Influence of Different Carbohydrates on Flavonoid Accumulation in Hairy Root Cultures of Scutellaria baicalensis.

Carbohydrate sources play important roles in energy and growth of plants. Therefore, in this study, we investigated the optimal carbohydrate source in hairy root cultures (HRCs) of Scutellaria baicalensis infected with Agrobacterium rhizogenes strain R1000. The hairy roots were cultured in half-strength B5 liquid medium supplemented with seven different carbohydrates sources (sucrose, fructose, glucose, galactose, sorbitol, mannitol and maltose), each at a concentration of 100 mM, in order to identify the best carbon sources for the production of major flavones, such as wogonin, baicalin and baicalein. Sucrose, galactose and fructose markedly influenced the production of major flavones and were therefore chosen for subsequent experiments. HRC growth and flavone accumulation were examined following culture with 30, 100 and 150 mM sucrose, galactose and fructose, respectively. From these data, 150 mM sucrose was found to be the optimal carbon source for the enhancement of baicalein production and growth of S. baicalensis HRCs. Fructose caused the greatest increase in baicalin accumulation. Additionally, galactose was the optimal carbon source for wogonin production. These results provide important insights into the optimal growth conditions, particularly the appropriate carbohydrate source, for S. baicalensis.

Frozen beauty: The cryobiotechnology of orchid diversity.

Orchids (Orchidaceae) are one of the most diverse plant groups on the planet with over 25,000 species. For over a century, scientists and horticulturalists have been fascinated by their complex floral morphology, pollinator specificity and multiple ethnobotanical uses, including as food, flavourings, medicines, ornaments, and perfumes. These important traits have stimulated world-wide collection of orchid species, often for the commercial production of hybrids and leading to frequent overexploitation. Increasing human activities and global environmental changes are also accelerating the threat of orchid extinction in their natural habitats. In order to improve gene conservation strategies for these unique species, innovative developments of cryopreservation methodologies are urgently needed based on an appreciation of low temperature (cryo) stress tolerance, the stimulation of recovery growth of plant tissues in vitro and on the 'omics' characterization of the targeted cell system (biotechnology). The successful development and application of such cryobiotechnology now extends to nearly 100 species and commercial hybrids of orchids, underpinning future breeding and species conservation programmes. In this contribution, we provide an overview of the progress in cryobanking of a range of orchid tissues, including seeds, pollen, protocorms, protocorm-like bodies, apices excised from in vitro plants, cell suspensions, rhizomes and orchid fungal symbionts. We also highlight future research needs.

Accumulation of phenylpropanoids and correlated gene expression in hairy roots of tartary buckwheat under light and dark conditions.

Differential expression patterns of flavonoid biosynthetic pathway genes in the hairy roots of tartary buckwheat cultivars "Hokkai T8" and "Hokkai T10" were studied over a time course of the light-dark cycle. The Agrobacterium rhizogenes-mediated transformation system was applied for inducing hairy roots. Further, a total of six phenolic compounds and two anthocyanins were analyzed in the hairy roots which were exposed to both light and dark conditions, and their amounts were estimated by HPLC. The gene expression levels peaked on day 5 of culture during the time course of both dark and light conditions. Notably, FtPAL, Ft4CL, FtC4H, FtCHI, FtF3H, FtF3'H-1, and FtFLS-1 were more highly expressed in Hokkai T10 than in Hokkai T8 under dark conditions, among which FtPAL and FtCHI were found to be significantly upregulated, except on day 20 of culture. Significantly higher levels of phenolic compound, rutin, along with two anthocyanins were detected in the hairy roots of Hokkai T10 under both conditions. Furthermore, among all the phenolic compounds detected, the amount of rutin in Hokkai T10 hairy roots was found to be ∼5-fold (59,01 mg/g dry weight) higher than that in the control (12.45 mg/g dry weight) at the respective time periods under light and dark conditions.

Differential stress-response expression of two flavonol synthase genes and accumulation of flavonols in tartary buckwheat.

