Contained and high-level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor

Chloroplast transformation is a promising approach for the commercial production of recombinant proteins in plants. However, gene containment still remains an issue for the large-scale cultivation of transplastomic plants in the field. Here, we have evaluated the potential of using tobacco transplastomic cell suspensions for the fully contained production of a modified form of the green fluorescent protein (GFP+) and, a vaccine antigen, fragment C of tetanus toxin (TetC). Expression of these proteins in cell suspension cultures (and calli) was much less than in leaves, reaching 0.5%-1.5% of total soluble protein (TSP), but still produced 2.4-7.2 mg/L of liquid culture. Much better expression levels were achieved with a novel protein production platform in which transgenic cell suspension cultures were placed in a temporary immersion bioreactor in the presence of Thidiazuron to initiate shoot formation. GFP+ yield reached 660 mg/L of bioreactor (33% TSP), and TetC accumulated to about 95 mg/L (8% TSP). This new production platform, combining the rapid generation of transplastomic cell suspension cultures and the use of temporary immersion bioreactors, is a promising route for the fully contained low-cost production of recombinant proteins in chloroplasts.     

A temporary immersion plant propagation bioreactor with decoupled gas and liquid flows for enhanced control of gas phase

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic‐driven TIB (Hy‐TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move the media that is a characteristic of other TIB designs. The advantage of utilizing low volumes of gas mixtures (that are more expensive than air) is shown by a doubling of the growth rate of plant root cultures under elevated (40%) oxygen in air, and with CO₂ supplementation showing improved phototrophic and photomixotrophic growth of seedless watermelon meristem cultures. The development of this bioreactor system involved overcoming contamination issues associated with utilizing very low gas flow rates and included utilizing microchip pressure sensors to diagnose unexpected changes in internal bioreactor pressure (± 20 Pa ～0.0002 atm) caused by flexing of non‐rigid plastic bag vessels. The overall design seeks to achieve versatility, scalability and minimum cost such that bioreactor technology can play an increasing role in the critical need to improve plant productivity in the face of increasing demand for food, reduced resources, and environmental degradation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:337–345, 2016     

Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system

Here, we evaluated the efficiency of shoot multiplication of Vanilla planifolia Jacks. ex Andrews using solid medium, partial immersion, and a temporary immersion system (TIS) to improve micropropagation in this species. Clusters of shoots were cultivated in vitro using Murashige and Skoog (MS) medium supplemented with 9.55 μM benzyladenine (BA) and 100 mL L^?1 coconut water. For the TIS, a RITA^® system was used and three immersion frequencies were evaluated (every 4, 8, and 12 h) with an immersion time of 2 min. After 30-d culture, the TIS produced the maximum multiplication rate (14.27 shoots per explant) when using an immersion frequency of 2 min every 4 h, followed by the partial immersion system (8.64 shoots per explant), and solid medium (5.80 shoots per explant). Next, the effect of the volume of culture medium per explant was also evaluated for TIS. The most suitable volume of culture medium for shoot formation was 25 mL per explant, which increased the rate of multiplication to 17.54 shoots per explant. Root initiation was 90% successful in TIS using half-strength MS medium supplemented with 0.44 μM naphthaleneacetic acid (NAA) and an immersion frequency of 2 min every 4 h. With this system, the shoot multiplication rate increased threefold compared to that obtained with solid medium. In addition, this system produced good results for the transplantation and acclimation (90% of survival) of in vitro-derived plants. These results offer new options for large-scale micropropagation of vanilla.     

