Search Results (21)

In vitro hedging; combined with the fed-batch liquid media process is an innovative system that generates multiple sterile plants without the use of exogenous cytokinin. This combined process was demonstrated with Cannabis sativa (‘Cherry1’, ‘BaOx’, ‘T1’, ‘Peach’) grown in vessels of three different physical states—stationary agar (A); stationary Oasis^® infused with liquid (OILs); and agitated Oasis^® infused with liquid (OILa). Vessels were pre-selected as control or supplemented; where supplement vessels received 15 mL DKW liquid media each cycle harvest. The number of shoot tips harvested; shoot length; and dry shoot mass from repeated cutting cycles was recorded. In a single harvest; ‘BaOx’ and ‘Cherry 1’ produced one shoot per plant from the original 15 planted on all treatments. ‘Peach’ and ‘T1’ produced less shoots on average; but the most in OIL treatments. All shoots harvested were longer in OIL compared to A; regardless of genotype. Over multiple cycles; ‘Peach’ and ‘T1’ were unable to reliably produce shoots on a repeated schedule and were, therefore, eliminated from the experiment. By cycle 3; maximum number of plants were produced; regardless of supplementation (‘Cherry 1’; 30; ‘BaOx’; 22). Shoot length was above 10 mm (planting standard) for both genotypes until after the third cycle (10 weeks) where number and quality decreased (nodes and internodes easily discerned). By the end of the experiment; the only shoots that remained productive for over 16 weeks and multiple repeated harvest cycles were those in OIL treatments with supplements. Full article

Blue and supplemental far-red light were observed to affect in vitro shoot growth with Cannabis sativa (‘BaOx’ and ‘Cherry 1’) in RV750 vessels. A modified “hedging” and fed-batch system for multiple harvests using Oasis^® foam and 120 mL DKW medium was used. Fifteen nodal and/or apical tips were planted and placed into PAR light treatments providing various red to blue ratios (polychromatic white 9:1 RB and dichromatic 2–15: 1 RB, with and without 5% far-red light). Treatments had similar light intensities (190–240 µmol · m⁻² · s⁻¹ PPFD) for a 16 h photoperiod. Shoot tips were harvested in vitro on five successive two-week cycles, with 15 mL of DKW media supplemented to each vessel following harvest. Shoot numbers, length, and fresh and dry mass were recorded at each cycle harvest. Five randomly selected shoot tips per vessel were rooted ex vitro on greenhouse mist bench for 16 days. Over multiple cycles, 5% far-red increased shoot numbers and length in both genotypes tested, regardless of polychromatic or dichromatic source. Shoots harvested per vessel increased from 15 to 28 in three cycles (6 weeks), but increased from 15 to 18 without far-red treatment. Shoot length in far-red-treated plants increased from 19 to 25 mm during cycles 1–3. Plants without far-red treatment were approximately 15 mm during the first three cycles. By cycle 5, both far-red- and non-far-red-treated plants decreased to 10 mm. Dry mass was greatest in cycle 1 for both genotypes (‘Cherry 1’ was 6 mg and ‘BaOx’ was 7 mg) under the highest amount of blue light, but 2 mg under the lowest amount of blue light. Dry mass decreased by 50% in cycle 3, to 4 mg, where it remained for the duration of the experiment. Sixty eight percent of shoots rooted ex vitro on the mist bench, regardless of any prior in vitro treatment. Full article

