Murashige-Skoog Basal Medium Research Articles

Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2mg/L and kinetin 2mg/L; for shoot initiation 6-benzylaminopurine 2mg/L, kinetin 0.2mg/L, and indole-3-acetic acid 0.2mg/L; for shoot multiplication 6-benzylaminopurine 3mg/L and indole-3-acetic acid 0.2mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

The impact of Ceftriaxone on Relative Copy Numbers of Mitochondrial and Chloroplast DNA in Grapevine Leaves

The cultivation of micropropagated grapevine plants in vitro is currently undergoing improvements in existing methods and the development of new cultivation techniques due to the problem of bacterial contamination caused by multidrug-resistant microorganisms. Explants (Vitis vinifera L., ‘Chardonnay’ variety) were cultured on Murashige-Skoog basal medium supplemented with different concentrations of ceftriaxone: 0 mg/L, 250 mg/L, and 1000 mg/L. After 30 days, morphometric characteristics of the micropropagated plants and relative copy numbers of mitochondrial and chloroplast DNA were evaluated. Leaf samples (5-10 mg) were randomly selected from each plant group for subsequent total DNA extraction. Quantitative RT-PCR was performed using LightCycler 480 SYBR Green I Master Mix (LifeScience, Roche) and analyzed with a LightCycler 96 automated analyzer (Roche Life Science). The relative copy numbers of NAD1 (mitochondrial DNA) and rps16 (chloroplast DNA) genes were determined using the GAPDH gene (chromosomal DNA) as the reference. The 2-DCt and 2-DDCt algorithms were used for quantitative assessment. Ceftriaxone at concentrations of 250 and 1000 mg/l reduces the relative number of copies of chloroplast and mitochondrial DNA, which indicates the suppression of photosynthesis and oxidative phosphorylation in grape microplants. The experimental scheme developed by us can be successfully used as a test system for assessing the degree of influence of various biogenic and abiogenic factors on plant objects in order to optimize their cultivation.

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.

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.

<i>In Vitro</i> Study of Callogenesis and Regeneration Potential of Elite Wheat (<i>Triticum aestivum</i> L.) Cultivars

The potential for biotechnological applications in crop improvement programs requires identifying genotypes that allow cell/tissue culture with predictable plant regeneration. In the past, many genotypes of wheat (Triticum aestivum L.) have been examined for potential use in tissue culture studies. The present research work has also been designed to study in vitro callogenesis expression and regeneration potential of wheat cultivars under controlled laboratory conditions. Seeds of four elite commercial high yielding cultivars of wheat namely: NARC-2011, AAS-2011, PAK-2013 and GAL-2013, were collected from the Crop Science Institute National Agricultural Research Center (CSI-NARC) Islamabad, as the source of plant material for in vitro studies. The seeds were surface sterilized in 10% sodium hypochlorite solutions for 10 minutes with continuous shaking under laminar air flow hood. After that seeds were placed on MS (Murashige & Skoog, 1962) based callus induction and regeneration medium with various concentrations of 2, 4-D and BAP in separate test tubes. Maximum callus induction frequency of 90% for Pak-13 and AAS-11, followed by 87% and 83% for Gla-13 and NARC-11, respectively, was recorded at 4 mg/l and 6 mg/l of 2, 4-D. Similarly, maximum regeneration of 90% for AAS-11 and Pak-13, followed by 80% and 87% for NARC-11 and Gla-13 respectively, was recorded on MS basal medium containing 1.5 mg/l of BAP. An increasing trend in regeneration from 0.5 to 1.5 mg/l of BAP was observed but it gradually decreased with increasing concentration of BAP from 1.5 mg/l for all wheat cultivars. The callus formed under light was golden brown, dry nodule and smooth compact and less embryogenic while under dark conditions, it was white to yellowish white, dry nodule and compact and more embryogenic. Best results for callus induction and regeneration were obtained at temperature (24°C ± 1°C) for all wheat cultivars.

