Murashige & Skoog Research Articles

Optimization of clonal micropropagation of Chrysanthemum

Micropropagation of Chrysanthemum is an alternative to the traditional method of reproduction. Thanks to this method, the Chrysanthemum reproduction time is reduced to 3-4 months. For clonal micropropagation, sterile microshoots of Chrysanthemums of the varieties Snow White, Stranger, and Baltica White were used. At the stage of the micropropagation, the Murashige-Skoog (MS) medium with the full and half composition of mineral salts and growth regulators (kinetin, 6-benzylaminopurine, β-indolylacetic acid) were used. A universal culture medium for clonal micropropagation of all varieties of Chrysanthemum and optimal mediums, taking into account variety-specificity were established. It was noted that under in vitro conditions, high proliferative activity was observed in Neznakomka variety.

Differential effects of synthetic media on long-term growth, starch accumulation and transcription of ADP-glucosepyrophosphorylase subunit genes in Landoltia punctata

Murashige & Skoog (MS) and Hoagland’s media were previously used for in vitro culture of Landoltia punctata. During subsequent ex vitro culture, the use of MS medium resulted in a higher growth rate, compared to Hoagland’s medium. Thus, a higher starch content of L. punctata in MS medium was previously hypothesized. Here, L. punctata strain 5632 was isolated and characterized using morphological characteristics and the atpF-atpH intergenic region. During early cultivation stage, fresh weight and relative growth rate in MS medium were lower than Hoagland’s medium. Conversely, starch content in MS medium was considerably higher than in Hoagland’s medium. Medium effects on expression of genes coding for starch-biosynthesis ADP-glucosepyrophosphorylase (AGPase) were determined. Genomic fragments of small (LeAPS) and large (LeAPL1) AGPase subunits were characterized. Differential expression between each AGPase subunit genes was observed in both media. Additionally, in MS medium, the highest correlation coefficients between starch content and gene expression was found with LeAPS (0.81) and followed by LeAPL3 (0.67), LeAPL2 (0.65) and LeAPL1 (0.28). In Hoagland’s medium, the coefficients of LeAPL3 (0.83) and LeAPL2 (0.62) were higher than LeAPS (0.18) and LeAPL1 (−0.62). This suggested different levels of contributions of these genes in starch biosynthesis in both media.

Effects of Methyl Jasmonate and Putrescine on Tryptanthrin and Indirubin Production in in vitro Cultures of Isatis demiriziana Mısırdalı

Tryptantrin and indirubin are pharmacologically active compounds used in treatment of disseases such as cancer and Alzheimer's. In this study, we investigated the influences of different concentrations of methyl jasmonate (MeJa) and putrescine (Put) on tryptanthrin and indirubin production in leaf explants and development of Isatis demiriziana Mısırdalı grown in vitro. In all media treated with methyl jasmonate, tryptanthrin production in leaves of plantlets showed an increase. The highest increase in tryptanthrin production was observed in solid Murashige-Skoog (MS) medium containing 1.0 mM MeJa (154.026 ± 0.11 µg g-1), about 2.85-fold higher than the control (untreated plantlets) (40.017 ± 0.031 µg g-1). Production of tryptanthrin decreased about 2.56-fold in the leaves of plantlets treated with Put, when compared to control. The highest indirubin production was obtained in the leaves of plantlets grown in the MS medium containing 0.1 mM MeJa (11.274 ± 0.035 µg g-1) but treatments with Put didn’t show any positive affect on the indirubin production. Analysis of tryptanthrin and indirubin were performed using high performance liquid chromatography (HPLC).

