Schenk And Hildebrandt Research Articles

Development of Haploid Plants by Shed-Microspore Culture in Platycodon grandiflorum (Jacq.) A. DC.

Anther and microspore cultures are efficient methods for inducing haploids in plants. The microspore culture by chromosome-doubling method can produce double haploid lines, developing pure lines within the first or second generations. This study aimed to induce haploid plants in Platycodon grandiflorum using the shed-microspore culture method. P. grandiflorum floral buds (n = 1503) were cultured in six types of medium to induce haploids. Anthers were placed on a solid-liquid double-layer medium and cold pre-treated at 9 °C for one week, followed by incubation in the dark at 25 °C. Embryogenesis was observed after approximately 70 days of culture, producing haploid plants through regeneration. Of the 1503 floral buds, embryos developed in 120 buds, resulting in the induction of 402 individuals. Among the media used, Schenk and Hildebrandt (SH) and 1/2SH exhibited high efficiency, with embryogenesis ratios of 12% and 13.4%, respectively. Additionally, the highest embryogenesis ratio (15.3%) was observed in flower buds sized 10 mm or less. Therefore, we established shed-microspore culture conditions to induce haploids in P. grandiflorum. Using this method, haploids can be efficiently induced in P. grandiflorum, shortening the breeding period by enabling the rapid development of inbred lines.

Efficient production and enhanced accumulation of Valerenic acid in Valeriana officinalis: Early identification of high-performing hairy root clones and Pioneering use of hydrogen peroxide as an elicitor

Valeriana officinalis (valerian) roots and rhizomes possess a long history of medicinal use due to their sedative, antiepileptic, and anticonvulsant properties. Valerenic acid, a bioactive sesquiterpene with therapeutic potential, is present in limited quantities within these tissues. This study explores the application of hairy root cultures for enhanced valerenic acid production. Hairy root induction was attempted on valerian leaves and petioles using three Rhizobium rhizogenes strains (ATCC15834, A4, and MSU440) across three culture media (Murashige and Skoog (MS), Gamborg's B5 (B5), and Schenk and Hildebrandt (SH)). A non-destructive imaging system and periodic analyses were employed to identify superior hairy root clones exhibiting increased branching frequency. Leaf explants co-cultured with R. rhizogenes strain ATCC15834 yielded the most promising clones, characterized by the highest dry weight (1.03 mg) and valerenic acid content (0.384 mg/g dry weight) when grown in a half-strength SH liquid medium. Following strain and media optimization, the impact of 50 mM hydrogen peroxide as an elicitor on valerenic acid production was investigated. This treatment resulted in a significant 1.76-fold increase in valerenic acid accumulation compared to the control group at the first day post-treatment. This approach presents a valuable strategy for the early identification of high-yielding hairy root lines. Moreover, the utilization of hydrogen peroxide, a safe and cost-effective elicitor, offers a rapid method for enhancing valerenic acid production in the selected superior clone. This study establishes a promising platform for the sustainable production of valuable plant compounds within both research and industrial settings.

Adventitious root culture of Lessertia frutescens for the production of triterpenoid saponins and polysaccharides

Lessertia frutescens is a perennial shrub of commercial importance in South Africa, but the scarcity of plant resources has limited current product production. In this study, to provide an alternative approach for obtaining L. frutescens material, adventitious roots (ARs) were induced from sterilized seedlings and cultured in a suspension culture system. During this process, selection tests were conducted to find a suitable auxin and its concentration for AR induction and a suitable basal medium for AR growth and metabolite accumulation; a kinetic study was then performed to constructure kinetic models. The results showed that compared to other auxins and concentrations, indole-3-butyric acid at 3 mg/L was suitable for increasing the number and length of ARs during AR induction. In AR suspension culture, Schenk and Hildebrandt (SH) was better than other basal media, and the maximum AR fresh (86.9 g/L) or dry weight (5.5 g/L), total triterpenoid saponin (92.6 mg/g DW), and polysaccharide (114.7 mg/g DW) contents were determined in the 1.5×SH medium. In addition, AR biomass and metabolite contents reached the maximum on day 42. The kinetic models for AR growth and triterpenoid and polysaccharide production were constructed, providing the basis for further optimization of culture conditions and large-scale culture.

Shoot Cultures of Vitis vinifera (Vine Grape) Different Cultivars as a Promising Innovative Cosmetic Raw Material-Phytochemical Profiling, Antioxidant Potential, and Whitening Activity.

