Preculture Step Research Articles

Phase-Separating Peptides Recruiting Aggregation-Induced Emission Fluorogen for Rapid E. coli Detection.

Rationally designed biomolecular condensates have found applications primarily as drug-delivery systems, thanks to their ability to self-assemble under physico-chemical triggers (such as temperature, pH, or ionic strength) and to concomitantly trap client molecules with exceptionally high efficiency (>99%). However, their potential in (bio)sensing applications remains unexplored. Here, we describe a simple and rapid assay to detect E. coli by combining phase-separating peptide condensates containing a protease recognition site, within which an aggregation-induced emission (AIE)-fluorogen is recruited. The recruited AIE-fluorogen's fluorescence is easily detected with the naked eye when the samples are viewed under UV-A light. In the presence of E. coli, the bacteria's outer membrane protease (OmpT) cleaves the phase-separating peptides at the encoded protease recognition site, resulting in two shorter peptide fragments incapable of liquid-liquid phase separation. As a result, no condensates are formed and the fluorogen remains non-fluorescent. The assay feasibility was first tested with recombinant OmpT reconstituted in detergent micelles and subsequently confirmed with E. coli K-12. In its current format, the assay can detect E. coli K-12 (108 CFU) within 2 h in spiked water samples and 1-10 CFU/mL with the addition of a 6-7 h pre-culture step. In comparison, most commercially available E. coli detection kits can take anywhere from 8 to 24 h to report their results. Optimizing the peptides for OmpT's catalytic activity can significantly improve the detection limit and assay time. Besides detecting E. coli, the assay can be adapted to detect other Gram-negative bacteria as well as proteases having diagnostic relevance.

SUCCESSFUL PRESERVATION OF BANANA EMBRYOGENIC CALLUS BY MINIMAL GROWTH AND CRYOPRESERVATION TECHNIQUE

This paper reports successful preservation methods by minimal growth and cryopreservation of banana embryogenic callus. The minimal growth and cryopreservation techniques can be applied for medium-term and long-term storage. The embryogenic callus of banana cv Dwarf Parfitt was used as the plant material. Different kind of medium formulations, temperature, gelling agents, and ABA concentrations were tested in minimal growth study. The type of explants, dehydration periods, and pre culture media were tested in cryopreservation study. The result showed that the use of WIC medium was better than BM2G for maintaining embryogenic callus. The application of 0.005 mg L-1 ABA combined with reduced temperature (150C) could preserve the cultures longer than 4 months with high survival rate (95%), high recovery rate (90%), and high number of recovered embryos indicating high embryogenic potential. For cryopreservation study, the best dehydration period was 20 minutes. The combined treatments of 150 g/l sucrose and 2 M glycerol at preculture step was the most suitable treatment for successful cryopreservation with 80% survival rates; and the recovered cultures maintained their somatic embryogenic capacity.

A milliliter to picoliter-level centrifugal microfluidic concentrator for fast pathogen detection and antimicrobial susceptibility testing

The available pathogenic bacteria detection and antimicrobial susceptibility testing (AST) are still time-consuming and tedious because of the lengthy culture procedures. Various kinds of systems have promised a great reduction in AST time, however the high-efficiency and user-friendly bacteria enrichment is always a challenge to tackle. Here we introduce a centrifugation-based microfluidic concentrator for milliliter to picoliter bacteria suspension enrichment to achieve a fast and real-time pathogen detection and AST. The concentrator consists of a milliliter-level loading chamber and a picoliter-level enrichment channel. By centrifuging, all pathogen cells in chamber can be condensed into enrichment channel, achieving a milliliter to picoliter enrichment. Thus, the pathogen cells can be detected directly by microscopic observation. More importantly, the AST can be started from original sample without traditional pre-culture steps for the high enrichment efficiency. Since all the cells were confined at the picoliter-level channel tip, detectable growth of phenotype can be easily observed in a few hours, and the different inhibition mechanism on cells from different antibiotics can be clearly observed. The proposed concentrator shows a detection limit of 102 cell/mL with 0.5 h of enrichment for detection and another 3 h of micro-culture for AST.