The optimal sterilizing compound and culture medium in Elaeocarpus grandiflorus L. in vitro shoot induction

Elaeocarpus grandiflorus L. is an identity plant of Salatiga City. Wherein, the plant is difficult to naturally propagation. This study would obtain propagation technique of E. grandiflorus by tissue culture. It was experimentally conducted, consists of two sub-experiments. The first aimed to find the optimal sterilization agent was conducted with a completely randomized design, namely sterilizing compound. There were two compounds studied, i.e., NaOCl and Ca(OCl)2. The second sub-experiment aimed to determine the optimal medium type and antioxidant compounds for inducing in vitro shoots. It was conducted with a completely randomized design with two factors, namely the medium type [i.e. Murashige & Skoog (MS) and Wood Plant Medium (WPM)], and antioxidant agents (i.e. activated charcoal and polyvinyl pyrrolidone/PVP). Data were analyzed by descriptive percentage, analysis of variance, and least significant difference test. The results showed that Ca(OCl)2 and NaOCl were effective to obtain the sterile and alive explants. The MS media added by PVP resulted in the most explants forming shoots, and WPM supplemented with PVP or active charcoal caused most explants regenerating callus. The results could be used as a basis for the development of in vitro shoot multiplication to reach an efficient propagation of E. grandiflorus.

Phenolic acid and flavonoid production in agar, agitated and bioreactor-grown microshoot cultures of Schisandra chinensis cv. Sadova No. 1 – a valuable medicinal plant

In vitro cultures and raw materials (fruits and leaves) of the valuable medicinal plant species – Schisandra chinensis cultivar Sadova No. 1 (SchS) – were evaluated for the production of two groups of phenolic compounds, phenolic acids and flavonoids, and their antioxidant potential. A series of experiments was conducted, aimed at optimizing culture conditions for maximum growth and phenolic production in SchS microshoots. Different concentrations of plant growth regulators (6-benzyladenine – BA and 1-naphthaleneacetic acid – NAA, from 0 to 3 mg/l) in Murashige-Skoog (MS) medium were tested in several cultivation systems (agar, agitated, bioreactor) over various growth periods (10, 20, 30, 40, 50 and 60 days). Furthermore, an elicitation experiment was conducted in which the bioreactor-grown microshoots were exposed to yeast extract. HPLC-DAD analyses confirmed the presence of eight phenolic acids – chlorogenic, cryptochlorogenic, gallic, neochlorogenic, protocatechuic, salicylic, syringic and vanillic, and two flavonoids: kaempferol and quercitrin, in the in vitro biomasses. The highest total phenolic acid (357.93 mg/100 g DW) and flavonoid (105.07 mg/100 g DW) contents were obtained in agar culture extracts cultivated for 30 days on MS medium containing 2 mg/l BA and 0.5 mg/l NAA and for 50 days on MS medium containing 0.1 mg/l BA and 2 mg/l NAA, respectively. These amounts were 1.59- and 5.95-fold lower than in parent plant leaf extracts (569.66 mg/100 g DW), and 4.30- and 1.25-fold higher than in fruit extracts (83.17 mg/100 g DW), respectively. Microshoots grown in a Plantform bioreactor also proved to be a good source of phenolic compounds, however, the elicitor treatment had no noticeable effect on their accumulation. Antioxidant capacity assessed by the Folin-Ciocalteu, FRAP, DPPH and CUPRAC assays revealed significantly higher potential in extracts from in vitro biomass and leaves of the parent plant, as compared to the parent plant fruit extracts.

A Study on In Vitro Propagation Possibilities of Some Clone Rootstocks of Prunus Species

In vitro techniques which is one of the modern breeding methods offer alternative methods to meet the growing food demands in parallel with the increasing world population by allowing rapid and intensive mass propagation of varieties and rootstocks in fruit cultivation under laboratory conditions. Clone rootstocks which are economically important and early yielding with maximum yield per unit area are preferred in fruit production. In this study, the propagation possibilities of Marianna GF 8/1, Myrobolan B, Myrobolan 29-C and St Julien A clone rootstocks used in the production of stone fruit species were investigated. In this study, shoot tips were used as explants. MS (Murashige-Skoog) media was used as the propagation media and 30 g L-1 sucrose and 7 g L-1 agar were added. The shoot tips were planted into the media and then placed in the temperature and light controlled culture chamber. Samples were placed to the subculture at 1-month intervals. Explant length, number of leaves and number of tillings were observed every 20 days. Nine different MS media were tested during the shoot growth stage. The best propagation was obtained in the 1 mg L-1 BAP + 0.5 mg L-1 IBA + 0.25 mg L-1 GA3 media. Eight different MS media were tested during the rooting stage. The best rooting was achieved in a media containing 1/2 MS + 2 mg L-1 IBA. Keywords: Marianna GF 8/1, Myrobolan B. Julien A, Myrobolan 29-C, micropropagation, plant growth regulators. DOI: 10.7176/JSTR/5-9-10