Use of Chlorella vulgaris for CO(2) mitigation in a photobioreactor

Carbon dioxide (CO₂) is a colorless gas that exists at a concentration of approximately 330 ppm in the atmosphere and is released in great quantities when fossil fuels are burned. The current flux of carbon out of fossil fuels is about 600 times greater than that into fossil fuels. With increased concerns about global warming and greenhouse gas emissions, there have been several approaches proposed for managing the levels of CO₂ emitted into the atmosphere. One of the most understudied methods for CO₂ mitigation is the use of biological processes in engineered systems such as photobioreactors. This research project describes the effectiveness of Chlorella vulgaris, used in a photobioreactor with a very short gas residence time, in sequestering CO₂ from an elevated CO₂ airstream. We evaluated a flow-through photobioreactor's operational parameters, as well as the growth characteristics of the C. vulgaris inoculum when exposed to an airstream with over 1850 ppm CO₂. When using dry weight, chlorophyll, and direct microscopic measurements, it was apparent that the photobioreactor's algal inoculum responded well to the elevated CO₂ levels and there was no build-up of CO₂ or carbonic acid in the photobioreactor. The photobioreactor, with a gas residence time of approximately 2 s, was able to remove up to 74% of the CO₂ in the airstream to ambient levels. This corresponded to a 63.9-g/m³/h bulk removal for the experimental photobioreactor. Consequently, this photobioreactor shows that biological processes may have some promise for treating point source emissions of CO₂ and deserve further study. Received 25 April 2002/ Accepted in revised form 27 July 2002     

Photosynthetic performance of a cyanobacterium in a vertical flat-plate photobioreactor for outdoor microalgal production and fixation of CO2

A vertical flat-plate photobioreactor was developed for the outdoor culture of microalgae using sunlight as the light source. The ability for biomass production and CO₂ fixation was evaluated by using a cyanobacterium, Synechocystis aquatilis SI-2. The average areal productivity was 31 g biomass m^–2 d^–1, which corresponded to a CO₂ fixation rate of 51 g CO₂ m^–2 d^–1, sustainable in the northern region of Japan during the winter time (January and February). The relationships between the efficiency of solar energy utilization of the reactor and its effect factors (cell concentration and irradiation) were investigated.     

Genetic transformation of hop (Humulus lupulus L. cv. Tettnanger) by particle bombardment and plant regeneration using a temporary immersion system

The efficiency of micropropagation of double-node shoots of hop (Humulus lupulus L. cv. Tettnanger) was evaluated using semi-solid and liquid culture medium in RITA® temporary immersion bioreactors. The highest fresh and dry weight of shoots, average number of shoots, and multiplication rate were obtained using the RITA® system, whereas the longest shoots were obtained on semi-solid medium. Moreover, shoot length was affected significantly by the inoculum density of double-node shoots in RITA® vessels. In addition, the RITA® bioreactors were suitable for shoot induction from organogenic calli. The percentage of shoot induction and the shoot fresh and dry weights were significantly higher in the RITA® system than in semi-solid medium. The age of organogenic calli and inoculum density significantly affected the induction of shoots from organogenic calli. The optimum conditions for DNA delivery into hop organogenic calli using the biolistic particle delivery system were also determined. Organogenic calli were bombarded with the plasmid pSR5-2 (gusA and nptII) varying helium pressure (900, 1,100, or 1,350 psi) and target distance (6, 9, or 12 cm). The highest gusA transient activity was obtained using a pressure of 900 psi and a target distance of 6 cm. For stable genetic transformation, 3-wk-old organogenic calli were bombarded with the plasmid pCAMBIA1303 (gusA, mgfp5, and hpt) using these optimum conditions. Stable gusA expression was observed in organogenic calli and shoots after 4 wk of culture on selection medium containing 2.5 mg l^?1 hygromycin. The presence of the mgfp5 gene in the hop genome was confirmed by PCR.     