Callus and cell suspension culture of Chelidonium majus L

Chelidonium majus L has long history as a being useful for the treatment of many diseases in Asian and European countries. Aim of this study is to cultivate callus and cell suspension culture in vitro using plant phytohormones. The proliferative capacity was tested on shoot and root explants, cultivated on Murashige-Skoog basal medium testing two auxins: 2,4-diclorphenoxiacetic acid (2.4D) and napheteline acetic acid in combination with cytokinine: kinnetine (K). Calluses were developed on MS medium with 0.5 mg/l Kin, 0.5 mg/l IAA from root explants, as well when added with 0.5 mg/l NAA and 0.5 mg/l Kin from shoot explants. More biomass of cell suspension culture of shoot and root callus was accumulated on MS medium added with 0.1mg/l Kin. DOI: http://dx.doi.org/10.5564/mjas.v11i2.238 Mongolian Journal of Agricultural Sciences Vol.11(2) 2013 pp.150-154

In vitro culture of Hypericum rumeliacum Boiss. and production of phenolics and flavonoids

The Balkan endemic species, Hypericum rumeliacum, Guttiferae was introduced in vitro for the first time with the aim to study the type of morphogenetic response to plant growth regulators and ability to produce phenolics and flavonoid compounds. The morphoregulatory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphtaleneacetic acid (NAA), 6-benzyladenine (BA) and combination of BA with NAA in Murashige–Skoog's basal medium on leaf lamina, internode stem segment, stem node and root cuttings was studied. Histological analysis of the structures regenerated from the primary explants proved the presence of both, embryoids and meristemoids. The node explants cultivated on BA-supplemented medium were the most favourable for regeneration through meristemoids. Therefore a double-stage culture approach, allowing an effective multiplication of large quantities of plant shoots in vitro along with maintenance of the biosynthetic capacity of the culture was developed. It comprised one subculture of three-nodal stem explants derived from the stock shoot cultures on MS medium supplemented with 0.2 mg/l BA followed by subculture of the induced multiple shoots on cytokinin-free MS medium. Determination of the total phenolics and flavonoids showed that the decrease of the levels of these secondary metabolites is transitional, as the exclusionof BA from the medium resulted in an increase of their total content.

Construction of genetically modified tobacco (Nicotiana tabacum cv. Petit Havana) harboring ompH(A:3) from Pasteurella multocida (A:3)

Fowl cholera, caused by Pasteurella multocida (A:3), is a fearsome disease leading to a nonproductive influence upon poultry industry. It has been known that outer membrane protein H (OmpH) in the bacterium is a strong candidate to bring on the notorious ailment. Genetically modified (GM) tobacco (Nicotiana tabacum cv. Petit Havana) harboring ompH(A:3) was constructed to develop a plant expression system for the protein, OmpH(A:3). Some 987 bp-long (ORF with the stop codon, TAA) of the ompH(A:3) excluding the nucleotide for signal peptide, was amplified by RT-PCR with the gene specific primers and pGEM-T-ompH(A:3) as template DNA. The PCR-amplified DNA was ligated into BamHI/Sacl-cut pBI121 to obtain a recombinant plasmid, pBI121-ompH(A:3). It was then transformed into Agrobacterium tumefaciens (LBA 4404) by liquid nitrogen method to generate a recombinant clone of Agrobacterium LBA4404/pBI121-ompH(A:3). The Agrobacterium LBA4404/pBI121-ompH(A:3) was inoculated into leaf discs of tobacco (2 day old). The gene-transfected leaves were cultured on Murashige-Skoog basal medium containing kanamycin (50 mg/mL) to generate numerous calli, from which some GM tobacco plants were obtained. Transgenicity of the tobacco plant was confirmed by PCR screening along with the DNA sequencing. Also, its expression in the GM-tobacco was examined qualitatively as well as quantitatively by ELISA/Western blot. These results suggest that the genetically modified tobacco plant can be potentially used as a model system to develop plant-based vaccine against the fowl cholera.

Increased calcium availability improves potato micropropagation and microtuberization

Liquid scintillation counting (LSC) was used to screen six potato cultivars (Alpha, Bintje, Green Mountain, Kennebec, Russet Burbank, and Shepody) and two wild species(S. microdontum andS. kurtzianum) for ability to take up the tracer45Ca2+ from treatment solutions containing high (15 mM) or low (5 mM) Ca2+ levels. In vitro potato micropropagation, microtuberization, and tissue calcium content, determined by flame atomic absorption spectrophotometry (FAAS), were compared for the six cultivars when Murashige-Skoog basal medium Ca2+ level was increased from 3, to 5 or 15 mM. All aspects of growth were improved when medium Ca2+ level was 15 mM. Microtuber induction occurred earlier, leading to improved yield (19–31%), and microtuber tissue Ca2+ concentration was greater (38–226%). Cv. Bintje was the most efficient genotype at accumulating Ca2+ from treatment solutions or growth media containing high or low Ca2+ levels. It could be identified as a calcium-packer using either LSC or FAAS screening.