Multiplicação in vitro de Xylopia aromatica em diferentes meios de cultura e concentrações de BAP

Este estudo teve por objetivo desenvolver uma metodologia de multiplicação in vitro para Xylopia aromatica, visando a produção de mudas, avaliando-se meios de cultura e concentrações de reguladores de crescimento mais adequados. Foram utilizados, como explantes, segmentos nodais, provindos de brotações de mudas produzidas via seminal e mantidas em viveiro. Foram testados os meios de cultura Murashige Skoog (MS) e Wood Plant Medium (WPM) suplementados com 800 mg L-1 de polivinilpirrolidona (PVP), 30 g L-1 de sacarose, 0,054 µM de ácido naftalenoacético (ANA) e 6 g L-1 de ágar e concentrações de 2,22 e 3,55 µM de benzilaminopurina (BAP) para a multiplicação in vitro. Aos trinta dias, avaliou-se o número de brotações laterais emitidas e o percentual de explantes formando calos na base. As maiores taxas de multiplicação foram obtidas com o meio MS acrescido de 3,55 µM de BAP. Em relação à formação de calos na base dos explantes, o meio MS promoveu maior formação quando comparado ao WPM, no entanto não comprometeu a formação de brotos. Foi possível estabelecer as fases de introdução e multiplicação, definindo assim o meio de cultura e concentrações de reguladores de crescimento mais adequados ao cultivo in vitro de Xylopia aromatica.

Efficient low cost seesap (CSUP) technique for micropropagation of newly introduced sweetener plant, Stevia rebaudiana Bertoni

Stevia rebaudiana is a non-caloric sweetening herb, which has a high potential for commercialization. During this study low cost media sterilization, seesap (CSUP) technique which applies sodium hypochlorite was used as an alternative to autoclaving. In vitro cultures were established using nodal segments collected from stevia mother plants. These in vitro shoots were cultured on Murashige & Skoog (MS) medium supplemented with 16 combinations of 6- benzyl amino purine (BAP) and kinetin (KN) with 3% sugar at 5.8 pH, to investigate the in vitro shoot multiplication. The developed shoots were transferred to half MS medium supplemented with 0, 1 and 2 mg/L Indole butyric acid (IBA) with 3, 4 and 5% sugar in nine combinations at 5.8 pH, to investigate the rooting. The well rooted stevia plantlets were transferred to Jiffy™ bags for hardening. The results showed that the combination of 1.0 mg/L BAP and 1.5 mg/L KN resulted in the highest number of shoots (11.8 shoots/shoot). The combination of 2.0 mg/L IBA with 5% sugar in half MS medium resulted in the highest root induction (7.2 roots / plant) and root elongation (1.02 cm). The plants grown in culture medium containing 5% sucrose gave 100% survival rate at acclimatization. Key words: Stevia, micropropagation, shoot induction, root induction, acclimatization, low cost seesap (CSUP).