Flavonoids are ubiquitously present in plants and play important roles in these organisms as well as in the human diet. Flavonol synthase (FLS) is a key enzyme of the flavonoid biosynthetic pathway, acting at the diverging point into the flavonol subclass branch. We isolated and characterized a FLS isoform gene, FtFLS2, from tartary buckwheat (Fagopyrum tataricum). FtFLS2 shares 48% identity and 67% similarity with the previously reported FtFLS1, whereas both genes share 47-65% identity and 65-69% similarity with FLSs from other plant species. Using quantitative real-time PCR and high-performance liquid chromatography (HPLC), the expression of FtFLS1/2 and the production of 3 main flavonols (kaempferol, myricetin and quercetin) was detected in roots, leaves, stems, flowers and different stages of developing seeds. The relationship between the expression of the 2 FLS genes and the accumulation of the 3 basic flavonols was analyzed in 2 tartary buckwheat cultivars. FtFLS1 and FtFLS2 exhibited differential transcriptional levels between the tartary buckwheat cultivars 'Hokkai T10' and 'Hokkai T8'. Generally, higher transcript levels of FtFLS1 and FtFLS2 and a higher amount of flavonols were observed in the 'Hokkai T10' cultivar than 'Hokkai T8'. The content of flavonols showed tissue-specific accumulation between the 2 cultivars. The transcription of FtFLS1 was inhibited by the exogenous application of abscisic acid (ABA), salicylic acid (SA) and sodium chloride (NaCl), while FtFLS2 was not affected by ABA but up-regulated by SA and NaCl. These data indicate that the 2 FtFLS isoforms of buckwheat have different functions in the response of buckwheat to environmental stress.

Characterization of genes for a putative hydroxycinnamoyl-coenzyme A quinate transferase and p-coumarate 3'-hydroxylase and chlorogenic acid accumulation in tartary buckwheat.

Tartary buckwheat ( Fagopyrum tataricum Gaertn.) contains a high level of flavonoid compounds, which have beneficial and pharmacological effects on health. In this study, we isolated full-length cDNAs encoding hydroxycinnamoyl-coenzyme A quinate hydroxycinnamoyltransferase (HQT) and p-coumarate 3'-hydroxylase (C3H), which are involved in chlorogenic acid (CGA) biosynthesis. We examined the expression levels of HQT and C3H using real-time RT-PCR in different organs and sprouts of two tartary buckwheat cultivars (Hokkai T8 and T10) and analyzed CGA content using high-performance liquid chromatography. Among the organs, the flowers in both cultivars showed the highest levels of CGA. We concluded that the expression pattern of FtHQT and FtC3H did not match the accumulation pattern of CGA in different organs of T8 and T10 cultivars. Gene expression and CGA content varied between the cultivars. We presume that FtHQT and FtC3H levels might be controlled by multiple metabolic pathways in different organs of tartary buckwheat. Probably, FtC3H might have a greater effect on CGA biosynthesis than FtHQT. Our results will be helpful for a greater understanding of CGA biosynthesis in tartary buckwheat.

Ethylene inhibitors enhance shoot organogenesis of gloxinia (Sinningia speciosa).

Shoot organogenesis and plant regeneration in Sinningia speciosa were improved using ethylene inhibitors. The leaf explants were cultured on initial shoot regeneration media (MS media with BAP at 2 mg/L + NAA at 0.1 mg/L) supplemented with different concentrations of aminoethoxyvinylglycine (AVG), cobalt chloride (CoCl2), and silver thiosulphate (STS). The addition of AVG, CoCl2, and STS significantly improved the regeneration frequency giving higher shoots per explant and longer shoot length. The highest shoot growth was found when STS at 5 mg/L was incorporated with generation medium, performing highest regeneration frequency with highest number of shoots. This treatment (STS at 5 mg/L) produced 40% more shoots per explant compared to control followed by STS at 10 mg/L with increasing 37% more shoots compared to control. In the cases of AVG and CoCl2 the highest shoot number per explant was found at 1 mg/L. Treated with AVG and CoCl2 at 1 mg/L increased shoot number by 16 and 12%, respectively, compared to control. Ethylene inhibitors could be used as a possible micropropagation and plant transformation protocol in S. speciosa for plant regenerations.

'Personalisation' of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects.

Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process relies on trial and error. Among the vitrification-based cryopreservation techniques, droplet-vitrification produces high post-cryopreservation recovery. However, the protocol itself cannot solve the problems engaged in plant cryopreservation, prominently due to dehydration with cytotoxic vitrification solutions. This paper proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions. The proposed standard protocol consists of a progressive preculture with 0.3 M sucrose for 31 h and with 0.7 M for 17 h, loading with vitrification solution C4-35% (17.5 percent glycerol + 17.5 percent sucrose, w/v) for 20 to 40 min, dehydration with vitrification solutions A3-90 percent (37.5 percent glycerol + 15% DMSO + 15 percent EG + 22.5 percent sucrose) for 10 to 30 min or B1-100 percent (PVS3) for 40 to 120 min at room temperature, cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40 degree C) unloading solution (0.8 M sucrose) and further unloading for 20 to 60 min, depending on size and permeability of the materials. Using this systematic approach we can identify whether the material is tolerant or sensitive to chemical toxicity and to the osmotic stress of dehydration with vitrification solutions, thus revealing which is the main barrier in solution-based vitrification methods. Based on the sensitivity of samples we can design a droplet-vitrification procedure, i.e. preculture, loading, dehydration with vitrification solutions, cooling and rewarming. Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.

Carotenoid accumulation and characterization of cDNAs encoding phytoene synthase and phytoene desaturase in garlic (Allium sativum).

Phytoene synthase (PSY) and phytoene desaturase (PDS), which catalyze the first and second steps of the carotenoid biosynthetic pathway, respectively, are key enzymes for the accumulation of carotenoids in many plants. We isolated 2 partial cDNAs encoding PSY (AsPSY-1 and AsPSY-2) and a partial cDNA encoding PDS (AsPDS) from Allium sativum. They shared high sequence identity and conserved motifs with other orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of AsPSY1, AsPSY2, and AsPDS in the bulbils, scapes, leaves, stems, bulbs, and roots of garlic. High-performance liquid chromatography demonstrated that carotenoids were not biosynthesized in the underground organs (roots and bulbs), but were very abundant in the photosynthetic organs (leaves) of A. sativum. A significantly higher amount of ß-carotene (73.44 µg·g(-1)) was detected in the leaves of A. sativum than in the other organs.

Molecular cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in the phenylpropanoid biosynthesis pathway in garlic (Allium sativum).

The cDNAs encoding phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were cloned from garlic (Allium sativum) using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE) PCR. Amino acid sequence alignments showed that AsPAL and AsC4H have more than 70% amino acid identity with their homologues in other plants. The expression of AsPAL and AsC4H transcripts was highest in the roots but surprisingly low in the bulbils, where phenylpropanoid compounds are most concentrated. These results suggest that some phenylpropanoids are synthesized in the roots and subsequently transported to the bulbils of A. sativum .

Development of alternative plant vitrification solutions in droplet-vitrification procedures.

This study aimed at developing alternative vitrification solutions, modified either from the original PVS2 vitrification solution by increasing glycerol and sucrose and/or decreasing dimethylsulfoxide and ethylene glycol concentration, or from the original PVS3 vitrification solution by decreasing glycerol and sucrose concentration. The application of these vitrification solutions to two model species, i.e. garlic and chrysanthemum in a droplet-vitrification procedure, revealed that PVS3 and variants were superior to PVS2 and variants and that most PVS2 variants were comparable to the original PVS2. Both species were sensitive to chemical toxicity of permeating cryoprotectants and chrysanthemum was also sensitive to osmotic stress. The lower recovery of cryopreserved garlic shoot apices dehydrated with PVS2 and variants compared with those dehydrated with PVS3 and variants seemed attributed to cytotoxicity of the vitrification solutions tested as well as to insufficient protection against freezing injury. Chrysanthemum shoot tips were very sensitive to both chemical toxicity and osmotic stress and therefore, induction of cytotoxity tolerance during preconditioning was required for successful cryopreservation. The present study revealed that some of the PVS2 variants tested which have increased glycerol and sucrose and/or decreased dimethylsulfoxide and ethylene glycol concentration can be applied when explants are of medium size, tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.

Desiccation sensitivity and cryopreservation of Korean ginseng seeds.