The primary purpose of this work was the initiation and optimization of shoot cultures of different Vitis vinifera L. cultivars: cv. Chardonnay, cv. Hibernal, cv. Riesling, cv. Johanniter, cv. Solaris, cv. Cabernet Cortis, and cv. Regent. Cultures were maintained on 30-day growth cycles using two media, Murashige and Skoog (MS) and Schenk and Hildebrandt (SH), with various concentrations of plant growth regulators. Tested media ('W1'-'W4') contained varying concentrations of 6-benzylaminopurine (BA) in addition to indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA). High performance liquid chromatography coupled with mass spectrometry (UPLC-MS) was used for metabolomic profiling. In all tested extracts, 45 compounds were identified (6 amino acids, 4 phenolic acids, 13 flavan-3-ols, 3 flavonols, and 19 stilbenoids). Principal component analysis (PCA) was performed to assess the influence of the genotype and medium on metabolic content. PCA showed that metabolic content was mainly influenced by genotype and to a lesser extent by medium composition. MS media variants induced the amino acid, procyanidin, and flavan-3-ol production. In addition, the antioxidant potential and anti-tyrosinase activity was measured spectrophotometrically. The studies on antioxidant activity clearly reveal very high efficiency in reducing free radicals in the tested extracts. The strongest tyrosinase inhibition capacity was proved for shoots cv. Hibernal cultured in SH medium and supplemented with NAA, with an inhibition of 17.50%. These studies show that in vitro cultures of V. vinifera cvs. can be proposed as an alternative source of plant material that can be potentially used in cosmetic industry.

Influence of elicitation and precursors on major secondary metabolite production in cultures of purple Orthosiphon aristatus Blume Miq

Major compound of interest in purple varieties of Orthosiphon aristatus Blume Miq (O. aristatus) are sinensetin and rosmarinic acid. Unfortunately, the content of these two compounds in this plant is only in small amount. An approach to produce more of these compounds is a plant tissue culture. Hence, leaf shoot explants of a purple variety O. aristatus were inoculated on Schenk and Hildebrandt (SH) media with variations of the growth regulator 2,4-dichlorophenoxyacetis acid (2,4-D). The calli formed were used as material for making cell suspension cultures. The precursors cinnamic acid, coumaric acid, caffeic acid, and ferulic acid with respective concentrations of 0.1; 0.5; and 1 mM and salicylic acid (14, 70, and 140 mg/L) were added. The media also contained Cu2+ ions (30, 40 and 50 μM), pectin (0.05; 0.1; 0.2% w/v) and AgNO3 (80, 100, 120 μM) respectively. Callus that grew on SH media with 2,4-D 0.4 ppm had a good profile, since it could produce callus faster than 2,4-D 1 ppm and 2 ppm with a friable texture on this medium. The cell suspension culture added with cinnamic acid 1 mM (S3) and Cu2+ 40 μM (C2) had a good growth curve compared to other elicitor and precursors. The highest levels of rosmarinic acid and sinensetin were found in cell suspension cultures adding with 1 mM cinnamic acid precursor. This study is believed to be the first report on the production of rosmarinic acid and sinensetin from suspension cultures of O. aristatus produced on SH media.

In Vitro Culture of Eremurus spectabilis (Liliaceae), a Rare Ornamental and Medicinal Plant, through Root Explants

Eremurus spectabilis M. Bieb, a perennial herbaceous wild species, is commonly used in the horticultural, ornamental, and pharmaceutical markets. Studies on the tissue culture systems for this species would be beneficial for mass multiplication as well as for future breeding programs. An in vitro propagation technique was established here using tuberous root explants as unique and responsive starting materials for culture initiation. The proliferated calli were sub-cultured on shoot proliferation media and regenerated microshoots were assessed. The shoot proliferation rate, leaf number, leaf length, and chlorophyll and carotenoid contents were recorded. The highest callus induction per explant (76.67%), callus dry weight (10.25 mg), callus firmness ratio (3.97), and callus color intensity ratio (2.83) were observed in explants inoculated on Murashige and Skoog (MS) medium supplemented with 10.0 mgL−1 6-benzylaminopurine (BAP). The highest shoot proliferation rates were obtained when calli were sub-cultured on MS or Schenk and Hildebrandt (SH) basal media supplemented with 2.0 mgL−1 BAP. The half-strength MS medium fortified with 4.0% sucrose + 2.0 mgL−1 indole butyric acid (IBA) + 200 mgL−1 activated charcoal was a superior combination for root emergence and rooting parameters. Regenerated plantlets were then successfully adapted to ex vitro conditions. The reported protocol can be exploited at a commercial scale following minor modification, or could be beneficial in the production of secondary metabolites in bioreactors where callus is required as fresh plant material.