Influence of Ribavirin on Prunus Domestica L. Regeneration, Genome Stability and Virus Eradication In Vitro

Abstract Prunus necrotic ring spot ilarvirus (PNRSV) and Apple chlorotic leaf spot trichovirus (ACLSV) are common in plum orchards. The aim of the study was to obtain virus-free planting material of Prunus domestica L. by chemotherapy in vitro. Ribavirin at concentrations of 10 to 50 mg·l−1 was added to Murashige–Skoog (MS) nutrition medium for virus eradication from microshoots. After a two-week period of chemotherapy, meristems were subcultured monthly on MS medium and proliferation index of shoots was estimated. Microshoots were retested by reverse transcription polymerase chain reaction for presence of virus. At lowest concentrations of 10 mg·l−1 ribavirin was entirely ineffective for ACLSV and 10 to 30 mg·l−1 was ineffective for PNRSV elimination. Ribavirin concentrations of 40 and 50 mg·l−1 destroyed both pathogens. However, at higher concentrations of 40 and 50 mg·l−1 ribavirin exhibited some signs of phytotoxicity on microshoots in the first sub-cultivation period. In order to test the genetic stability of the microplants after chemotherapy the amplified fragment length polymorphism (AFLP) method was applied. Plant genome stability in ‘Magna Glauca’ at concentrations of 40 mg·l−1 was damaged, as the presence of polymorphic AFLP markers were observed.

Growth regulators induced shoot regeneration and volatile compound production in Lippia rotundifolia Cham., a threatened medicinal plant

Lippia rotundifolia Cham. is an endemic and endangered plant found in the savanna that is aromatic and rich in monoterpenes. The aims of the study were to evaluate the influence of plant growth regulators on the growth and chemical analysis of L. rotundifolia by headspace/GC–MS. Nodal segments (1.0 cm) were cultured on basal Murashige & Skoog (MS) medium supplemented with the following amounts of growth regulators: 0.0, 2.22, 6.66, 11.10, and 15.54 μM 6-benzylaminopurine (BAP) combined with 0.0, 2.68, and 5.36 μM naphthaleneacetic acid (NAA) or 0.0, 2.27, 4.54, 6.81, and 9.08 μM thidiazuron (TDZ) combined with 0.0, 2.68, and 5.36 μM NAA. The most effective concentrations were 11.10 μM BAP + 2.68 μM NAA, which induced 11 shoots explant−1, and 15.54 μM BAP + 2.68 μM NAA, with 11.6 shoots explant−1. The TDZ at a concentration of 6.81 μM with 2.68 μM NAA resulted in 20 shoots explant−1, 9.08 μM TDZ without NAA in 22.8 shoots explant−1, and 9.08 μM TDZ + 2.68 μM NAA in 18.1 shoots explant−1. Rooting took place on MS medium free of plant growth regulators, and acclimatization was successful (95%). The influence of plant growth regulators on the fraction of volatile compounds appeared to be quite variable. The cytokinin concentration was found to significantly influence the production of volatile compounds in L. rotundifolia shoot cultures. These findings indicate that shoots regenerated from nodal segments on MS medium containing higher concentrations of BAP and NAA have increased myrcenone and limonene. Ocimenone contents increased only at low concentrations of BAP and NAA.

In vitro proliferation of Lebanese Lemna minor and Lemna gibba on different nutrient media

This study aimed to assess the ability of two Lebanese duckweed species, (Lemna minor and L. gibba), to grow under in vitro conditions on three nutritive solutions, Murashige Skoog (MS), Schenk-Hildebrand (SH) and Algal Assay Procedure (AAP). Plant growth of both species, expressed as doubling time, differed significantly (p < .05) between the tested media with best results obtained on SH for L. minor after 7 days of culture and on AAP for L. gibba after 2.7 days for both species. Growth index was significantly higher (P < .05) for L. minor on SH reaching 41.6 after 21 days of culture whereas L. gibba exhibited its highest growth index of 15.51 on AAP. These results indicate the efficiency of SH and AAP media in promoting the vegetative proliferation of L. minor and L. gibba to further provide a good source of duckweed material for phytoremediation applications.

