Axenic Tissue Cultures Research Articles

Establishing an inexpensive, space efficient colony of Bemisia tabaci MEAM1 utilizing modelling and feedback control principles.

A stable, synchronized colony of whitefly (Bemisia tabaci MEAM1 Gennadius) was established in a single ~30 cu.ft. reach‐in incubator and supported on cabbage host plants which were grown in a 2 × 2′ mesh cage without the need for a greenhouse or dedicated growth rooms. The colony maintenance, including cage cleaning and rotation of plants, was reduced to less than 10 h per week and executed by minimally experienced researchers. In our hands, this method was approximately 10‐fold less expensive in personnel and materials than current typical implementations. A predator‐prey model of whitefly colony maintenance that included whitefly proliferation and host plant health was developed to better understand and avoid colony collapse. This quantitative model can be applied to inform decisions such as inoculum planning and is a mathematical framework to assess insect control strategies. Extensive measurements of colony input and output (such as image analysis of leaf area and whitefly population size) were performed to define basic ‘feedback control’ parameters to gain reproducibility of this inherently unstable scaled‐down whitefly colony. Quantitative transfer of ~100 whiteflies repeatedly produced more than 5000 adult whiteflies over a 6‐week, two‐generation period. Larger scale experimentation could be easily accommodated by transferring adult whiteflies from the maintenance colony with a low flow vacuum capture device. This approach to colony maintenance would be useful to programs that lack extensive plant growth room or greenhouse access and require a “clean” implementation that will not contaminate an axenic tissue culture laboratory.

Hydroponic cultivation conditions allowing the reproducible investigation of poplar root suberization and water transport

BackgroundWith increasing joint research cooperation on national and international levels, there is a high need for harmonized and reproducible cultivation conditions and experimental protocols in order to ensure the best comparability and reliability of acquired data. As a result, not only comparisons of findings of different laboratories working with the same species but also of entirely different species would be facilitated. As Populus is becoming an increasingly important genus in modern science and agroforestry, the integration of findings with previously gained knowledge of other crop species is of high significance.ResultsTo ease and ensure the comparability of investigations of root suberization and water transport, on a high degree of methodological reproducibility, we set up a hydroponics-based experimental pipeline. This includes plant cultivation, root histochemistry, analytical investigation, and root water transport measurement. A 5-week-long hydroponic cultivation period including an optional final week of stress application resulted in a highly consistent poplar root development. The poplar roots were of conical geometry and exhibited a typical Casparian band development with subsequent continuously increasing suberization of the endodermis. Poplar root suberin was composed of the most frequently described suberin substance classes, but also high amounts of benzoic acid derivatives could be identified. Root transport physiology experiments revealed that poplar roots in this developmental stage have a two- to tenfold higher hydrostatic than osmotic hydraulic conductivity. Lastly, the hydroponic cultivation allowed the application of gradually defined osmotic stress conditions illustrating the precise adjustability of hydroponic experiments as well as the previously reported sensitivity of poplar plants to water deficits.ConclusionsBy maintaining a high degree of harmonization, we were able to compare our results to previously published data on root suberization and water transport of barley and other crop species. Regarding hydroponic poplar cultivation, we enabled high reliability, reproducibility, and comparability for future experiments. In contrast to abiotic stress conditions applied during axenic tissue culture cultivation, this experimental pipeline offers great advantages including the growth of roots in the dark, easy access to root systems before, during, and after stress conditions, and the more accurate definition of the developmental stages of the roots.

Area from image analyses accurately estimates dry-weight biomass of juvenile tissue from the moss Ceratodon purpureus.

PREMISEMosses have long served as models for studying many areas of plant biology. Investigators have used two‐dimensional measurements of juvenile growth from photographs as a surrogate for dry‐weight biomass. The relationship between area and biomass, however, has not been critically evaluated.METHODSHere we grew axenic tissue cultures of 10 Ceratodon purpureus isolates to study the relationship between these parameters. We measured area and biomass on replicate cultures with two distinct starting inoculum sizes each week for three weeks. We then examined the correlation between area and biomass as well as the influence of variation in inoculum size on both parameters.RESULTSWe found a strong correlation between area and biomass after two weeks of growth. Furthermore, we found inoculum size affected biomass during the first week of growth but not in subsequent weeks and inoculum size had no detectable effect on area.DISCUSSIONThese analyses provide experimental confirmation that area is a suitable proxy for biomass and provide clear guidelines for when inoculum size variation may affect downstream growth estimates.

Huperzine A and Huperzine B Production by Prothallus Cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart.