A green K-fertilizer using mechanical activation to improve the solubilization of a low-reactivity potassium mineral by Aspergillus niger

Here, we investigated process engineering strategies to improve the biological solubilization of K-feldspar, a low-reactivity potassium mineral, aiming to obtain a green K-fertilizer for agricultural applications. The results using submerged cultivation of Aspergillus niger showed that the biological K-solubilization of pristine K-feldspar rock released up to 15.87% (reaching 220 mg L−1 after 3 days) when using 5% glucose as carbon source and initial pH 4, without a preculture step. Besides medium acidification, the microorganism-mineral physical interaction played a key role in promoting K-solubilization. Furthermore, prior mechanical activation of the K-feldspar led to substantial increases of up to 172% in K release (reaching 326.86 mg L−1), compared to the original mineral, and reduced the K-solubilization cultivation time to only 24 h. The results demonstrated the potential of integrating mechanical activation with biological solubilization to advance on the use of low-solubility potassium minerals as K-fertilizers, thus contributing to a more sustainable agriculture.

Performance of molecular methods for the detection of Salmonella in human stool specimens.

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture and the lack of validated molecular diagnostic tests for the detection of Salmonella in the stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimised an in-house monoplex real-time polymerase chain reaction (PCR) for the detection of Salmonella ttr and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same ttr and InvA genes and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: Ttr and InvA primers were both able to detect all the different Salmonella serovars tested and had superior limits of detection when DNA was extracted after selenite pre-culture. T tr sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99%, respectively. Culture showed the highest PPV (99.73%), and monoplex- ttr had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance, although stool culture and monoplexed ttr primers had superior specificity and sensitivity, respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

A multiplexed nanoliter array-based microfluidic platform for quick, automatic antimicrobial susceptibility testing.

Antimicrobial resistance stemming from indiscriminate usage of antibiotics has emerged as a global healthcare issue with substantial economic implications. The inefficacy of commonly used antibiotics combined with superfluous consumption has worsened the issue. Rapid antimicrobial susceptibility testing (AST) to antibiotics can be advantageous in thwarting bacterial infections. Therefore, this study developed a simple nanoliter array-based microfluidic platform for performing rapid AST, which can handle and manipulate liquids both in nanoliter and microliter volumes. The platform consisted of two microfluidic devices, one for performing AST and another for diluting antibiotics and these two were suitably integrated. The microfluidic device used for generating microarrays for AST experiments is single-layered (no air layer) and has no active microvalves and air hole, which makes the device easy to fabricate and use. The loading process ensures uniform distribution of bacteria and relies on displacing the air from microarrays through porous polydimethylsiloxane membranes. Furthermore, the chip for dilution consisted of active microfluidic components, and could prepare and test seven different concentrations of antibiotics, which make the platform multiplexed and be capable of evaluating minimum inhibitory concentrations (MICs), a clinically relevant parameter. MIC determination requires less number of bacteria (∼2000) and hence shortens the pre-culture step, i.e. bacteria culture in blood and urine. This automated system demonstrated AST and evaluated MICs using Escherichia coli and two antibiotics, including ampicillin and streptomycin, and the results were ascertained using a gold standard method. It only took 8-9 h to perform AST, which is substantially less compared to a conventional process and hence is of high clinical utility.

Performance of molecular methods for the detection of Salmonella in human stool specimens

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture, and the lack of validated molecular diagnostic tests for the detection of Salmonella in stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimized an in-house monoplex real time polymerase chain reaction (PCR) for the detection of Salmonella TTR and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction, and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same TTR and InvA genes, and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over a period of 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: TTR and InvA primers were both able to detect all the different Salmonella serovars tested, and had superior limits of detection if DNA was extracted after selenite pre-culture. TTR sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99% respectively. Culture showed the highest PPV (99.73%) and mono-TTR had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance although stool culture and monoplexed TTR primers had superior specificity and sensitivity respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

Culture-free Antibiotic-susceptibility Determination From Single-bacterium Raman Spectra

Raman spectrometry appears to be an opportunity to perform rapid tests in microbiological diagnostics as it provides phenotype-related information from single bacterial cells thus holding the promise of direct analysis of clinical specimens without any time-consuming growth phase. Here, we demonstrate the feasibility of a rapid antibiotic-susceptibility determination based on the use of Raman spectra acquired on single bacterial cells. After a two-hour preculture step, one susceptible and two resistant E. coli strains were incubated, for only two hours, in the presence of different bactericidal antibiotics (gentamicin, ciprofloxacin, amoxicillin) in a range of concentrations that included the clinical breakpoints used as references in microbial diagnostic. Spectra were acquired and processed to isolate spectral modifications associated with the antibiotic effect. We evidenced an “antibiotic effect signature” which is expressed with specific Raman peaks and the coexistence of three spectral populations in the presence of antibiotic. We devised an algorithm and a test procedure that overcome single-cell heterogeneities to estimate the MIC and determinate the susceptibility phenotype of the tested bacteria using only a few single-cell spectra in four hours only if including the preculture step.

