μM Benzyladenine Research Articles

Cytotoxic activity of callus extract from Vachellia farnesiana (L) Wight & Arn.

The in vitro cultures of Vachellia farnesiana (L) Wight & Arn. have demonstrated cytotoxic activity through callus extract on the HeLa cell line. Explants excised from in vitro-grown seedlings from seeds of two different locations were inoculated on Murashige and Skoog (MS) culture media containing various concentrations of N-6 benzyladenine (BA) or kinetin with 2,4-dichlorophenoxyacetic acid (2,4-D). Optimal efficiency in friable callus induction (100%) was achieved in leaf explants cultured on MS media containing 2.32µM BA + 13.57µM 2,4-D. Plant tissues (callus and leaf) were extracted and subjected to quantitative phytochemical analysis, revealing the highest total alkaloid and phenolic content in leaf extracts from Queretaro adult specimens (339.5 ± 20.9mg atropine equivalents (AE) per g dry extract (DE) and 158.4 ± 12.5mg gallic acid equivalents (GAE) per g DE, respectively). In contrast, callus cultures exhibited significantly higher total triterpene content (356-381mg ursolic acid equivalents (UAE) per g DE) compared to leaf extracts (208-243mg UAE/g DE). Both leaf and callus extracts displayed cytotoxic activity against the HeLa cell line, with a significantly lower half-maximal inhibitory concentration (IC50) for leaf extracts (28-32µg/mL) compared to callus cultures (43-66µg/mL), suggesting that alkaloids were primarily responsible for the cytotoxic activity. Furthermore, this study provides valuable insights into the controlled production of bioactive compounds with cytotoxic activity, with callus serving as a rich source.

Effect of abiotic and biotic elicitors on vincristine accumulation in endosperm derived in vitro cultures in Catharanthus roseous (L.) G. Don

Catharanthus roseus (L.) G. Don (Apocynaceae) is a well-studied herb renowned for its in vitro culture as a source of the anti-cancer alkaloid, vincristine. However, despite the recognized advantages of triploid cells over diploid cells in terms of productivity, the triploid endosperm tissue of this important medicinal plant has not been utilized for in vitro culture initiation. In this investigation, zygotic embryos and endosperm tissues were cultured on Murashige and Skoog (MS) medium supplemented with various combinations of auxins and cytokinins. The medium containing 2.50 µM 6-Benzyladenine (BA) and 1.25 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) proved to be the most effective for callus and cell culture formation. Ploidy analysis using ploidy analyzer confirmed that the endosperm-derived callus exhibited mixoploid, while the embryo-derived callus remained diploid. Liquid Chromatography Mass Spectrometry (LC–MS) analysis of callus and cell cultures grown on MS media with different combinations of auxins, cytokinins, elicitors, and precursors (both biotic and abiotic) revealed the accumulation of vincristine. Notably, treatment with a biotic elicitor derived from Aspergillus niger (300 mg/l) demonstrated superior efficacy in promoting the maximum accumulation of vincristine in endosperm-derived callus and cell biomass. These findings hold promise for the sustainable production of the anti-cancer alkaloid vincristine from endosperm-derived callus and cell cultures of Catharanthus roseus.

Agrobacterium-Mediated Genetic Transformation of Withania coagulans (Dunal) with rol A Genes and Its Antioxidant Potential.

