Murashige-Skoog Medium Research Articles

In vitro callus induction and plantlet regeneration of Achyranthes aspera L., a high value medicinal plant

ObjectiveTo study callus induction from different explants (internode, leaf, root) and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L. MethodsSterilized explants were prepared by using 0.1% HgCl2 and 0.5% Bavistin and callus was obtained when cultured onto Murashige Skoog's (MS) medium by using different concentrations and combination of 2,4-D, NAA, BAP, IAA, IBA with 3% sucrose and 0.8% agar. Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively. ResultsSterilization treatment of 0.1% HgCl2 for 2–3 min and Bavistin 0.5% for 10–12 min showed the highest percentage of asepsis and survival rate. Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf. Highest shootlets number (4.83±0.17) and length (3.8±0.16) cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L. Concerted efforts of BAP 2.0 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number (6.77±0.94). In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations. Experimentally, 3.0 mg/L IBA was enabled to induce maximum rootlets number (10.0±9.82) on full strength MS medium. Afterwards, regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized. The survived plantlets showed 66.67% survival frequency without any morphological abnormality. ConclusionsThe results demonstrated that different explants were good source of callus induction, morphology analysis as well as indirect plantlets regeneration.

In vitro cultivation of zygotic embryos from Murici (Byrsonima cydoniifolia A. Juss.): establishment, disinfection, and germination

The objective of this study is to establish an in vitro germination and cultivation protocol for murici (Byrsonima cydoniifolia A. Juss.) using zygotic embryos. Therefore, three assays were performed: in assay I, embryo asepsis was tested at exposure times of 5, 10, 15, 20, 25, and 30 minutes in 2.5% sodium hypochlorite, with or without immersion in 70% alcohol; in assay II, MS (MURASHIGE; SKOOG, 1962) e WPM (LLOYD; McCOWN, 1980) culture media were tested at salt concentrations of 25, 50, and 100%, with or without the addition of sucrose, to germinate the buds; in assay III, seedling growth was evaluated in MS and WPM culture media at salt concentrations of 25, 50 and 100%. Sodium hypochlorite (2.5%) with or without 70% alcohol was used to avoid contamination because it was not toxic to murici embryos. Water-agar was the most appropriate culture medium for bud germination, and 50% WPM was appropriate for seedling growth.

Conservation through in vitro propagation and restoration of Mahonia leschenaultii, an endemic tree of the Western Ghats

Conservation of Mahonia leschenaultii was achieved through multiple axillary shoot formation in single node cultures, in vitro and ex vitro rooting of the shoots, and successful reintroduction and establishment of the rooted plants in the forests of Palani hills in Southern India. A preliminary experiment on the culture of young nodal explants (1.0-1.5 cm) from field-grown plants in different basal nutrient media revealed high frequency bud beak in Schenk and Hildebrandt (SH) medium followed by Woody Plant medium and Murashige Skoog medium in 4 weeks. Subsequent culture of both shoot tips (0.5-0.8 cm) and nodes for 6 weeks in SH medium supplemented with various concentration of cytokinins resulted in optimal formation of 3.6 shoots in 80% of the nodes against 1.35 shoots in 75% of the shoot tips in the presence of 2.0 and 1.0 mg/l benzyl adenine (BA), respectively. Invariably, the resident apical and axillary meristems grew into a single shoot (1.0-1.8 cm) in 1-2 weeks followed by the characteristic differentiation of additional buds from its base. A synergistic combination of 1.0 mg/l BA and 0.02 mg/l indole-3-acetic acid (IAA) induced the maximum number (5.9) of axillary shoot formation which were relatively high (75%) when the explant was collected during May-June and the fifth nodes from the top of the growing shoots were used. Repeated subculture of the nodes from shoot cultures at 5-6 week intervals in medium supplemented with reduced concentrations of the growth regulators (0.5 mg/l BA, 0.01 mg/l IAA) through at least 10 passages enabled consistent production of 6-7 shoots per node at 92% success rate without loss of vigour, growth and morphological abnormalities. Shoots of 3-6 cm were rooted in vitro in the presence of 1.0 mg/l indole-3-butyric acid (IBA) and hardening in the mist house at 76-78% and these rooted plants established in a potting medium of river sand and top soil (1:1) under constant mist irrigation. The plants reared in the nursery for 5-8 weeks were successfully reintroduced into the natural forest segment of the Institute's campus (MSL 200 m) at Palode and Vattakanal shola forests of Palani hills (MSL 2500 m) with establishment frequencies of 78.8 and 90.6, respectively, after 18 months. Growth characteristics of the plants (height, number of leaves and branches) reintroduced into native habitat at Vattakanal were better than those at Palode. Genetic fidelity of the 12-month old plants established in the nursery/field was confirmed by analysis of amplified products of genomic DNA obtained with random primers.