Cannabis has been cultivated for thousands of years for a variety of purposes, including fiber, seeds, oil, and medicinal compounds. The cannabis industry is growing rapidly because several countries have recently legalized the use of cannabis. In these countries, the industry related to cannabinoid ingredients such as cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC) is steadily increasing every year. High concentrations of cannabinoids are mainly produced in unfertilized female flowers. Maintaining plants with high cannabinoid content is essential for producing uniform substances in large quantities. This study established an in vitro micropropagation protocol that can maintain the mother plant characteristics of Cannabis sativa ‘Cheungsam’. As a result of this experiment, the shoot tips and nodes of Cannabis sativa ‘Cheungsam’ at various concentrations (0, 0.25, 0.5, 1.0 mg/L) of 2iP, BA, and mT plant growth regulators (PGRs), and all concentrations of 2iP showed better results compared to two other hormones. However, the cut surfaces turned black, and excessive hyperhydricity occurred. Based on these symptoms, activated charcoal was added to the medium with the assumption that necrosis and hyperhydricity occur due to the accumulation of reactive oxygen species (ROS). When treated with 0.5 g/L charcoal, hyperhydricity was not overcome, and there was no difference compared to the control. As a new alternative, we divided the experiments into MS (Murashige and Skoog) and DKW (Driver and Kuniyuki Walnut) medium, which were commercially available. As a result, the rate of hyperhydricity was reduced, the cut surface did not turn black, and the growth conditions were also improved. Subsequently, ½ MS medium and ½ DKW medium were treated with various concentrations of IBA alone and with combinations of IBA and NAA for rooting. As a result, ½ DKW with IBA 0.5 mg/L showed the highest rooting rate and the best root condition for Cheungsam. After 4 weeks, when considering rooted plants with a height above 5 cm that were acclimatized, the acclimatization rate reached 100%. In conclusion, the Cannabis sativa ‘Cheungsam’ plants used in this study produced healthy shoots on DKW medium containing 1.0 mg/L 2iP and 0.5 mg/L of IBA in ½ DKW medium showed the best rooting rate. Full article

Worldwide, oak species are threatened with extinction due to habitat loss, pathogens, and changing fire regimes. Ex situ conservation through tissue culture may protect the remaining genetic diversity of Quercus dumosa, or the coastal sage scrub oak, from further loss. We designed three basal salt formulations based on the mineral composition of shoot tips and first leaves from mature Q. dumosa and explored carbohydrate source, stress-mitigating compounds, and plant growth regulator concentrations to develop a method of cultivating many Q. dumosa culture lines in vitro. All three novel basal salt formulations led to decreased necrosis compared with commercial basal salt formulas WPM, MS, and DKW. Substitution of 30 g L⁻¹ sucrose with glucose and adding 250 mg L⁻¹ ascorbic acid, 5.2 mg L⁻¹ SNP sodium nitroprusside, and 103 mg L⁻¹ y-aminobutyric acid improved culture health overall. In an experiment involving 115 culture lines, 0.66 mg L⁻¹ 6-benzylaminopurine produced the highest average shoots per explant, but 0.33 mg L⁻¹ produced the greatest proportion of shoots 2 cm or greater. Incubation for 24 h in 20 mg L⁻¹ indole-3-butyric acid led to the most rooting. These methods show promise for the ex situ conservation of many genotypes of endangered Q. dumosa. Full article

Cistus creticus L. (rockrose), a species of ecological and medicinal significance, constitutes a valuable component of the Mediterranean ecosystem. The present study investigated the effect of the inorganic salt concentration of Murashige and Skoog medium (MS), woody plant medium (WPM), and Driver and Kuniyaki Walnut medium (DKW) at several strengths (1/8×, 1/4×, 1/2×, 1×, and 2×) on the in vitro growth and organogenesis of rockrose. Significant interactions were observed throughout the experiments between pairs of plant origins, medium types, and strengths, and we also examined the extent to which they affected the studied traits was examined. The types of nutrient medium affected all studied traits except shoot and root percentages. The maximum growth percentage (143.49%) was gained using full-strength WPM. The best performance in shoot percentage was obtained using MS (100%) at several strengths along with 1× WPM (100%). The topmost rooting percentage values (98.61%) were obtained using 1× WPM and 1/2× DKW. The highest number of shoots and roots were observed using full-strength MS (9.39) and half-strength WPM (6.49), respectively. The maximum values for shoot and root length were achieved using 1/2× MS (0.78 cm) and 1/8× WPM (1.55 cm), respectively. The origin of the plant material did not influence any studied trait. Moreover, the genetic relations among the populations used in the in vitro culture were assessed using simple sequence repeats (SSR) markers. Twenty-eight alleles were identified across all five STR loci. The different and effective alleles per locus were 5.60 and 4.72, respectively. The average observed and expected heterozygosity was estimated at 0.52 and 0.72, respectively. Shannon’s information index and the inbreeding coefficient (F) were assessed at 1.48 and 0.30, respectively, revealing a narrow genetic base and high genetic similarity among origins, suggesting that they belong to the same population. Full article