In ancient times, Withania coagulans Dunal was used as a therapeutic plant for the treatment of several diseases. This report aims to examine the effect of Agrobacterium tumefactions-mediated transformation of W. coagulans with the rolA gene to enhance secondary metabolite production, antioxidant activity, and anticancer activity of transformed tissues. Before transgenic plant production, the authors designed an efficient methodology for in vitro transformation. In this study, leaf explants were cultured on Murashage and Skoog (MS) media containing different amounts of naphthalene acetic acid (NAA) and benzyl adenine (BA). The best performance for inducing embryogenic callus was in MS medium containing 4 μM NAA and 6.0 μM BA, while the best results for shooting (100%) were obtained at 8 μM benzyl adenine. On the other hand, direct shooting was attained by subculturing leaves on MS medium supplemented with 8 μM benzyl adenine. Prolonged shoots showed excellent in vitro rooting results (80%) with 12 μM indole-3-butyric acid (IBA). The samples were precultivated for 3 days and were followed by 48 h infection with A. tumefaciens strain GV3101 having pCV002. Then, a vector expressed the rol A gene of strain Agrobacterium rhizogenes. Furthermore, three independent transgenic shoot lines and one callus line (T2) were produced and exhibited stable integration of transgene rol A genes, as revealed by PCR analysis. Transgenic strains showed a significant increase in antioxidant potential as compared to untransformed plants. Additionally, LC-MS analysis showed that the transformed strains have a higher withanolide content as compared to untransformed ones. Moreover, the reduced proliferation of prostate cancer cells was observed after treatment with extracts of transgenic plants. Furthermore, these transformed plants exhibited superior antioxidant capability and higher withanolide content than untransformed ones. In conclusion, the reported data can be used to select withanolide-rich germplasm from transformed cell cultures.

Optimization of Plant Growth Regulators for In Vitro Mass Propagation of a Disease-Free 'Shine Muscat' Grapevine Cultivar.

This study addresses the propagation challenges faced by 'Shine Muscat', a newly introduced premium grapevine cultivar in South Korea, where multiple viral infections pose considerable economic loss. The primary objective was to establish a robust in vitro propagation method for producing disease-free grapes and to identify effective plant growth regulators to facilitate large-scale mass cultivation. After experimentation, 2.0 µM 6-benzyladenine (BA) exhibited superior shoot formation in the Murashige and Skoog medium compared with kinetin and thidiazuron. Conversely, α-naphthaleneacetic acid (NAA) hindered shoot growth and induced callus formation, while indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) demonstrated favorable root formation, with IBA showing better results overall. Furthermore, inter simple sequence repeat analysis confirmed the genetic stability of in vitro-cultivated seedlings using 2.0 μM BA and 1.0 μM IBA, validating the suitability of the developed propagation method for generating disease-free 'Shine Muscat' grapes. These findings offer promising prospects for commercial grape cultivation, ensuring a consistent supply of healthy grapes in the market.

In vitro micropropagation and tiliroside production in Paratecoma peroba (Record) Kuhlm, an endemic and endangered Brazilian tree

In vitro tissue culture can be an alternative method for endangered species propagation, biodiversity conservation and secondary metabolite studies. Paratecoma peroba (Record) Kuhlm. (Bignoniaceae) is an endemic and endangered Brazilian species. This work aimed to establish in vitro morphogenesis and callus induction and to perform a phytochemical analysis of P. peroba callus extract. Higher seed germination (43%) was obtained in Wood Plant Medium culture without activated charcoal (AC). Combination of 5 µM benzyladenine + 10 µM gibberellic acid, without AC, resulted in a higher number of shoots (2 shoots/explant). A callus culture was stabilised from zygotic embryos using 2,4-dichlorophenoxyacetic acid. A callus methanolic extract was used for phytochemical analysis. The isolated substance was identified as tiliroside (kaempferol 3-O-β-D-(6’’-O-E-p-coumaroyl)-glucopyranoside) by NMR and quantified in callus and leaf extracts by HPLC. This study adds to the chemical knowledge of this species and it is the first report of a flavonol in Paratecoma.

In vitro regeneration, phytochemical profiling and antioxidant activity in Ruta chalepensis plants established from alginate encapsulated synthetic seeds

