Laminar Air-flow Cabinet Research Articles

The Medicinal Fungus Cordyceps militaris: Academia to Industry in Thailand

The medicinal mushroom (Cordyceps species) is a highly valued source of useful natural products having diverse biological activities. Cordyceps militaris (commonly known as orange caterpillar fungus), a kind of herbal drug and food additive mushroom, were used as a tonic food in Asia from ancient times. Their products have been developed from cultivated fruit bodies and fermented mycelia of C. militaris. Various studies of culture techniques will be expected to reduce contamination. A traditional methodology (autoclave) to sterilize culture media was compared with a protocol that included hydrogen peroxide to sterilize culture media (without autoclaving and inoculating cultures without the laminar airflow cabinet) for growing C. militaris CMRU strain. The results showed that the two methods had no significant differences in cordycepin production and biomass production. Production of C. militaris products is now becoming a large industry in Thailand.

In vitro propagation, cold preservation and cryopreservation of Taxus baccata L., an endangered medicinal and ornamental shrub

In vitro propagation, cold preservation, and cryopreservation are three essential approaches to preserve the genetic resources of red-listed plants, including English yew (Taxus baccata L.). Different concentrations of plant growth regulators (PGRs) and different pre-treatments of cold preservation and cryopreservation are the prerequisites of these three approaches. Apical bud as explant and Murashige and Skoog (MS) as the culture medium for all three sections of the research, kinetin (Kin) and indole-3-butyric acid (IBA) as PGRs for the micropropagation section, and encapsulation-dehydration as pre-treatment for the sections of cold preservation and cryopreservation were used. The results of the micropropagation section indicated that the highest number of shoots (5.40 per explant) and roots (5.98 per explant) were obtained from the culture of the explants on the media containing 1 mg L–1 IBA together with 1 and 2 mg L–1 Kin, respectively. The results of the cold preservation section revealed that the highest percentage of survival of germplasms (100%) after storage in the refrigerator was observed in the apical buds pre-treated by dehydration of encapsulated explants with 0.75 M sucrose for two hours, followed by dehydration under a laminar airflow cabinet for two hours. The results of the cryopreservation section demonstrated that the highest percentage of survival of germplasms (100%) after storage in liquid nitrogen was obtained in the apical buds pre-treated by encapsulation-dehydration under a laminar airflow cabinet for two hours. At the acclimatization stage, 100% of the plantlets acclimatized suitably with ex vitro conditions.

Validation of an automated dispensing system for subsequent dose dispensing of different radionuclides

BackgroundAutomated dispensing systems (ADSs) for radiopharmaceuticals have been developed to reduce the radiation exposure of personnel, to improve the accuracy of the dispensed dose and to limit the microbiological contamination. However, before implementing such systems, validation according to various applicable guidelines is necessary to ensure safety and quality. Here we present the selection, validation and implementation of the PT459R2 from manufacturer Lynax s.r.o. as a guidance protocol for validation according to GMP and GRPP guidelines. Validation included linearity accuracy and precision of the internal scintillation detector for different isotopes and microbiological validation for aseptic procedures.ResultsThe ADS can dispense accurate doses in the following linear range: 1000–10,000 MBq for lutetium-177, 20–74 MBq for zirconium-89, 100–1000 MBq for gallium-68 and 100–2000 MBq for fluorine-18. The maximum bias is 2.35% and the maximum coefficient of variation is 3.03% which meets the acceptance criteria of < 5%. Furthermore, the ADS does not affects the GMP class A environment in a laminar airflow cabinet and can dispense aseptically. In addition, radiation exposure is acceptable and data integrity is preserved.ConclusionThe PT459R2 ADS met all the requirements from our performance qualification and is therefore suitable for daily routine use in our center. Our approach can be used as a guidance for PQ of an ADS in a Radiopharmacy according to GMP and GRPP guidelines.