Bovine blastocysts obtained from reconstructed cytoplast and karyoplasts using a simple portable CO2 incubator

To enable both the multiplication of elite livestock and the engineering of transgenic animals for various agricultural and biochemical purposes, scientists around the world are intensively studying efficient ways of improving developmental competency of bovine embryos reconstructed by somatic cell nuclear transfer. Because it is widely accepted that culture conditions along with many other factors contribute to the developmental competency of reconstructed embryos, this preliminary study was designed to test whether or not bovine reconstructed embryos could develop in vitro using a simple portable CO(2) incubator. CO(2) and O(2) gas tensions and air pressure can be varied using this system. The parameters used in the five conducted trials were low CO(2) (2-5%) and O(2) (8-10%) gas tensions, and negative air pressure (of 300 mm Hg). Chamber temperature was maintained at 38.5 degrees C. Bovine fetal fibroblasts were used as donor karyoplasts and were fused into microsurgically enucleated M II oocytes followed by activation and culture. From the 250 enucleated oocytes, 217 (86.8%) fused, 183 (73.2%) cleaved, and 43 (17.2%) developed to the blastocyst stage. While relatively low developmental rates were achieved, technical proficiency may have been a contributing factor. Further studies using this system are needed to determine optimal levels of O(2), CO(2), and air pressure.     

Microbial cell disruption for improving lipid recovery using pressurized CO2: Role of CO2 solubility in cell suspension,sugar broth,and spent media

The study of in situ gas explosion to lyse the triglyceride‐rich cells involves the solubilization of gas (e.g., carbon dioxide, CO₂) in lipid‐rich cells under pressure followed by a rapid decompression, which allows the gas inside the cell to rapidly expand and rupture the cell from inside out. The aim of this study was to perform the cell disruption using pressurized CO₂ as well as to determine the solubility of CO₂ in Rhodotorula glutinis cell suspension, sugar broth media, and spent media. Cell disruption of R. glutinis was performed at two pressures of 2,000 and 3,500 kPa, respectively, at 295.2 K, and it was found from both scanning electron microscopy (SEM) and plate count that a substantial amount of R. glutinis was disrupted due to the pressurized CO₂. We also found a considerable portion of lipid present in the aqueous phase after the disruption at P = 3,500 kPa compared to control (no pressure) and P = 2,000 kPa, which implied that more intracellular lipid was released due to the pressurized CO₂. Solubility of CO₂ in R. glutinis cell suspension was found to be higher than the solubility of CO₂ in both sugar broth media and spent media. Experimental solubility was correlated using the extended Henry's law, which showed a good agreement with the experimental data. Enthalpy and entropy of dissolution of CO₂ were found to be ?14.22 kJ mol^?1 and 48.10 kJ mol^?1 K^?1, 9.64 kJ mol^?1 and 32.52 kJ mol^?1 K^?1, and 7.50 kJ mol^?1 and 25.22 kJ mol^?1 K^?1 in R. glutinis, spent media, and sugar broth media, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:737–748, 2017     

A simple method for mass propagation of potato (Solanum tuberosum L.) using a bioreactor without forced aeration

We have developed a simple system for mass propagation of plant organs using a bioreactor without forced aeration. In this system, explants were cultured in bottles equipped with an air-permeable membrane on the cap and these bottles were slowly rotated on a bottle roller. Microtubers of potato were induced using a two-step culture method. In the first step, potato plantlets were cultured under static conditions. After shoot proliferation, the culture medium was replaced with a medium containing a higher concentration of sucrose and the bottles were rotated at 1 rpm. The number of tubers was clearly increased in this system compared to the culture without rotation. The results indicated that our system can be applied for mass propagation of potato tubers at low cost. Received: 3 April 1998 / Revision received: 22 July 1998 / Accepted: 21 August 1998     

Na2Zn3(CO3)4.3H2O: a potentially useful compound for zinc supplementation.

The infrared and Raman spectra of the title compound were recorded and are briefly discussed on the basis of its structural characteristics. Its thermal behaviour was investigated by means of TG and DTA measurements. Several dissolution tests were also performed. The results support the potential usefulness of this double carbonate as a useful compound for Zn(II) supplementation.     