Pepper plants response to metal nanoparticles and chitosan in nutrient media

Neutral nanoparticles (NPs) of iron, zinc, copper or their compositions were introduced into nutrient Murashige-Skoog (MS) medium instead of traditional metal salts with/without chitosan supplements to obtain healthy and crop productive plants. Metal NPs concentrations were: Fe NPs (0.06, 0.3, 3.0); Zn NPs (0.016, 0.08, 0.4); Cu NPs (0.004, 0.0008, 0.00016) (mg L-1). Pepper (Capsicum annuum) cultivar LJ-King was grown on modified media in aseptic conditions. After 45 days, root and stem length, root activity, green mass gain and chlorophyll content in leaves were measured. Pepper plants grown on media with metal NPs showed increased root length and activity (for Fe, Zn, Cu NPs and their composition) and chlorophyll content in leaves (for NPs Fe and Cu). However, addition of Chitosan modified the effects. Monitoring of pepper grown on MS with NPs composition (Fe: Zn: Cu as 3.0:1.0:0.004 mg L-1) with electron microscope demonstrated no NPs accumulation in roots, sprouts and leaves.

An Efficient Somatic Embryo Liquid Culture System for Potential Use in Large-Scale and Synchronic Production of Anthurium andraeanum Seedlings.

Anthurium andraeanum Lind. is the second most important tropical flower in the world flower market. Somatic embryogenesis and plant regeneration in Anthurium has been reported previously; however, a stable and effective method for its commercial use has not been available. In this study, an efficient somatic embryogenesis and liquid culture system for large-scale production of A. andraeanum seedlings was achieved. Building on previous research, this study investigated the main factors for proembryogenic mass (PEM) proliferation, somatic embryo (SE) development, and SE germination in Anthurium. The results showed that relatively low concentrations of plant growth regulators, mineral nutrition, and sucrose promoted PEM proliferation, SE formation, and germination in a liquid culture system. This system can be described as follows: PEMs were induced from leaf blade explants on Murashige & Skoog (MS) medium with half-strength MS macronutrients (1/2 MS) containing 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L-1 kinetin (KT), and 3% sucrose and were proliferated in ½ MS liquid medium containing 1.0 mg L-1 2,4-D, 0.5 mg L-1 KT, and 3% sucrose. The highest proliferation coefficients were 5.11–5.16. PEMs were then transferred to MS medium with 1/8 MS macronutrients (1/8 MS) liquid medium containing 1% sucrose to develop into globular embryos and mature embryos. Finally, the mature embryos were placed on four layers of absorbent filter paper saturated with 1/8 MS liquid medium containing 1% sucrose for germination, and an average of 60 seedlings per gram SEs was obtained. This liquid culture system can be used in large-scale and synchronic production of Anthurium seedlings.

Effects of chemical substances on the rapid cultivation of moss crusts in a phytotron from the Loess Plateau, China

In this study, typical moss crusts, which were dominated by the species Didymodon vinealis (Brid.) Zand., were collected from the Loess Plateau and a 65-day cultivation experiment was performed to study the effects of five kinds of nutrient solutions (Knop, Murashige-Skoog (MS), Benecke, Part and Hoagland), two kinds of carbohydrates (glucose and sucrose) and three kinds of plant growth regulators (thidiazuron (TDZ), 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA)) on the coverage, plant density, and plant height of moss crusts. The main conclusions are as follows. (1) All Knop, MS, Benecke, Part and Hoagland nutrient solutions improved the coverage and plant density of moss crusts to different degrees and the promotional effects of the Hoagland nutrient solution were most significant. (2) Glucose and sucrose could promote the formation of moss crusts, but they inhibited the development of moss crusts at concentrations greater than 10 g/L. (3) With an increase in concentration, the effects of TDZ on the development of moss crusts changed from “enhanced” to “inhibited”. Regardless of whether the concentration was high or low, 6BA had no significant effects on the growth of moss crusts, and NAA reduced the development of moss crusts. Results suggest that nutrient solutions (e.g. Hoagland), low concentration carbohydrates solutions, and some plant growth regulators (e.g. 1 mg/L TDZ) enhance the development of moss crusts in Loess Plateau under the appropriate environmental conditions.

Storage and in vitro germination of orchids (Orchidaceae) seeds from Atlantic Forest - Brazil.