A Novel, Closed-Line Mini Extracorporeal Photopheresis (ECP) Technique Using the Therakos TM CellEx TM Photopheresis System, Developed for Patients with Contraindications Against Conventional ECP Therapy

Introduction: Conventional ECP has proven efficacy for treating several diseases, most significantly steroid-refractory chronic GVHD. Unfortunately, body weight and sufficient venous access limit the availability of this treatment modality. Children, in particular, usually have limited access to ECP. A simplified mini buffy coat ECP technique that allows treating small children and patients with apheresis contraindications has been tested in selected cases during the last 20 years. However, this method relies on an open-line system that requires a longer time and a cell processing laboratory, making this approach challenging. A new -closed-line- method, using the Therakos TM CellEx TM Photopheresis System, has been developed and evaluated in our Center. This method requires no processing laboratory or other special equipment and takes only 60-120 minutes, making the procedure potentially usable in routine practice. Design and methods: A 7-year-old girl had been treated at our Pediatric Bone Marrow Transplantation Unit with grade IV chronic steroid-refractory GVHD (skin, and less severe liver involvement). As all other options were exhausted, ECP became a desirable form of treatment. However, the patient's low body weight and serious venous access problems hindered conventional ECP treatment. We developed a solution that requires 200-250 ml whole blood collection from the patient, takes less than 2 hours, does not need special cell processing equipment, and allows the ECP treatment to commence in the form of a regular autotransfusion. Consent forms were obtained from the patient's caregivers as well as from the local Ethical Committe. The patient's age at the start of treatment was seven years, and her body weight was 28 kg. A total of 17 mini-ECPs were performed weekly up until the submission of this abstract, and the number of treatments per patient was 33. The duration of mini-ECP treatment has been five months. 5 to 8 mL/kg of whole blood was drawn from the patient. Separation of the white blood cell (WBC) rich buffy coat of the patient's blood - with donor blood product supplementation as needed - was performed through an individualized use of the CellEx TM photopheresis kit and blood prime program. The buffy coat was ultraviolet A (UVA)-irradiated after adding 8-methoxypsoralen (8-MOP) within the Therakos TM CellEx TM Photopheresis System and divided into two fractions under a laminar air flow cabinet. The autologous residual blood with the first fraction of the UVA irradiated buffy coat was transfused immediately. The second buffy coat fraction was stored at 4 °C and transfused the following morning through a peripheral vein. Results : The median volume of the collected whole blood was 250 mL (203-250 mL). The median volume was 72 ml (48-88 mL) for the first fraction of the irradiated buffy coat and 71 ml for the second fraction (48- 88 mL). Apoptosis and cell death were analyzed by annexin V staining; apoptosis and lymphocyte proliferation was measured by flow cytometry. UVA irradiation of the 8-MOP infused buffy coat resulted in significant induction of WBC apoptosis at 24 hours. No clinical side effects were observed during mini ECP treatment; all microbiological control tests were negative. Partial response was observed within three months (skin), and the tapering of systemic immunosuppression (methylprednisolone and ruxolitinib) began at five months. Conclusion: Mini whole blood ECP induces apoptosis and lymphocyte proliferation inhibition, both occurring after standard ECP. The closed-line method requires no special equipment and is not demanding for the patient, providing a viable solution to the technical difficulties children with the need for ECP treatment face.

Design and Construction of a Mini Laminar Airflow Cabinet to Support Laboratory Activities in Aseptic Condition

Design and Construction of a Mini Laminar Airflow Cabinet to Support Laboratory Activities in Aseptic Condition

The Development and Implementation of Airflow Visualization Studies ("Smoke" Studies) as a Training Tool in Aseptic Hospital Compounding Facilities.