Temporary immersion systems for the mass propagation of sweet cherry cultivars and cherry rootstocks: development of a micropropagation procedure and effect of culture conditions on plant quality

Sweet cherry (Prunus avium L.) varieties and cherry rootstocks are an important part of the fruit industry, and difficulties associated with mass propagation provide an opportunity for the use of temporary immersion systems (TIS). We show the establishment of culture procedures for four genotypes: the rootstocks Maxma-14 and Colt and the varieties ‘Van’ and ‘Rainier.’ The starting explants were internodal segments from seedlings kept in solid propagation medium (PM) (Driver-Kuniyuki Walnut (DKW) base supplemented with indole butyric acid, benzyl amino purine; ascorbic acid, myo-inositol, and agar). Segments were cultured under TIS for 14 d and led to whole plant generation after 30 d of culturing in solid rooting media, which depended on whether they are varieties or rootstocks. A 15-d acclimatization phase led to establishment in greenhouse. The efficiency of TIS was specifically analyzed for the two best PM-derivative media and compared to cultures using solid medium. A number of shoots (P _x), biomass (Q _x), and sucrose consumption (SC) were evaluated for these purposes. The results showed that Maxma-14, Colt, and ‘Van’ TIS cultures had improved performance in comparison to solid cultures, whereas ‘Rainier’ showed no differences. The number of immersions influenced all of the productive parameters (P _x, Q _x, and SC), whereas genotype affected P _x, and the time of immersion influenced SC. The best Q _x and P _x values were obtained with the rootstocks Maxma-14 and Colt, as well as the variety Van; these showed no hyperhydration. Physiological studies show that 14-d TIS-produced shoots represented an intermediate stage between solid-derived and adult plants, although the photosynthetic efficiencies of these materials revealed a lack of autotrophic ability at this point.     

Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100

A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.     

Construction of a genetically engineered microorganism for CO2 fixation using a Rhodopseudomonas/Escherichia coli shuttle vector

The CO2 fixation ability of Rhodopseudomonas palustris DH was enhanced by introducing the recombinant plasmid pMG-CBBM containing the form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbM) isolated from Rps. palustris NO. 7. Sequencing of a 3.0-kb PstI fragment containing the cbbM gene revealed an open reading frame encoding 461 amino acids, homologous to known cbbM genes, with a ribosome binding site upstream of cbbM and a terminator downstream of cbbM, without promoter. pMG-CBBM, a Rhodopseudomonas/Escherichia coli shuttle expression plasmid, was derived from the Rhodopseudomonas/E. coli shuttle cloning vector pMG105, by inserting the promoter of the pckA gene and the cbbM gene into its multiple cloning site. Plasmid pMG-CBBM was transformed into Rps. palustris DH by electroporation, and was stably maintained when transformants were grown either photoheterotrophically or photolithoautotrophically in the absence of antibiotics. This is the first report of an expression plasmid containing a Rps. palustris-specific promoter that allows stable expression of a foreign gene in the absence of antibiotic selection.     

Effects of elevated CO2 on the capacity for photosynthesis of a single leaf and a whole plant, and on growth in a radish

The atmospheric concentration of CO2 will probably rise to about 700 micromol mol(-1) by the end of this century. The effects of elevated growth CO2 on photosynthesis are still not fully understood. Effects of elevated growth CO2 on the capacity for photosynthesis of a single leaf and a whole plant were investigated with the radish cultivar White Cherish. The plants were grown under ambient ( approximately 400 micromol mol(-1)) or elevated CO2 ( approximately 750 micromol mol(-1)). The rates of net photosynthesis per leaf area with a whole plant and a single leaf of plants of various ages (15-26 d after planting) were measured under ambient and elevated CO2. The rates of photosynthesis were increased by 20-28% by elevated CO2. There was no effect of elevated growth CO2 on the rate of photosynthesis, clearly indicating no downward acclimation of photosynthesis to elevated CO2. Elevated CO2 increased dry weight accumulation by >27%. The effect of elevated CO2 on other growth characteristics will also be shown.     