Expression Analysis of Phenylpropanoid Pathway Genes and Metabolomic Analysis of Phenylpropanoid Compounds in Adventitious, Hairy, and Seedling Roots of Tartary Buckwheat.

Tartary buckwheat (Fagopyrum tataricum) is an important crop that belongs to the Polygonaceae family, whose roots have received considerable attention due to the presence of compounds with high nutritional and medicinal value. In this study, we aimed to develop an efficient protocol for the culture of adventitious (ARs) and hairy (HRs) roots on a half-strength Schenk and Hildebrandt (SH) medium containing different concentrations of the auxins, α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA). The highest percentage of root induction (91.67%) was achieved with 0.5 mg/L IAA, whereas the greatest number of roots was found in 1 mg/L IAA. In contrast, 0.1 mg/L IBA returned the longest roots. As expected, HRs were obtained from in vitro leaf explants infected with Agrobacterium rhizogenes R1000. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 11 phenolic pathway genes revealed that five genes (FtPAL, FtC3H, FtHQT, FtCHS, and FtANS) were highly expressed in HRs, whereas only four (FtC4H, FtFLS2, FtDFR, and FtANR), and three (Ft4CL, FtCHI, and FtF3H) were recognized in the ARs and seedling roots (SRs), respectively. HPLC analysis of phenolic compounds in different root cultures showed that the majority of the phenolic compounds (both individual and total) were significantly accumulated in the HRs. Principal component analysis (PCA) identified differences among the three root types, whereby HRs were separated from ARs and SRs based on the amount of phenolic compounds present. Analysis of the metabolic pathway revealed that among the identified metabolites, the 3, 2, and 1 pathways were associated with flavonoid, flavone and flavonol, and phenylpropanoid biosynthesis, respectively. Hierarchical clustering analysis and the heat map showed that the different root cultures presented unique metabolites.

Induction and submerged cultivation of Valeriana jatamansi adventitious root cultures for production of valerenic acids and its derivatives

In vitro adventitious roots were induced from leaves of Valeriana jatamansi to assess their potential as a sustainable alternative to extract pharmaceutically important phytoconstituents. Among the different media used, a significantly (p ≤ 0.05) high root induction (90%) was achieved on Schenk and Hildebrandt (SH) medium fortified with 9.84 µM indole-3-butyric acid (IBA). In addition, various process parameters i.e. IBA concentration, sucrose and medium strength were also optimized under submerged cultivation. The maximum fresh root biomass (144.09 ± 11.36 g/L) with a high relative growth rate (2.01 ± 0.04) and growth index (13.41) was achieved in half-strength SH medium having 2% sucrose and 4.92 µM IBA. Further, a significantly high yield of total valerenic acid derivatives [1525.14 µg/g dry weight (DW)] was recorded in adventitious roots as compared to donor plant parts. Individually, valerenic acid (506.27 µg/g DW) was accumulated higher in plant rhizomes, while acetoxyvalerenic (534.91 µg/g DW) and hydroxyl valerenic acid (919.57 µg/g DW) in adventitious roots. Interestingly, hydroxy valerenic acid was unmeasurable in donor plant parts. The phenolic compounds were also found maximum in adventitious roots (451.85 µg/g DW) with the dominance of pharmaceutically important kaempferol and rutin. A substantial increase in phytochemicals was evident at subsequent culture stages with shortened in vitro cultivation cycle (2 months) than field-grown plants (24 months). Moreover, adventitious roots also accumulated 0.059% essential oil with patchouli alcohol (24%) as a key constituent. Conclusively, an enriched metabolic profile and substantially shorter growth cycle under submerged cultivation undoubtedly demonstrated the potential of induced V. jatamansi adventitious roots as a feasible source of phytoconstituents. In vitro adventitious roots induced leaf of Valeriana jatamansi showed improved metabolic profile and shorter cultivation cycle, thereby exhibiting potential as a sustainable alternative for extraction of industrially important phytoconstituents.

Agrobacterium-Mediated Genetic Transformation of Taiwanese Isolates of Lemna aequinoctialis.