The orchid seed banks of Atlantic Forest may be considered a key strategy for the conservation of species threatened with extinction by indiscriminate collection or habitat destruction. The aim of this study was to evaluate the seed viability, to choose the best culture medium for the asymbiotic germination and evaluate germination, after storage for different periods and temperatures for the Brazilian native orchids: Gomesa praetexta (Rchb.f.) M.W.Chase & N.H.Williams, Gomesa forbesii (Hook.) M.W.Chase & N.H.Williams, Gomesa recurva R.Br. and Grandiphyllum divaricatum (Lindl.) Docha Neto. Knudson C (KC), Murashige & Skoog (MS), half-strength MS (1/2 MS macro- and micro-nutrients) and Woody Plant Medium (WPM) culture media were tested for germination. The WPM culture medium was the best for asymbiotic germination of all species evaluated, with high germination percentages and improved seedling development. Seeds of G. divaricatum, G. praetexta, G. recurva and G. forbesii indicated orthodox behavior, with high viability rates after 12 months of storage, being recommended the storage temperature of -80°C for the first three species and -20°C for G. forbesii. The protocol developed in the present study was efficient for seed bank storage, in vitro germination and seedling production of G. divaricatum and G. praetexta, contributing to conservation strategies of these species.

Multiple Shoot Regeneration in Seed-derived Immature Leaflet Explants of Red Dragon Fruit (Hylocereus costaricensis)

Dragon fruit which belongs to Cacteceae family was once widely grown as ornamental plant and is now considered as fruit crop. Though there were many reports on propagating dragon fruit inadequate information is available on obtaining best quality pant material. Hence the present work is carried out to find the effect of phyto harmones, 6-Benzylaminopurine (BAP) and Naphthalene acetic acid (NAA) on callus formation, shoot and root regenerations from dragon fruit in vitro using stem cuttings as explants. Stem explants in Murashige Skoog (MS) medium containing 3mg/l BAP and 0.5mg/l NAA exhibited highest in vitro response for callus formation. Further, maximum number of shoots (13.2) was regenerated from explants when MS basal medium was supplemented with 3mg/l BAP and 0.5mg/l NAA. Within four weeks somatic shoots were raised from explants. It took 70 days for shoots to grow 2.0cm long. Subsequently regenerated shoots were transferred to rooting medium supplemented with 3mg/l BAP and 0.5mg/l NAA which proved to be effective concentrations for the growth and development of roots (4.8) in vitro.

Comparison of Volatile Profile and Antioxidant Activity of Piper divaricatum G. Meyer (Piperaceae) Using Cuttings and Cell Tissue

In this study, P. divaricatum (Piperaceae) plants were subcultured by cuttings and cell tissue in order to carry out a comparison of the volatile profile, phenolic compounds, and antioxidant activity. Propagation by cuttings used vermiculite substrate and Murashige-Skoog (MS) medium in the absence of growth regulators and rooting started at 15 days of growth providing a higher number of plants after 90 days. In vitro propagation was performed using shoot apices as explants in MS media supplemented with 0.5 mg mL-1 BA (6-benzyladenine). Volatile profiles analyzed by gas chromatography-mass spectrometry (GC-MS) showed as main compounds methyl eugenol, E-β-ocimene, and β-elemene in the in vivo and in vitro cultures. Phenolic contents determined by the Folin-Ciocalteu method had no significant difference at the end of 90 days of growth displaying a good linear correlation with antioxidant activity and phenylpropanoids amounts (r > 0.7). However, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging was higher for in vitro plants grown in comparison to in vivo plants cultured.

In Vitro Propagation of Wetland Medicinal Plant Lythrum salicaria L.