Micropropagation of Santolina canescens Lagasca and in vitro volatiles production by shoot explants

Shoot-tips of Santolina canescens, an aromatic species producing a novel diacetylene derivative, were in vitromultiplied on Murashige-Skoog basal medium (MS) containing different concentrations of 6-benzyladenine (BA) and kinetin (KIN). Rooting phase was performed on media supplemented with different auxins: 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA) and 1-naphthaleneacetic acid (NAA). The best axillary-bud proliferation was recorded on MS medium containing 1.33 μM BA plus 0.32 μM NAA. Plantlets rooted easily on the different tested media, but more abundant and stronger roots occurred on media containing 2.68 μM NAA. The composition of oil obtained through the cultured explants was analyzed by headspace gas chromatography (HS-GC), and santolindiacetylene (SDA) in vitro production was tested. In order to stimulate the production of this product, different concentrations of linoleic acid were supplemented to the media. The best santolindiacetylene production was obtained when the rooting media was supplemented with 0.36 μM linoleic acid.

Effect of genotype, explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l−1 2,4-D and 0.5 mg l−1 BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l−1 BA. Supplementation with 0.1 mg l−1 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l−1 indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well.

Indirect Organogenesis from the Leaf Explants of Medicinally Important Plant Curculigo orchioides Gaertn

Curculigo orchioides Gaertn is an important medicinal plant of commercial value. A protocol for indirect regeneration from the leaf explants was developed. The leaf explants on MS (Murashige & Skoog) basal medium fortified with 6-benzylaminopurine (9μM) gave rise to proliferating green callus. The calli regenerated shoots on subculturing in the MS medium with 1.1μM 6-benzylaminopurine which was the optimum concentration for multiplication. Rooting was observed in MS medium supplemented with α-napthaleneacetic acid (5.3μM), indole-3-butyric acid (1.2μM), and on MS basal medium free of plant growth regulators. The rooted plants were hardened and transferred to field with 80% survival.

In vitro induction of multiple shoots and plant regeneration inVigna

Cotyledonary nodes, excised cotyledons, and hypocotyl segments of six varieties ofVigna mungo andV. radiata have been tested for their morphogenic potential on media containing a range of hormonal combinations including benzyladenine, kinetin, thidiazuron (TDZ), and zeatin. Multiple shoots developed on cotyledonary node explants in all varieties tested on basal medium containing cytokinin. Presence of both the cotyledons, either full or half, resulted in a maximum number of shoots produced. Shoot bud regeneration was achieved via meristem formation on excised cotyledons on Murashige-skoog basal medium with B5 vitamins supplemented with TDZ. Mature plants had normal phenotypes.V. mungo var. PS1 andV. radiata var. Pusa 105 were found to be the most responsive varieties for shoot regneration. The histology ofin vitro organogenesis was studied.

Tissue culture of Aloe arborescens Miller var. natalensis Berger

AbstractWe examined the culture conditions for callus induction in the tissue of Aloe arborescens. When incubated in the dark at 25°C, for about 30 days callus was induced. The frequency of callus formation was in the order of superior stalk > middle stalk > inferior stalk. No callus was formed in the leaf. When the Murashige–Skoog (MS) basal agar medium was supplemented with 3% sucrose, 0.1–0.5 μM kinetin (a plant growth regulator) and 10–50 μM alpha‐naphthalenacetic acid, callus formation occurred at an incidence of 20–52% (mean: 39%). When the MS basal agar medium was supplemented with 3% glucose and 10% Alpha Modification of Eagle's Medium instead of 3% sucrose, callus formation was promoted, but the protein and aloin concentrations, and carboxypeptidase activity in the Aloe callus decreased more than those observed in the sucrose‐containing medium.

トマトはい軸切片の不定芽形成に及ぼすポリエチレングリコールの効果

The effect of polyethylene glycol (PEG) on adventitious bud formation on excised hypocotyl segments of tomato seedling cv. KNVF-R, an interspecific hybrid rootstock between Lycopersicon esculentum x L. hirsutum, was investigated.1. Segments excised from hypocotyls shorter than 8 cm, and incubated under light intensity exceeding 3 klx formed buds; the optimum segment length was 4 mm. About 95% of the segments formed adventitious buds in 1/2 strength Murashige-Skoog basal medium supplemented with 50 g•liter-1 PEG-6000 and 30 g•liter-1 sucrose. However, when the number of segments per flask (containing 40 ml of medium) exceeded 50, the percentage of segments which initiated buds decreased.2. Mannitol and PEG-1500 were not effective at all. Abscisic acid (ABA) at 10 mg• liter-1 stimulated bud formation.3. Segments with buds of 2 mm length rooted within 2 weeks on a basal medium (1/2 MS supplemented with 30 g•liter-1 sucrose and 8 g•liter-1 agar)All regenerated plants were established by transplanting to a vermiculite potting mixture.

