanga L. by encapsulation-dehydration

Kaempferia galanga L. is an endangered multi-purpose medicinal plant in Family Zingiberaceae, the rhizomes of which are used for several ayurvedic formulations. Encapsulation-dehydration (ED) method was optimized for cryopreservation of shoot tips of K. galanga. Shoot tips (STs) bearing the apical meristem dissected from the established in vitro shoot cultures were preconditioned in MS+0.4 M sucrose prior to encapsulation in calcium alginate and the beads subsequently transferred to MS liquid+0.3 M sucrose for 3 days afterward dehydration inside the laminar airflow for 4 hours upon rapid freezing in LN and rapid thawing produced maximum 62.2% survival and 46.7% regeneration rates. Shoot regeneration was observed from the apical meristems exclusive of intermediary callus phase. The plantlets regenerated from cryopreserved STs transferred to the field were phenotypically analogous with the mother plant.

The effect of different light treatments on morphogenesis, phenolic compound accumulation and antioxidant potential of Dracocephalum forrestii transformed shoots cultured in vitro.

The aim of this study was to evaluate the effects of wavelengths of light emitted from LEDs on cultured in vitro transformed shoots of Dracocephalum forrestii. The shoots were grown on MS agar medium with 0.5mg/l BPA (N-benzyl-9-(tetrahydropyranyl)-adenine) and 0.2mg/l IAA (indole-3-acetic acid) under four light environments: blue, red, red/blue (7:3) and white (control). After four weeks of culture, shoot multiplication rate, biomass and morphology were evaluated, as well as bioactive phenolic content, antioxidant capacities and antioxidant enzyme activities. The hydromethanolic extracts from shoots were analyzed using UHPLC method, and antioxidant potential was evaluated using radical scavenging (1,1-diphenyl-2-picrohydrazyl and superoxide anion), and ferric reducing antioxidant power (FRAP), and enzymatic methods, i.e. sodium dismutase (SOD), catalase (CAT) and peroxidase (POD) activity. It was found that the blue and red/blue light had the strongest effect on morphogenesis and shoot propagation; in these conditions, more than five new shoots were obtained per explant. The blue light cultures demonstrated the highest fresh (0.41g/tube FW) and dry weights (0.045g/tube DW), the highest levels of polyphenols (99.7mg/g DW), i.e. almost three times greater than under white light (35.4mg/g DW), as well as the highest antioxidant potential. Therefore, LED culture appears to be a beneficial strategy for enhancing the production of the medicinal value of transformed D. forrestii shoot culture.

Growth response of diploid and tetraploid taro (Colocasia esculenta (L.) Schott) shoot culture to drought stress using polyethylene glycol

Taro genetic improvement through polyploidy induction is expected to be tolerant to abiotic stress. Several studies have shown that polyploidy plants have a higher adaptability to dry environments. In vitro selection technique for evaluation of plant tolerance to drought stress can be done by applying polyethylene glycol (PEG) as a selection agent. The aim of the research was to investigate the growth response of diploid and tetraploid taro shoot culture to drought stress using PEG. The experiment was conducted using a completely randomized design with two factors. The first factor was the concentration of PEG at 0, 5, 10, 15 and 20%. The second factor was Bentul taro clones which were 1 diploid clone and 2 tetraploid clones (clones 4.6.3 and 5.4.4). PEG was added to liquid MS medium containing 2 mg/l BAP. Observations of growth variables were carried out every week until the six weeks of culture. Fresh and dry weights, mortality percentage and proline content were determined at six weeks of culture. The results showed that the addition of PEG in liquid medium significantly affected the number of leaves, petiole length, number of roots, fresh and dry weights, as well as shoot mortality percentage. Differences in clones significantly affected the number of leaves, fresh and dry weights. The two factors tested (PEG and clones), gave an interaction on the number of leaves, fresh and dry weights. The proline content in all diploid and tetraploid clones showed an increase with increasing PEG concentration. The LC50 value in diploid clones was 9.82%, in tetraploid clones 4.6.3 and 5.4.4 were 14.14 and 15.45%, respectively. The results showed that PEG at 10% and 15% could be used for in vitro selection to drought stress both for diploid and tetraploid taro.

