MS Liquid Medium Research Articles

Accumulation of podophyllotoxin in the root culture of Hyptis suaveolens (L.) Poit: A potential natural lignan for clinically useful anticancer drugs

Podophyllotoxin (PTOX) is a natural antiviral, antirheumatic and anticancer molecule, but it is expensive to chemically synthesize. The present study aimed to evaluate growth and PTOX accumulation in root cultures of Hyptis suaveolens treated with different concentrations of auxins, vitamins and myo-inositol. Root cultures grown in liquid medium supplemented with different concentrations of indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), vitamins and myo-inositol were used to measure biomass production and PTOX content. PTOX quantitation was performed via high-performance liquid chromatography with diode-array detection (HPLC-DAD) using a newly developed analytical method after successful validation. Root culture in MS medium supplemented with 1 mg L−1 IBA +0.5 mg L−1 NAA resulted in the highest root dry weight (248.76 mg) and the highest PTOX concentration in the roots (179.97 μg g−1 root). The roots cultured in liquid MS medium supplemented with 1 mg L−1 IBA +0.5 mg L−1 NAA presented the greatest increase in root biomass and PTOX content. This adequate balance of vitamin and myo-inositol supplementation in liquid MS culture medium increased the root dry weight and PTOX content.

In Vitro Rooting of <em>Gyrinops walla</em> in Activated Charcoal-Containing Semi-Solid Culture and Filter Paper-Bridged Liquid Culture Systems

Plant tissue culture is the best option in producing planting material for cultivation programmes of Gyrinops walla. Perfection of in vitro rooting protocol for this species is the most decisive step in micropropagation. In the present study the effect of physical state of substrate, different concentrations of sucrose, IBA and NAA in media containing activated charcoal on in vitro rooting was investigated. In experiment-1, 0.2% (w/v) activated charcoal was added in to the ½ MS medium fortified with 30 or 40 g/L sucrose and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). In experiment-2, filter paper-bridged liquid medium was used with 40 g/L sugar and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). The treatments were arranged in completely randomized design with three replicates. Percentage of rooted microshoots and number of roots per shoot were recorded. The ½ MS semi-solid medium with 0.2% activated charcoal, 40 g/L sucrose and 0.1 mg/L NAA resulted in the significantly highest rooting percentage (50%)while the significantly highest number of roots (6.0) were noted in ½ MS charcoal added (0.2%) basal medium with 40 g/L sucrose and 0.5 mg/L NAA. Shoots supported with filter paper bridge on ½ MS liquid medium, supplemented with 0.5 mg/L IBA, had the highest rooting percentage (66.7%) and the highest mean number of roots per shoot (3.0) (P<0.05). Results confirmed that in vitro rooting of G. walla could be perfected by optimizing the culture medium and its constituents.

A protocol for the development and maintenance of Coffea arabica (L.) cell suspension cultures

Coffea spp. are remarkable sources of phytochemicals, but the lack of a well-defined culture medium aimed at the induction of non-embryogenic and friable callus hampers the establishment of plant cell suspension cultures for large-scale production of valuable compounds. In this paper, we describe a one-medium protocol suitable to obtain both callus and cell suspension cultures from leaves of two elite cultivars of C. arabica. The protocol was developed through an iterative process involving the determination of the best concentration of auxin and cytokinin, their optimal ratio, as well as the most effective molecule of either hormone class. Young leaves were found to be a good and easy-to-use explant source for callus induction and proliferation, provided that a cytokinin was present in association with a chlorinated auxin in a full strength, semi-solid MS medium. The best results were obtained by hormone concentration and combination of 1 mg/L of both kinetin and 2,4,5-trichlorophenoxyacetic acid. The same ratio of these growth regulators was conveniently used for the development and stabilization of cell suspension cultures in liquid MS medium. When grown in darkness, stabilized suspension cultures showed a fine and homogeneous texture, with a 10-fold biomass increase within 25 days and a cell viability > 90%. In addition, the phytochemical profile revealed the presence of the most widely studied coffee compounds. The protocol can be applied to obtain adequate amounts of cell biomass for use in physiological studies concerning the production of secondary metabolites.