Duckweed (Lemna aequinoctialis) is one of the smallest flowering plants in the world. Due to its high reproduction rate and biomass, duckweeds are used as biofactors and feedstuff additives for livestock. It is also an ideal system for basic biological research and various practical applications. In this study, we attempt to establish a micropropagation technique and Agrobacterium-mediated transformation in L. aequinoctialis. The plant-growth regulator type and concentration and Agrobacterium-mediated transformation were evaluated for their effects on duckweed callus induction, proliferation, regeneration, and gene transformation efficiency. Calli were successfully induced from 100% of explants on Murashige and Skoog (MS) medium containing 25.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μM thidiazuron (TDZ). MS medium containing 4.5 μM 2,4-D and 2.0 μM TDZ supported the long-lasting growth of calli. Fronds regenerated from 100% of calli on Schenk and Hildebrandt (SH) medium containing 1.0 μM 6-benzyladenine (6-BA). We also determined that 200 μM acetosyringone in the cocultivation medium for 1 day in the dark was crucial for transformation efficiency (up to 3 ± 1%). Additionally, we propose that both techniques will facilitate efficient high-throughput genetic manipulation in Lemnaceae.

In Vitro Propagation, Huperzine A Content and Antioxidant Activity of Three Genotypic Huperzia serrata.

Huperzia serrata is a traditional herb and endangered Chinese medicinal material, which has attracted much attention due to its production of Huperzine A (HupA). In vitro propagation of H. serrata is considered a new way to relieve the resource pressure of H. serrata. In this study, three different genotypic wild H. serrata were used for in vitro propagation. Then, the antioxidant activity and the content of HupA in the regenerated H. serrata were investigated. The results showed the survival rate of the explant was increased to 25.37% when using multiple sterilization processes. The best induction medium for H. serrata was the Schenk and Hildebrandt (SH) medium supplemented with 0.5 mg·L−1 Naphthalene acetic acid (NAA) and 0.1 mg·L−1 2,4-Dichlorophenoxyacetic acid (2,4-D), where the regeneration rate of the explant was to 57.04%. The best proliferation medium was the SH medium with NAA (1.0 mg·L−1), as the biomass of in vitro tissue increased 164.17 ± 0.41 times. High-performance liquid chromatography analysis showed that the in vitro culture of three genotypes could produce HupA and the content of HupA was 53.90–87.17 µg·g−1. The antioxidant experiment showed that the methanol extract of in vitro H. serrata had higher antioxidant activity than that of wild H. serrata. This study provides a reliable in vitro H. serrata culture protocol and laid an important foundation for the antioxidant capacity of the thallus and the content of HupA.

A culture system for the stable and high-efficiency proliferation of adventitious roots of Panax notoginseng and ginsenoside accumulation

Panax notoginseng is an important medicinal plant that belongs to the Araliceae family. In this study, a stable and high-efficiency system for the proliferation of P. notoginseng adventitious roots was established via two steps, and their growth and accumulation of three ginsenosides (Rg1, Re and Rb1) was analyzed in suspension culture. Adventitious roots were firstly induced from the cotyledons of P. notoginseng seeds, in 1/2 Schenk and Hildebrandt (SH) medium with 2.0 mg L−1 indole-3-butyric acid (IBA) and 30 g L−1 sucrose. Using these adventitious roots as explants, the influence of 1/2 SH and 1/2 Murashige and Skoog (MS) medium without NH4NO3 (1/2MS-N), and IBA (0–4.0 mg L−1) and naphthalene acetic acid (NAA) (0–2.0 mg L−1) on the induction of new adventitious roots was investigated. Culture conditions of 1/2 SH with 2.0 mg L−1 IBA and 2.0 mg L−1 NAA were optimal for the formation of new adventitious roots, and led to an induction rate of 100 % and 12.62 new adventitious roots on average for each explant after 3 weeks of culture. In culture conditions of 1/2MS-N with 2.0 mg L−1 IBA and 50 g L−1 sucrose, induced adventitious roots rapidly elongated to a mean length of 14.23 mm within 3 weeks, demonstrating that a two-step process of adventitious root regeneration involving steps of initiation and elongation was most effective. Suspension cultures of adventitious roots were analyzed to evaluate the effects of culture cycle time, concentrations of sucrose and IBA and amount of inoculation on adventitious roots. After 7 weeks, the total yield of three ginsenosides (11.13 mg 100 mL-1) was highest in root cultures with 1/2 MS-N, 30 g L−1 sucrose, 4.0 mg L−1 IBA, and 3.5 g 100 mL−1 root inoculation culture. The highest total concentration of ginsenosides (18.58 mg g−1 DW) was obtained from adventitious roots cultured from 3.0 g 100 mL−1 inoculation material and the concentration of ginsenoside Re (8.05 mg g−1 DW) was 1.8-fold higher than that in 3-year-old roots cultivated in the field (4.44 mg g−1 DW). The establishment of this culture system will be beneficial for the scale-up culture of adventitious roots and ginsenoside production in P. notoginseng.