Reduced nitrogen influences somatic embryo quality and plant regeneration from suspension cultures of orchardgrass

Production of somatic embryos in suspension cultures ofDactylis glomerata L. (orchardgrass) was stimulated by the addition of various compounds containing reduced nitrogen to Schenk and Hildebrandt (SH) basal medium. Equimolar concentrations of combinations of proline and either serine or threonine supported embryogenesis, whereas, these amino acids individually did not promote embryogenesis. SH medium supplemented with 6 to 25 mM ammonium ion or Murashige-Skoog basal medium also supported embryo production. Ammonium ion did not act synergistically with either proline or serine to enhance embryogenesis. Embryos produced in media containing amino acid combinations were formed singly, did not exhibit secondary embryogenesis, and had significantly higher conversion rates compared to those formed in either SH medium supplemented with ammonium ion only or in combination with amino acids.

Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium

Explants excised from strawberry (Fragaria x ananassa Duch.) plantlets were cultured in vitro for 21 days on half-strength MS (Murashige & Skoog 1962) basal liquid medium with 20gl 1 sucrose and without sugar in the vessels capped with gas permeable microporous polypropylene film. The experiments were conducted under CO 2 nonenriched (350-450/zmol mol 1 in the culture room) and CO 2 enriched (2,000/xmolmol -I during the photoperiod in the culture room) conditions with a PPF (photosynthetic photon flux) of 200/xmol m-: s -1. The CO 2 concentration in the vessels decreased to approximately 200/xmol mol -~ during the photoperiod on day 21 under CO 2 nonenriched conditions. The fresh and dry weight, net photosynthetic rate (NPR) per plantlet, NPR per g leaf fresh weight, NPR per g leaf dry weight, the number of unfolded leaves, and ion uptake of PO]-, NO3, Ca2+, Mg2+ and K + on day 21 were the greatest under photoautotrophic (no sugar in the medium) and CO 2 enriched conditions. The residual percent of PO]- was 3% on day 21 under photoautotrophic and CO 2 enriched conditions.

In vitro micropropagation of the tropical fruit tree Syzygium cuminii L.

Multiple shoots were obtained from nodal and shoot tip segments of 10 to 15-day-old seedlings of Syzygium cuminii L. on Murashige & Skoog (MS) revised medium supplemented with 6-benzyladenine (BA) (0.23–8.90 μM) singly or in combination with α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). Excised shoots were placed for root induction on MS medium containing NAA and/or IBA and then transferred to MS basal medium to form complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred into the soil.

Maturation of Soybean Somatic Embryos and the Transition to Plantlet Growth

The maturation of soybean (Glycine max L. Merr.) somatic embryos was characterized. Maturation was assayed by evaluating the ability of somatic embryos to make the transition to a plantlet through a germination-like process. Somatic embryos were organized from cotyledons of immature soybean embryos. Maturation of somatic embryos occurred on a Murashige-Skoog basal medium supplemented with activated charcoal and 0.28 molar sucrose. After 8 weeks on this medium, somatic embryos exhibited vigorous, high frequency development to plantlets. The "germination" frequency (conversion) of somatic embryos, and plantlet recovery frequency varied concurrently with maturation period. Conversion and plant recovery required no exogenous growth regulators. Desiccation of immature somatic embryos under controlled humidity regimes resulted in increased frequency of conversion of immature somatic embryos. Morphological abnormalities appeared in the somatic embryos, but few were detrimental to conversion velocity. There was little effect of genotype on conversion velocity or frequency.

Secretory production of ascorbate oxidase by cultured cells of cucumber

Peelings of cucumber ( Cucumis sativus) were induced to form callus, which in turn produced ascorbate oxidase, on Murashige-Skoog basal medium with plant hormones. Cell lines which produced high levels of ascorbate oxidase and had a high growth rate were selected. Addition of 100 times more copper, a prosthetic metal of ascorbate oxidase, than that in the basal medium resulted in a significant increase in enzyme activity. A suspension culture of the cells was also established, which was found to produce ascorbate oxidase in the medium. The secretion of the enzyme from the cells was stimulated by addition of calcium ions. Maximum cell yield and maximum levels of ascorbate oxidase were obtained at about the 20th day by shaking culture, but similar growth and enzyme activity were found at the 10th day in an air-lift type jar fermentor.