Peroxidase Activity in Native and Callus Culture of Moringa Oleifera Lam

Manscript received May 13, 2013; revised July 11, 2013. Abstract—In vitro Moringa oleifera Lam. seedlings were successfully produced through seed culture on Murashige & Skoog (MS) agar medium containing 3% (w/v) sucrose and 0.2% (w/v) Gelrite in the absence of growth regulators under 1,500 lux of light density, 16 hour photoperiod light at temperature of 25 ± 2°C. Shoot-derived callus and rootderived callus of M. oleifera were established via culture of shoot and root on the MS medium supplemented with 0.5 mg/l of 2,4-dichlorophenoxy acetic acid (2,4-D) in the dark at 25 ± 2°C. Stem, leaf and root of native M. oleifera and M. oleifera callus were assayed for peroxidase activity using guaiacol as a substrate of the enzyme. In native plant, crude extract from root provided the highest peroxidase specific activity, followed by those from stem and leaf with the specific activity of 19.73 ± 0.18, 16.56 ± 1.43 and 13.38 ± 1.04 unit/mg protein, respectively. Crude extract of root-derived callus and shoot-derived callus of M. oleifera possessed specific activity of 167.25 ± 16.12 and 103.99 ± 10.64 unit/mg protein. These values are significantly higher than their counter parts from native M. oleifera suggesting the potential use of the callus cultures as new and improved sources of peroxidase.

In vitro micropropagation of chive (Allium schoenoprasum L.)

The investigations on micropropagation of <i>Allium schoenoprasum</i> embraced shoot formation from cultured shoot tips, shoot multiplication, root formations, and cold storage of rooted plants. All explants were cultured on modified Murashige-Skoog medium. Surface disinfection of shoots derived from stock plants was not necessary to obtain no infection culture. The highest shoot multiplication was obtained on medium with 20 g l<sup>-1</sup> sucrose and 1.0 mg 1<sup>-1</sup> 6-benzylo-arninopurine (BA). Root formation was observed in both indoleacetic acid (IAA) and indolebutyric acid (IBA). Rooted plants were successfully kept in cold storage (5°C ± 1°C darkness). Only 20% of plants died after 6 months of storage.

The Dynamics of Cell Division in Some Local Potato Varieties Cultivated in Vitro on Micropropagation Medium

Abstract This study was performed to reveal the changes in cell division, as a result of the prolonged period of subculture on micropropagation medium, of five local varieties of Solanum tuberosum L. maintained on in vitro collection at Suceava Genebank, Romania. For this purpose it was used the Murashige-Skoog medium (MS- 1962) with addition of 40 g/l sucrose, and 6 mg/l daminozide. The effect of prolonged period of subculture up to two and 12 months was expressed as mitotic index and frequency of cells with abnormal division. Mitotic index ranged from 20.1 to 22.1% after 12 days, between 15.5 - 17.7% after two months and between 17.7 - 19.2% after 12 months of subculture. The results obtained showed that the frequency of aberrant cells increased with the preservation time on the in vitro cultures and their accumulation rate depended on the genotype. Were identified interphases with micronuclei, metaphases with retarded chromosomes, ana-telophases with chromosomal bridges, retarded chromosomes and chromosomal fragments, but their percentage was low in all the genotypes.

