Membranes to reduce adherence of somatic embryos to the cell lift impeller of a bioreactor (original) (raw)
Related papers
Bioreactor design for propagation of somatic embryos
Six identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (“clonal or somatic seeds”). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a slow-speed stirrer that regularly changes direction of rotation to prevent “quiet” zones in the suspension in which cells can ssettle and grow. In addition, the oxygen is provided, bubble-free, through thin silicone tubing loops that are hanging loose, moving with the liquid to prevent cell growth on these tubes. We used off-the-shelf components whenever possible, to reduce the costs to a minimum, which was another aim of the construction. The result was a suite of relatively inexpensive computer-controlled bioreactors that could control temperature, oxygen, pH, stirrer speed and stirrer direction. In addition, we have provided different light spectral qualities by simple means of filtering the light. Using the present software, the parameters can be set up to alter every hour during the 24 h day/night cycle. All our cultures have improved growth in the bioreactors compared to identical cultures in Erlenmeyer flasks. The cultures used are: embryogenic cultures of carrot (Daucus carota), Norway spruce (Picea abies), birch (Betula pendula), cyclamen (Cyclamen persicum) and shoot cultures of Christmas begonia (Begonia x cheimantha). The paper also discusses recommendations for improvements of the current system for future revisions.
From cells to embryos to rooted plantlets in a mist bioreactor
Plant Cell, Tissue and Organ Culture (PCTOC), 2014
A mist bioreactor using a disposable bag as culture chamber was used to propagate carrot embryogenic cells into rooted plantlets. The best operating configuration was akin to a vertical hanging garden using 50-90 lm nylon mesh for explant attachment. Cells spray inoculated into the reactor were 51.2 % viable. Misting cycle and aeration conditions were studied and showed that under the same hourly volumetric nutrient feed and 0 VVM, embryo development in the reactor was best using a 0.3 min on/ 2.7 min off misting cycle, yielding about 23 % post heart stage embryos. Compared to 0 VVM, 3 % CO 2 enrichment improved embryo development in reactor culture. Spray inoculated cells also attached to several vertically hung poly-L-lysine coated strips and then developed in situ into embryos. Cell attachment was significantly improved when they were suspended in salt-free sucrose solution during spray inoculation. Almost 90 % of the originally attached cells remained on the nylon mesh 24 h later after spraying with B5 medium in the mist reactor. Strip grown embryos had the same post heart stage ratio but shorter overall length compared to those developed on a horizontal platform. Young plantlets developed uniformly up and down the hanging strips and did not detach after 3 weeks of culture suggesting this technology may prove useful for improving micropropagation.
Induction of cassava somatic embryogenesis in liquid medium associated to floating membrane rafts
2000
The objective of this study was to examine the effect of two culture systems, liquid medium associated to floating membranes and solid medium, both supplemented with different concentrations of 2,4-D, in the induction of somatic embryogenesis of cassava (Manihot esculenta Crantz). Only 28% of the young leaf lobes (with 9 µM 2,4-D) were induced to form organized embryogenic structures (OES) with membrane rafts, compared to 50% of the explants presenting this type of tissue in solid medium with 36 µM of 2,4-D. Despite the lower response observed in liquid medium with membrane, the amount of OES/explant in all 2,4-D concentrations was higher than solid medium. Based on the results and considering the high cost of the membrane rafts, this system was not distinctly superior than solid medium for inducing somatic embryogenesis in cassava.
Liquid Culture Systems for Plant Propagation
Acta Horticulturae, 2003
Manual handling is required during conventional micropropagation. Labour costs may represent 65-85 % of the total costs, and methods of automation can reduce costs. Scaling-up in micropropagation can increase the number of explants handled and thereby decrease the labour costs and can be achieved in several different ways: 1) Homogenisation of plant tissue in blenders rather than manual cutting; 2) Automation through use of liquid cultures and bioreactors; and 3) Robotics. This paper focuses on the use of two liquid systems: 1) Temporary immersion systems; and 2) Permanent submersion of the plant cells/tissue that requires oxygen supply (rotary shakers or bioreactors). Somatic embryos and shoot cultures can be grown in both liquid systems, embryogenesis possibly being the most suited for full automation through a synthetic seed scheme. Inclusion of an automatic cutter would facilitate almost full automation of shoot cultures as well. Both temporary immersion and bioreactor applications have been reported for several agricultural, horticultural or forest plants. So far, commercial applications have been rather limited. Temporary immersion systems range from being simple, home-made devices managing the ebb and flow of liquid medium through peristaltic pumps, to rather expensive sophisticated equipment. Bioreactors have the additional challenge of oxygen supply. The challenges of bioreactor cultures have been described in detail by Heyerdahl et al. (1995). This paper uses our own bioreactors as examples of rather sophisticated bioreactors built in Norway. In 1992, the Department of Agricultural Engineering of the Agricultural University of Norway (NLH) constructed and built six identical, computer controlled two-litre bioreactors. These were made according to specifications from the Plant Cell Laboratory and used for scaling up plant propagation in liquid cultures. We were mainly interested in somatic embryogenesis, but also shoot cultures that could be scaled up in such vessels. Our bioreactors have been used to cultivate carrot, birch, Norway spruce, cyclamen and begonia. The paper will briefly describe the different liquid systems for an overview. The NLH bioreactor system is described in more detail and some results presented.