In the present study, the effects of different types of sugars and cultivation medium pH levels on the micropropagation of Krymsk 5^® cherry rootstock were investigated. During the proliferation stage, the effects of four sugars (sucrose, fructose, glucose, and sorbitol) both separately and in two combinations were studied, along with the effects of pre-adjusted pH (4.5, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, or 6.5) on shoot proliferation parameters, growth medium’s post-autoclaving and post-cultivation pH, and their relations. Similarly, during the rooting stage, the effects of four sugars (sucrose, glucose, fructose, or sorbitol) at three concentrations (1% w/v, 2% w/v, or 3% w/v) without any auxin inclusion were studied as well as the effects of two sugars (sucrose or fructose) at six pre-adjusted pH levels (4.8, 5.2, 5.8, 6.2, or 6.5), also in the absence of auxin, on rooting parameters. Explants cultivated in fructose-supplemented growth mediums exhibited superior proliferation performance, characterized by the highest values of shoots per explant, shoot length, and nodes per explant. Generally, the medium’s pH decreased after autoclaving, and proliferation performance was favored by low pH values (either pre-adjusted or post-autoclaving). As far as rooting is concerned, fructose inclusion induced a higher rooting percentage (88%) compared to sucrose. The highest rooting was obtained in fructose-supplemented rooting mediums at concentrations of 2% or 3% w/v (95% rooting in both cases), in the absence of auxins. Post-autoclaving pH in fructose-supplemented rooting mediums was lower and buffered in low pH levels than in sucrose-supplemented ones, and the rooting of explants in all pH combinations with fructose exceeded 75%. In addition, rooting was negatively correlated with the post-autoclaving pH. These findings underscore the significance of both the sugar type and the post-autoclaving pH of the medium in both proliferation and rooting stages, highlighting their possible physiological, biochemical, or hormonal effects. Additionally, rooting without the use of auxin, but with the correct choice of sugar, emerges with both financial and environmental benefits, whereas fructose could be potentially used as a buffering agent. Full article

The Fengfeng mining area is in the transition zone between the North China Plain and the Taihang Mountains, and groundwater is the main source of water supply in the district. Under the combined influence of human activities and natural geological conditions, the quality of different types of groundwater varies greatly, posing a potential threat to the safety of drinking water. In this study, hydrogeochemical processes in different types of groundwater were analyzed using multivariate statistical analysis methods with ion–ratio relationships, and a groundwater quality and health risk assessment model was developed. The research findings show that the main chemical components and TDS in the groundwater have obvious spatial distribution characteristics, i.e., the content of deep karst water (DKW) in the west is significantly lower than that of shallow pore water (SPW) in the east, and the hydrochemical type has changed from HCO₃–Ca to SO₄–Ca. The chemical components of SPW and DKW are mainly derived from silicates and carbonates, accompanied by weathering dissolution of sulphidic minerals, especially SPW. The chemical components of the groundwater was also influenced by the cation exchange reaction and human activities. The quality of the SPW was significantly worse than that of the DKW, and the nitrates in SPW carry a high non-carcinogenic risk, especially to children. The shallow pore water is not suitable for drinking water. This study can provide guidance on the safety of drinking water in the Fengfeng coal mining area and other areas with intensive industrial, mining, and agricultural activities. Full article