Polyamine elicited aristolochic acid production in in vitro clonally fidel Aristolochia indica L.: An ISSR and RAPD markers and HPTLC based study

The present investigation was conducted to find out the influence of different polyamines (PAs) viz. spermine (spm), spermidine (spd) and putrescine (put) and plant growth regulators (PGRs) on multiplication, rooting, callusing and regeneration in Aristolochia indica L. in vitro. Firstly, the A. indica plants were collected, surface sterilized and cultured in Schenk and Hildebrandt (SH) media fortified with various PGRs and PAs in different concentrations and combinations. SH media fortified with benzylaminopurine (BAP) (2.0 mg/l)+put (0.5 mM) and in another experiment, BAP (2.0 mg/l)+spd (1 mM) produced best results with 41.00 and 39.2 axillary shoots/ nodal explant respectively. Following 4-6 weeks of incubation, explants propagated in SH media augmented with various combinations and concentrations of BAP and kinetin (kin) (0.5, 1, 1.5 and 2 mg/l), spm, spd and put (0.5 and 1 mM) generated white friable callus. SH media supplemented with BAP (2.0 mg/l)+spd (0.5 mM) displayed best response with an average number of 47.5 base callus derived shoots . Following 6 weeks of incubation, SH+ indole-3-acetic acid (IAA) (1.0 mg/l)+spd (0.5 mM) resulted maximum average number (7) of roots per shoot. The genetic homogeneity of the mother plant (MP), hardened plant (HP) and in vitro regenerated plants was ascertained employing random-amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. In this present study, in randomly selected A. indica plants, by using 6 ISSR and 5 RAPD primers, 63 scorable bands were derived, 59 of which were recorded as monomorphic with a degree of monomorphism of 90%. High-performance thin layer chromatography (HPTLC) revealed aristolochic acid (AA) content in in vitro grown A. indica roots and the roots from the mother plant (MP) as 6074.54 µg/g and 5891.14 µg/g respectively.

The novel paclitaxel-producing system: establishment of Corylus avellana L. hairy root culture

Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA4, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g−1 (DW)) in hairy root extracts.

Effects of shoot tip removal, wounding manipulation, and plant growth regulators on shoot regeneration and plantlet development in Paphiopedilum species

The micropropagation procedure of three endangered Paphiopedilum species (Paphiopedilum callosum, Paphiopedilum gratrixianum, and Paphiopedilum delenatii) by removing shoot apex and wounding method is reported in this study. The results showed that shoot regeneration of P. callosum, P. gratrixianum, and P. delenatii was at high frequency (5.61; 5.48; and 6.00 shoot/explant, respectively) when decapitated shoot apex was cultured on liquid Schenk and Hildebrandt (SH) medium added with thidiazuron (TDZ, 0.4–0.6 mg L−1). Multiple shoots were also induced in vitro shoots by wounding method, when cultured on medium containing TDZ (0.4–0.6 mg L−1) which were 4.48; 5.37; and 5.31 shoots/explant, respectively in each species. The shoots regenerated via both shoot apex decapitation and wounding method were well-developed to plantlets on SH medium supplement with 1.0 mg L−1 Naphthaleneacetic acid (NAA) after 90 days of culture. High-quality plants of P. callosum, P. gratrixianum, and P. delenatii. were observed when the plantlets were transferred onto peat moss with 100% survival rate after 24 months in greenhouse.