In vitro acute gamma radiation on tissue of pink and white lotus (Nelumbo nucifera Gaertn.) in Thailand

This research aimed to investigatethe age of embryos of pink and white lotus for in vitropropagation and examine the sensitivity of young lotus plants derived from tissue culture to acute gamma radiation. The seeds of both lotus varieties were 4-week, 6-week and 8-week-old embryos cultivated on solid MS medium. They were then transferred to solid MS medium supplemented with 3 mg/L BA. After culturing, the 6-week-old pink lotus embryos had an average maximum petiole length of 16.78 ± 1.01 cm and an average of 11.84 ± 0.44 roots/explant, while the 6-week-old white lotus embryos had an average maximum petiole length of 15.48 ± 0.68 cm and an average of 11.76 ± 0.70 roots/explant. Each young lotus plant produced one shoot. After treating the young lotus plants with liquid MS medium supplemented with 3 mg/L BA, both lotus varieties showed increased shoot numbers at 3.54 ± 0.13 and 3.40 ± 0.91 shoots/explant. Young lotus plants of both varieties were exposed to gamma radiation levels of 0, 20, 30 and 40 Gy. The LD50 value for pink lotus was 26.138 Gy, while the LD50 value for the white lotus was 32.031 Gy.The LD50 value will offer vital information for determining the optimal dosage of gamma radiation for both pink and white lotus in vitro.

Establishment of Physalis alkekengi cell suspension culture: time-dependent behavior of genes related to the steroidal compounds, key enzymes, and physalins under static magnetic field.

Cell suspension culture has the potential to be a valuable source for the bioactive compound productions. In this study, an optimized procedure was established for callus and cell suspension culture of Physalis alkekengi for the first time, and the impact of static magnetic field (SMF, 6 mT) was studied on the high-value metabolic compounds through investigation of signaling molecules and gene expressions at the late log-to-stationary phase. Results showed that the growth regulators of 6-benzyl amino purine (BAP, 1.5mg-1 L) and 1-naphthaleneacetic acid (NAA, 0.4mg-1 L) induced the highest fresh weight, callus rate, callus index, and total withanolides. Cell suspension culture was established in the liquid MS medium supplied with BAP (1.5mg-1 L) and NAA (0.1mg-1 L). SMF application decreased slightly the cell growth and viability and enhanced the number of round-shaped cells. The hydrogen peroxide (H2O2) and nitric oxide (NO) levels increased at an all-time series after SMF exposure, and their maximum contents were observed after 12h. A significant alteration of malondialdehydecontent was also identified after 12h of SMF exposure. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxyD-xylulose 5-phosphate synthase (DXS), squalene synthase (SQS), sterol Δ7-reductase (DWF5), and C-7,8 sterol isomerase (HYD1) genes was upregulated significantly after 24 and 48h. An increase in the total withanolides was related to more activity of HMGR and DXS enzymes in SMF-exposed cells and the maximum physalin A (12.8mgg-1 DW) and physalin B (1.92mgg-1 DW) obtained after 24h compared to controls. Findings suggest that SMF can play a supportive factor in inducing steroidal compounds in P. alkekengi through modulating H2O2 and NO levels and the related-gene expressions.

Multi‐omics reveal the molecular basis of LrAN2 overexpression in black goji callus promoting the excessive accumulation of petanin, a promising food blue colorant

AbstractLycium ruthenicum fruit (LRF) is a potential source of natural colorant in a wide pH range due to richness in monoacylated anthocyanin petanin. However, the yield of LRF largely lags behind the market demand in food industry. In this study, overexpressing LrAN2 (LrAN2OE), an anthocyanin activator in LRF, produced excessive acylated anthocyanins including petanin in L. ruthenicum callus (LRC). Physiological analysis indicates that LrAN2OE extensively orchestrate the redox homeostasis, including antioxidant enzymes system and antioxidants with low molecular weight. KEGG enrichment analysis indicates that LrAN2 hierarchically orchestrate the acylated anthocyanin biosynthesis in LRC at multi‐omics level, such as small RNAome, transcriptome, and metabolome. The anthocyanin yield of LrAN2OE callus cultured by 5% sucrose or glucose is significantly enhanced. Furthermore, the anthocyanin yield approach to 96.23 mg/g dry weight, approximately 3.5 folds of that in LRF, when LrAN2OE suspension cells are cultured in liquid MS medium supplement with 5% sucrose and 100 mM NaCl. Notably, anthocyanins extracted from LrAN2OE callus are stable in pH 1.0–2.0, pH 7.0, and pH 9.0, showing red or blue color within 30 days at 4°C. Our findings suggest that LrAN2 is perfect gene resource for metabolic engineering of acylated anthocyanins in LRC, a promising chassis producing acylated anthocyanins. Taken together, LRC metabolically engineered by LrAN2 is a potential source of natural food color, which facilitates to make up the shortfall of LRF as source of natural colorant in future food industry.

