Vitrification is one of the most significant issues encountered in plant tissue culture applications. It diminishes the quality of in vitro plants, causing their leaves and stems to appear watery and ...translucent, and it may impede the success of the acclimatization step. In this respect, this study investigates the impact of sucrose and 6-Benzylaminopurine (BAP) concentrations on shoot regeneration and vitrification development in
Aronia melanocarpa
, known for its high antioxidant content and health benefits. Initially, the presence of BAP, in combination with varying sucrose concentrations, leads to a substantial increase in shoot number, and the largest number (7 shoots per nodal explant) was observed in the Murashige and Skoog (MS) medium containing 3% sucrose and 5.0 mg/L BAP. Furthermore, sucrose concentration plays a crucial role in shoot growth, with higher concentrations promoting more extensive shoot development. However, when 3% sucrose was combined with higher BAP (from 1.0- to 5.0 mg/L), an increased incidence of vitrification was observed over time. Interestingly, lower sucrose concentrations (1% or 2%) combined with 0.5 mg/L or 2.5 mg/L BAP initially delayed vitrification but eventually led to its occurrence. Microscopic analysis of leaf samples with varying levels of vitrification indicates significant differences in the density of stomata, further confirming the detrimental impact of vitrification on cellular structures and physiological processes. The recovery of vitrified plants was evaluated using different growth media combinations. The absence of BAP in the medium led to higher recovery percentages (min 96%) without necrosis, while the addition of 0.5 mg/L BAP promoted shoot growth but potentially inhibited root development. It has been found that media with 1 mg/L BAP and either 10 g/L or 20 g/L sucrose, as well as media with 30 g/L sucrose and 0.5 mg/L BAP, are the most suitable for efficient shoot regeneration with minimal vitrification risk. However, increasing BAP levels for faster shoot regeneration also raises the risk of vitrification. During acclimatization, vitrified plants exhibited stunted shoot growth, shorter and narrower leaves, reduced root numbers and lengths, and decreased survival rates, particularly under lower humidity conditions. The cultivation period required for the recovery of the crop was determined to be 6 weeks under greenhouse conditions for a sustainable plant propagation.
Key message
Optimizing
Aronia melanocarpa
shoot regeneration: Balance sucrose and BAP for vigorous growth. Beware vitrification! Tailor medium and conditions for successful recovery.
This study aims to address the effects of different concentrations (0, 0.1, 1.0, 10, 100 or 1000 mg L−1) of engineered zinc oxide (ZnO) nanoparticles (34 nm in size) on growth parameters, steviol ...glycosides (rebaudioside A and stevioside) production and antioxidant activities in the tissue culture grown shoots of Stevia rebaudiana Bertoni. The highest percentage of shoot formation (89.6%) at 1 mg L−1 of ZnO nanoparticles concentration suggests a positive influence of ZnO nanoparticles on S. rebaudiana growth as compared to other treatments with or without ZnO nanoparticles. Additionally, HPLC results illustrate a significant enhancement of steviol glycosides (almost doubled as compared to the control) in micropropagated shoots grown under an oxidative stress of 1 mg L−1 of ZnO nanoparticles. This finding is further affirmed by an increased 2,2-diphenyl-1-picryl hydrazyl (DPPH) scavenging activity, total anti-oxidant capacity, total reducing power, total flavonoid content and total phenolic content, with an ascending oxidative pressure and generation of reactive oxygen species (ROS). However, the antioxidant activities, formation of secondary metabolites and the physiological parameters showed a sudden decline after crossing a threshold of 1 mg L−1 concentration of ZnO nanoparticles and falls to a minimum at 1000 mg L−1, elucidating maximum phytotoxic effect of ZnO nanoparticles at this concentration. This is the first study evaluating both the favorable and adverse effects of ZnO nanoparticles employed to a highly valuable medicinal plant, S. rebaudiana.
•Maximum rebaudioside-A and stevioside at 1 mg/L concentration of ZnO nanoparticles.•ZnO nanoparticles affects physiological parameters and steviol glycoside content.•ROS as a key factor for physiology and biochemical aspects of Stevia rebaudiana.