Evolution of C4 plants: a new hypothesis for an interaction of CO2 and water relations mediated by plant hydraulics

C(4) photosynthesis has evolved more than 60 times as a carbon-concentrating mechanism to augment the ancestral C(3) photosynthetic pathway. The rate and the efficiency of photosynthesis are greater in the C(4) than C(3) type under atmospheric CO(2) depletion, high light and temperature, suggesting these factors as important selective agents. This hypothesis is consistent with comparative analyses of grasses, which indicate repeated evolutionary transitions from shaded forest to open habitats. However, such environmental transitions also impact strongly on plant-water relations. We hypothesize that excessive demand for water transport associated with low CO(2), high light and temperature would have selected for C(4) photosynthesis not only to increase the efficiency and rate of photosynthesis, but also as a water-conserving mechanism. Our proposal is supported by evidence from the literature and physiological models. The C(4) pathway allows high rates of photosynthesis at low stomatal conductance, even given low atmospheric CO(2). The resultant decrease in transpiration protects the hydraulic system, allowing stomata to remain open and photosynthesis to be sustained for longer under drying atmospheric and soil conditions. The evolution of C(4) photosynthesis therefore simultaneously improved plant carbon and water relations, conferring strong benefits as atmospheric CO(2) declined and ecological demand for water rose.     

Results of testing a new method of using antibiotics in nutrient media for the isolation of Shigella: the 2-streak method]

A new variant of media with antibiotics for isolation of Shigella, i.e. a method of 2 streaks each containing different antibiotics was tested in analysis of excrements from patients with acute dysentery. It was found that the new method is more effective than the well known method of gradient plates (isolation of Shigella in one series of the experiments amounted to 85.2 and 64.7 per cent respectively, and in the other series of the experiments the respective figures were 95.4 and 89.3 per cent). Its efficiency was lower as compared to the procedure of inoculation onto 2 plates, i.e. onto the media with and without an antibiotic (isolation of Shigella was 67.5 and 77.4 per cent respectively). The new method provided a higher frequency of Shigella isolation as compared to inoculation onto the media without an antibiotic, as well as onto any of the media used with one antibiotic. The method of 2 streaks offers wider possibilities for choosing the antibiotics for adding to the nutrient medium, as well as estimation of the antibioticograms and species structures of Shigella distributed in a concrete area.     

High efficient conversion of CO2-rich bio-syngas to CO-rich bio-syngas using biomass char: a useful approach for production of bio-methanol from bio-oil

A novel approach for high efficient conversion of the CO₂-rich bio-syngas into the CO-rich bio-syngas was carried out by using biomass char and Ni/Al₂O₃ catalyst, which was successfully applied for production of bio-methanol from bio-oil. After the bio-syngas conditioning, the CO₂/CO ratio prominently dropped from 6.33 to 0.01-0.28. The maximum CO yield in the bio-syngas conditioning process reached about 1.96 mol/(mol CO₂) with a nearly complete conversion of CO₂ (99.5%). The performance of bio-methanol synthesis was significantly improved via the conditioned bio-syngas, giving a maximum methanol yield of 1.32 kg/(kg_catalyst h) with a methanol selectivity of 99%. Main reaction paths involved in the bio-syngas conditioning process have been investigated in detail by using different model mixture gases and different carbon sources.     

A Battery-Operated Instrument for Non-destructive Measurements of Photosynthesis and Transpiration of Ears and Leaves of Cereals using 14CO2 and a Lithium Chloride Hygrometer