Organogenesis on apical buds in common fig (Ficus carica) var. Black Jack

Plant tissue culture involves the use of explants obtained from plants to induce organogenesis with the help of plant growth regulators (PGRs). Micropropagation techniques provide a faster and economical solution to the limitations associated with traditional methods of plant cultivation. The present study focuses on the multiple shoot induction and proliferation of Ficus carica var. Black Jack. Factors that influence the growth of in vitro multiple shoots on the apical buds, which include growth media and PGRs, were investigated in this study. Different concentrations of cytokinins like 6-benzylaminopurine (BAP), Thidiazuron (TDZ), and Kinetin (Kin) were used on woody plant medium (WPM) for the optimization of media for multiple shoot induction and proliferation. Apical buds of Ficus carica var. Black Jack growing in WPM supplemented with BAP produced the healthiest plantlets, with the highest number of multiple shoots. The most efficient medium composition which produced the highest number of multiple shoots (37.8) per growing explant was WPM supplemented with 20 µM BAP. Proliferated multiple shoots were efficiently rooted using WPM + 20 µM BAP + 8 µM indole-3-acetic acid (IAA). This optimized medium composition significantly enhanced the production of multiple, disease-free plantlets using single apical bud explants of Ficus carica var. Black Jack. In the present study the observations indicate that WPM supplemented with 20 µM BAP is the best-suited medium for organogenesis and multiple shoot culture of Ficus carica var. Black Jack, and this technique can be potentially applied for commercialization of the plant. How to cite: Parab AR, Chew BL, Yeow LC, et al. Organogenesis on apical buds in common fig ( Ficus carica ) var. Black Jack. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2020.01.010

Impact of Metallic Nanoparticles on In Vitro Culture, Phenolic Profile and Biological Activity of Two Mediterranean Lamiaceae Species: Lavandula viridis L'Hér and Thymus lotocephalus G. López and R. Morales.

Nanoparticles (NPs) recently emerged as new chemical elicitors acting as signaling agents affecting several processes in plant metabolism. The aim of this work was to investigate the impact of the addition of copper oxide (CuO), zinc oxide (ZnO) and iron oxide (Fe3O4) NPs (<100 nm) at different concentrations (1, 5 and 10 mg/L) to the culture media on several morphological, physiological and -biochemical parameters of in vitro shoot cultures of Lavandula viridis L’Hér and Thymus lotocephalus G. López and R. Morales (Lamiaceae), as well as on phenolic profile and bioactivity (antioxidant and enzyme inhibition capacities). Although some decreases in shoot number and length were observed in response to NPs, biomass production was not affected or was improved in both species. Most NPs treatments decreased total chlorophyll and carotenoid contents and increased malondialdehyde levels, an indicator of lipid peroxidation, in both species. HPLC-HR-MS analysis led to the identification of thirteen and twelve phenolic compounds, respectively, in L. viridis and T. lotocephalus extracts, being rosmarinic acid the major compound found in all the extracts. ZnO and Fe3O4 NPs induced an increase in total phenolic and rosmarinic acid contents in T. lotocephalus extracts. Additionally, some NPs treatments also increased antioxidant activity in extracts from this species and the opposite was observed for L. viridis. The capacity of the extracts to inhibit tyrosinase, acetylcholinesterase and butyrylcholinesterase enzymes was not considerably affected. Overall, NPs had a significant impact on different parameters of L. viridis and T. lotocephalus in vitro shoot cultures, although the results varied with the species and NPs type.