In this present study, seed vigor was tested after different storage durations (0, 60, 120, 180 days; maximum 420 days) in air-conditioning cabinets set to various temperatures (4, 10, 20 °C) and ...free-temperature at room condition. After seven days for each of the storage durations, germination rate (GR), mean germination time (MGT), germination speed (GS), seed vigor index (SVI), shoot and root length (SL, RL), shoot and root dry weight (SDW, RDW), root number (RN), electrical conductivity (EC) and pH for all seed lots were recorded. The average GR at room temperature storage decreased from 86% recorded on the 60th day, to 59% by the end of the 420th day, while germination rate was still around 70% with the seeds stored at 4 °C. The decrease in GS was recorded after the 300th day till the end of 420 days of storage (ranging between 9.8 and 9.4 seed day−1). The lowest SVI was 7.9 at the beginning of storage (day 0) and increased gradually within 60 days of storage for all temperatures tested. Irrespective of storage duration, the SL and RN showed the highest overall mean of 10.7 cm and 4.8 root for the seeds stored at 4 °C. EC sharply decreased overall to a mean of 18.9 μScm−1 g−1 for all temperatures within the first 60 days of storage. The highest correlation (r = 0.937) was determined between GS and MGT. This study provides a new insight to the farmers for better understanding that concerns practical implications of einkorn farming as follows: storage at 4 °C is best way of the maintanence of highest seed vigor. For the storage duration regardless of the temperatures, it can be kept minimum 60 days, since the seed deterioration can be effective after 180 days of storage depending on storage temperature.
•The germination rate at room temperature storage decreased from 86% recorded in 60th day, to 59% by the end of the 420th day.•Mean germination time was ranging between 3.4 and 3.8 days for all temperatures.•Storage under room temperatures and 20 °C, germination loss is higher than other conditions.•Storage can be achieved up to 180 days for all storage temperatures.•60 days of storage at 4 °C is needed for the maintenance of seed vigor and 120 days of storage is needed at higher temperatures.
Quinoa (Chenopodium quinoa Willd.) cultivated since more than 5000 BC. Because the gluten-free and high nutritious grains have been of commercial interest over last two decades, studies have focused ...on mainly adaptation and commercial production. In this study, seed vigor was tested after different storage periods (60, 120, 180, 240, 300 and 360 days) and temperatures (4-, 10- and 25 °C) for Mint Vanilla and Titicaca varieties at the temperature-humidity-controlled environments. Seed testing and growth parameters for the grains raised from each storage conditions were recorded after 7 days of seed incubation in each treatment. It was shown that the maintenance of seed vigor can vary significantly depending on genotype selection (i.e. seed vigor was the highest for Titicaca grains), as a result of kinetic modeling and constellation plot analysis. The most effective temperature that can be used in storage for the longer durability for quinoa seeds is 4 °C and 10 °C. Seed deterioration and vigor loss were a clear patent at 25 °C for both quinoa varieties. As a result, the storage temperature and duration show the change of germination parameters and may be used as a reference for quinoa quality and consumption safety.
Display omitted
•Seed vigor of Minta Vanilla was lost after 120-day of storage at 25 °C.•Seed vigor of Titicaca was lost after 180-day of storage at 25 °C.•Moisture content significantly was declined after 180-day of storage at 4 °C but increased at 10- and 25 °C.•Constellation plot analysis showed that Titicaca were more durable for the prolong storage at 4 °C.•Kinetic analysis at 4 and 10 °C became a standout to prevent germination loss.
Hydrogen peroxide (H
2
O
2
) functions as an abiotic stress elicitor for plants. In this study, the effect of oxidative stress caused by H
2
O
2
on steviol glycosides (SGs) production and antioxidant ...activities of an in vitro grown antidiabetic plant,
Stevia rebaudiana
, has been investigated. Direct shoot organogenesis was performed, and after 4 weeks of culture, the shoots of
S. rebaudiana
were transferred to MS medium containing H
2
O
2
(10 or 20 mM) and incubated in growth room for 6 h. It resulted in increased SGs production, such as rebaudioside A (Reb-A) and stevioside (ST), and enhancement of total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power and DPPH free radical scavenging activity when compared with control. The results clearly showed highest amount of SGs produced in MS medium containing 10 mM H
2
O
2
producing 2.97% Reb-A and 2.32% ST (w/w), followed by the medium employing 20 mM H
2
O
2
and control group. It is also evident that H
2
O
2
pre-treatment caused significant increase in non-enzymatic antioxidants that play defensive role against an oxidative stress induced by H
2
O
2
. The described protocol has a great potential to be utilized for Reb-A production on commercial scale.