Induction and characterization of induced tetraploid, mixoploid and control diploid teak seedlings (Tectona grandis L.f.) from shoot cultures treated with oryzalin*

Teak, known for its high-quality timber and economic value, holds significant potential as the major component of an agroforestry system to combat climate change and support sustainable development goals. The challenge of slow teak growth can be addressed by introducing fast-growing teak varieties amenable to vegetative propagation. Furthermore, the improvement of teak through somatic cell genetics, such as inducing polyploidy, can result in more vigorous, faster-growing, and drought-tolerant trees. While a report exists on a tetraploid teak accession with enhanced less water tolerance, information on the induction and characterization of tetraploid teak is lacking. In this study, we report the induction of polyploid teak by immersing 1 – 1.5 cm nodal stem segments and shoot tips obtained from teak shoot cultures in a modified MS liquid medium containing 5 − 30 µM oryzalin for one week, followed by subculture for 6 passages. The resulting shoots were acclimatized and grown in the nursery for 10 months. Based on flow cytometry for ploidy analysis of pre-selected plants having morphologies typical of polyploid followed by chromosome counting, we identified two tetraploid plants from one accession and 18 mixoploid teak plants from four accessions. Chromosome counting analysis confirmed that a diploid plant had a chromosome number of 2n = 2x = 36, tetraploid plant had a chromosome number of 2n = 4x = 72, and mixoploid plant exhibited both chromosome number of 2n = 2x = 36 and 2n = 4x = 72 in root tip cells. Morphological characterization revealed that tetraploid and mixoploid teak plants displayed typical polyploid characteristics, such as larger, thicker, and greener leaves, increased root diameter, increased cell size in root tip region, and larger stomata size but with reduced density. The development of tetraploid teak holds the potential to provide faster-growing and more drought-tolerant varieties, facilitating breeding efforts, including the development of triploid varieties.

Establishment of feijoa (Acca sellowiana) callus and cell suspension cultures and identification of arctigenin - a high value bioactive compound.

Feijoa (Acca sellowiana (O. Berg.) Burret), also known as pineapple guava, is a member of the Myrtaceae family and is well known for its fruit. Chemical profiling of the different tissues of the feijoa plant has shown that they generate an array of useful bioactive compounds which have health benefits such as significant antioxidant activities. In this study, an in vitro culture system has been developed, which could be explored to extract high-value bioactive compounds from feijoa. Feijoa tissue culture was initiated by the induction of callus from floral buds. Sections of floral buds were plated on MS medium supplemented with 2,4-D and BAP at 2.0mg/L and 0.2mg/L concentrations, respectively. Cell suspension cultures of feijoa were established using a liquid MS medium with different concentrations of 2,4-D and BAP and cultured on a rotary shaker. The growth of the cell suspension was evaluated with different parameters such as different carbohydrate sources, concentration of MS media, and inoculum density. When the cell suspensions were treated with different concentrations of MeJA at different time points, phytochemicals UPLC - QTOF MS analysis identified extractables of interest. The main compounds identified were secondary metabolites (flavonoids and flavonoid-glucosides) and plant hormones. These compounds are of interest for their potential use in therapeutics or skin and personal care products. This report investigates essential methodology parameters for establishing cell suspension cultures from feijoa floral buds, which could be used to generate in vitro biomass to produce high-value bioactive compounds. This is the first study reporting the identification of arctigenin from feijoa, a high-value compound whose pharmaceutical properties, including anti-tumour, anti-inflammatory and anti-colitis effects, have been widely reported. The ability of feijoa cell cultures to produce such high-value bioactive compounds is extremely promising for its use in pharmaceuticals, cosmeceuticals and nutraceuticals applications.