Efficient removal of heavy metals from artificial wastewater using biochar

Increase environmental pollution, especially water contaminated with heavy metals has led to various problems in human life. Accumulation and magnification of heavy metals in human tissues through consumption of contaminated water can cause hazardous impacts on health. In the present study, bio-char was prepared from the shoot and root culture of in-vitro grown Plumbago zeylanica. Effect of bio-char (prepared from both the plant materials) on removal of chromium and cadmium from synthetic water was evaluated. Change in cadmium and chromium absorbance was recorded at every hour interval up to six hours using UV–Visible spectrophotometer. It was observed that after six-hour of incubation there was no further removal of heavy metals. Further optimization of different parameters such as concentration of heavy metal, solution pH and bio char concentration were performed. At 100 ppm concentration of heavy metals maximum removal was obtained. Furthermore, it was observed that pH 7 and 2 mg/ml bio char concentration showed maximum removal efficiency. The result showed that shoot derived bio char has more potential to remove cadmium and chromium as compared to the root derived bio char. In-vitro grown shoot culture provides an effective method to remove pollutants as it grows faster compared to wild type culture. Therefore, this method is useful for heavy metals removal from wastewater.

Protocol for micropropagation of strawberry (Fragaria × ananassa) cv. ‘Sweet Charlie’ and ‘Winter Dawn’

The objective of this study was to develop a protocol for large-scale propagation of strawberry cv. ‘Sweet Charlie’ and ‘Winter Dawn’ using the micropropagation technique. Shoot cultures were obtained from shoot tips on Murashige and Skoog (MS) medium with 4% table sugar, 0.75% agar, 5 mg L−1 6-benzyladenine and 0.01 mg L−1 kinetin. These shoots were multiplied and maintained on MS medium with 1 mg L–1 6-benzyladenine and 0.1 mg L–1 kinetin. Rooting of in vitro raised shoots was successfully conducted by pulse treatment with 500 mg L–1 indole-3-butyric acid for 30 s and subsequent culturing in MS medium with 1 mg L–1 indole-3-butyric acid, 0.1 g L–1 activated charcoal and 6% table sugar. Plants were successfully acclimatized and survived in field conditions.

Gamma irradiations induced morphological and biochemical variations in in vitro regenerated ginger (Zingiber officinale rosc.)- an invaluable medicinal spice

Purpose To evaluate the effect of gamma irradiations on morphological and biochemical variations of in vitro shoots, regenerated plants and rhizomes of ginger (Zingiber officinale Rosc.) var. Himgiri. Materials and methods In vitro established shoot cultures were treated with different doses of gamma irradiations (10–100 Gy). Nonirradiated shoots were used as controls. Radio sensitivity of shoots and in vitro growth parameters were recorded. Surviving shoots along with controls were multiplied, rooted and hardened. Irradiation effect on chromosomes was evaluated by comparing with controls. Various morphological and biochemical observations were recorded in regenerated plants and rhizomes and compared to controls. Results Irradiated shoots showed decline in survival percentage (95% in 10 Gy to 12.33% in 100 Gy) with increase in dose rate after 4 weeks of irradiation. Shoot survival significantly decreased with irradiation dose rate above 20 Gy. Shoot multiplication rate showed an increase from 1:2 in 1st subculture to 1:9 after 5th subculture, in 10 Gy irradiated cultures with 4.5 cm average length of shoots and 8.19 number of leaves after 5th subculture. Average rooting showed an increase from 15.55% after 2nd subculture to 100% after 5th subculture, in 10 Gy irradiated shoots; however, no multiplication and rooting was observed in higher doses. In vitro rooted shoots were subjected to hardening with 100 percent survival in a mixture comprised of soil, cocopeat and FYM. One-year-old hardened plants when observed for morphological variations showed nonsignificant variations in comparison to controls. Rhizomes obtained from 10 Gy irradiated plants showed enhanced concentration of 6-gingerol and reduced crude fiber content when compared with controls. Conclusions Gamma irradiation doses up to 10 Gy resulted in nonsignificant phenotypic variations but can positively affect biochemical composition of rhizomes leading to development of biochemically improved mutant lines of ginger with increased 6-gingerol and lowered crude fiber content. Therefore, suggesting the lower doses of gamma irradiations for improvement of biochemical constituents in vegetatively propagated plants.

Biotechnological production of diterpenoid lactones from cell and organ cultures of Andrographis paniculata.