In the compounding facilities of hospital pharmacies, extemporaneous preparations for parenteral administration are produced using aseptic handling. The designated environment for this practice is a clean area, such as a laminar airflow (LAF) cabinet placed in a classified cleanroom complying with good manufacturing practices (GMP) and International Organization for Standardization (ISO) 14644-1 guidelines. The European GMP Annex 1 (Revision 2020) and United States Pharmacopeia (USP) <797> monograph state that airflow visualization studies (“smoke” studies) should be performed to substantiate the cleanroom and LAF cabinet performance and their qualification status. Even though smoke studies are required by these guidelines, current literature does not describe detailed practical protocols and acceptance criteria. The objective of this study was to develop and implement a practical smoke study protocol to ensure compliance with aseptic handling guidelines in hospital pharmacies. First, a literature search was performed to collect information about smoke study protocols and acceptance criteria. Subsequently, a smoke study protocol was developed for a downflow and crossflow LAF cabinet as well as for grade C/B cleanroom areas. As a proof of concept, the smoke study protocol for the downflow LAF cabinet was executed in the at-rest and in-operation states. Video recordings of the smoke studies were analyzed to assess the performance of the cabinet. Finally, the video recordings obtained from the smoke studies were used in a training program for hospital pharmacy operators, which showed that smoke studies might aid in operators’ aseptic handling awareness. To the best of our knowledge, the present study provides for the first time a practical approach for the development of smoke study protocols in a hospital pharmacy setting and shows potential for training operators, process optimization, and continuous quality improvement.

Microbiological controls of aseptic handling in Dutch hospital pharmacies: Results, limits, and methods for assessing.

Introduction‘Aseptic handling is the procedure to enable sterile products to be made ready to administer using closed systems’ (EU Resolution CM/Res(2016)2). Microbiological controls are an important part of the overall assurance of process and product quality of aseptic handling. They consist of the End-of-Session Broth Test (ESBT) using Tryptone Soya Broth, Microbiological Monitoring (MM) using Ø 55- and 90-mm agar plates and a periodical Operator Broth Transfer Validation Test (OBTVT) using Tryptone Soya Broth. This study describes the results of these controls over a 7-year period, involving between 44 and 49 pharmacies (mostly hospital pharmacies). All pharmacies use a web-based programme for processing, evaluation and assessing microbiological controls (‘Microbio’). Aggregated results in Microbio are used for benchmarking and feedback information. ObjectiveThe objective of this study is to analyse the results of the 7-year period and to develop methods for assessing of, and determining realistic limits for, microbiological controls during aseptic handling. As secondary objective the role of Microbio is highlighted. Results and discussionResults of ESBT are expressed as Contamination Rate (CR), which is the percentage of units filled in a process simulation that are positive for microbial growth after incubation. Compared with the first 3 years of the study, the results in the last 4 years were significantly better: mean CRs are 0.20 and 0.11, respectively (p-value <0.01). For assessing CRs of ESBT, the approach ‘the more frequent samples with growth, the stronger the corrective actions’ was adopted. Levels of investigative and corrective actions, based on the 95% Upper Confidence Limit, are suggested. Microbiological Monitoring (MM) during aseptic handling into a laminar airflow cabinet or safety cabinet consists of settle plates, glove prints, contact plates of the worktop and surface bioburden determination of disinfected ampoules and vials. The results are expressed as the Contamination Recovery Rate (CRR), which is the rate at which MM samples contain any level of contamination. During the study period, the results of glove prints and contact plates improved substantially. The most probable explanation of this finding is improved disinfection procedures of the gloved hands, the worktop inside LAF/SC and the ampoules and vials. Results of settle plates did not change. There were too few results available to evaluate the surface bioburden of disinfected ampoules and vials. Benchmarking and feedback information from Microbio may have contributed to the improved ESBT and MM results. Results of the Operator Broth Transfer Validation Test (OBTVT) are expressed as Contamination Rate (CR). The target is zero samples with growth (CR = 0). The overall CR result over the study period is 0.50%. This is worse than ESBT (overall CR is 0.14%). This is probably due to the high number of critical steps in OBTVT compared to ESBT. ConclusionResults of microbiological controls improved during the study period. Realistic limits as well as methods for assessing ESBT and MM results are given and discussed.