Strategies for the Regeneration of Paphiopedilum callosum through Internode Tissue Cultures Using Dark–light Cycles

Paphiopedilum spp. is one of the most commercially popular orchids because of its variety of shapes, sizes, and colors. However, it is at risk for extinction because of its exploitation. Regeneration of orchid plants using internode segments is extremely difficult. In this study, young P. callosum plants (1.5 cm) were exposed to eight dark–light cycles (14 days of dark and 1 day of light) for stem elongation to increase the number of nodes to obtain internode tissues. After 75 days of culture, the highest callogenesis (31.25%) was achieved when internode tissue was cultured on liquid Schenk and Hildebrandt (SH) medium containing 30 g·L−1 sucrose, 1.0 mg·L−1 Thidiazuron (TDZ), 1.0 mg·L−1 2,4-Dichlorophenoxyacetic acid (2,4-D), and cotton wool as the support matrix. The optimal media for induction of protocorm-like bodies (PLBs) were the same compositions as previously mentioned and were supplemented with 9 g·L−1 Bacto agar as the gelling agent. PLB clumps (5–6 PLBs/clump) produced the best shoots on medium containing 0.5 mg·L−1 α-Naphthaleneacetic acid (NAA) and 0.3 mg·L−1 TDZ. Among the organic substances tested, 200 g·L−1 potato homogenate (PH) added to Hyponex N016 medium supplemented with 1.0 mg·L−1 NAA, 30 g·L−1 sucrose, 170 mg·L−1 NaH2PO4, 1.0 g·L−1 peptone, and 9 g·L−1 Bacto agar resulted in the best rooting. The rooted plantlets with four to five leaves were acclimatized and had a 100% survival rate. The method presented in this research provides a strategy for the development of highly effective propagation of Paphiopedilum species using ex vitro explants for both conservation and horticultural purposes.

Biological diversity of Lemna aequinoctialis Welw. isolates influences biomass production and wastewater phytoremediation

Duckweeds are promising biomass producers for bioenergy and other biotechnological applications such as phytoremediation. Genetic diversity of duckweed species could influence their biotechnological potential. Nine isolates of Lemma aequinoctialis Welw. that are native of the Recife metropolitan area, Pernambuco state, in northeastern Brazil, were characterized in vitro under 1X and 0.5X Schenk and Hildebrandt (SH) culture media. Molecular identification by plastid DNA atpF-atpH barcoding sequences revealed very low genetic diversity. In contrast, exposure to environmental stimuli under 0.5X SH growth medium revealed phenotypic differentiation between the isolates. Three of the nine isolates were evaluated further for their ability to remediate the effluent from a domestic sewage treatment plant. They were able to reduce up to 94.8% of the chemical oxygen demand and to remove up to 39.3% of total nitrogen and 57.1% of phosphorus in the effluent, while producing biomass that has potential as feedstock for bioenergy production.

Establishment of a high-frequency plant regeneration system from rhizome-derived embryogenic cell-suspension cultures of Curcuma longa L.

We developed an efficient plant regeneration technique for turmeric (Curcuma longa L.) via somatic embryogenesis derived from embryogenic cell suspensions cultured from petioles. We initiated in vitro callus cultures from the plantlets of turmeric cultured on Murashige and Skoog (MS) medium. Then leaf, petiole, stem, and root explants of in vitro-grown plantlets were cultured on Schenk and Hildebrandt (SH). The stem and root explants formed white nodular calluses at a frequency of 86.7% and 85.8%, respectively, when cultured on SH medium; however, we used the calluses derived from the root explants for initiating a cell-suspension culture. In this study, the actively growing cell suspension culture comprised small, highly cytoplasmic cell aggregates and large vacuolated cells. On being transferred to a basal SH medium (without growth regulators), a few of these calluses differentiated into somatic embryos, which indicated that the initial calluses were embryogenic cells. We stimulated additional shoot differentiation by adding 0.5 mg L−1N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ) and various concentrations of N6-benzyladenine (BA) to the embryogenic cell suspension. The highest frequency at which shoot primordia developed from embryogenic cell clusters was 70% (when cultured in the dark on the supplemented MS medium), with more than 83.3% of regenerated shoot primordia producing roots. We successfully transplanted rooted plantlets into a soil mixture of sterile vermiculite and potting soil (1:1) and grew them to maturity in a growth chamber, achieving a survival rate of > 95%. To our knowledge, this is the first report of complete plant regeneration of turmeric from embryogenic cell-suspension cultures. The embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and gene manipulation for improving turmeric quality.