This is the first report of an efficient and effective procedure to optimize the biosynthesis of huperzine A (HupA) and huperzine B (HupB) in vitro from Huperzia selago gametophytes. Axenic tissue cultures were established using spores collected from the sporophytes growing in the wild. The prothalia were obtained after 7–18 months. Approximately 90 up to 100% of the gametophytes were viable and grew rapidly after each transfer on to a fresh medium every 3 months. The best biomass growth index for prothallus calculated on a fresh (FW) and dry weight (DW) basis, at 24 weeks of culture, was 2500% (FW) and 2200% (DW), respectively. The huperzine A content in the gametophytes was very high and ranged from 0.74 mg/g to 4.73 mg/g DW. The highest yield HupA biosynthesis at >4 mg/g DW was observed on W/S medium without growth regulators at 8 to 24 weeks of culture. The highest HupB content ranged from 0.10 mg/g to 0.52 mg/g DW and was obtained on the same medium. The results demonstrate the superiority of H. selago gametophyte cultures, with the level of HupA biosynthesis approximately 42% higher compared to sporophyte cultures and 35-fold higher than when the alkaloid was isolated from H. serrata, its current source for the pharmaceutical industry. Moreover, the biosynthesis of HupB was several-fold more efficient than in H. selago sporophytes growing in the wild. HPLC-HR-MS analyses of the extracts identified eight new alkaloids previously unreported in H. selago: deacetylfawcettine, fawcettimine, 16-hydroxyhuperzine B, deacetyllycoclavine, annopodine, lycopecurine, des-N-methylfastigiatine and flabelline.

Evidence for Methylerythritol Pathway (MEP) Contributions to Zerumbone Biosynthesis as Revealed by Expression Analysis of Regulatory Genes and Metabolic Inhibitors Studies

Sesquiterpenoid Zerumbone, the principal secondary metabolite in Zingiber zerumbet Smith, has been identified as the putative molecule conferring resistance against soft-rot causative, Pythium myriotylum. Metabolic precursors for sesquiterpenoid biosynthesis namely, isopentenyl diphosphate (IPP) and DMAPP, are generated either from cytosolic mevalonic acid (MVA) and/or the plastidal methylerythritol phosphate (MEP) pathway. Evaluation of expression pattern of regulatory genes of MEP and MVA pathway following P. myriotylum infection revealed that while transcripts of MVA regulatory gene, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), did not show significant changes, biphasic expression pattern was observed for MEP pathway regulatory genes, viz., 1-deoxy-D-xylulose-5-phosphate reductase (DXR) and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) as well for ZzTPS (terpene synthase) and Farnesyl phosphate synthase (FPS) compared with uninfected control. Contribution of the two pathways in zerumbone biosynthesis was evaluated in axenic tissue culture raised Z. zerumbet plantlets using MVA and MEP pathway specific inhibitors, mevinolin (MEV) and fosmidomycin (FOS) respectively. Results generated by regulatory gene and metabolite analysis are informative with respect to the role of plastidial IPP pool generated via MEP pathway in zerumbone biosynthesis and the possible redirection of flux in the event of metabolic perturbations mediated by chemical inhibitors.

PENGARUH TEKNIK STERILISASI EXPLAN TERHADAP TINGKAT PEROLEHAN KULTUR JARINGAN AKSENIK RAMIN (Gonystylus bancanus)

Since 2005, ramin has been included in CITES Appendix II and according to IUCN red list, ramin is also regarded as vulnerable (VU A1cd). The Lack of information on explant sterilization techniques in ramin tissue culture is one reason why there has not been enough initiation to develop ramin micro propagation. Plant tissue culture contamination has economic impact due to its direct influence in losses during in vitro culture of plants. The aim of this study was to observe the influence of explants sterilisation technique on acquisition rate of ramin axenic tissue culture with the rate of contamination increase, acsenic culture acquisition and shoot elongation as parameters. Ramin seedlings as explants source were collected from Tumbang Nusa, Central Kalimantan. Fifty replicating explants of one nodule each in 3 techniques were used in this study based on in vitro incubation time: sterilization 1 (24 hours incubation), sterilization 2 (48 hours incubation) and sterilization 3 (72 hours incubation) with non metallic antimicrobial compounds namely detergent, ditiocarbamate with mankozeb active ingredient (compound A), a compound containing 5.25% NaOCl added sodium hypochlorite and hydrogen oxide (compound B), 70% alcohol (compound C) as explants surface sterilants. Murashige-Skoog was used as media for evaluation of sterilization technique and explants regeneration on one year incubation. The results of this study indicate that the lowest rate of contamination was found in sterilization 3 (28%) with the success number of axenic tissue culture at 46%. The average shoot elongation of the axenic culture was 5.91 cm after 12 months subcultured in every month.