The design and evaluation of a battery-operated cuvette suitablefor non-destructive measurements of photosynthesis and transpirationin the field are described. The ear or leaf of wheat was sealedin a clear acrylic tube of 2 cm diameter and ¹⁴CO₂-enrichedair was circulated in the closed system which had a total volumeof 1.17 x 10^–3 m³; a boundary layer conductance of 5.2cm s^–1 was measured with an ear in the tube. Photosynthesiswas calculated from the decrease in activity of 2.5 cm3 samplesof air taken from the chamber at 15, 60, 120, and 180 s afterassimilation began. The gas samples were injected into scintillationvials containing 0.5 cm³ of strong base and subsequently countedin a scintillation counter. Transpiration was calculated fromthe time required to increase the humidity of the circulatinggas by a known amount as measured by a lithium chloride hygrometer.The cuvette was flushed between measurements and recharged froma cylinder of air enriched with ¹⁴CO₂; ten measurements an hourwere possible with two operators. Comparisons between the lossin activity of the gas samples and the activity gained by astainless steel mesh saturated with strong base, or activityextracted from exposed leaves, indicated the method of gas-samplingwas sensitive, reproducible, and accurate. Transpiration ofears of wheat measured with the lithium chloride hygrometerin the closed cuvette gave comparable values to those obtainedwhen the same ears were measured in an open system with a dewpointhygrometer. Formulae for calculating photosynthesis and transpirationare given.     

Achieving Target 2 of the Global Strategy for Plant Conservation: building a preliminary assessment of vascular plant species using data from herbarium specimens

The Global Strategy for Plant Conservation calls for a preliminary assessment of the conservation status of all known plant species by the year 2010. To date insufficient progress has been made on meeting this target. New efforts to develop a preliminary list beyond using the full IUCN criteria in plant assessments are needed. Here we present an algorithm that provides a preliminary assessment of the conservation status of plant species using data from herbarium specimens. We use Hawaiian specimen data from the United States National Herbarium to calibrate the parameters of the algorithm and then use specimen data from the Arecaceae, Commelinaceae, Gesneriaceae and Heliconiaceae as examples of the application of the algorithm. The algorithm was calibrated to insure 95% accuracy in placing the Hawaiian plant species into previously and independently determined threatened categories. Our results indicate that 28% of the Hawaiian taxa, 27% of the species of Arecaceae, 45% of the species of Commelinaceae, 32% of the species of Gesneriaceae, and 35% of the species of Heliconiaceae are Not Threatened and will not need any further evaluation for the preliminary assessment. Species identified here as Potentially Extinct and Potentially Threatened can be further assessed by additional herbarium material and/or conservation specialists for final evaluation using other assessment strategies (e.g., regional and national lists, taxonomic expert assessment, etc.).     

High-density SNP linkage map construction and QTL mapping for flavonoid-related traits in a tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) using 2b-RAD sequencing

Background

Flavonoids are important components that confer upon tea plants a unique flavour and health functions. However, the traditional breeding method for selecting a cultivar with a high or unique flavonoid content is time consuming and labour intensive. High-density genetic map construction associated with quantitative trait locus (QTL) mapping provides an effective way to facilitate trait improvement in plant breeding. In this study, an F1 population (LJ43×BHZ) was genotyped using 2b-restriction site-associated DNA (2b-RAD) sequencing to obtain massive single nucleotide polymorphism (SNP) markers to construct a high-density genetic map for a tea plant. Furthermore, QTLs related to flavonoids were identified using our new genetic map.

Results

A total of 13,446 polymorphic SNP markers were developed using 2b-RAD sequencing, and 4,463 of these markers were available for constructing the genetic linkage map. A 1,678.52-cM high-density map at an average interval of 0.40 cM with 4,217 markers, including 427 frameset simple sequence repeats (SSRs) and 3,800 novel SNPs, mapped into 15 linkage groups was successfully constructed. After QTL analysis, a total of 27 QTLs related to flavonoids or caffeine content (CAF) were mapped to 8 different linkage groups, LG01, LG03, LG06, LG08, LG10, LG11, LG12, and LG13, with an LOD from 3.14 to 39.54, constituting 7.5% to 42.8% of the phenotypic variation.

Conclusions

To our knowledge, the highest density genetic map ever reported was constructed since the largest mapping population of tea plants was adopted in present study. Moreover, novel QTLs related to flavonoids and CAF were identified based on the new high-density genetic map. In addition, two markers were located in candidate genes that may be involved in flavonoid metabolism. The present study provides valuable information for gene discovery, marker-assisted selection breeding and map-based cloning for functional genes that are related to flavonoid content in tea plants.