Assays of PCB congeners and organochlorine insecticides with the transgenic Arabidopsis and tobacco plants carrying recombinant guinea pig AhR and GUS reporter genes

Certain congeners of polychlorinated biphenyls (PCBs) and organochlorine insecticides are ligands of aryl hydrocarbon receptors (AhRs) in animals. A recombinant guinea pig (g) AhR, XgDV, was constructed by fusing the ligand-binding domain of gAhR, the DNA-binding domain of LexA, and the transactivating domain of VP16. Then, the expression unit of β-glucuronidase (GUS) reporter gene regulated by XgDV was introduced into Arabidopsis and tobacco plants. When the transgenic Arabidopsis XgDV plants were cultured on Murashige–Skoog (MS) medium containing PCB congeners, the GUS activity in the plants increased toxic equivalent (TEQ)-dependently. The GUS activity in the transgenic Arabidopsis XgDV plants cultured on MS medium containing the organochlorine insecticide dieldrin was also induced. On the other hand, in the case of DDT, the GUS activity induced by 3-methylcholanthere in the plants decreased. The transgenic Arabidopsis XgDV plants detected 1000 ng g−1 PCB126 in 1 g of soils. Thus the XgDV plants seemed to be useful for convenient assays of PCB congeners and organochlorine insecticides, without any extraction and purification steps.

THE INFLUENCE OF 3-(6,11-DIOXO-6,11-DIHYDRO-12H-BENZO[b]PHENOXAZIN-12-YL)BUTANOIC ACID ON RAPESEED MORPHOGENESIS IN VITRO AND GLUCOSINOLATE AND THIOCYANATE QUANTITIES

Naphthoquinones are widely distributed in plants, fungi, and some animals. Their biological activities have been studied including their effects on prokaryotic and eukaryotic cells. Vitamin K is a derivative of 1,4-naphthoquinone. Other natural naphtoquinones include juglone, plumbagin, droserone. Naphthoquinone derivatives have significant pharmacological properties: they are cytotoxic, have a significant antibacterial, antifungal, antiviral, insecticidal, anti-inflammatory and antipyretic properties. Menadione sodium bisulphite (MSB) increased the growth of tomato plants and alfalfa callus and stimulated rooting of mungbean cuttings. The levels of IAA were increased by about 3 to 4 fold following application of MSB to tomato, cucumber, corn and capsicum plants. Plumbagone, juglone and lawsone are naturally occurring naphthoquinones of plant origin that have antibacterial effects on several species of both aerobic and anaerobic organisms, and toxins derived from naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) are produced by Fusarium solani and attack plants, other fungi and bacteria. Oilseed rape, Brassica napus L., is one of the most important crops for production of vegetable oils in the world. Traditional rapeseed cultivars contain high amounts of glucosinolates. Although glucosinolates have antibacterial, antifungal properties and cancerchemoprevention activity, their antinutritional effects have limited the use of meals from oilseed rape for human food and animal feed. Besides, these compounds provide the bitter flavour and sulfurous aroma due to breakdown. Therefore, one of the main biotechnology objective is reduction of glucosinolates content in seeds to trace amounts. Rapeseed and its products are rich sources of bioactive compounds such as: polyphenols, phytosterols, tocopherols and other antioxidants, which are important in the prevention and treatment of coronary heart diseases, cancers, diabetes, hypertension, neurodegenerative and autoimmune diseases. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. The regeneration of plants in vitro is interesting not only for studies of the regeneration process itself but also for success of in vitro manipulations of crop improvement. The main object was to explore the influence of 3-(6,11-dioxo-6,11-dihydro-12H-benzo[b]phenoxazin-12-yl)butanoic acid on rapeseed morphogenesis in vitro and glucosinolate and thiocyanate quantities. 3-(6,11-Dioxo-6,11-dihydro-12H-benzo[b]phenoxazin-12-yl)butanoic acid was synthesized at the Department of Organic Chemistry. We used Murashige-Skoog medium with 6-benzylaminopurine (BAP) (2 mg/l) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0.2 mg/l) for obtaining rapeseed callus cultures in vitro. Compounds were examined at different concentrations 0.5 mg/l; 1.0 mg/l; 1.5 mg/l; 2.0 mg/l in the medium. It was determined, that the growth of rapeseed „Sponsor“ sprouts stimulated and biomass increased of rapeseed, which were grown on MS medium with 3-(6,11-dioxo-6,11-dihydro-12H-benzo[b]phenoxazin-12-yl)butanoic acid (1 mg/l). It was explored, that the lowest amount of glucosinolates was in rapeseed overground parts, which were grown on MS medium with 3-(6,11-dioxo-6,11-dihydro-12H-benzo[b]phenoxazin-12-yl)butanoic acid (2 mg/l) and the lowest amount of thiocyanates was in rapeseed roots, which were grown on MS medium with 3-(6,11-dioxo-6,11-dihydro-12H-benzo[b]phenoxazin-12-yl)butanoic acid (1.5 mg/l).DOI: http://dx.doi.org/10.5755/j01.ct.60.2.1948