Korean ginseng germplasm is maintained as clonal germplasm since there is no practical method for long-term seed conservation. The aim of this study was to establish a cryopreservation protocol for Korean ginseng seeds. Desiccation of undehisced ginseng seeds to a moisture content (MC) of 7.1 % did not decrease their dehiscence and germination. After cryopreservation, the dehiscence percentage of desiccated seeds decreased for MC above 12.5%; it was 26% for 22.6% seed MC and nil for 41.9% seed MC. Germination percentage did not decrease significantly between 12.5-22.6% seed MC, while germination percentage of dehisced seeds decreased below 7.2% MC, reaching 25.8% at 3.8% MC. After cryopreservation, the germination percentage decreased from 90.5-92.9% at 8.3-10.6% MC to 84.8% at 12.5% MC. At MCs below 8.3%, germination rapidly decreased from 85.0% at 7.2% MC to 34.9% at 5.3% MC. Therefore, the hydration window for cryopreservation of ginseng seeds is around 8-11% MC. Undehisced Korean ginseng seeds were characterized by their high lipid and protein content (lipids, 42.6% FW; proteins, 41.0% FW). When using thermal analysis, during the cooling phase, exothermic ice crystallization peaks were observed with dehisced ginseng seeds above 13.5% MCs (3.3 J/g FW). A second crystallization peak was detected following ice crystallization peaks.

Cryopreservation of cultivated and wild potato varieties by droplet vitrification: effect of subculture of mother-plants and of preculture of shoot tips.

In this paper, we studied the effect of subculture of mother-plants and of preculture of shoot tips of two potato varieties (Dejima, cultivated and STN13, wild) cryopreserved using the droplet-vitrification technique. The subculture conditions (light intensity, aeration and planting density) significantly affected survival of both non-cryopreserved and cryopreserved shoot-tips in both varieties. The subculture duration and the position of the shoot tips on the axis of the in vitro plantlets had a significant (P<0.0001) effect on survival of cryopreserved shoot tips. The optimal subculture duration was 7 and 5 weeks and the optimal size of shoot tips was 1.5-2.0 and 1.0-1.5 mm for var. Dejima and STN13, respectively. Survival of cryopreserved shoot tips was influenced by the sucrose concentration in the preculture medium and the preculture duration. The highest survival of cryopreserved shoot tips was observed after preculture with 0.3 M sucrose for 8 h followed by 0.7 M sucrose for 18 h. These results indicate that the parameters of the subculture of mother-plants and of preculture of shoot tips should be carefully optimized, especially in the case of wild species.

Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure.

The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.

Changes in sucrose and glycerol content in garlic shoot tips during freezing using PVS3 solution.

Changes in moisture content (MC), sucrose and glycerol concentration in garlic shoot tips were monitored during loading and unloading with PVS3 solution. Upon PVS3 treatment, shoot tip MC decreased rapidly and sucrose and glycerol concentrations increased rapidly during the first 30 min. Sucrose and glycerol concentrations increased more slowly thereafter. Shoot tip MC in after PVS3 treatment was affected by their size, but not by sucrose concentration of the preculture medium. As the size of shoot tips increased, so their MC increased after PVS3 treatment. However, sucrose and glycerol concentrations decreased after PVS3 incubation, and concentrations in dehydrated shoot tips were much lower than those measured in non-air dried controls. During unloading with 1.2 M sucrose medium, shoot tip MC increased rapidly during the first 10 min, whereas glycerol concentration decreased steadily over 90 min. Loading and unloading of PVS3 solution in garlic shoot tips follows the principle of solute bulk flow.

Thermal analysis of garlic shoot tips during a vitrification procedure.

The thermal behavior of garlic shoot tips was analyzed during the course of a vitrification protocol using the PVS3 vitrification solution. The size of shoot tips did not significantly influence the thermal behavior of garlic shoot tips. Though there was no significance, endo-thermal enthalpy from melting of crystalline ice increased as preculture duration increased to 6 days. Preculture on medium with 0.5 M sucrose significantly lowered exo- and endothermal enthalpies of dehydration-control shoot tips. By contrast, after dehydration with PVS3 solution, the concentration of sucrose in preculture medium had no significant effect on the value of enthalpies. A big thermal event was observed in garlic shoot tips air-dried for 1-3 h before dehydration. Both vitrification solution and dehydration duration significantly (P < 0.0001) influenced exo- and endothermal enthalpies. After dehydration with PVS1, PVS2, Fahy or Steponkus solutions for 120 min, only a small peak was detected in some shoot tips, but recovery of cryopreserved shoot tips was low. Dehydration duration with PVS3 solution significantly (P < 0.0001) influenced exo- and endothermal enthalpies and onset temperatures during cooling and warming. After dehydration for 150 and 180 min with PVS3 vitrification solution, no crystallization was observed during cooling and warming in most replicates, and recovery of cryopreserved shoot tips was highest (> 80%). There was a significant (P < 0.001) negative correlation between moisture content of shoot tips and concentration of sucrose and glycerol, and regeneration of cryopreserved shoot tips. By contrast, there was a significant (P < 0.001) positive correlation between MC and enthalpy of ice melting, and onset temperature of crystallization. Overall, the results of the analysis of the thermal behavior of garlic shoot tips coincide very well with their recovery after cryopreservation and provide a very useful tool for the establishment and optimization of cryopreservation protocols.