Indução de calos, regeneração de brotos e conservação de tamareiras (Phoenix dactylifera) cultivadas in vitro

Abstract Date palm (Phoenix dactylifera( cv. Medjool is a significant plant, grown in Jordan. In vitro propagation gives operative resources for the significant propagation of date palms. Maximum callus induction was achieved from MS media supplemented with benzyl amino purine (BA) and naphthalene acetic acid (NAA). The highest plant regeneration was recorded on MS medium supplemented with dichlorophenoxyacetic acid (2,4-D) at 3.0 mg/L, and BA at 2.0 mg/L. A significant positive impact on shoot formation was recorded on MS medium supplemented with 1.0 mg/L BA with 0.5 to 1.5 mg/L NAA in both liquid and solid MS medium. To maintain survival and regrowth capacity, sucrose could be used for medium-term conservation at lower concentrations (0.1 - 0.2 M). In addition, sorbitol might be used at 0.1 M to maintain the quality of explants. The vitrification technique for long-term preservation was experimented. Embryogenic callus was used as explants for conservation. The survival as well as regrowth percentages of non-cryopreserved and cryopreserved tissue cultures were affected by their duration of treatment with the vitrification solution plant vitrification solution 2 (PVS2) and modified plant vitrification solution 2 (MPVS2). Results showed that using PVS2 for 60 minutes for cryopreserved calli was more effective than other treatments. After storage in liquid nitrogen, the highest survival rate (65%) and regrowth rate (40%) were achieved.

Production of Benzyl Isothiocyanate from Moringa peregrina (Forssk.) Fiori via Suspension Cultures

Benzyl isothiocyanate (BITC) is one of the main constituents of Moringa peregrina, an endangered species in the flora of Egypt. The purpose of this study was to establish a procedure to enhance the in vitro production of BITC in Moringa peregrina suspension cultures. This could then aid in the mass production of this useful pure active compound and avoid the exploitation of natural resources of this valuable plant. Suspension cultures of Moringa peregrina was established from callus cells in liquid MS medium supplemented with 10.74 μM naphthalene acetic acid, 2.325 μM kinetin, in addition to salicylic acid as an elicitor and/or L-phenylalanine as a precursor of BITC. The combination of both salicylic acid and L-phenylalanine gave the highest accumulation of BITC after two weeks with 10.42-fold increase, compared to its content in the mother plant. This study is promising for the large-scale production of BITC, which has been identified as an anticancer agent with possible applications in the pharmaceutical industry. Plant Tissue Cult. & Biotech. 33(2): 85-95, 2023 (December)

In vitro regeneration, transplantation and phytochemical profiles of Kaempferia angustifolia Roscoe

Kaempferia angustifolia Roscoe is a medicinal plant in the family Zingiberaceae, with wild occurrence decreasing as a result of both natural and human threats. Tissue culture techniques provide an alternative method of propagation for mass production. Microshoots of K. angustifolia (1 cm in length) were cultured on MS medium supplemented with various concentrations of cytokinins (BA and Kinetin) and auxins (NAA and IAA) for 8 weeks. Phytochemical profiles were evaluated by total phenolic contents (TPC), total flavonoid contents (TFC), antioxidant activity (2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing ⁄ antioxidant power (FRAP) assay) and Gas chromatography–mass spectrometry (GC–MS) analysis. Maximum number of shoots was observed in medium supplemented with 2 mg/l BA plus 0.5 mg/l NAA (6.33 shoots/explant) and optimal rooting was induced in MS medium with 2 mg/l Kinetin plus 1 mg/l NAA (35.70 roots/explant). Highest numbers of shoots and roots were obtained when cultured on liquid MS medium supplemented with 2 mg/l BA, 2 mg/l Kinetin and 1 mg/l NAA (8.73 shoots/explant and 29.67 roots/explant, respectively). Regenerated plantlets of K. angustifolia were transferred to pots containing different types of plant materials under natural conditions for 8 weeks. Optimal survival rate was 100% when transplanting K. angustifolia to soil, sand, soil and sand (1:1) or soil and small pieces of rock (1:1) ratio. The methanol extract of leaves K. angustifolia from natural plants and in vitro derives plants showed a significantly higher amount of TFC and antioxidant activity. GC-MS analysis identified 52 phytochemical compounds in leaves of K. angustifolia. This study may be helpful to increase the value of commercial production of K. angustifolia, pharmaceutical and medicinal purpose.

A liquid culture system for plantlet conversion and slow growth storage of encapsulated shoot tips of Justicia adhatoda L.