There is an increasing trend in forest production towards planting rapid-growing trees as attractive, environmentally friendly energy sources. This study aimed to establish an alternative to the traditional propagation of a number of selections of Robinia pseudoacacia L. by developing an in vitro culture protocol. This study’s topic is of great importance, and it reflects an ongoing concern at the University of Oradea’s Faculty of Environmental Protection’s sustainable research program. The explants from four forms (called S1, S2, S3, and S4), selected for their phenotypic characteristics, were inoculated on four culture media (Murashige–Skoog (MS), Anderson, Chée–Pool, and Driver and Kuniyuki Woody (DKW)) with the same phytohormonal balance. DKW medium proved to be the better support of morphogenic activity, and it was further tested under different phytohormonal balances. Different results were observed depending on the hormone content in the DKW environment. In the presence of 0.5 mg/L benzylaminopurine (BAP) and 0.04 mg/L aminoisobutyric acid (AIB), 91.5% of the explants developed an average of 4.45 ± 0.18 shoots, whereas the average upper shoot height (3.82 cm) was recorded on DKW medium with 0.5 mg/L BAP and 0.04 mg/L α-naphthaleneacetic acid (NAA). Auxin, 0.05 mg/L AIB, promoted root production (5.27 ± 0.15 roots/explant), while 0.1 mg/L NAA promoted root length. In conclusion, the S4 selection produced the greatest outcomes of all environmental variables in terms of both the number of shoots and their heights. Full article

Catalpa bungei C. A. Mey is a unique and precious multi-purpose tree species that possesses great timber-related, ornamental and medicinal values. In this study, MS, N6 and DKW were used as basic media, and different concentrations of 6-BA and NAA were added for callus induction. The induction rate and total flavonoid content of callus tissue showed that the best callus induction medium was DKW + 2.0 mg·L⁻¹ 6-BA + 0.5–1.0 mg·L⁻¹ NAA. The leaves of different bark phenotypes of C. bungei C. A. Mey and the callus tissue extracted from young leaves of C. bungei C. A. Mey were used as experimental materials to construct metabolomic profiles of widely targeted flavonoids. Based on the metabolomic databases, the predominant flavonoids were screened from the callus tissues. Eight flavonoid metabolites increased in callus, and diosmetin-7-O-rutinoside (diosmin) was the flavonoid constituent with the shortest retention time, most efficient detection and best medicinal functions among these 8. The optimal medium for callus induction was supplemented with different concentrations of elicitors (salicylic acid SA and yeast extract YE). The optimal elicitor and the amount to be added were determined by analyzing the induction rate of callus, as well as the total contents of flavonoids and diosmin. The addition of SA and YE in appropriate amounts increased the total flavonoid content in the callus, but only the addition of YE promoted the formation of diosmin in the callus. The optimal medium formulation to promote the formation of callus was DKW + 2.0 mg·L⁻¹ 6-BA + 1.0 mg·L⁻¹ NAA + 30 g·L⁻¹ sucrose + 6.8 g·L⁻¹ agar + 10 μmol·L⁻¹ SA. The medium formulation to optimally increase the content of geraniol glucoside was DKW + 2.0 mg·L⁻¹ 6-BA + 1.0 mg·L⁻¹ NAA + 30 g·L⁻¹ sucrose + 6.8 g·L⁻¹ agar + 200 mg·L⁻¹ YE. The results of the present study will provide a scientific basis for the subsequent increase in the content of the active components of the suspension cells via the addition of elicitors, and for the production of diosmin in factory settings. Full article

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication of the derived shoots in liquid culture in a temporary immersion system (TIS). The best explants were found to be 30 cm long immature inflorescences, preferably collected in spring. Such an explant type was easy to decontaminate, and the spikelets isolated from it provided over 100 embryogenic callus lines. Among the callus induction media tested, gelled MS medium supplemented with 1.1 mg/L 2,4-D provided the highest percentage of responsive spikelets and the highest density of embryogenic callus. Maturation of the embryogenic callus was easily triggered on gelled MS medium devoid of plant growth regulators. The obtained shoots could be further multiplied on previously optimized gelled DKW medium supplemented with 30 g/L sucrose, 5 mg/L BA, 0.1 mg/L IBA, and 6.8 g/L plant agar. Subsequent high multiplication of the developed shoots was achieved in liquid culture in TIS using a Plantform™ bioreactor, with an immersion cycle of 12 min every 8 h. Full article