Assessment of desiccation sensitivity of tea embryos for cryopreservation.

Tea (Camellia sinensis (L.) O. Kuntze) has been reported as a species with recalcitrant seeds. The seeds can be stored for less than one year under high humidity conditions in a refrigerator at 5-7 degrees C. An efficient cryopreservation protocol for tea embryos using embryonic axes with portions of cotyledons still attached as drying material was established, which led to survival percentages around 92%. However, understanding the pattern of desiccation sensitivity, which is the key-limiting factor for cryopreservation, is of importance for implementation of cryopreservation using this protocol. In this study, the degree of desiccation sensitivity of tea seeds and cotyledonary embryonic axes (CEAs) was studied as a function of dehydration velocity, repeated dehydration-rehydration cycles, storage temperature, duration of storage of dried CEAs at room temperature, and seed harvesting date. This study suggests that there are no less than two mechanisms involved in desiccation sensitivity of tea seeds and embryos. Firstly, desiccation sensitivity of tea embryos occurs predominantly in a quantitative manner with continuous variation under intermediate dehydrated status rather than because of desiccation itself to a critical moisture content (MC). Secondly, desiccation sensitivity is due to the removal of the structural water at MCs of lower than 11.5%, when the EAs are flash-dried.

Evolution of DMSO concentration in garlic shoot tips during a vitrification procedure.

In this paper, the evolution of dimethylsulfoxide (DMSO) concentration and moisture content (MC) of garlic shoot tips was studied during the course of a vitrification protocol using the PVS2 vitrification solution. DMSO concentration of shoot tips increased rapidly, reaching 34.1 mg per g fresh weight after 20 min of PVS2 treatment and remained stable afterwards, while moisture content decreased from 82 to 60 percent, reaching 53 percent after 60 min. A reverse process was observed during unloading. There was a highly significant negative correlation between shoot tip moisture content and DMSO concentration during the dehydration and unloading treatments. Using unloading solutions with osmolarities between 0.42 and 2.29 Osm led to very different shoot tip MCs, between 63.55 and 81.24 percent, while DMSO concentration was between 14.83 and 19.97 mg per g fresh weight. After 24 h on recovery medium, DMSO concentration of shoot tips had decreased to 3.2 mg per g fresh weight.

Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions.

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.

Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification.

This paper investigates the effect of the origin and size of the explants employed and of the preconditioning (cold acclimation, preculture) and loading treatments on survival and regeneration of cryopreserved garlic shoot apices using vitrification with the PVS3 vitrification solution. Both the origin and size of explants had a significant effect on regeneration of cryopreserved apices. Higher regeneration was generally observed with apices excised from bulbs and bulbils, followed by cloves, and those originated from larger propagules regrew more rapidly. Smaller apices (1.5 or 3.0 mm in diameter) displayed higher regeneration than large ones (4.5 mm in diameter). Cold acclimation at 5 degree C of apices before freezing had no positive effect on regeneration after cryopreservation. Preculture of apices at 10 or 23 degree C for more than 3 days had a detrimental effect on regeneration. The optimal sucrose concentration in the preculture medium was 0.3-0.5 M. Loading apices for 30 or 60 min at 23 degree C in medium containing 2 M glycerol + 0.4 M sucrose or 1 M glycerol + 0.8 M sucrose had no effect on regeneration after cryopreservation, in comparison with apices cryopreserved without loading treatment. Under optimal conditions, regeneration of cryopreserved apices sampled from large cloves was above 90 percent.