We developed a highly efficient protocol for the short-term conservation and in vitro regeneration of Ruta chalepensis L. This involved encapsulating nodal segments (NS) in a sodium-alginate solution (3% w/v) and calcium chloride (100 mM) to produce synthetic seeds. The effect of various concentrations of plant growth regulators (PGRs) on the conversion of synthetic seeds into plantlets was examined and the best results, with a conversion rate of 96.8%, were obtained on Murashige and Skoog (MS) medium supplemented with 5.0 μM 6-benzyladenine (BA) and 0.5 μM α-naphthalene acetic acid (NAA) after 12 weeks of culture. When stored at 4 °C for different durations (2-12 weeks), encapsulated and non-encapsulated NS displayed distinct conversion responses. Encapsulated NS stored at 4 °C for 10 weeks exhibited a regeneration rate of 72.7%, while non-encapsulated NS did not survive under the same conditions. The proliferated shootlets derived from cold-stored NS were successfully rooted in agar-solidified MS medium containing 0.5 μM indole-3-butyric acid (IBA) and successfully acclimatized to ex vitro conditions. UV-Visible spectroscopy (UV), gas chromatography-mass spectrometry (GC–MS), and high-performance liquid chromatography (HPLC) were used to identify bioactive chemicals in the aqueous methanolic extracts of the acclimatized plants and compared with the donor plant. The bioactive chemicals were found considerably higher in the acclimatized plants (Phenols 95.0 mg GAE/g DW, flavonoids 99.2 mg QE/g DW, tannins 38.7 mg TAE/g DW, hesperidin 94.5 µg/mg, and quercetin 25.4 µg/mg). The antioxidant potential was also evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and demonstrated high scavenging activity at a concentration of 800 µg/mL in the acclimatized plants (83.0%). This study represents the first successful demonstration of in vitro regeneration, phytochemical profile analysis, and assessment of antioxidant activity in R. chalepensis plants established from synthetic seeds following short-term cold storage.

Screening of Cork Oak for Resistance to Phytophthora cinnamomi and Micropropagation of Tolerant Seedlings

Massive propagation of cork oak (Quercus suber) individuals tolerant to Phytophthora cinnamomi (Pc) is probably the most important challenge for cork production. Screening for resistance to Pc of ca. 200 seedlings obtained from a single cork oak tree that has survived the epidemic was performed by soil infestation. Twenty months after Pc inoculation, 33 seedlings survived from Pc infection and the four most vigorous seedlings were selected. The plants were forced to produce new shoots under controlled climatic conditions, and the new shoots were used to establish the plants in vitro by axillary budding. High axillary shoot proliferation rates were achieved by culturing the new shoots on Lloyd and McCown (WPM) medium, followed by subculturing for 2 weeks on 0.22 µM benzyladenine (BA) and for 2 weeks further on 0.04 µM BA. Addition of 20 µM silver thiosulphate (STS) increased the proliferation rates and improved the appearance and development of shoots. Rooting rates of 80–100% were obtained by culturing the shoots for 24 or 48 h on Gresshoff and Doy medium with ⅓ macronutrients plus 122.5 µM indole-3-butyric acid and subsequent transfer to root expression medium containing 20 µM STS. The results of this study optimize the micropropagation of a relevant and recalcitrant tree species in forestry.

Plant growth regulators improved cutting induced physio-biochemical responses to postponement senescence in chrysanthemum

The marketability of cut flowers relies on its postharvest longevity, making it crucial to develop technologies that canextend its vase life. The vase life in chrysanthemum is dependent upon floret senescence and leaf yellowing. In view ofextending the vase life, the present study elucidates the implication of plant growth regulators viz. benzyl adenine (BA),thidiazuron (TDZ) and salicylic acid (SA) on postharvest performance and flower longevity of chrysanthemum stems. Theharvested stems were treated with various concentrations viz. BA and SA at 50,100,150 and 200 μM and TDZ at 5,10,15and 20 μM. Treatments containing 10 μM TDZ and 100 μM BA were most effective in improving the longevity (23.35 and20.32 days respectively) of cut chrysanthemum stems. However, TDZ outplayed BA followed by SA extends the flowerlongevity through the maintenance of higher physiological and biochemical characteristics. Cluster analysis inferred that thetreatments of 50 and100 μM BA and 5 and10 μM TDZ were found to be efficient in delaying floret senescence (24.01 days)and leaf yellowing (20.54 days) in cut chrysanthemum stems. Postharvest longevity exhibited a positive correlation withantioxidant activities, total soluble sugars and proteins, while it showed a negative correlation with anthocyanin andcarotenoid contents. The results have demonstrated that use of growth regulators such as benzyl adenine, thidiazuron andsalicylic acid may preserve the quality of cut chrysanthemum stems and delay their senescence.