Agrobacterium tumefaciens-mediated transformation of drumstick (Moringa oleifera Lam.)

ABSTRACTDrumstick (Moringa oleifera Lam.) is an industrially important tree species. Due to the availability of an efficient regeneration system, the present study was undertaken to develop a reliable and stable Agrobacterium tumefaciens-mediated transformation system for the genetic improvement of drumstick. A. tumefaciens strain EHA105, harbouring the plasmid pCAMBIA1301 containing the hygromycin phosphotransferase II and ß-glucuronidase genes driven by the CaMV 35S promoter, was used to establish a transformation system in drumstick. The use of vacuum infiltration-assisted Agrobacterium infection (excluding a pre-culture step) and co-cultivation for two days significantly increased the transformation frequency. Genomic integration and transgene expression were confirmed by β-glucuronidase (GUS) assays, polymerase chain reaction (PCR), Southern blot analysis and real-time quantitative PCR (RT-qPCR). This transformation protocol provides a basis for the future development of genetic engineering techniques to improve the performance of drumstick.

A rapid mitochondrial toxicity assay utilizing rapidly changing cell energy metabolism.

Drug-induced liver injury is a major cause of safety-related drug-marketing withdrawals. Several drugs have been reported to disrupt mitochondrial function, resulting in hepatotoxicity. The development of a simple and effective in vitro assay to identify the potential for mitochondrial toxicity is thus desired to minimize the risk of causing hepatotoxicity and subsequent drug withdrawal. An in vitro test method called the "glucose-galactose" assay is often used in drug development but requires prior-culture of cells over several passages for mitochondrial adaptation, thereby restricting use of the assay. Here, we report a rapid version of this method with the same predictability as the original method. We found that replacing the glucose in the medium with galactose resulted in HepG2 cells immediately shifting their energy metabolism from glycolysis to oxidative phosphorylation due to drastic energy starvation; in addition, the intracellular concentration of ATP was reduced by mitotoxicants when glucose in the medium was replaced with galactose. Using our proposed rapid method, mitochondrial dysfunction in HepG2 cells can be evaluated by drug exposure for one hour without a pre-culture step. This rapid assay for mitochondrial toxicity may be more suitable for high-throughput screening than the original method at an early stage of drug development.

Enhanced cellulase production by Trichoderma harzianum by cultivation on glycerol followed by induction on cellulosic substrates.

The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.

Development of an efficient, genotype independent plant regeneration and transformation protocol using cotyledonary nodes in safflower (Carthamus tinctorius L.)

Safflower is an important oilseed crop with a nutritionally desirable oil composition comprising low levels of saturated fatty acids and high levels of unsaturated fatty acids. In this study, a robust, genotype-independent plant regeneration protocol was developed for geographically diverse safflower genotypes, including one accession each from America, Australia, Egypt, Germany, Kazakhstan and three important Indian genotypes (Sharda, Bhima and PBNS-12). Use of cotyledonary nodes as explants resulted in genotype-independent regeneration on BAP (6-Benzylaminopurine), NAA (Naphthalene acetic acid) and ascorbic acid supplemented MS medium. Histological analysis revealed that multiple shoot apical meristems originated independently from peripheral cortical regions of explants. We developed a highly efficient in vitro micrografting method which enabled successful rooting of 85–90 % of regenerated shoots. An efficient genetic transformation system was also established for three Indian genotypes viz., Sharda, Bhima and PBNS-12 using the Agrobacterium strain, LBA4404 and phosphinothricin as the selection agent. This is the first report on use of phosphinothricin-based selection and cotyledonary nodes as explants for Agrobacterium-mediated transformation of safflower. Use of vacuum infiltration-assisted Agrobacterium infection and inclusion of a pre-culture step significantly increased transformation frequencies in all the three genotypes as seen by GUS assays on transformed calli. Genomic integration and transgene expression were confirmed by PCR, Southern hybridization and GUS assays. Most transgenic plants (90 %) exhibited a normal phenotype when grown under controlled conditions and produced viable seeds. This protocol would be useful for introduction of desirable traits in diverse genotypes of safflower.