Improved axenic hydroponic whole plant propagation for rapid production of roots as transformation target tissue

BackgroundPlant roots are used as an efficient target tissue for plant transformation assays. In root propagable species transformed roots are able to regenerate into whole plants without the addition of exogenous hormones, thus avoiding somaclonal variation associated with many plant transformation protocols. Plants grown in soil or soilless solid medium have roots that tend to be extremely delicate and are difficult to sterilize in advance of plant transformation experiments. Axenic tissue culture plants grown on semi-solid media are slow to produce large amounts of biomass compared to plants grown in solution-based media.MethodsSeeds were germinated and grown for 14 days on half-strength semi-solid Murashige and Skoog medium containing 1% sucrose. Seedlings were then transferred to Magenta™ GA7 vessels containing either liquid or semi-solid ½ MS medium with 0.25, 0.5, 1, 2 or 3% sucrose. In the hydroponics (liquid medium) treatments, expanded clay balls were used to anchor seedlings. Hydroponic vessels were fitted with a sterile air aeration hose and filled ¾ full (100 mL) with liquid ½ MS media. Liquid media were replaced after 7 days. All plants were grown under fluorescent lights for 14 days.ResultsWe have developed an improved axenic hydroponic propagation system for producing large quantities of plant roots for use in transformation assays using Taraxacum kok-saghyz as a model for root propagable species. Plants grew significantly faster in liquid media than on solid media. Addition of sucrose from 0.25 to 2% was correlated with an increase in biomass accumulation in plants grown in liquid media.ConclusionsOur improved axenic hydroponic method yields sufficient quantities of roots for extensive plant transformation/molecular studies.

Tissue culture production of hazelnut – disinfestation and impact of agar content

Australian hazelnuts are a new and emerging industry with the potential annual value in Tasmania alone estimated to be in the order of $ 10-15 million. Currently tree propagation is a slow and inefficient process with suckers collected from mature trees and grown on in nurseries. This parent material may harbour diseases and pests and surviving progeny may have inconsistent growth rates, undesirable suckering and non-uniform growth habits, making orchard establishment challenging. The development of tissue culture technologies for hazels has been pioneered in Oregon, USA, resulting in new cultivars and improved planting stock underpinning successful industry establishment in the USA. There has been little success with tissue culture production of local cultivars in Australia. This project examined strategies to optimise the transfer of glasshouse and field grown cuttings (explants) into axenic tissue culture, by testing different disinfestation treatments with sodium hypochlorite (NaOCl). Further, once in tissue culture, we tested the impact of varying agar content in media on key growth parameters. Outcomes indicated fungal and bacterial contamination from field-sourced explants was greater than those from glasshousesourced and that a greater NaOCl concentration (2.1% compared to 1.05%) was required to obtain axenic tissue cultures. Also, low agar content (0.2-0.4%) in tissue culture media produced improved hazel explant growth with greater shoot fresh weights and node numbers than with high agar content. These findings may help underpin the development of micro-propagation as a useful source of quality planting material for the hazelnut industry in Australia.

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

SUMMARY The effects of auxins and cytokinin on callus formation, growth and regeneration of Gracilaria tenuistipitata Chang et Xia and G. perplexa Byrne et Zuccarello (Gracilariales, Rhodophyta) are reported. Plant growth regulators (PGR) in concentrations ranging from 0.1 to 100.0 μmol of indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (K) were added to the ASP 12-NTA solid medium (0.7% agar), and apical and intercalary segments (5 mm long) were inoculated as initial explants. K stimulated growth rates of intercalary segments of G. tenuistipitata in a linear relation, and 2,4-D (1.0 μmol) and K (10.0 μmol) stimulated growth rates of apical and intercalary segments of G. perplexa, respectively. The simultaneous formation of apical, basal, and intermediate calluses is reported for the first time in axenic tissue cultures of red algae. With intercalary segments of G. tenuistipitata, basal callus induction rates were higher than those of apical and intermediate calluses in the majority of treatments, and auxins had stimulatory effects on the formation of all callus types. In apical segments of G. perplexa, intermediate callus formation was stimulated only by treatment with 1.0 μmol of K, while apical callus formation was stimulated by indole-3-acetic acid (1.0–10.0 μmol), 2,4-D (10.0–100.0 μmol), or K (0.1 μmol). Intercalary segments of G. perplexa developed only intermediate calluses, and the majority of treatments with PGR stimulated higher rates than those presented by apical segments. Potential for regeneration (development of adventitious plantlets originated from callus cells) was higher in apical calluses than in basal and intermediate calluses developed in intercalary segments of G. tenuistipitata. Moreover, auxins and cytokinin were essential to the induction of regeneration in intermediate calluses, while specific concentrations stimulated regeneration from basal and apical calluses. Plant regeneration in G. perplexa was observed only after transferring calluses from solid to liquid medium, and the majority of treatments with PGR had stimulatory effects. Regenerating plants of G. perplexa developed tetrasporangia, and released tetraspores giving rise to adult gametophytes. Our results indicate that auxins and cytokinin have a regulatory role in the growth and morphogenesis in G. tenuistipitata and G. perplexa, and diversity of responses presented by both species is related to specific developmental systems.