In vitro propagation and quality evaluation of long-term micro-propagated and conventionally grown Fagopyrum dibotrys Hara mutant, an important medicinal plant

Fagopyrum dibotrys (D. Don) Hara is an important plant species of medicinal value. An efficient protocol for in vitro propagation of an F. dibotrys mutant that contains a higher level of (-)-epicatechin was developed. Adventitious buds were directly regenerated from stem nodal explants cultured in Murashige-Skoog (MS) basal medium containing 3% sucrose and appropriate combinations of plant growth regulators. In the presence of 2.0 mg/l 6-benzylaminopurine (6-BA), 0.5 mg /l thidiazuron (N-phenyl-N’-1,2,3-thidiazol-5-urea) (TDZ), and 0.2 mg/l naphthaleneacetic acid (NAA), the frequency of organogenesis was 90%, with a mean of 9.09 shoots per explants. The greatest rooting frequency, root number and length were attained using 1/2 MS medium containing 0.5 mg/l NAA and 2% sucrose. After two years of in vitro propagation and conservation, the regenerating plantlets were successfully transplanted into the field, and none showed morphological variations after one year of field growth. The regenerated rhizomes were assessed for drug-yielding potential through evaluation of (-)-epicatechin contents by HPLC, and then compared with those grown conventionally. The genetic basis of the stable drug yielding potential of the micro-propagated perennial buckwheat was revealed by random amplified polymorphic DNA-based profiling. Key words: Fagopyrum dibotrys, Shoot organogenesis, (-)-Epicatechin content, High performance Liquid Chromatography (HPLC), RAPD analysis.

Effect of phosphate deficiency on the content and biosynthesis of anthocyanins and the expression of related genes in suspension-cultured grape (Vitis sp.) cells

The effect of phosphate (Pi) starvation on anthocyanin biosynthesis was examined in suspension-cultured grape cells derived from Bailey Alicante A grapes. Cyanindin-3-O-glucoside, peonidin-3-O-glucoside, cyanidin-3-O-(6-O״-p-coumaroyl)-glucoside and peonidin-3-O-(6-O״-p-coumaroyl)-glucoside were detected in cells in both the complete (+Pi)- and Pi-deficient Murashige-Skoog (-Pi) medium but accumulation was much more substantial cells in-Pi cultures. Highest incorporation of [ring-(14)C]phenylalanine into anthocyanins was found at days 3-14 in the cells in-Pi medium. Time-course expression profiles of anthocyanin biosynthetic enzyme genes and a regulatory gene were examined after cells were transferred to the+Pi and-Pi medium. The transcript levels of UFGT and VvmybA1 in-Pi cells were higher than in+Pi cells 24h after transfer coinciding with the start of anthocyanin accumulation in-Pi cells.