Pyrus spinosa Forssk. (almond-leaved pear) is a wild-growing native tree of the hilly countryside of Greece, which has recently gained interest for its valuable endogenous substances. In the present work, the determination of the total phenol content (TPC) and antioxidant capacity (AC) was carried out during 2021 and 2022 in leaves from 32 genotypes of mature wild-growing trees at four different locations of Central Macedonia District, Greece: Agia Anastasia, Chalkidiki, Lagadas and Pieria. The measurements taken from the leaf samples of all genotypes revealed a strong positive linear correlation between TPC and AC (R² = 0.772). Two genotypes from Agia Anastasia, coded as AA2 and AA3, were comparatively the richest in TPC (44.86 for AA2 and 46.32 mg GAE/g fresh weight for AA3) and AC (70.31 and 71.21 μg AAE/g fresh weight for the same genotypes). For these two genotypes of high TPC and AC, an efficient micropropagation protocol was developed to preserve and multiply this valuable germplasm. Newly emerged shoot tips were excised from winter shoots and, after disinfestation, they were established on a modified Murashige and Skoog (MS) nutrient medium with 5 μΜ of 6-Benzyloaminopurine (BAP). For shoot multiplication, explants from clean cultures were transferred to Pear Medium 1 with 5 μΜ of BAP. The effect of the nutrient medium was tested by using five different nutrient media (modified MS, Pear Medium 1, Pear Medium 2, DKW and WPM) supplemented with 5 μΜ of BAP. Pear Medium 1 was the most effective in shoot formation. Among the four different BAP concentrations (0, 5, 10 and 20 μΜ) used in Pear Medium 1, 5 μΜ of BAP resulted in the production of the significantly highest number of shoots (22.7 shoots per explant, 2.4 cm long). The exposure of cultures to 10 μmol·m⁻²·s⁻¹ for one week under light irradiance followed by 35 μmol·m⁻²·s⁻¹ for four weeks increased both the number and length of the shoots produced. A 20-s dip of the shoot bases into 49.0 μΜ of Indole-3-butyric acid (IBA) in EtOH equally affected rooting in a modified rooting MS (79%) and Pear Medium 1 (80%). The root system developed better in the modified rooting MS medium (mean number of roots of 2.7 with a length of 3.9 cm). The acclimatization of the plantlets was successfully carried out (high survival rates five weeks after their establishment) on a perlite substrate under controlled environmental conditions. Full article

With the continuous development of society, the output of kitchen waste (KW) is fast increasing. De-oiled kitchen water (DKW) and kitchen waste treatment residue (KWTR), two main by-products of the KW treatment industry, are produced accordingly on a large scale. The need to develop an effective technique for the utilization of DKW and KWTR is attracting wide attention. In the present study, black soldier fly larvae (BSFL) were employed as a biological treatment method to treat KWTR with the addition of DKW. The influence of DKW (0–140 mL) on the efficiency of BSFL treatment evaluated by the growth and development of BSFL, the body composition of BSFL, the nutrient content of bioconversion residue (BR), and the bioconversion efficiency of KWTR, was investigated. The results showed that the growth and development of BSFL, the body composition of BSFL, and the conversion rate of KWTR were initially promoted and then inhibited with the addition of DKW. Notably, the amount of DKW added in the T110 group was the most suitable for the growth of BSFL and the accumulation of body composition. Compared with the blank comparison group, the content of crude protein (CP), crude ash (CA), salinity, total phosphorus (TP), and dry matter (DM) of BSFL in the T110 group increased by 3.54%, 6.85%, 0.98%, 0.07% and 2.98%, respectively. However, the addition of DKW could steadily increase the nutrient content of BR, with the highest amount at 140 mL DKW. Following DKW addition, the contents of CP, ether extract (EE), crude fiber (CF), organic matter (OM), total nitrogen (TN), TP, and total potassium (TK) were increased by 4.56%, 3.63%, 10.53%, 5.14%, 0.73%, 0.75%, and 0.52%, respectively, compared with those of the blank comparison group. The study showed that DKW could be used as a nutrient additive in the bioconversion process of KWTR by BSFL, which provided a new method for the resource utilization of DKW. Full article