The present article demonstrates the applicability of liquid medium in propagation and short-term storage of synthetic seed of Justicia adhatoda L., a medicinally important plant used in many ayurvedic drugs. Shoot tips and nodal segments excised from shoots regenerated on meta-topolin (mT) containing medium were encapsulated in 3% Na-alginate and 100 mM CaCl2.2 H2O for the preparation of artificial seeds. Irrespective of media composition, shoot tips performed better than nodal segments with respect to shoot emergence from alginate beads and further conversion into plantlets in a single step. Morphogenic responses of encapsulated shoot tips in liquid as well as on agar-solidified medium were evaluated and maximum plantlet conversion was observed on PGR-free MS liquid medium. Encapsulated shoot tips were stored for 60 days using slow-growth storage approaches which were induced by a reduction of sucrose concentration (1%) in the medium and reducing MS medium strength to half. After storage, more than 50% synthetic seeds were converted into plantlets when transferred to a regrowth medium. This slow-growth approach may be applied for the short-term storage of synthetic seeds for a reasonable period that is sufficient for the exchange of germplasm for distant places.

Development of efficient and sustainable droplet-vitrification cryoconservation protocol for shoot tips for long-term conservation of Dahlia germplasm

Dahlias are the one amongst the tuberous herbaceous plants, valued for their attractive colorful flowers with varied size cultivated due to its ornamental value as a potted plant or cut flower in many countries. Dahlia germplasm was being conserved in the in vitro Genebank of ICAR- NBPGR, New Delhi since 2005–06 under normal growth and slow growth with a regular subculturing in every 6 months and 1 year respectively, which is laborious and time-consuming. Hence, in the current study we attempted to develop an efficient droplet-vitrification cryopreservation protocol for safe and long-term conservation of dahlia germplasm. Significant effect of type and position of explants, plant vitrification solution-2 (PVS2) dehydration duration, pregrowth period and loading solution treatment duration on post-thaw regrowth after cryoconservation was observed. Shoot tips, each being 1.0 mm in length and 0.5 mm in width positioned at the tip of in vitro shoots with a similar developmental stage (similar shoot length) were excised from in vitro cultures pregrown for 6 wks on B5 basal medium supplemented with 0.5 mg/l BAP, 30 mg/l silver nitrate and 3% sucrose. The excised shoot tips were precultured on MS + 0.3 M sucrose for overnight followed by loading solution (2.0 M glycerol and 0.4 M sucrose in liquid MS medium) treatment for 80 min, PVS2 dehydration for 30 min and then transferred onto PVS2 droplets on aluminum foil strips and cryostored in liquid nitrogen (LN). An average of 44.44% post-thaw regrowth was obtained in six accessions tested and no significant difference among the accessions was observed. There was no significant loss in regrowth of cryostored shoot tips after 1 year and 6 years of cryobanking. This justifies the genotype-wide and sustainability of the protocol for cryoconservation of Dahlia germplasm using droplet-vitrification cryopreservation procedure in the cryobanks. To the best of our knowledge, this is the first report on cryoconservation of Dahlia germplasm.

Establishing a simple selection method of high hypericin-producing adventitious root lines from leaf explants of Hypericum perforatum var. Topaz

ABSTRACT Hundreds of adventitious root lines from Hypericum perforatum L. were examined for their hypericin content in order to establish a system for studying the production of hypericin in adventitious root lines. The effects of the medium consistency (solid and liquid) and the effects of three different auxins (Indole-3-butyric acid (IBA), Indole-3-acetic acid (IAA), and 1-Naphthaleneacetic acid (NAA)) on the induction of adventitious roots in leaf explants were studied. In a half-strength MS liquid medium with 2 mg/L IBA concentration, adventitious roots developed from H. perforatum leaves, whereas NAA and IAA caused callus-like and low branch roots. HPLC-UV was used to profile hypericin content variations in 100 H. perforatum adventitious root lines. Hierarchical component analysis (HCA) was used for selecting elite lines. After cluster analysis and selecting high-performance lines in terms of hypericin, five elite lines were chosen and the amount of hypericin production, as well as the phenol and flavonoid content, was analysed.