In vitro selection of salt-tolerant Ailanthus altissimaSwingle

Salt-tolerant cell lines of Ailanthus altissima were selected from callus derived protoplasts. Murashige–Skoog (MS) liquid medium incorporated with various concentrations of NaCl was employed to enrich salt-tolerant A. altissima cell lines. Salt-resistant A. altissima cells were transferred on MS solid medium supplemented with 2.5 μM 2,4-dichlorophenoxy acetic acid (2,4-D), 0.5 μM benzyl adenine (BA) and various NaCl concentrations. The callus was cultured on MS medium containing NaCl for 5 months, to determine the survival rate as an index of salt tolerance. The measurement of growth parameters for salt-tolerant cells showed that the selected plant cell lines grew better than the unselected ones at all levels of NaCl tested. The salt-tolerant callus accumulated proline in correlation to the concentration of salts. Media supplemented with BA induced shoot differentiation of salt-resistant A. altissima cells.

Antioxidative and antitumor properties of in vitro-cultivated broccoli (Brassica oleracea var. italica)

Context: Broccoli [Brassica oleracea L. var. italica Plenck. (Brassicaceae)] contains substantial quantities of bioactive compounds, which are good free radical scavengers and thus might have strong antitumor properties. Enhancing production of plant secondary metabolites could be obtained with phytohormones that have significant effects on the metabolism of secondary metabolites. In that manner, in vitro culture presents good model for manipulation with plant tissues in order to affect secondary metabolite production and thus enhance bioactive properties of plants.Objective: Estimation of the antioxidative and antitumor properties of broccoli cultivated in different in vitro conditions.Materials and methods: In vitro germinated and cultivated broccoli seedlings, as well as spontaneously developed calli, were subjected to Soxhlet extraction. Antioxidative activity of the herbal extracts was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical method. Antitumor properties of the extracts were determined using crown-gall tumor inhibition (potato disc) assay.Results: Three, 10, 20, and 30 days old broccoli seedlings, cultivated in vitro on three different Murashige–Skoog media, two types of callus, and seedlings from sterile filter paper were used for extraction. In total, 15 aqueous extracts were tested for antioxidative and antitumor potential. Three day-old seedlings showed the highest antioxidative activity. Eleven out of 15 aqueous extracts demonstrated above 50% of crown-gall tumor inhibition in comparison with the control. Tumor inhibition was in association with types and concentrations of phytohormones presented in growing media.Discussion and conclusions: It is demonstrated that phytohormones in plant-growing media could affect the bioactive properties of broccoli either through increasing or decreasing their antioxidative and antitumor potential.

In vitro organogenesis and plant regeneration from leaves of Actinidia eriantha Benth. cv White (kiwifruit)

Plantlets were regenerated from leaf explants of Actinidia eriantha Benth. cv White (kiwifruit). The best medium for adventitious shoot formation from leaf blade strips was Murashige-Skoog (MS) medium with 0.1 mg L−1 NAA and 2.0 mg L−1 BA. The frequency of shoot regeneration (%) was dependent on the size and the part of the leaf from which the explant was taken. The highest frequency of shoot regeneration was 7.30±2.61 shoots per explant, for explants taken from within 0–3 cm of the leaf blade base, near a vein and not on the leaf edge. When using petiole-derived explants, a maximum frequency of adventitious shoot regeneration of 79.17% with 6.54±2.31 shoots per explant was obtained using 0.1 mg L−1 NAA and 2.0 mg L−1 ZT in MS medium. The highest rate of rooting obtained was 96.67% when using 1/2 MS medium containing 0.6 mg L−1 IBA and 93.75% when using wet moss without IBA. This is the first report to study the effect of explants taken from different parts of the leaf blade on shoot regeneration in A. eriantha. All plantlets were transferred to pots of peat/perlite and grown on before being planted in the field. Plantlets were successfully acclimatized to field conditions and produced healthy plants.