Steviol glycosides (SGs) and gibberellic acids share a part of their biosynthesis pathways. Despite the widespread studies on the effect of gibberellic acid 3 (GA3), the effect of gibberellic acid 4 ...and 7 (GA4/7) on Stevia rebaudiana has never been studied. This study aimed at a comparative evaluation of different hormone effects, i.e., 1 mg L−1 GA4/7, 1 mg L−1 GA3, or 0.5 mg L−1 kinetin and 0.5 mg L−1 6-benzylaminopurine (BAP) (KB 0.5), on in vitro propagation, growth, morphological properties, and content of SGs in leaf samples of stevia. In comparison with the control group (hormone-free), the treatments of KB 0.5 or GA3 produced the highest biomasses and largest leaf areas. The three hormonal treatments produced a similar number of leaves, the ratio of fresh to dry weight, and leaf length. GA4/7-treated explants produced the highest ratio of leaf area to leaf length. The effect of GA4/7 on shoot elongation was greater than that of the control or even GA3. While the effect of GA3 on rebaudioside-A (Reb-A) production was similar to that of the control (16.2 and 18.04 mg g−1, respectively), GA4/7 resulted in a lower amount of it (13.31 mg g−1). Except for GA4/7, which induced more stevioside accumulation, the treatments’ effects were comparable to that of the control. The ratio of stevioside to Reb-A was the highest for GA4/7 (2.62), followed by GA3 (1.93), and then the two others. Sum of Reb-A and stevioside content was not changed by the use of any of the treatments.
•GA3 and GA4/7 effects on stevia growth were similar, except for auxiliary shoot length, which elongated with GA4/7.•The ratio of Stevioside to Reb-A content significantly increased in GA4/7 treated plants.•The effects of kinetin and BAP on each of the SG contents, ratio, and the total amount, were similar to the control.•GAs application in stevia plants has a negative effect on Reb-A accumulation, but not on Stevioside amount.•The results suggest that the GAs may change the ratio of Reb-A to Stevioside but not the total SG contents.
Silybum marianum L. is a medicinal plant used in the treatment for jaundice and liver diseases. In this study, an adventitious root culture was developed for the production of health promoting ...phytochemicals. Adventitious roots were induced from nodal explants on solid Murashige and Skoog medium supplemented with 1.0 mg l⁻¹ of α-Naphthalene acetic acid. Growth kinetics of the roots was investigated every week, for 8 weeks of culture period. Highest fresh biomass formation (153 mg l⁻¹) was observed in 6-week old cultures. Adventitious roots were harvested from different growth stages as control (CTR), lag phase (LAG), logarithmic phase (LOG) or stationary phase (STN). Metabolite profiling of the samples was investigated using electro spray ionization time of flight mass spectrometry. Significant phenylpropanoids such as cinnamic acid and di-hydro kaempferol were predominantly found in LOG phase, whereas the highest amount of malonic acid was detected in STN as compared to other growth phases. More sucrose content was detected in CTR, while the tryptophan content was higher in LOG phase. Among the vital fatty acids, prostaglandin A1 and phenyl acetic acid were at highest levels in STN phase. However, more brassicasterols were observed in LAG phase than other growth phases. Punicic acid and lignan pinoresinol were detected abundantly in the LOG phase. Biochemical characterization revealed significant correlations between silymarin content and DPPH as well as TPC and TFC in the growth curve. Interestingly, among all growth stages there was no correlation of PAL activity with TFC and silymarin content.
Nanoparticles are known to play remarkable role as abiotic stress elicitors in plants. This study reports the comparative analysis of effects produced by capped zinc oxide (ZnO)-polyethylene glycol ...(PEG), ZnO-polyvinyl pyrrolidone (PVP), copper oxide (CuO)-PEG, CuO-PVP and uncapped (ZnO and CuO) nanoparticles on the medicinal plant, Stevia rebaudiana raised in vitro for the production of commercially important sweetener compounds. In context of shoot organogenesis, ZnO-PEG, ZnO-PVP, CuO-PEG, CuO-PVP were employed to the growth medium that resulted in increased growth parameters, and larger content of steviol glycosides as compared to the shoots raised in medium containing ZnO and CuO, revealed by high-performance liquid chromatography. In the meanwhile, non-enzymatic antioxidant activities including total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power and 2,2-diphenyl-1-picryl hydrazyl-free radical scavenging activity were calculated and showed comparatively greater amounts in shoots grown in medium containing capped ZnO or CuO nanoparticles. Furthermore, the ZnO and its derivatives revealed to be more reactive at 1 mg/l of concentration. Whereas, the CuO and its derivatives produced greater response on Stevia at 10 mg/l concentration of nanoparticles. This study paves the way for more such studies encompassing capped and uncapped nanoparticles and their ultimate effect on in-vitro grown plant tissues for the production of active metabolites on industrial scale.