Establishment of Direct Organogenesis Protocol for Arachis hypogaea cv. Virginia in Liquid Medium by Temporary Immersion System (TIS)

Peanuts (Arachis hypogaea L.) are a rich source of herbal oil, proteins, minerals, vitamins, fibers, essential amino acids, as well as bioactive compounds, and are thus widely used for human nutrition and animal feed, and for prevention from certain diseases. However, the in vitro regeneration response of the species is generally low, and it also displays a significant variability among its varieties. Thus, the development of advanced protocols and approaches for the in vitro propagation of peanut is still of immense importance. A recently developed in vitro propagation technique, TIS; Temporary Immersion Bioreactor System, provides a new approach for the mass propagation of plants. Accordingly, the present study provides an efficient de novo regeneration protocol for Arachis hypogaea L. cv. Virginia by using a TIS. Different concentrations of cytokinins, i.e., benzyladenine (BA) or thidiazuron (TDZ), were tested with several combinations of dry and medium immersion periods of TIS, corresponding to a total of 8, 12, 16, 24, 32, 36, 48, 64, 72, and 96 min daily immersions for the induction of direct organogenesis. The study exhibited that an MS medium added to 110 µM BA or 10 µM TDZ are the most appropriate medium formulations in TIS, when applied for 16 min every 16 h. The application of optimized procedures to cv. NC7 and two valuable Turkish autochthonous varieties, 7 × 77 and Com74, is also reported. To the best of our knowledge, the present study draws attention also for being the first study in which a TIS was used for peanuts.

Micropropagation of Hermaphrodite Papayas: Increased Root Induction and Plant Survival during Acclimatization

In Hawaii, the commercial papaya industry is based on cultivars that segregate as females or hermaphrodites. Multiple seedlings are planted and then thinned at flowering to single hermaphrodites at each site. The aim of this study was to increase propagation efficiency by improving our procedure for micropropagation of hermaphrodite plants only. Initially, shoots were multiplied in vented jars on M2 medium, a Murashige and Skoog formulation containing 0.25 μM 6-benzyladenine (BA) and 0.1 μM α-naphthalene acetic acid (NAA). At weekly intervals, micropropagated shoots were either incubated for 4 to 7 days in IBA2 medium containing 20 μM indole-3-butyric acid (IBA) or were dipped in autoclaved rooting powder containing 0.8% IBA (DIP); then, they were placed in M2 until root initials or small roots were visible. After root induction in both treatments, plants were transferred to an in vitro medium containing ½ MSO and 30 g⋅L−1 sucrose in vermiculite (VER). The IBA2 treatment produced 467 potted plants compared to 475 produced by the DIP treatment; however, the average number of days that each treatment required from root induction to potting of rooted plants was not significantly different (IBA2: 52.42 ± 5.65 days; DIP: 51.94 ± 3.61 days). Plants from both treatments were grown in either wet potting medium (500 mL water/300 g potting medium) or damp potting medium (120 mL water/300 g potting medium) to test the effect of moisture content on plant survival and growth after potting. Use of damp rather than wet potting medium resulted in significantly higher plant survival and growth. These results could facilitate more efficient commercial practice for papaya growers.

Thidiazuron Induced In Vitro Clonal Propagation of Lagerstroemia speciosa (L.) Pers.—An Important Avenue Tree

A high throughput regeneration protocol has been developed for Lagerstroemia speciosa through node explants under the regime of various plant growth regulators (PGRs). This protocol can provide an alternative mode to seed-grown plants and minimize the cost–time of regeneration, significantly. Murashige and Skoog (MS) medium containing various combinations of PGRs exhibited a marked stimulatory effect on morphogenesis. Of the various combinations tried, node explant pretreated with thidiazuron (TDZ; 5.0 µM) for 4 weeks and followed with transfer into MS medium containing 1.0 μM 6-benzyladenine (BA) and 0.25 μM α-naphthalene acetic acid (NAA) was reported to be the best treatment as it resulted in a maximum number of 24.5 shoots with an average shoot length of 7.1 cm per explant in 90% of cultures after 12 weeks of incubation. The in vitro-generated shoots rooted satisfactorily in the adopted ex vitro method of rooting, which saves time and cost. Among the different treatments, the greatest rooting percentage (85%) was observed in the 200 μM IBA-treated shoots, with the highest root number (8.7) and length (3.4 cm) occurring after 4 weeks. Four months after being transferred to ex vitro, some of the physiological attributes of the in vitro-propagated plants were examined and compared to the ex vitro plants. Further, analysis of the genetic integrity in tissue culture-raised plantlets along with the parental tree was accomplished through DNA-based RAPD technique. The monomorphic banding pattern obtained by the RAPD primers resulted in a high level of genetic uniformity in regenerated plants.