Production of lignans in calluses of Schisandra chinensis

Calluses were induced from leaves of Schisandra chinensis Baillon (Schisandraceae). Murashige-Skoog (MS) and Woody Plant (WP) media were used for the induction, in full and half strength (1/2 MS or 1/2 WP) salt formulations. Test media were solidified with 0.25% gelrite and supplemented with 2% sucrose and various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), 3-indolebutyric acid (IBA), and 6-benzylaminopurine (BAP). Optimal conditions for callus induction and growth were found to be 1/2 MS medium containing 0.02 mg/l Kin and 0.2 mg/l 2,4-D. Chloroform extracts of all induced calluses contained gomisin A and F as major components. Gomisin A and F contents of calluses that were cultured under the optimal conditions mentioned above were highest compared to the calluses incubated with other combinations of plant hormones and media. Subculture, by repeated transfer of cultured calluses to fresh medium, caused no decrease in the production of gomisin A and F. Optimal conditions for lignan production were found to be 1/2 MS medium supplemented with 0.05 mg/1 Kin and 0.2 mg/l 2,4-D. Under these conditions, gomisin A and gomisin F contents were 0.05 and 0.04% of callus dry weight, respectively.

In vitro cultures of Drosera aliciae as a source of a cytotoxic naphthoquinone: ramentaceone

A protocol for the in vitro propagation of Drosera aliciae to increase the yield of the naphthoquinone, ramentaceone, was developed. The highest micropropagation coefficient was obtained using half-strength Murashige-Skoog medium supplemented with 0.4 μM 6-benzyladenine (BA). The genetic fidelity and stability of the regenerated plants was confirmed with RAPD markers. The activity of the isolated ramentaceone was determined against four human tumor cell lines: U937, HeLa, MCF-7, HCT-116 with the highest cytotoxic activity towards the leukemic U937 cell line with an IC(50) value of 3.2 μM.

Fontes de silício no desenvolvimento de plântulas de bananeira 'Maçã' micropropagadas

O objetivo deste trabalho foi avaliar o efeito de diferentes fontes de silício durante a fase de enraizamento in vitro de bananeira 'Maçã' por meio de análise fisiológica, fitotécnica e ultraestrutural. Foram utilizados propágulos já estabelecidos in vitro e inoculados em meio MURASHIGE & SKOOG (MS), adicionado de 30g L-1 de sacarose, 1mg L-1 de ANA (ácido naftalenoacético) e solidificado com 1,8g L-1 de PhytagelTM. Foram testadas três fontes de silicato acrescidas ao meio MS, silicato de sódio, silicato de potássio e silicato de cálcio, na dosagem de 1g L-1 e o meio MS sem silicato como testemunha. O delineamento experimental foi o inteiramente casualizado, com quinze repetições. Os propágulos foram mantidos por 45 dias em sala de crescimento, sob condições controladas. Verificou-se aumento nos teores de clorofila a, b e total na presença de silicato de cálcio. A suplementação do meio de cultura com silicato de sódio promoveu aumento de comprimento, massa fresca e seca de parte aérea. O silício proporciona adequado desenvolvimento das plântulas.

Polygala paniculata: um recurso de salicilato de metila produzido por cultura de tecidos vegetais

O estabelecimento de cultura in vitro de Polygala paniculata L. permitiu a propagação rápida de matéria-prima vegetal sob condições assépticas e padronizadas. As plantas in vitro foram submetidas à extração e destilação simultâneas (EDS) e a composição química do óleo essencial foi analisada por CG/DIC e CG/EM. Foram avaliados os efeitos de BAP (6-benzilaminopurina) sobre o desenvolvimento in vitro e produção de voláteis em P. paniculata. A principal característica do óleo essencial consistiu na presença marcante de salicilato de metila. Plantas cultivadas em meio controle MS (Murashige & Skoog) produziram mais do que 80% de salicilato, enquanto o uso de BAP, reduziu a produção de salicilato de metila a 71% e 21% para as concentrações de 2 e 4 mg L-1, respectivamente. A cultura de tecidos de P. paniculata pode ser indicada como fonte de salicilato de metila.