The aim of this experiment is to develop a protocol to establish the in vitro propagation potential of Lythrum salicaria L (purple loosestrife). Seeds of L. salicaria were germinated and grown in Murashige Skoog (MS) medium for 30 days. At the end of 30 days, shoot tips of L. salicaria were cultured on MS medium supplemented with different combinations and concentrations of 6-benzylaminopurine (BAP; 1, 2 and 3 mg/L) and 1-naphthaleneacetic acid (NAA; 0, 0.1 and 0.5 mg/L). Regenerated shoots were transferred to MS supplemented with varying concentrations of different auxins: NAA (0.5 and 1 mg/L) and indole-3-butyric acid (IBA; 0.5 and 1.0 mg/L) for rooting. Among the cytokinins investigated, BAP indicated the best response to initiate shoot elongation and multiple shoot formation alone. The highest number of shoot per explant (37±3.38) was obtained in MS medium with 1 mg/L BAP. Although, the highest shoot length (3.1 cm ± 0.48) was found on medium with 1 mg/L BAP+0.5 mg/L NAA, there were no statistically differences among all types of medium. Percentage of rooting, shoot length and root length were altered significantly with different concentrations of NAA and IBA. Thus, shoot length (9.5 cm±0.46), root length (4.1 cm±0.13) and the percentage of rooting (100%) increased significantly with a concentration of 1 mg/L NAA. It was concluded that MS medium with 1 mg/L NAA has been superior for root development compared to other hormone concentrations.

Culture Medium and Varietal Selection for Establishment of Callus Culture of Artemisia annua

Plant tissue culture technology offers the potential of producing medicinally important secondary metabolites such as anti-malarial artemisinin from Artemisia annua. In this study, callus induction of three different varieties of A.annua, namely T1, T2 and Hi varieties was carried out using leaf explants on Murashige & Skoog (MS) and Litvay (LV) media with three different supplementations. MS medium added with 0.5 mg/L BA, 0.5 mg/L NAA and 0.5 g/L of casein hydrolysate (CH) induced the highest yield of callus biomass compared to the effect of picloram- or 2,4-enriched MS medium. T2 variety was found to be the highest yielding variety in this medium. Picloram-enriched MS medium induced better callus in term of callus biomass and friability than 2,4-enriched medium. Hi variety induced in MS medium added with 0.5 mg/L picloram produced highest callus biomass among the three varieties. Callus formed on 0.5 mg/L picloram was also much more easily dispersed than callus of all three varieties cultured in the other two MS medium. LV-based medium was generally shown to be poor in inducing callogenic response from leaf explants. Therefore Hi callus sourced from MS medium supplemented with 0.5 mg/L picloram was selected to initiate liquid cell culture of A. annua which can further be explored for production of artemisinin, an anti-malarial compound. Abbreviations: MS – Murashige and Skoog’s salts and vitamins [1]; LV – Litvay medium [2]; BA – 6- benzyladenine; NAA – alpha-napthaleneacetic acid; 2,4-D – 2,4-dichlorophenoxyacetic acid; picloram – 4-amino-3,5,6-trichloropicolinic acid, CH – casein hydrolysate

Asymbiotic seed germination and in vitro seedling development of Acianthera prolifera (Orchidaceae)

The effect of the culture medium and of the addition of sucrose and activated charcoal on in vitro germination and protocorms development of Acianthera prolifera was analysed in order to establish a protocol for the seedlings propagation and ensure their conservation. Murashige & Skoog (MS), MS with half the salt concentration (MS/2), Knudson C (KC), and Woody Plant Medium (WPM) culture media were tested for asymbiotic seed germination and plantlet development for 16 weeks. MS/2 was more efficient in the beginning of germination and WPM was more suitable for plantlet formation. Protocorms and plantlets developed more efficiently in WPM containing 1 g L−1 activated charcoal. Culture media supplemented with sucrose promoted plantlet growth after a 12-week subculture and the recommended concentration of sucrose was 60 g L−1 for WPM, and 15 g L−1 for MS/2. Seed and protocorm anatomy was characterized until plantlet formation. The seedlings were successfully acclimatized (95%) in a commercial mixture of pine bark, coconut fibre, and activated charcoal combined with vermiculite (1:1) after 12 weeks of culture in a greenhouse.