Andrographis paniculata (AP) is a medicinal plant that is traditionally used in Indian, Chinese, Malay, Thai, and Oriental system of medicines to treat various disorders. AP consists of andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), and neoandrographolide (NAD) as major diterpene lactones which has extremely bitter properties; therefore, AP is commonly called "King of bitters." AD, DDAD, and NADare reported to possess therapeutic values such as antioxidant, immunostimulatory, hepatoprotective, anti-cancer, anti-inflammatory, anti-rheumatoidal, anti-malarial, anti-leishmanial, anti-fertility, anti-obesity, antipyretic, and antimicrobial attributes. According to the Indian Pharmacopoeia, the leaves and tender shoots of AP yield up to 1%, 0.16%, and 0.11% of AD, DDAD, and NAD, respectively, on a dry-weight basis. However, variability in the accumulation of AD, DDAD, and NADin plants has been reported with respect to species, genotype, season, phenological stage, plant part used, and geography of a region of cultivation. Therefore, cell and tissue culture systems especially cell, shoot, and adventitious root cultures are explored as alternatives for constant and higher production of AD, DDAD, and NAD. This review explores the prospects of exploiting the plant cell and tissue culture systems for the controlled production of AD, DDAD, and NAD. Various strategies such as elicitation by using biological and chemical elicitors are explored for the enhancement of accumulation of AD, DDAD, and NAD in cell and organ cultures. KEY POINTS: • This review explores the possibilities of diterpene lactone production from cell and organ cultures. • Various strategies are explored for the enhanced accumulation of AD, DDAD, and NAD in cell and organ cultures. • Prospects of diterpene lactone production are highlighted.

Effect of Elicitation with (+)-Usnic Acid on Accumulation of Phenolic Acids and Flavonoids in Agitated Microshoots of Eryngium alpinum L.

The present work was aimed at studying the potential of elicitation on the accumulation of phenolic compounds in in vitro shoot cultures of Eryngium alpinum L., a protected plant from the Apiaceae family. The study examined the influence of (+)-usnic acid on the biomass growth as well as on the biosynthesis of the desired flavonoids and phenolic acids in the cultured microshoots. The phenolic compound content was determined by HPLC-DAD. The flavonoid of the highest concentration was isoquercetin, and the phenolic acids of the highest amount were rosmarinic acid, caffeic acid and 3,4-dihydroxyphenylacetic acid, both in the non-elicited and elicited biomass. Isoquercetin accumulation was efficiently increased by a longer elicitation with a lower concentration of lichenic compound (107.17 ± 4.67 mg/100 g DW) or a shorter elicitation with a higher concentration of acid (127.54 ± 11.34 and 108.37 ± 12.1 mg/100 g DW). Rosmarinic acid production generally remained high in all elicited and non-elicited microshoots. The highest content of this acid was recorded at 24 h of elicitation with 3.125 µM usnic acid (512.69 ± 4.89 mg/100 g DW). The process of elicitation with (+)-usnic acid, a well-known lichenic compound with allelopathic nature, may therefore be an effective technique of enhancing phenolic compound accumulation in alpine eryngo microshoot biomass.

Preliminary study on in vitro shoot culture of Hibiscus coddii subsp. barnardii, an indigenous South African flowering plant

Abstract In vivo and in vitro grown plants of Hibiscus coddii subsp. barnardii were used as explant source for establishment of in vitro cultures. Nodal shoot explants derived from in vivo grown plants, both naturally and under controlled environmental conditions, showed high sensitivity to the surface disinfection treatment and poor survival in in vitro culture. In vitro grown seedlings proved successful as aseptic source of apical and basal shoot explants to establish contamination-free in vitro cultures. Sprouting of axillary buds was observed on 90% of apical shoot explants after four weeks of culture on full strength, plant growth regulator (PGR)-free Murashige and Skoog (MS) medium. However, further proliferation of short shoots, limited to the bud sprout at the explant base, occurred on only 50% of these explants. In contrast, all basal shoot explants attained 3-5 single primary axillary shoots (30-40 mm in length) while a clump of short (5-10 mm) shoots also formed at the base in 60% of these explants. In both explant types, addition of 0.25-1 mg L-1 6-Benzylaminopurine (BAP) to the MS medium resulted in a low frequency (10%-60%) of explants with short shoots (5-10 mm) that showed no further elongation. Moreover, explants cultured in the presence of BAP showed a high frequency of callus formation (up to 90%) and low survival (20%-60%). A lower frequency of callus formation (30%-40%) and higher survival (90%-100%) of both explant types occurred on BAP-free medium. Further subculturing of primary and secondary axillary shoots onto fresh MS medium (with and without BAP) did not improve shoot multiplication. Regenerated plantlets from PGR-free MS medium were successfully acclimatized and hardened-off.