Juniperus drupacea Labill. (Cupressaceae) is a species with ecological and medicinal value. In Europe, it is native only in southern Greece, and is listed as endangered. Due to its uniqueness, this study attempted, for the first time, an in vitro propagation effort of Syrian juniper. Explants of the lateral shoot tips were surface-sterilized and cultured on Murashige and Skoog (MS) medium. The cultures were subcultured on MS, woody plant medium (WPM), and Driver and Kuniyaki Walnut (DKW) supplemented with different concentrations of 6-benzylaminopurine (BA), thidiazuron (TDZ), or meta-topolin [6-(3-hy-droxybenzylamino)purine] for shoot induction. Explants derived from female trees exhibited 54.17% bud proliferation on DKW medium with 4 μM meta-topolin or 4 μM TDZ and on WPM with 4 μM meta-topolin or 4 μM BA. A total of 62.50% of the male tree derived explants produced multiple shoots on DKW with 4 μM BA. The maximum average number of shoots per explant were 1.17 per explant in both cases. The length of the shoot derived from explants of female origin was 2.94 mm compared to 2.69 mm of the in vitro shoots from the explants of male trees. Overall, the best medium and plant growth regulator combination for the explants derived from both female and male trees, for the traits under study, was proven to be DKW + 4 µM TDZ. Our experiments show that Juniperus drupacea, under in vitro conditions, shows recalcitrance in rooting, as the applications of IBA, NAA, and IAA concentrations were proven to be ineffective treatments. Although the results show low values, this avant-garde study provides a foundation for further research on the in vitro regeneration of Juniperus drupacea. Full article

Difficult-to-root plants often perform poorly during acclimatization and in vitro rooting can increase the survival and quality of plants. The influence of auxin application and mineral nutrition on in vitro rooting and subsequent effects on plant quality in eight Prunus genotypes were investigated. Microshoots were rooted in vitro on Murashige and Skoog (MS), ½ MS, Driver and Kuniyuki (DKW), or New Prunus Medium (NPM) media formulations in combination with 15 µM indole-3-butyric acid (IBA), 4-day 15 µM IBA pulse, 1 mM 30 s quick-dip, or IBA-free treatments. Shoots were observed pre- and post-acclimatization to determine rooting methods to maximize quality and minimize labor. A genotype-specific response to auxin application was observed with seven of eight genotypes achieving 100% survival when paired with the recommended IBA treatment. Peaches performed best when treated with 4-day IBA pulse or 30 s quick-dip. Rooting of P. cerasifera, it’s hybrid to P. persica, and P. munsoniana all benefitted from IBA application. Shoots rooted with 15 µM IBA were smaller and lower quality in most genotypes. DKW maximized size and quality in six genotypes. Better shoots and larger root systems during in vitro rooting produced better plants in the greenhouse with no detrimental effect of callus growth. Rooting techniques to maximize plant quality while reducing labor are specified. Full article

Castanea sativa cv. ‘Garrone Rosso’ and ‘Marrone di Castel del Rio’ are two of the most prized varieties in Italy due to their valuable and healthy nuts used for fresh consumption and in the confectionery industry. Despite the growing demand for chestnuts, there are constraints regarding plant propagation that hamper the renewal and new planting of orchards in different areas. Castanea sativa is susceptible to diseases that have caused a reduction in its area of production. For this reason, in vitro culture represents a valuable technique for germplasm preservation and plant multiplication enabling production of a high number of plants for use in breeding programs. Here we present an in vitro micropropagation protocol for Italian Castanea sativa cv. ‘Marrone di Castel del Rio’ and cv. ‘Garrone Rosso’ to contribute to the preservation and enhancement of the Italian germplasm. Nodal explants were used as the starting material for in vitro establishment. The cv. ‘Marrone di Castel del Rio’ showed a high percentage of survival explants (92%) when subjected to long bleach exposure (25 min), in contrast to what was observed for the ‘Garrone Rosso’ cultivar. Ascorbic acid was found to be the best compound to counteract phenol exudation. The MS3B and DKW media supplied with 0.5 mg/L BAP were effective for in vitro establishment, while the DKW medium (0.1 mg/L BAP and 0.05 mg/L IBA) was preferable for the proliferation phase. A double-layer rooting methodology was used and 35% rooting was observed with 25 mg/L IBA rooting treatment. Full article