In vitro propagation of Alkanna tinctoria Tausch.: a medicinal plant of the Boraginaceae family with high pharmaceutical value

Alkanna tinctoria Tausch. (Boraginaceae), commonly known as alkanet or dyers’ bugloss/alkanet, is a perennial plant rich in naphthoquinone enantiomers, such as Alkannin and Shikonin (A/S), which possess a wide range of pharmaceutical properties and are used as cosmetics, food additives, and natural dyes. This plant is mostly exploited from the wild, increasing the risk of its extinction as reported for other A/S producing plants extracted from their natural environment. Its cultivation under controlled conditions remains difficult and the need for alternative production systems both for preserving this endangered species and for increasing the production of A/S at a marketable level, has become a necessity. In the present study, a protocol for the in vitro production of A. tinctoria plants using shoot-tip explants was developed. Several culture media, concentrations of hormones, sugar, and gelling agents were tested to improve proliferation, rooting, and acclimatization of micropropagated shoot-tip explants from plants collected in the wild. Surface disinfection was optimal after immersion of shoot-tips and/or nodal explants in Signum® fungicide (30 min), ethanol 70% (1 min), and sodium hypochlorite 3% (10 min). Shoot proliferation was the highest on Murashige-Skoog basal medium enriched with 1.1 μM 6-benzyladenine, 0.15 μM α-naphthalene acetic acid, and 0.3 μM gibberellic acid with a proliferation rate of 3.9 every two weeks. For rooting, the Root Culture 1 (RC1) modified medium free of ammonium nitrate and enriched with 2.85 μΜ indole-3-acetic acid was the more adequate with 80% of roots formation after 30 days. Finally, acclimatization was optimal (100% survival rate) following transfer of the rooted explants in pots containing a peat moss:perlite (1:1, v/v) mixture, kept under a 90% relative humidity fog system for 10 days, followed by a decrease in relative humidity of 5% every day until 40% and a gradual increase in light intensity. The protocol developed allowed the production under in vitro culture conditions of a sufficient number of A. tinctoria plants with high levels of ex vitro survival, opening the door to industrial exploitation of its secondary metabolites and to the conservation of this important medicinal plants.

Efficient plant regeneration via meristematic nodule culture in Paeonia ostii ‘Feng Dan’

Tree peony (Paeonia sect. Moutan) is an economically important multipurpose woody plant in terms of its medical, ornamental and oil values, but its breeding and industrial development are severely limited due to inefficient traditional propagation methods and existing in vitro regeneration systems. Meristematic nodules (MNs) are an attractive alternative to solve this problem. This study first presented a protocol for in vitro regeneration of P. ostii ‘Feng Dan’ via MN culture with four consecutive steps, including embryogenic callus (EC) formation, MN induction and leaf cluster differentiation, shoot elongation, rooting and acclimatization. The highest EC induction rate (81.25%) was achieved when cotyledons were cultured on modified Murashige and Skoog (mMS) medium with 4.04 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 5.37 µM α-naphthylacetic acid (NAA) for 30 days. The optimal MN induction rate (100%) and leaf cluster differentiation rate (45.83%) were obtained when ECs were cultured on modified woody plant medium (mWPM) supplemented with 2.02 µM CPPU + 2.27 µM thidiazuron (TDZ) for a subculture time of 10 days. The combination of 1.29 µM 6-benzyladenine (BA) + 0.58 µM gibberellin (GA3) yielded the best shoot elongation (13.40 shoots per nodule), rooting rate (43.33%) and consequently survival rate (45.83%). The study will be beneficial to the mass propagation, breeding and genetic improvement of tree peony.