First Record of Geotrichum candidum Causing Root Rot to Sugar beet (Beta vulgaris. L) in Wyoming and Michigan, USA

Sugar beet (Beta vulgaris L.) root rotten is caused by a number of soil-borne pathogens included Rhizoctonia solani, Aphanomyces cochlioides, Clonostachys rosea, Globisporangium ultimum, Rhizopus stolonifera, and Fusarium equiseti [1-4]. In September 2019, watersoaked soft rot with brown irregular lesions with a fermented smell, and thick foamy white mycelial growth symptoms were observed on sugar beet roots collected from Wyoming and Michigan (Figure 1 and 2). The symptoms covered approximately 40% of the sugar beet root surface. Diseased beet root tissues were excised from the junction of diseased and healthy tissue. Small pieces (10mm2) were surface sterilized with 10% sodium hypochlorite for 1min, rinsed thrice with sterile distilled water, air-dried on laminar airflow cabinet for 5 minutes, and transferred to corn meal agar (CMA), and clarified V8 (CV8)- incubated at 25°C with a 12-h photoperiod for 7 days. White, thin, flatted, and feathery colonies appeared on both media (Figure 3 and 4). Conidia were arthrosporous, mostly like chains, segmented, hyaline, subglobose, and single-celled (Figure 5). The dimension of conidia varied from 5-12 x 2-5 μm [5]. Based on microscopic and macroscopic characters, the fungus was speculated to be Geotrichum species. Ten isolates were developed by the single spore isolation technique. To confirm the species of the fungus, Genomic DNA was extracted from two representatives of the isolates and the internal transcribed spacer (ITS) region was amplified using the ITS1/ITS4 primers [6]. The amplified PCR products were cleaned and sent for Sanger sequencing by GenScript (GenScript, Piscataway, NJ). A Blastn search of the ITS sequences from the isolates showed 100% identity to those of Geotrichum candidum isolates KF713519.1 and MF782775.1.

Improving the aseptic transfer procedures in hospital pharmacies part C: evaluation and redesign of the transfer process

ObjectivesTo transfer sterile medical devices (SMD), infusion bags (IB), ampoules (A), injection vials (V) and infusion bottles (B) into a laminar airflow cabinet (LAF) or safety cabinet (SC) with a...

АПТЕЧНОЕ ИЗГОТОВЛЕНИЕ И КОНТРОЛЬ КАЧЕСТВА ЛЕКАРСТВЕННЫХ СРЕДСТВ ЗА РУБЕЖОМ

The purpose of this work was to study foreign experience in pharmacy manufacture and quality control of medicinal preparations (MP). Scientific literature and internet sources analysis, the author's own observations from 2005 to 2020 were made. It has been established that pharmacy manufacture of MPs abroad is carried out for the special needs of patients when pharmaceutical industry cannot produce them for various reasons. There are various approaches to standardization of the number of manufacturing pharmacies abroad: from complete prohibition of pharmacy manufacture of medicinal preparations in Portugal to mandatory license requirement in Austria and Germany. To manufacture medicinal preparations at a pharmacy the laboratory is established where necessary equipment, inventory, pharmaceutical substances and excipients are placed and the technological process is carried out. Pharmacy manufacture of sterile medicinal preparations is conducted under aseptic conditions in a specially equipped room or in a laminar-airflow cabinet. MPs must not be registered in most countries of the world. In the US and the European Union (EU) medicinal preparations are classified into medicines for immediate use and for prolonged storage. Expiration date must be stated for prolonged storage medicinal preparations, a dossier is made for them in the EU pharmacies. Pharmacies in most countries of the world manufacture medicinal preparations from tablets and capsules. Much attention abroad is given to ensure the quality of pharmacy manufacture of medicinal preparations: in the US, pharmacy manufacture of medicinal preparations is carried out in accordance with the requirements of Pharmacopoeia, in the EU according to the EU Council Resolution. A number of countries have developed guidelines or standard operational procedures for pharmacy manufacture of medicinal preparations. Constant training and assessment of employees’ competencies engaged in pharmacy manufacture of medicinal preparations is performed.