Characterization of the Populus PtrCesA4 promoter in transgenic Populus alba × P. glandulosa

An efficient shoot regeneration method was developed using leaf explants of Populus alba × P. glandulosa, and the optimized shoot regeneration medium contained 0.1 mg L−1 NAA, 0.5 mg L−1 6-BA and 0.002 mg L−1 TDZ. The factors for Agrobacterium-mediated transformation of this hybrid were further optimized as follows: no preculture step, Agrobacterium cell suspension with an OD600 of 0.6, 20 min infection time and 2d co-cultivation duration. To investigate Populus trichocarpa cellulose synthase A4 promoter (PtrCesA4pro), we constructed the PtrCesA4pro:: β-glucuronidase (GUS) binary vector, in which the cauliflower mosaic virus 35S (CaMV35S) promoter of pBI121was replaced with the PtrCesA4pro to drive the expression of a GUS reporter gene. Forty-eight kanamycin-resistant plantlets were obtained through Agrobacterium-mediated transformation of this hybrid. Genomic DNA PCR and Southern blot analyses confirmed the integration of PtrCesA4pro::GUS into this hybrid and the expression of GUS gene was driven by the PtrCesA4 promoter. A histochemical GUS staining presented the activity of PtrCesA4 promoter in the transgenic lines. No GUS signal was observed in leaves, including the veins and petioles, and notably weak staining was detected in the secondary xylems of the roots. The PtrCesA4 promoter was highly active in the fibers, the vessels of developing xylems, and the fibers of mature phloems. Surprisingly, GUS staining was detected in the cambial cells of the highly lignified stems of these transgenic trees. Our results implicate PtrCesA4 promoter as a good genetic tool for controlling gene function in wood development and tree molecular breeding.

<b>Cryopreservation of the mangaba tree (<em>Hancornia speciosa</em> Gomes): a protocol for long-term storage

The aim of this study was to evaluate the efficiency of vitrification and droplet vitrification for the cryopreservation of Hancornia speciosa shoot tips. The shoot tips were subjected to four different periods of exposure (15, 30, 45 and 60 min.) to plant vitrification solution 2 (PVS2) before plunging into liquid nitrogen. We evaluated the regrowth of H. speciosa shoot tips that were cryopreserved by the classical vitrification technique and by the droplet vitrification technique. Shoot tips were submitted to different periods of pre-culture (absence, 24 or 48h) in a medium containing 0.3 M sucrose prior to cryopreservation. With a PVS2 exposure period of 60 min., significant differences were observed between the two techniques used in this study with respect to regrowth rates; however, both cryopreservation methods allowed for a shoot tip regrowth rate of over 70%. Use of a preculture step increased the regrowth rate of cryopreserved shoot tips. Shoot tip cryopreservation techniques employing vitrification and droplet vitrification proved to be viable for the long-term storage of mangaba tree samples.

A versatile, non genetically modified organism (GMO)-based strategy for controlling low-producer mutants in Bordetella pertussis cultures using antigenic modulation.

The uncontrolled presence of non-producer mutants negatively affects bioprocesses. In Bordetella pertussis cultures, avirulent mutants emerge spontaneously and accumulate. We characterized the dynamics of accumulation using high-throughput growth assays and competition experiments between virulent and avirulent (bvg(-) ) isolates. A fitness advantage of bvg(-) cells was identified as the main driver for bvg(-) accumulation under conditions of high virulence factor production. Conversely, under conditions that reduce their expression (antigenic modulation), bvg(-) takeover could be avoided. A control strategy was derived, which consists in applying modulating conditions whenever virulence factor production is not required. It has a wide range of applications, from routine laboratory operations to vaccine manufacturing, where pertussis toxin yields were increased 1.4-fold by performing early pre-culture steps in modulating conditions. Because it only requires subtle modifications of the culture medium and does not involve genetic modifications, this strategy is applicable to any B. pertussis isolate, and should facilitate regulatory acceptance of process changes for vaccine production. Strategies based on the same concept, could be derived for other industrially relevant micro-organisms. This study illustrates how a sound scientific understanding of physiological principles can be turned into a practical application for the bioprocess industry, in alignment with Quality by Design principles.

A simple way to overcome the recalcitrance of the water fern Ceratopteris thalictroides (L.) Brongn. to cryopreservation

<em>Ceratopteris thalictroides</em> is a water fern very sensitive to both dehydration and low temperature. This study focuses on the cryopreservation of this species by encapsulation-dehydration technique, in particular on the effects of pre-culture step, alginate bead size and the physical conditions of culture on the cryopreservation efficiency. Encapsulated and non-pre-cultured gametophytes did not survive cooling with liquid nitrogen. When cryopreservation was preceded by a 2-week period of pre-culture, regrowth reached 42.1%. Reduction in the size of the alginate bead, and culture in total darkness resulted in improved gametophyte regrowth capacity (75.5% or 81.7%, respectively). The best results (91.3%) were obtained when all factors tested occurred simultaneously. The gametophytes recovered very quickly and sporophytes were formed within 4 weeks after rewarming. These simple improvements can be used, not only for the cryopreservation of gametophytes in cryptogams but also for some recalcitrant species of seed plants.