Synthetic seed propagation of the therapeutic-honey plants

Context Increasing demand for therapeutic honey has driven establishment of Leptospermum nectar plantations. Methods for propagation involving synthetic seeds (artificially encapsulated miniature cuttings) may speed production of Leptospermum polygalifolium Salisb. and L. scoparium J.R.Forst. & G.Forst. Aims The study aimed to determine how nutrient strength of the encapsulation solution and the presence of benzyladenine (BA) in the emergence medium affect shoot and root emergence from synthetic seeds of L. polygalifolium and L. scoparium. Methods Nodes from in vitro shoots of three L. polygalifolium clones (P1, P6, P11) and two L. scoparium clones (S6, S12) were encapsulated in 3% sodium alginate with half- or full-strength Murashige and Skoog (MS) medium, and the synthetic seeds were placed on full-strength MS emergence medium containing 0 or 2.2 μM BA. Key results Full-strength MS in the encapsulation solution was effective for shoot emergence of both species. BA increased the percentage of synthetic seeds with shoot emergence in clone P6 but decreased the percentage in clone S12. BA stimulated shoot emergence through callus in clones P1, P6, S6 and S12, and increased the number of shoots per emergent synthetic seed in clones S6 and S12. Surprisingly, the simple use of full-strength MS medium without hormones was highly effective for adventitious rooting, stimulating root emergence and plantlet formation in 26–57% of L. polygalifolium and 100% of L. scoparium synthetic seeds. Conclusions These two Leptospermum species are highly amenable to propagation via synthetic seeds. A simple formulation of hormone-free, full-strength MS medium in the encapsulation solution and emergence medium provides high frequencies of plantlet conversion. Implications Synthetic seeds have potential to assist in mass production of Leptospermum plants for nectar plantations to meet demand for therapeutic honey.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Stimulation of Physiological Processes in St. John’s Wort (Hypericum perforatum L.) Seedlings by Treatments with Triacontanol and Benzyladenine

Abstract Treatment of St. John’s wort plantlets with 1 µM triacontanol and 2 µM benzyladenine stimulates growth and metabolic processes, being an environmental-friendly approach for optimizing the cultivation of these valuable medicinal plants under controlled conditions. When the two growth regulators (a bioactive cuticular wax constituent and a cytokinin) are applied simultaneously, they act synergistically, enhancing each other’s effect on the biomass accumulation and on certain parameters of the photosynthetic light use efficiency, such as the effective quantum yield of photosystem II and the overall vitality index of the photosynthetic apparatus which performs the conversion of light energy into usable forms for carbon dioxide assimilation. The results concerning the interactions between the two externally applied growth regulators during the early development of St. John’s wort plants may lead to a more efficient cultivation of this herbal medicinal product, including the possibility to modulate the production of pharmacologically active metabolites.

In vitro regeneration of the endangered cactus Turbincarpus mombergeri (Riha), a hybrid of T. laui × T. pseudopectinatus

Turbinicarpus mombergeri is a cacti species formed by a hybridization process between Turbinicarpus laui and Turbinicarpus pseudopectinatus. Under natural conditions, it is very difficult for two species be genetically compatible for hybridization, and to produce flowers at the same time. Thus, T. mombergeri is a very interesting and a rare species. Unfortunately, the current populations are decreasing and now it is considered critically endangered. The aim of this research was to develop a successful protocol for propagating T. mombergeri using the in vitro culture techniques. Seed disinfection was performed with Plant Preservative Mixture, and 80% of germination occurred at day 45 in Murashige-Skoog medium. The shoots were cut longitudinally, and the segments were transferred to media containing 2.22 or 4.44 µM benzyladenine to induce shooting. The generated shoots were highly hydrated, and presented abundant callus. The hyperhydricity was controlled by reducing salt medium concentration, by increasing calcium levels and by using polyethylenglycol. The reduction of callus was attained by adding tri-iodo benzoic acid. Vigorous and thick shoots were generated in medium containing urea, and rooting improved in the presence of 0.5 µM indoleacetic acid. Plantlets with normal morphology were obtained, and the survival rate of the plants in soil was 80%. The methodology developed represents an alternative for propagation of T. mombergeri under controlled conditions for commercial or conservation purposes.