Application of cryobanking for Platycodon grandiflorum in vitro axillary buds using cryo-plate methods

The application of the V cryo-plate method using either PVS2 or PVS3 and the D cryo-plate method to Platycodon grandiflorum in vitro axillary buds was investigated. Precultured buds (1 mm) were attached to cryo-plates using calcium alginate gel. Then, osmoprotection treatment (2 M glycerol and 1 M sucrose) was performed by immersing the cryo-plates for 30 min at 25°C. The procedures for each cryogenic method were as follows: in the V cryo-plate method, the buds on cryo-plates were exposed to PVS2 for 40 min (highest regrowth 71.1%) or PVS3 for 50 min (highest regrowth 82.2%). In the D cryo-plate method, the buds on the cryo-plates were dehydrated under the air current of a laminar air flow cabinet for 60 min (highest regrowth 80.0%) at 25°C. Then, the cryo-plates were plunged directly into liquid nitrogen. After cryopreservation, buds on the cryo-plates were rewarmed and unloaded by immersion in 1 M sucrose solution for 15 min at 25°C for subsequent plant regeneration. The average regrowth seen on the V cryo-plates exposed to PVS3 and in the D cryo-plate method was higher than that of the V cryo-plates exposed to PVS2. The regrowth after cryopreservation in the V cryo-plate method with PVS2 was not stable. Thus, the V cryo-plate method with PVS3 and the D cryo-plate method are considered to be practical cryopreservation methods for P. grandiflorum germplasm.

Microbiological monitoring during aseptic handling: Methods, limits and interpretation of results

Aseptic handling is the procedure to enable sterile products to be made ready to administer using closed systems (EU Resolution CM/Res(2016)2). Microbiological monitoring (MM) and media fills are used for environmental and process control.In this study, the application of MM methods during aseptic handling inside, or related to working in, a laminar airflow cabinet or safety cabinet in hospital pharmacies is described and evaluated. Results are expressed as colony forming units (cfu) and Contamination Recovery Rate (CRR; the rate at which MM samples contain any level of contamination -USP<1116>-). For trend analysis, a rolling CRR is developed (a rolling CRR calculates a CRR using a predetermined number of most recent samples).Of all MM methods, glove print is the most informative. The added value of air sampling is doubtful. Because of microbiological as well as statistical considerations, the use of CRR for assessing MM results is advised. Glove prints, in general, give the highest CRR. A CRR < 10% is a realistic limit for MM during aseptic handling in hospital pharmacies. A rolling CRR, calculated using the last 100 samples, is a good compromise between reliability of the CRR value and timely prediction of process changes.

Reducing the risk of non-sterility of aseptic handling in hospital pharmacies, part B: risk control

ObjectivesTo determine prospectively the risk reducing measures of non-sterility during aseptic handling and to develop a method for prioritising these measures.MethodsIn the first part of this series of articles, we...

Cryopreservation of oil palm (Elaeis guineensis Jacq.) polyembryoids via encapsulation\u2013desiccation

The current report assesses the efficiency of encapsulation–desiccation protocol to cryopreserve oil palm (Elaeis guineensis Jacq.) polyembryoids. Specifically identified polyembryoids, comprising of haustorium and torpedo-shaped structures, were encapsulated [comprising 3% (w/v) sodium alginate and 100 mM CaCl2]. Calcium alginate-encapsulated and sucrose-precultured polyembryoids were subjected to different spans of desiccation in a laminar air-flow cabinet, followed by freezing in liquid nitrogen. The effect of sucrose preculture (with gradual exposure to 0.3, 0.5, 0.75 and 1 M for 7 days) and dehydration periods (0–10 h) under sterile air-flow on post-freezing survival and regrowth of encapsulated polyembryoids were studied. Cryopreserved and thawed polyembryoids (initially precultured in sucrose, followed by 9 h air-desiccated to 23.3% moisture content) displayed the highest survival percentage (73.3%) and regeneration (of shoot, root and secondary somatic embryo) on Murashige and Skoog regrowth medium containing sucrose (0.3–1 M) and 0.2 mg/l 2,4-dichlorophenoxy acetic acid. In addition, ultrastructural study using scanning electron microscopy exhibited successful revival of cryopreserved polyembryoids, owing to retention of cellular membrane stability through optimized and protected (encapsulated) desiccation. The present study thus substantiates the potential of this encapsulation–desiccation procedure in cryopreservation of oil palm polyembryoids for long-term conservation programs.