1995
The influence of culture flask closures, i.e., cotton plugs and rubber and aluminum-foil caps, on headspace gas composition and growth of leaf petiole callus-derived GF 677 cell suspensions was comparatively tested. Oxygen concentration always remained comparable to that of the lab atmosphere and CO2 and C2H 4 levels were slightly higher when flasks were closed with cotton plugs. By contrast, the gas environment markedly changed within 72 to 96 h in culture inside rubber and aluminum-capped flasks. Under rubber caps, CO 2 increased up to 18%, with a net production of about 0.8 mmol CO 2, oxygen decreased to 6% within 72 h, and ethylene accumulated up to 9 M1 liter-1 after 96 h. When aluminum foil closures were used, C2H 4 and CO2 increased over the first 72 h, reaching concentrations of about 6 ~tl liter 1 and 7% (0.3 mmol produced), respectively, and decreasing after 192 h to 0.1 ~tl-1 and 2%, respectively. The gaseous environment markedly affected cell growth. The exponential growth period of suspensions cultured in flasks closed with cotton plugs and aluminum foil caps started after about 72 h and the stationary phase after 240 h, the cell dry weight reaching its maximum at about threefold the initial weight. Large cell aggregates were found in flasks closed with cotton plugs~ slight aggregation with aluminum closures, and no aggregates with rubber caps. However, hardly any growth, progressive browning of the suspensions, and the death of most ceils occurred in rubber-capped flasks. A type of closure allowing low gas exchange rates, like aluminum caps, and frequent subcultures thus seems most conducive to rapid growing, more homogeneous GF 677 cell suspension cultures, and the prevention of aggregates, if undesired.
Biotechnology Progress, 1999
Growth of plant cell cultures is demonstrated in an uncontrolled, simple, and inexpensive plastic-lined vessel. Sustained specific growth rates of 0.22 day -1 for Hyoscyamus muticus cell suspension cultures are achieved in a low-cost gas-sparged bioreactor configuration (6.5 L working volume, wv) which is comparable to an "optimized" 5 L wv mechanically agitated fermentor. In an effort to reduce bioreactor costs, the need for an autoclavable vessel was eliminated. Sterilization is achieved by separate autoclaving of the plastic liner and by gas-phase sterilization using ethylene oxide. The initial run sterilized with ethylene oxide displayed a long lag, apparently due to residual sterilant gas. Because ethylene oxide could eliminate costs associated with autoclave rated vessels, a quantitative basis for aeration time was developed by experimental measurements and modeling of diffusion in the polymer liner. Operational techniques to eliminate toxicity are implemented to grow 0.2 kg dry weight of plant cells in 13 days in a 40 L (28.5 L wv) air-lift bioreactor without autoclave sterilization. The biomass yields for all reactors were statistically indistinguishable from shake flask culture.
A novel method for the immobilisation and culture of plant cells
FEBS Letters, 1983
A novel technique is described for the immobilisation of plant cells. The method is simple and does not require the use of potentially toxic gels for the entrapment process. Liquid-suspended cells of two species, Capsicum frutescens Mill. and Daucus carota L. were found to invade, and were strongly retained in, polyurethane foam particles over a 21 day culture period. The viability of the immobilised cells was high (70-80% were alive after 21 days). On agitation of the loaded foam particles in fresh liquid medium, at least 95% of the cells remained immobilised after 3 or 4 days. Cell immobilisation Secondary metabolite production Cell differentiation Polyurethane foam Capsicum frutescens Mill. Daucus carota L.
Industrial Crops and Products, 2009
a b s t r a c t Commercial micropropagation of plants is enhanced with the use of liquid media cultures; however the presence of hyperhydricity is commonly observed in cultures of the succulent plant Agave tequilana Weber cultivar azul, this phenomenon persists even with the use of temporary immersion systems (TIS). Thin cell suspension layer technology is proposed to solve this problem. This technology fuses the advantages of a liquid culture made through cellular dissociation, and the use of solid medium for somatic embryogenesis expression of the species. The technology was evaluated by means of two experiments in order to know the influence of gelling agent phytagel ® , and of sucrose concentrations through interaction with three cellular suspension densities. It was clear that concentrations of phytagel at 6, 8, 10 or 12 g l −1 are not significant for embryoid expression of A. tequilana. On the other hand, sucrose at 30 and 60 g l −1 have statistically superior values than concentration of 120 g l −1 . A larger cellular density (161 × 10 3 cells ml −1 ) gave a statistical difference in number of embryoids. The advantages of thin cell suspension layer were remarkable: it encouraged complete expression of embryoids without transfer to extra media cultures, and a higher number of generated embryoid was obtained. Absence of hyperhydricity was observed in all regenerants.