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.

Application of Plant Extracts in Micropropagation and Cryopreservation of Bleeding Heart: An Ornamental-Medicinal Plant Species

Lamprocapnos spectabilis (L.) Fukuhara (bleeding heart) is valued both in the horticultural and pharmaceutical markets. Despite its great popularity, information on the in vitro tissue culture technology in this species is limited. There is also little knowledge on the application of plant extracts in the tissue culture systems of plants other than orchids. The aim of this study is to compare the utility of traditional plant growth regulators (PGRs) and natural extracts—obtained from the coconut shreds, as well as oat, rice, and sesame seeds—in the micropropagation and cryopreservation of L. spectabilis ‘Gold Heart’ and ‘White Gold’. The biochemical analysis of extracts composition is also included. In the first experiment related to micropropagation via axillary buds activation, the single-node explants were cultured for a 10-week-long propagation cycle in the modified Murashige and Skoog medium fortified either with 1.11 µM benzyladenine (BA) and 1.23 µM indole-3-butritic acid (IBA) or with 10% (v/v) plant extracts. A PGRs- and extract-free control was also considered. In the cryopreservation experiment, the same 10% (v/v) extracts were added into the medium during a seven-day preculture in the encapsulation-vitrification cryopreservation protocol. It was found that the impact of natural additives was cultivar- and trait-specific. In the first experiment, the addition of coconut extract favoured the proliferation of shoots and propagation ratio in bleeding heart ‘Gold Heart’. Rice extract, on the other hand, promoted callus formation in ‘White Gold’ cultivar and was more effective in increasing the propagation ratio in this cultivar than the conventional plant growth regulators (4.1 and 2.6, respectively). Sesame extract suppressed the development of the explants in both cultivars analysed, probably due to the high content of polyphenols. As for the second experiment, the addition of plant extracts into the preculture medium did not increase the survival level of the cryopreserved shoot tips (sesame and oat extracts even decreased this parameter). On the other hand, coconut extract, abundant in simple sugars and endogenous cytokinins, stimulated a more intensive proliferation and growth of shoots after rewarming of samples. Analysing the synergistic effect of conventional plant growth regulators and natural extracts should be considered in future studies related to L. spectabilis.

Micropropagation and cryopreservation by vitrification of the Spanish endemic medicinal plant Sideritis leucantha Cav. subsp. leucantha (Lamiaceae)

Sideritis leucantha Cav. subsp. leucantha is a Spanish endemic medicinal plant that faces conservation problems. Therefore, this work is aimed to develop a protocol for the in vitro propagation and cryopreservation of shoot tip explants of this species. The morphogenic responses were recorded using Murashige and Skoog medium containing 6-benzyladenine, 2-isopentenyladenine, kinetin, or thidiazuron for multiplication and 1-naphthaleneacetic acid or indole-3-butyric acid for rooting. Maximal shoot proliferation was observed with medium containing 0.088 M sucrose plus 0.22 or 0.44 μM 6-benzyladenine. The longest shoots and greatest number of nodes per shoot were obtained from cultures incubated on medium with 0.44 μM 2-isopentenyladenine. Optimal rooting was achieved with 1-naphthaleneacetic acid at concentrations of 0.05 μM (highest percentage of rooted shoots) and 0.26 μM (highest number of roots). The morphological traits of the regenerated plantlets did not differ from those of wild-type plants, and these plantlets were successfully adapted to ex vitro conditions. After cryopreservation by vitrification, the highest percentage of regenerated shoot tips (70 ± 6%) was obtained with the treatment consisting of 60 min of loading solution (0.4 M sucrose plus 2 M glycerol in liquid medium) followed by exposure to plant vitrification solution 2 for 30 min at room temperature and then immersion in liquid nitrogen for 60 min. This study constitutes the first work on the micropropagation of S. leucantha subsp. leucantha and the first work on the cryopreservation of Sideritis species. The results obtained could be applied commercially and also in the conservation for other species of Lamiaceae.