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

Effects of plant growth regulators and culture medium on morphogenesis of Solieria filiformis (Rhodophyta) cultured in vitro

Morphogenesis and plant regeneration were analyzed in axenic tissueculture of the red alga Solieria filiformis (Kutzing)Gabrielson. Thallus segments cultured in ASP 12-NTA synthetic medium showedgrowth of filaments formed by divisions of cortical, subcortical and medullarycells (filamentous explants), whereas in seawater enriched with Von Stosch'ssolution, thallus segments developed branches. Filamentous explants were abletoregenerate plants when transferred from a solid to a liquid medium. Plantregeneration was significantly promoted by treatment with plant growthregulators on filamentous explants formed from intercalary segments, up to 67plantlets per explant in treatments with 6-benzylaminopurine (5.0 mgl−1), in contrast to three plantlets in controlslackingplant growth regulators. These adventitious plantlets developed into plantsmorphologically similar to those originated from germinating spores. Theseresults indicate that plant growth regulators play a role on the regulation ofmorphogenesis, and could be useful for micropropagation of colloid-producingredalgae.

Sterilization of Plants for Phytoremediation Studies by Bleach Treatment

ABSTRACT Bleach treatment of plants was studied as a simple alternative to axenic tissue cultures for demonstrating phytodegradation of aqueous and gas-phase environmental contaminants. Parrotfeather (Myriophyllum aquaticum), spinach (Spinacia oleracea), and wheat (Triticum aestivum) were exposed to 0.525% NaC10 solutions for 15 s, then rinsed in deionized water. Plate counts indicated that 97 to 100% of viable bacteria were removed from parrotfeather and spinach. Transformation rates for 2,4,6-trinitrotoluene (TNT) by bleached and untreated parrotfeather were virtually identical. Similarly, treated and untreated spinach, wheat heads, and wheat leaves removed methyl bromide (MeBr) from air at the same rates. However, wheat root with attendant adhering soil was rendered inactive by bleach treatment. Parrotfeather roots examined by dissecting microscope and by electron microscope showed no significant damage caused by bleach treatment.

AXENIC TISSUE CULTURE AND MORPHOGENESIS IN PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)

To better understand developmental phenomena in macroalgal tissue culture, we examined the morphogenesis of Porphyra yezoensis Ueda (strain TU‐1) cultured aseptically in defined synthetic media. Generally, the filamentous thalli (sporophyte; conchocelis phase) of P. yezoensis were densely tufted with uniseriate filaments. The foliose thalli (gametophyte) were monolayered. In this study, axenic filamentous thalli retained their characteristic morphogenesis; there were no obvious differences between morphogenetic traits in unialgal and axenic conditions. However, conchospores, which might have developed into the foliose form under unialgal conditions, germinated into calluslike masses under axenic conditions. Most of the gametophytes gradually lost their typical morphogenesis after the first longitudinal cell division. Some of the calluslike masses developed rhizoidlike structures in several places or along the entire mass. Therefore, we concluded that P. yezoensis, in axenic cultures, loses its typical morphogenesis only during the gametophytic phase. The axenic tissue culture of Porphyra established in this study is a promising assay system for the identification of growth and morphogenetic factors.