In Vitro Propagation Method for Production of Phenolic-Rich Planting Material of Culinary Rhubarb 'Malinowy'.

Culinary rhubarb is a popular vegetable crop, valued for its long, thickened stalks, very rich in different natural bioactive ingredients. Tissue cultures are a useful tool for vegetative propagation of virus-free rhubarb plants and rapid multiplication of valuable selected genotypes. The aim of this study was to develop an effective method for in vitro propagation of selected genotypes of Polish rhubarb ‘Malinowy’ characterized by high yield and straight, thick and intensive red stalks. Identification and quantification of anthocyanins and soluble sugars by the HPLC method in shoot cultures and ex vitro established plantlets were also performed. Shoot cultures were established from axillary buds isolated from dormant, eight-year-old rhizomes. Effective shoot multiplication of rhubarb ‘Malinowy’ was obtained in the presence of 6.6 µM benzylaminopurine or 12.4 µM meta-topolin. Both cytokinins stimulated shoot formation in a manner that depended on sucrose concentration. Increasing the sucrose concentration from 59 to 175 mM decreased the production of shoots and outgrowth of leaves by 3-fold but enhanced shoot length, single shoot mass and callus formation at the base of shoots. This coincided with increased accumulation of soluble sugars (fructose, glucose) and anthocyanins-cyanidin-3-O-rutinoside (max. 208.2 mg·100 g−1 DM) and cyanidin-3-O-glucoside (max. 47.7 mg·100 g−1 DM). The highest rooting frequency (94.9%) and further successful ex vitro establishment (100%) were observed for shoots that were earlier rooted in vitro in the presence of 4.9 µM indole-3-butyric acid. Our results indicated that anthocyanin contents in leaf petioles were influenced by developmental stage. Under in vitro conditions, it is possible to elicit those pigments by sucrose at high concentration and meta-topolin.

Plant Tissue culture and Ultra High Diluted studies: suggesting a novel model using in vitro techniques

Plant tissue culture techniques have been used to evaluate the effects of many different substances and/ or conditions in plant growth and development. It provides information of great value about problems related to basic and applied aspects of plant as well as contributed to understanding of factors responsible for growth, metabolism, synthesis of secondary compounds, stress response. Considering all this wide range of applications and as all plant tissue culture techniques are undergone under axenic and controlled conditions (culture medium composition, light and temperature, for instance), it seems to be a value model for Ultra High Diluted (UHD) studies. Lippia alba is a Brazilian plant that tissue cultures protocols and in vitro essential oil production have already been described in scientific literature. None of all scientific papers evaluated the effects of UHD substances on in vitro development or secondary metabolic production. The main goal was to evaluate the use of plant tissue culture to investigate the effects of UHD benzilaminopurine (BA) on Lippia alba shoot culture. Nodal segments obtained from plants growth in vitro was subcultured to Murashigue &amp; Skoog semi-solid medium added with 2ml of these different solutions: BA 3µmol, BA 12CH (10-24), water 12CH and water (no dilution and succussion). Weekly 1 ml of solutions were added to cultures. The experiment was repeated twice and each one consisted in 3 culture vessel with 5 nodal segments per treatment (n=30). All plants were maintained in growth room under controlled temperature (25°C), light and photoperiod (16L/8D). The tested substances were prepared according to the method of stepwise dilution and succussion as describe in Brazilian Homeopathic Pharmacopoeia. The experiment was blinded all the time. After 60d, plantlets were evaluated for number of shoots, shoot length, rooted plants (%), callus development (%) and fresh biomass. Data were submitted to ANOVA following by Duncan’s and t-test. Plants from water 12CH and BA 12CH increased the number of new shoots and promoted the highest shoot length. By adding BA 3µmol the organogenetic response was inhibited since neither shoot nor root were developed. However, it was observed a significant basal callus development. Plant tissue culture could be adapted for UHD studies. More studies are being conducted in way to analyze other experimental conditions and biochemical/phytochemical parameters.