A rapid assay for assessing bacterial effects on Arabidopsis thermotolerance

BackgroundThe role of beneficial microbes in mitigating plant abiotic stress has received considerable attention. However, the lack of a reproducible and relatively high-throughput screen for microbial contributions to plant thermotolerance has greatly limited progress in this area, this slows the discovery of novel beneficial isolates and the processes by which they operate.ResultsWe designed a rapid phenotyping method to assess the effects of bacteria on plant host thermotolerance. After testing multiple growth conditions, a hydroponic system was selected and used to optimize an Arabidopsis heat shock regime and phenotypic evaluation. Arabidopsis seedlings germinated on a PTFE mesh disc were floated onto a 6-well plate containing liquid MS media, then subjected to heat shock at 45 °C for various duration. To characterize phenotype, plants were harvested after four days of recovery to measure chlorophyll content. The method was extended to include bacterial isolates and to quantify bacterial contributions to host plant thermotolerance. As an exemplar, the method was used to screen 25 strains of the plant growth promoting Variovorax spp. for enhanced plant thermotolerance. A follow-up study demonstrated the reproducibility of this assay and led to the discovery of a novel beneficial interaction.ConclusionsThis method enables rapid screening of individual bacterial strains for beneficial effects on host plant thermotolerance. The throughput and reproducibility of the system is ideal for testing many genetic variants of Arabidopsis and bacterial strains.

Mass Propagation of Dendrocalamus Giganteus Wall. Ex Munro Through in Vitro Culture

The present experiment was conducted to establish an efficient protocol for mass propagation of Dendrocalamus giganteus Wall. ex Munro through in vitro culture using nodal segments as explants. The highest percentage of shoot induction (86.67 %) was obtained in the liquid MS medium supplemented with 1.5 mg/l BAP and 1.0 mg/l TDZ with maximum number of shoots (3.91 ± 0.87) per explant. The highest number and length of shoots were 22.83 ± 1.30 shoots/culture and 7.71 ± 0.96 cm, respectively induced on agitated liquid MS medium supplemented with 2.0 mg/l BAP and 1.0 mg/l TDZ. Incorporation of 8 % coconut water in the above mentioned medium resulted satisfactory shoot growth and development. The best response towards root induction (90 %) was observed when 3-4 shoots clump were transferred onto halfstrength of gelled MS medium fortified with 4.0 mg/l IBA with 9.31 ± 1.45 roots per unit shoot. Successful acclimatization of well-rooted clumps of 4-5 shoots was achieved in a mixture of soil, sand, and compost (1 : 1 : 1) with 90% survival rate. Bangladesh J. Bot. 52(2): 337-343, 2023 (June)

Survival and State of Garlic Explants of Two Lithuanian Cultivars after Cryopreservation

Cryopreservation features of Lithuanian garlic (Allium sativum L.) cultivars ‘Žiemiai’ and ‘Dangiai’ were investigated. The survival rate and state of explants, depending on the type of explant, and dehydration conditions were evaluated in the experiment. Explants isolated from cloves and bulbils were dehydrated with a plant vitrification solution 3 (PVS 3) containing 50% w/v glycerol and 50% w/v sucrose in liquid MS medium. Three different exposure durations of 1.5, 2, and 3 h in PVS3 solution were applied at 0 °C. Unloaded stem domes were planted in Petri dishes with MS medium supplemented with 1 mg L−1 naphthaleneacetic acid (NAA) and 6–dimethylallylamino purine (2-iP) on 0.8% plant agar. The obtained results showed that the cryopreservation in liquid nitrogen reduced the survival rate of explants by 20–40%. The average number of surviving explants after freezing reached 64.3% for both cultivars. Cultivar ‘Žiemiai’ showed a 24% higher capability for survival than ‘Dangiai’. The total number of surviving explants of ‘Žiemiai’ reached 76.1%. Explants from bulbils were 23% more effective for cryopreservation compared to cloves. Evaluation of the effect of dehydration duration showed that the survival rate of the explants from bulbils of both cultivars was highest after treatment with PVS3 for 3 h and reached 91%. Treatment in PVS3 solution for 2 h was sufficient for survival of explants from cloves. Obtained results confirm that the efficiency of garlic cryopreservation depends on complex factors.

The effect of plant growth regulators on the antioxidant enzyme activity and secondary metabolite production in the cell suspension cultures of Melia azedarach L.