In vitro studies of the submerged angiospermRuppia maritima: Auxin and cytokinin effects on plant growth and development

Axenic tissue cultures ofRuppia maritima L. were established and propagated clonally in vitro from terminal rhizome segments collected from Tampa Bay, Florida, USA. Cultures were maintained in a base medium consisting of synthetic seawater supplemented with half-strength Murashige and Skoog salts and 1% sucrose at pH 5.6. The effects of five cytokins [6-furfurylaminopurine (kinetin), 6-benzylaminopurine (BAP), 2-isopentyladenine (2iP), 6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin), andn-phenyl-n′-1,2,3-thidiazol-5′yl urea (thidiazuron)] and one auxin [napthalene acetic acid (NAA)] on explant growth and development were investigated. Cytokinin additions resulted in a 3- to 4-fold increase in nodal production, branching, and biomass ofR. maritima after 12 wk in culture. Cultures responded in a dose-dependent manner to 2iP but exhibited broad dose-response curves to kinetin, BAP, zeatin, and thidiazuron. NAA addition resulted in increased leaf and internodal lengths, but reduced the number of leaves per node and the rhizome biomass. The addition of NAA almost completely suppressed root growth in media without cytokinins and had an antagonistic effect on nodal production and branching in cytokinin-supplemented media.

Axenic tissue culture and callus formation of the marine brown alga Laminaria angustata.

A method for axenic tissue culture and callus formation of the marine brown alga Laminaria angustata were established for the first time. Axenic medullary tissues were obtained from the stipes of the sporoplytes by the punching method using cork borer. Callus tissues have been induced from the medullary tissues on ASP 12-NTA medium solidified with 1.0% agar. These cultures were maintained in an incubator at 14°C and illuminated with cool white fluorescent lamps at ca. 2000 1x with 14 hr light and 10 hr dark cycle. This method for tissue culture will contribute to advances in breeding and cultivation of Laminaria plants.

Germination of Spores of Cantharellus Cibarius

Cantharellus cibarius Fr., one of the most well-known, edible, wild mushrooms of Europe and North America, is generally considered refractory to cultivation under sterile conditions. While some, even experienced, mycologists have failed in their efforts for axenic tissue culture (4, 5), others have been more successful (1, 6, 7, 9, 10). However, to the author's knowledge spore germination has never been observed in' this fungus. By means of a combination of activated charcoal and growing colonies of Rhodotorula glutinis (Fres.) Harrison spore germination in vitro could be induced in Laccaria laccata (Scop. ex Fr.) Berk. et Br. (2), as well as in a few other mycorrhizaforming hymenomycetes (3). The present paper reports the application of this method to the germination of Cantharellus cibarius. Basidiospores of C. cibarius and two other, related species were obtained under sterile conditions from fruit-bodies collected in the neighborhood of Uppsala, during the fall of 1977. They were kept in Petri dishes in darkness at 4 C. The germination tests were made on plates of an autoclave-sterilized agar medium, the spores suspended in sterile, distilled water, and the suspension then spread out over the agar surface, ca. 50,000 spores per plate, by means of a glass rod. The composition of the medium was the same as that earlier described (3), where also some more technical details are given. In certain experimental series activated charcoal and/or living Rhodotorula glutinis cells were added to the plates as shown in FIG. 1. The plates were incubated in darkness at 25 C and inspected for possible germinations once a wk. Just as in the experiments with Laccaria laccata (2), the spores of C. cibarius germinated only in the plates containing both activated charcoal and a Rhodotorula colony. The spores had been kept for only 2 da after deposit. The germination process commenced by the spore

Procedures and media for obtaining tissue cultures of 12 conifer species

Procedures for obtaining axenic tissue cultures of Abies grandis, Larix occidentalis, Picea abies, Picea engelmanni, Picea pungens, Pinus albicaulis, Pinus contorta, Pinus flexilis, Pinus nigra, Pinus ponderosa, Pseudotsuga menziesii, and Thuja plicata are described. All species were cultured on a basal medium containing calcium nitrate, magnesium sulphate, potassium phosphate (monobasic), ammonium sulfate, ferric sulfate, manganese sulfate, glucose, 0.8% Bacto agar, and one of three auxins (indoleacetic acid (IAA), naphthaleneacetic acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D)) at various concentrations. Growth of most species was further enhanced by the addition of several vitamins and amino acids. These species were relatively specific in their auxin requirements. The type of sterilant (H2O2, NaOCl) and manipulation required in the preparatory procedures were also specific for individual species.

TISSUE CULTURE OF PINUS MONTICOLA ON A CHEMICALLY DEFINED MEDIUM

Procedures for obtaining and maintaining axenic tissue cultures of Pinus monticola are described. Western white pine tissue was cultured on a chemically defined medium containing calcium nitrate, magnesium sulfate, potassium phosphate (monobasic), ammonium sulfate, ferric sulfate, manganese sulfate, glucose, and one of three auxins, IAA, NAA, or 2,4-D. Addition of several B vitamins, amino acids, and kinetin increased growth on these media but were not required.