An efficient plant regeneration from Rhaponticum carthamoides transformed roots, enhanced caffeoylquinic acid derivatives production in pRi-transformed plants and their biological activity

Rhaponticum carthamoides (Willd.) Iljin (Asteraceae) is an endemic plant species whose roots and rhizomes have been used for many years in traditional Siberian medicine. In this study, the transformed roots of R. carthamoides obtained after Agrobacterium rhizogenes transformation were able to spontaneously regenerate green buds and shoots. Bud and shoot regeneration was observed in more than 90% of the cultures grown in Schenk and Hildebrandt (SH) or Gamborg (B5) liquid medium with full micro- and macronutrient concentrations, under light conditions. The regeneration of shoots from transformed roots may be improved by supplementation of Murashige and Skoog (MS) agar medium with indole-3-acetic acid (IAA) and benzyladenine (BA). The shoots were multiplied on MS medium supplemented with 0.1 mg/L IAA and 0.2 mg/L BA and then rooted successfully. The transformation of the plants derived from transformed roots was confirmed by PCR analysis and Southern hybridization. The three-month-old pRi-transformed plants (TR-plants) were able to biosynthesize caffeoylquinic acid derivatives such as chlorogenic acid, and 3,5- or 4,5-di-O-caffeoylquinic acids. Plants obtained directly from seeds (SD-plants) being in the same age and grown under the same conditions as the transformed plants were used as compared material. The transformed plants demonstrated higher secondary metabolite levels and greater biomass accumulation than SD-plants. It was found that extracts from TR-plants exhibited antioxidant properties in the model of human blood plasma and inhibited the growth of A2058 human melanoma cells depending on the tested concentration. The present study demonstrated that the pRi-transformed plants of R. carthamoides may be an alternative to conventional field crops and these plants can be used as the promising source of the valuable compounds, such as caffeoylquinic acid derivatives.

Enhancing Astragaloside Production in Astragalus membranaceus through Growth Media and Auxins

The secondary metabolites of triterpenoid saponins, isoflavonoids and polysaccharides are present in Astragalus membranaceus those are biologically active. Astragalosides are a major group of identified compounds that have various pharmacological properties. The present study was considered to enhance astragaloside biosynthesis in hairy root cultures of A. membranaceus by using different growth media and concentrations of different auxins. Our findings revealed that Schenk and Hildebrandt (SH) medium responded greatly for the production of the highest dry weight. However, the highest astragaloside production was detected while using SH medium of its half strength. The growth of hairy roots and accumulation of astragaloside in response to different auxinlike Indole-3-Acetic Acid (IAA), Indole-3-Butyric Acid (IBA) and 1-naphthaleneacetic acid (NAA) were different. Hairy root growth rate was increased by the treatment of IBA and NAA, but was unaffected by IAA. Accumulation of Astragaloside was enhanced in response to the treatment of IBA and NAA. In most cases, treatment with the 0.5 mg L-1 NAA accumulated the highest level of stragaloside in the hairy root of A. membranaceus, which might, therefore, be utilized as an ideal mass-scale production system for astragalosides.

Polyethylene glycol and chilling overcome Somatic embryogenesis obstacle in Pyrus communis

Abstract The purpose of this investigation is to achieve the procedure of somatic embryogenesis (SE) as an efficient propagation way in four pear cultivars: Natanzi, Ghosi, Williams and Dar Gazi, by focusing on two strategies: One is the application of competent explants, and the other one is stress employment. The results were variable because of cultivars behavior was highly genotype specific. However, the results identified that the highest frequency of embryogenic callus induction was obtained from the endosperm and immature cotyledonary segment explants of P. communis cultivars on Murashige and Skoog medium (MS) containing 8.05 μM 1-naphthaleneacetic acid (NAA), 13.32 μM N6-benzyladenine (BA) supplemented with 250 mg/L glutamine as a reduced nitrogen source and 30 g/L (w/v) sucrose in Ghosi (75.46%). The optimized media were different for each cultivar in the induction phase, namely, Natanzi and Ghosi in MS, Williams in Schenk and Hildebrandt (SH) and Dar Gazi in Nitsch and Nitsch (NN). Then, it was observed that when embryogenic calli were transferred to the media containing 1% (w/v) Polyethylene glycol (PEG), calli of some cultivars further differed into embryos while other cultivars produced in chilling at 4 °C. Also, maturation of embryos occurred at 4 °C for 4 months. Then passages using MS media caused germination of embryos and plantlets were produced. To sum up, the greatest frequency of embryogenic callus (75.46%) occurred in Ghosi, and the highest embryo maturation and germination (77.06% and 37.66%, respectively in mentioned cultivars) were observed in Natanzi.