Cardiac glycosides are plant natural compounds used to treat cardiac insufficiency in humans. Recent findings suggest a further medical use in oncology and virology. Since species of the genus ...Digitalis are the major source of these compounds, in vitro culture techniques can be useful tools for the propagation and selection of elite genotypes. Digitalis mariana ssp. heywoodii, an endemic and endangered species in Portugal, is known to have a very high content of cardiac glycosides, especially glucoevatromonoside. Here, D. mariana ssp. heywoodii plants were cloned from a seed-derived shoot explant through direct or indirect in vitro propagation. Multiple shoot formation was induced from a seed grown on agar-solidified Murashige and Skoog (MS)-based medium supplemented with 9.8 μM indole-3-butyric acid (IBA). Shoots were used to regenerate plants directly, and some were then used as a source of root explants for callus induction and growth by transferring to MS medium supplemented with 9.3 μM kinetin (KIN), 5.7 μM indoleacetic acid (IAA), and 1.1 μM 2,4-dichlorophenoxyacetic acid (2,4–D). Callus that was 3, 6, or 24 mo old efficiently developed adventitious shoots when placed on MS medium containing 13.3 μM 6-benzylaminopurine (BAP) and 1.7 μM IAA. Plants regenerated from either callus (R plants) or shoot cultures (S plants) were transferred to the greenhouse and propagated for 3, 4, or 10 mo. Regenerated plants were analyzed for cardenolide content and accumulation pattern. The highest cardenolide content, about 2.0 mg/100 g DW in terms of digitoxin equivalents (Dteq), was found in S10 plants (10 mo in the greenhouse), whereas plants regenerated from 24-mo-old callus (R24/10; 10 mo in the greenhouse) had the lowest cardenolide content of about 1.2 mg Dteq/100 g DW.
Abstract Vitrification is one of the most significant issues encountered in plant tissue culture applications. It diminishes the quality of in vitro plants, causing their leaves and stems to appear ...watery and translucent, and it may impede the success of the acclimatization step. In this respect, this study investigates the impact of sucrose and 6-Benzylaminopurine (BAP) concentrations on shoot regeneration and vitrification development in Aronia melanocarpa , known for its high antioxidant content and health benefits. Initially, the presence of BAP, in combination with varying sucrose concentrations, leads to a substantial increase in shoot number, and the largest number (7 shoots per nodal explant) was observed in the Murashige and Skoog (MS) medium containing 3% sucrose and 5.0 mg/L BAP. Furthermore, sucrose concentration plays a crucial role in shoot growth, with higher concentrations promoting more extensive shoot development. However, when 3% sucrose was combined with higher BAP (from 1.0- to 5.0 mg/L), an increased incidence of vitrification was observed over time. Interestingly, lower sucrose concentrations (1% or 2%) combined with 0.5 mg/L or 2.5 mg/L BAP initially delayed vitrification but eventually led to its occurrence. Microscopic analysis of leaf samples with varying levels of vitrification indicates significant differences in the density of stomata, further confirming the detrimental impact of vitrification on cellular structures and physiological processes. The recovery of vitrified plants was evaluated using different growth media combinations. The absence of BAP in the medium led to higher recovery percentages (min 96%) without necrosis, while the addition of 0.5 mg/L BAP promoted shoot growth but potentially inhibited root development. It has been found that media with 1 mg/L BAP and either 10 g/L or 20 g/L sucrose, as well as media with 30 g/L sucrose and 0.5 mg/L BAP, are the most suitable for efficient shoot regeneration with minimal vitrification risk. However, increasing BAP levels for faster shoot regeneration also raises the risk of vitrification. During acclimatization, vitrified plants exhibited stunted shoot growth, shorter and narrower leaves, reduced root numbers and lengths, and decreased survival rates, particularly under lower humidity conditions. The cultivation period required for the recovery of the crop was determined to be 6 weeks under greenhouse conditions for a sustainable plant propagation.
PDF