The elimination of Plum pox virus in plum cv. Bluefree and apricot cv. Hanita by chemotherapy of in vitro cultures

In vitro cultures of plum cv. Bluefree and apricot cv. Hanita infected with Plum pox virus (PPV) were used for the virus elimination by chemotherapy. Low ribavirin concentrations of 5 and 10 mg/l in Murashige-Skoog medium were applied in the treatment. Plum pox virus was completely eliminated by 5 mg/l of ribavirin in plum cv. Bluefree within twenty weeks and in apricot cv. Hanita in twelve weeks of the application. Plum pox virus was completely eliminated by 10 mg/l of ribavirin both in plum cv. Bluefree and apricot cv. Hanita within twelve weeks. The presence of PPV was not proved by RT-PCR. Clones of plum cv. Bluefree and apricot cv. Hanita were re-tested by RT-PCR one year after the termination of the ribavirin treatment and negative results confirmed the elimination of Plum pox virus.

In vitro cultures and regeneration of Bienertia sinuspersici (Chenopodiaceae) under increasing concentrations of sodium chloride and carbon dioxide

To study the developmental transition of chloroplasts from C(3) to C(4) photosynthesis in the terrestrial single-cell C(4) species Bienertia sinuspersici, a regeneration protocol was developed. Stem explant material developed callus either with or without red nodular structures (RNS) when cultured on Murashige-Skoog (MS) salts and vitamins, supplemented with 5mM phosphate, plus 1mgL(-1) dichloropenoxy-acetic acid (2,4-D), and 87mM sucrose (Stage 1 media). Only calli having RNS were able to regenerate plantlets. MS media plus phosphate was used throughout regeneration, with the Stage 2 media containing 2mgL(-1) 6-benzylaminopurine, 43mM sucrose and 1.5% soluble starch. Stage 3 media had no hormones or organic sources of carbon, and cultures were grown under ambient (~400ppm) versus CO(2) enrichment (1.2% CO(2)). When calli without RNS were cultured under Stage 3 conditions with 1.2% CO(2), there was an increase in growth, protein content, and photosystem II yield, while structural and biochemical analyses indicated the cells in the calli had C(3) type photosynthesis. CO(2) enrichment during growth of RNS during Stage 3 had a large effect on regeneration success, increasing efficiency of shoot and root development, size of plantlets, leaf soluble protein, and chlorophyll concentration. Anatomical analysis of plantlets, which developed under 1.2% CO(2), showed leaves developed C(4) type chlorenchyma cells, including expression of key C(4) biochemical enzymes. Increasing salinity in the media, from 0 to 200mM NaCl, increased tissue osmolality, average plantlet area and regeneration success, but did not affect protein or chlorophyll content.

Germinação de embriões zigóticos e desenvolvimento in vitro de coquinho-azedo

Este trabalho foi desenvolvido com objetivo de avaliar os efeitos da luz, das concentrações de sais MS e de sacarose sobre a germinação, in vitro, de embriões, e o desenvolvimento de plântulas de coquinho-azedo (Butia capitata (Mart.) Becc.). Para o preparo do meio de cultivo, foram utilizados sais MS suplementados com vitaminas, mio-inositol, caseína hidrolizada, carvão ativado, sacarose e ágar. Para avaliação do efeito da luminosidade, embriões zigóticos foram cultivados na presença e na ausência de luz. Diferentes concentrações de minerais MS (0; 25; 50; 75 e 100% em relação à formulação original do meio MS) foram testadas em associação com duas concentrações de sacarose ( de0 a 2%). Em ambos os experimentos, avaliaram-se, após 30 dias da inoculação, percentuais de oxidação, germinação e emissão de raízes e bainhas foliares. Os resultados obtidos mostraram que condição de luminosidade não afetou a germinação, porém, a ausência de luz favoreceu a emissão de raízes. Ocorreu o alongamento na ausência de sacarose, indicando a existência de reservas energéticas no embrião. A adição de sacarose proporcionou menores níveis de oxidação, favoreceu o alongamento e mostrou-se imprescindível para o desenvolvimento inicial das plântulas. Concentrações de sais entre 50 e 75% da concentração original do meio MS proporcionaram menores níveis de oxidação. A concentração de 75 % da concentração original de sais do meio MS proporcionou maior enraizamento.