Gentianella lutescens subsp. carpatica J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production.

In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was established from epicotyl explants cultured on MS basal medium with 0.2 mg L−1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L−1 indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones—mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside—in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 μM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2–3-fold compared to the control shoots. Elicitors at 100–300 μM increased the accumulation of mangiferin, demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 μM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.

Tetraploidization Increases the Contents of Functional Metabolites in Cnidium officinale

Cnidium officinale is an important medicinal crop grown in Asia for its pharmacological properties. In this study, tetraploid breeding was conducted to increases the content of medicinal compound and tolerance to the environmental conditions using in vitro shoot culture of C. officinale. For this, we generated tetraploid C. officinale plants using oryzalin, a chromosome doubling agent, and compared the morphological traits, cytological characteristics, and heat stress-responsive gene expression levels between tetraploid and diploid genotypes. Chromosome doubling efficiency was the highest in plantlets treated with 4.0 mg∙L−1 oryzalin for 2 days. Compared with diploids, the plant height of tetraploids was reduced, while the petiole diameter was increased by approximately 39%. The dry matter of tetraploid leaves was significantly higher than that of diploid leaves. Compared with diploids, tetraploids showed higher chloroplast number and stomatal complex size but lower chlorophyll and carotenoid contents. The phenolic content of tetraploid plantlets was significantly higher than that of diploid plantlets. Contents of naringin as well as salicylic acid and gentisic acid, which are strong antioxidant compounds, were dramatically increased upon tetraploidization. Interestingly, liquid chromatography–mass spectrometry (LC–MS) analyses revealed increased levels of senkyunolide F and phthalide in tetraploid roots but not in tetraploid or diploid leaves.

Effect of factors on growth and development of Dendrobium officinale Kimura et Migo cultured in vitro and ex vitro

Dendrobium officinate Kimura et Migo, a species of orchid for beautiful flowers, is used in traditional medicine in many Asian countries because there are many important pharmaceuticals (chrysotoxene, erianin, confusarin, polysaccharide, alkaloid ...) in anti-cancer, anti-aging, boosting immunity and vasodilation, etc. In recent years, more than 4,000 hectares of Dendrobium officinate Kimura et Migo artificial planting has been available in China and the price of dry product was around ¥ 80,000/kg. Currently, plantlet quality is an important factor influencing the acclimatization stage, growth, and development of plants in the greenhouse. In this study, the effect of a number of factors medium, plant regulator, ventilation, and substrate to improve plantlet quality as well as further growth and development in the greenhouse conditions were investigated. The results showed that SH medium was suitable for in vitro shoot growth in terms of monitoring parameters after 90 days of culture. In shoot multiplication stage, the shoots culture on SH medium supplemented with 2 mg/L BA, 30 g/L sucrose, 9 g/L agar, 1g /L activated charcoal (AC) gave the best results with 4.53 shoots/per shoot. The SH medium supplemented with 1.0 mg/L NAA, 30 g/L sucrose, 9 g/L agar, 1.0 g/L AC combined with ventilation conditions was suitable for rooting stage with plant height (5.73 cm), number of roots (4.77), root length (5.00 cm), fresh weight (3.36 g), dry weight (0,31 g), and total chlorophyll (SPAD) (45.76 nmol/cm2). Plantlets derived from culture ventilation conditions cultivated on the mixture of pine bark and fern fiber (50:50) was the highest survival rate (100%) and growth after 12 months in the greenhouse.

The Protective Function and Modification of Secondary Metabolite Accumulation in Response to Light Stress in Dracocephalum forrestii Shoots.