ABSTRACT In the present study, we investigated the effect of plant growth regulators and explant type on callus induction, cell suspension culture growth, antioxidant enzyme activity, and accumulation of rutin, quercetin, and kaempferol in M. azedarach L. cell cultures. Our studies showed that the inflorescence and petiole explants had a high percentage of callus induction compared to the leaf explants, whereas the highest callus growth was obtained from the leaf explants cultured on the MS medium containing 5 mg l−1 NAA and 5 mg l−1 BAP and 5 mg l−1 NAA and 3 mg l−1 kinetin Kin. Furthermore, the highest cell growth was obtained from the leaf and inflorescence calli transferred to liquid MS medium supplemented with 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D)+1 mg l−1 Kin/BAP. The highest activity of antioxidant enzymes and the lowest protein content was related to the leaf cell suspension cultures in liquid MS medium containing 3 mg l−1 NAA and 3 mg l−1 BAP. The inflorescence cell suspension cultures in the MS medium containing 3 mg l−1 NAA+1 mg l−1 BAP exhibited the highest rutin accumulation (47.536 mg g−1 FW). However, the highest amount of quercetin (8.570 mg g−1 FW) and kaempferol (5.420 mg g−1 FW) accumulation were obtained from the petiole cell suspension cultures in the MS medium with 1 mg l−1 2,4-D +1 mg l−1 Kin.

Elicitation based enhancement of solasodine production in in-vitro cultures of different Solanum species

Aim: To study the enhancement of solasodine content using elicitors such as NaCl, pectin, salicylic acid and yeast extract in cell suspension cultures of Solanum incanum, Solanum nigrum, Solanum surattense and Solanum villosum. Methodology: In-vitro callus induction from leaf explants was carried out on MS media supplemented with auxin, 2, 4- Dichlorophenoxyacetic acid (2.0 mg l-1). MS liquid medium supplemented with 2, 4-D (2.0 mg l-1) and varied concentrations of different elicitors were used for cell suspension culture. Results: The elicitor NaCl (150 mM) indicated maximum increment in solasodine production in three Solanum species studied. Salicylic acid with 75 μM resulted in considerable elevation in solasodine content in Solanum spp. Response to elicitation by pectin was high at different concentrations for different species of Solanum. Biotic elicitor yeast extract at 3.0 g l-1 concentration considerably increased solasodine production in S. nigrum and S. villosum. Solanum villosum exhibited best results in terms of solasodine concentration enhancement in response to pectin and yeast extract elicitors whereas Solanum incanum responded best to pectin followed by NaCl. Interpretation: The current results indicated that NaCl, pectin, salicylic acid and yeast extract can be effectively applied as influential elicitors for the enhancing the production of solasodine in cell suspension culture of different Solanum species. Key words: Callus, Cell suspension culture, Elicitors, Solanum, Solasodine, Steroidal alkaloids

THE EFFECT OF HEAVY METAL IONS ON THE PEROXIDASE ACTIVITY IN ARABIDOPSIS THALIANA

The biosphere pollution with the heavy metals (HM) has increased significantly in recent decades due to human activity. Plants can accumulate and concentrate HM, which negatively affects their growth, productivity and quality of agricultural products. Some HM, such as copper, belong to the group of biogenic elements that, in low concentrations, are essential for the normal functioning of plant organisms. Other HM such as cadmium are toxic even in low concentrations. The toxicity of HM is related to oxidative damage. In the plant cell, the antioxidant system provides protection against this kind of stress. However, data on changes in antioxidant enzyme activities in the early stage of the cellular response to HM-induced stress remain scarce. Therefore, we focused our research on studying peroxidase (POD) activity changes in Arabidopsis thaliana under conditions of rapid uptake of copper and cadmium ions into leaf tissue. For the experiments, 4.5–5-week-old A. thaliana plants were used. The plants were incubated on 0.5x MS liquid medium containing copper or cadmium chloride at concentrations of 0.1, 0.5 and 5 mM. The HM salt treatment was carried out in the dark at 20 °C for 2 (short-term stress) and 12 (long-term stress) hours. After that, the leaves were frozen and the POD activity was measured. Evaluation of the effects of Cd2+ and Cu2+ ions shows that these HM cause a decrease in POD activity after 2 hours and its increase after 12 hours of treatment. Therefore, modulation of POD activity is a component of the HM stress response in A. thaliana. Analysis of the available data revealed that the enzymes POD and CAT, which eliminate hydrogen peroxide, can partially replace each other and thus provide cellular protection in different phases of the stress response.