Strategies for reliable and improved large-scale production of Pyrococcus furiosus with integrated purification of hydrogenase I

The hyperthermophilic archaeon Pyrococcus furiosus is an interesting organism for research and application, especially owing to its unique NADPH-dependent hydrogenase I. However, mass production of P. furiosus through fermentation is susceptible to fault because of its sensitivity to oxygen, a short exponential and stationary phase and a rapid cell lysis in typical cultivation process. In this study, significant improvement for pilot plant scale production processes for P. furiosus biomass was made by investigations of the fermentation process with subsequent hydrogenase I enzyme purification. Scale-up in a 300-L stirred tank bioreactor was successfully achieved. A repeated-batch cultivation process with high reproducibility and productivity was realized. Furthermore, the enzyme hydrogenase I was purified, and its activity tested and verified. The improvements in this production process for the production of large amount of P. furiosus biomass and hydrogenase I have been achieved, especially by successfully implementing the following key measures and steps: unsterile cultivation setup, skipping typical intermediate preculture and inoculation steps, accelerating the cultivation process by defining an optimal state of the inoculation, optimal time point of biomass harvesting and finally by choosing a one-step purification procedure for enzyme recovery.

Biolistic transformation of elite genotypes of switchgrass (Panicum virgatum L.)

Transformation of elite switchgrass (Panicum virgatum L.) genotypes would facilitate the characterization of genes related to cell wall recalcitrance to saccharification. However, transformation of explants from switchgrass plants has remained difficult. Therefore, the objective of this study was to develop a biolistic transformation protocol for elite genotypes. Three switchgrass genotypes (ST1, ST2, and AL2) were previously selected for tissue culture responsiveness. One genotype, SA37, was selected for further use due to its improved formation of callus amenable to transformation. Various medium sets were compared and a previously published medium set provided cultures with >96 % embryogenic callus, and data on transient and stable gene expression of RFP were used to optimize biolistic parameters, and further validate the switchgrass (PvUbi1) promoter. SA37 proved to be the most transformable, whereas eight transgenic calli on average were recovered per bombardment of 20 calli (40 % efficiency) when using a three-day day preculture step, 0.6 M osmotic adjustment medium, 4,482 kPa rupture disks and 0.4 μm gold particles which traveled 9 cm before hitting the target callus tissue. Regenerability was high, especially for ST2, for which it is possible to recover on average over 400 plants per half-gram callus tissue. It is now possible to routinely and efficiently engineer elite switchgrass genotypes using biolistic transformation.

Enzymatic ring‐opening polymerization of ε‐caprolactone by a new lipase from Yarrowia lipolytica

AbstractA new lipase from the yeast Yarrowia lipolytica was isolated and used in the enzymatic ring‐opening polymerization of lactones. The effect of used commercial oil from a vacuum pump (instead of olive oil) and in the presence of wheat flour was evaluated. It was found that lipase production is favored when wheat flour and used commercial oil is added instead of olive oil with an incubation time of 16.5 h at 29°C and 250 rpm. Lipase shows a specific activity of 3.47 nmoles of 4‐methyl umbelliferone released/mg of protein/min. In this way, preculture step can be avoided and an important reduction in production time can be obtained. In vitro polymerization of ε‐caprolactone at different temperatures in the presence of n‐heptane for 15 days produces low‐molecular‐weight polyesters with unique multiphase morphology. Polyesters were characterized by NMR (solution and solid‐state) spectroscopy, differential scanning calorimetry (DSC), Fourier‐transformed infrared, wide‐angle X‐ray scattering and MALDI‐TOF. Polyester crystallinities calculated by DSC were high, as expected for low‐molecular‐weight PCLs. Final polymer possesses carboxylic acid and hydroxyl end‐groups, as determined by 1H‐ and 13C NMR analysis. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci 2008

Physiological Changes in Gentian Axillary Buds During Two-step Preculturing with Sucrose that Conferred High Levels of Tolerance to Desiccation and Cryopreservation

Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing. In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0.1 m sucrose at 25 degrees C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0.4 m and 0.7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied. During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0.1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2.4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels. These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.