The aim of this work was to determine the effect of stress conditions caused by different light sources, i.e., blue LED (λ = 430 nm), red LED (λ = 670 nm), blue and red LED (70%:30%) and white LED (430–670 nm) on the growth and morphology of cultivated in vitro Dracocephalum forrestii shoot culture. It also examines the effects on bioactive phenolic compound production and photosynthetic pigment content, as well as on antioxidant enzyme activity (CAT, SOD, POD) and antioxidant properties. The most beneficial proliferation effect was observed under white LEDs (7.1 ± 2.1 shoots per explant). The white and blue lights stimulated the highest fresh weight gain, while red light induced the highest dry weight gain. The total phenolic acid content ranged from 13.824 ± 1.181 to 20.018 ± 801 mg g DW−1 depending on light conditions. The highest content of rosmarinic acid was found in the control shoots (cultivated under fluorescent lamps), followed by culture grown under red light. All LED treatments, especially red and blue, increased salvianolic acid B content, and blue increased apigenin p-coumarylrhamnoside biosynthesis. The greatest ferric reduction activity was observed in shoots cultivated under red light, followed by blue; this is associated with the presence of the highest total phenol content, especially phenolic acids. Similarly, the highest DPPH radical scavenging potential was observed under red light followed by blue. This study proves that LEDs have emerged as significant support for directed in vitro propagation, taking advantage of specific stress responses on various light spectra. This study also showed how stress induced by different LED light spectra increases in Dracocephalum forrestii the synthesis of pharmacologically-active compounds. Hence, light stress may turn out to be a simpler alternative to metabolic engineering for improving the production of secondary metabolites of therapeutic value.

Characterization of Green Pigment from Sauropus androgynus Shoot Cultures

The dark green leaves of Sauropus androgynus (Euphorbiaceae) have various nutritive values and are commonly used for human consumption as food, medicine, and natural dye substance in South-east Asia. Shoot cultures of this plant were established by adding various concentrations of kinetin (Kn) and benzyl adenine (BA) using nodal explants. The best results were recorded when Kn 0.1 mg/L was used with BA 1 mg/L (BA1Kn0.1). Spectrophotometric analysis showed two peaks of green pigment in shoot cultures, A pigment (λmax = 663.6 - 663.8 nm, absorbance 0.1111) and B pigment (λmax= 611.3 - 613.9 nm, absorbance 0.0390). Thin Layer Chromatography (TLC) analysis showed two green spots (Rf Y = 0.31 and Rf Z = 0.25) of shoot cultures on medium supplemented with BA1Kn0.1 for 10 days. Pigment profiles of shoot culltures were similar to their corresponding mother plants. Random Amplified Polymorphic DNA (RAPD) was used as a preliminary technique to evaluate the genetic similarity of the shoot cultures and their corresponding mother plants. It showed four similar DNA banding patterns to their leaves, ranging from 271-765 bp.

RNAi-mediated gene knockdown of progesterone 5\u03b2-reductases in Digitalis lanata reduces 5\u03b2-cardenolide content

Key messageStudying RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) in 5β-cardenolide formation.Progesterone 5β-reductases (P5βR) are assumed to catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-cardenolides. P5βRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5βRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5βR1 (AY585867.1) and DlP5βR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5β-cardenolide formation, we here established several RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5βR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5βR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5βR2-RNAi lines but not in Dl P5βR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5βR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).

Robust in vitro culture tools suitable for sustainable bioprospecting of the genus Hypericum

This article reports simple, robust, and species-independent methods for establishing in vitro cell suspension and shoot cultures useful for sustainable bioprospecting of Hypericum, a genus containing several medicinal plants commonly used in treating mild and moderate depression. Loose cell masses or shoot buds were directly induced by culturing seeds on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 6-benzyladenine (BA). Homogeneous cell suspension cultures could be achieved when cells were transferred to liquid culture medium, and robust shoot cultures could be established by transferring cultures containing shoot buds to basal medium. Stable cell suspension and (or) shoot cultures could be established for all Hypericum spp. tested (H. androsaemum L., H. linariifolium Vahl, H. elodes L., H. pulchrum L., H. humifusum L., H. undulatum Schousb. ex Willd, H. perfoliatum L., H. canariense L., H. tomentosum L., H. perforatum L. and H. maculatum Crantz), regardless of their habits and habitats.