Cryopreservation of Habenaria radiata and Habenaria rhodocheila seeds using the aluminum cryo-plate vitrification method

The genus Habenaria consists of 600 species of terrestrial orchids distributed in Asia, America and Africa. Habenaria rhodocheila Hance is found in Thailand, Myanmar, Cambodia and Laos. Habenaria radiata (Thunb.) K. Spreng is native to Japan. The number of both species has decreased in their habitats because of overcollecting and environmental changes in the habitats. Conservation of the surviving plants is an urgent issue to maintain the genetic diversity of the species. Seeds of H. radiata and H. rhodocheila were surface sterilized, desiccated in a laminar air-flow cabinet, and then subjected to the cryo-plate vitrification method. Viability of the seeds with or without immersion in liquid nitrogen (LN) was estimated by seed germination. The seeds of H. radiata showed high viability after immersion in LN without PVS2 treatment, and the viability decreased when exposure to PVS2 exceeded 40 min. On the other hand, viability of the H. rhodocheila seeds after immersion in LN increased with PVS2 treatment for 10 to 30 min, stayed at high levels with 40 to 60 min of PVS2 treatment, and then decreased with more than 90 min of PVS2 treatment.

Efficiency of aluminum cryo-plates for cryopreservation of Dendrobium signatum Rchb. f. pollinia

The efficiency of V cryo-plate and D cryo-plate methods for cryopreservation of pollinia of Dendrobium signatum Rchb. f., a Thai orchid was investigated. Pollinia were collected from flowers and then placed on the aluminum cryo-plate containing 12 wells embedded with 3% sodium alginate gel. In V cryo-plate method, cryo-plates with pollinia were immersed in 0.6 M sucrose + 2 M glycerol loading solution (LS) for 15 min, then dehydrated with PVS2 solution for 40 min at room temperature (29±2°C). In D cryo-plate method, cryo-plates with pollinia were treated with LS for 15 min, then dehydrated in a laminar air-flow cabinet for 3 h at room temperature (29±2°C). In both methods, cryo-plates were directly plunged into liquid nitrogen for 40 min, and rapidly warmed in 1.2 M sucrose for 15 min. The cryopreserved and non-cryopreserved pollinia were used to pollinate flowers of the same species. The results showed that cryopreserved pollinia retained fertilizing ability, and had similar pod formation as those pollinated with non-cryopreserved pollinia. The pod formation from cryopreserved pollinia in V cryo-plate and D cryo-plate methods were 55.6 and 50%, respectively. Seeds from non-cryopreserved and cryopreserved pollinia were successfully produced and germinated into plantlets with well-formed leaves and roots. Multiple stems (4.8 stems plant(-1)) produced from one plantlet when cultured on modified VW agar medium (1949) supplemented with 100 g L(-1) banana, 150 mL coconut water, 50 g L(-1) potato, and 20 g L(-1) sucrose for 120 d at 25±2°C. Cryopreserved pollinia using aluminum cryo-plate can be used successfully for pollination.

Cryopreservation ofArundina graminifolia(D. Don) Hochr. seeds using D cryo-plate method

In this study, cryopreservation of Arundina graminifolia seeds by D cryo-plate and encapsulation dehydration technique were investigated. The results showed that the D cryo-plate method gave the highest regrowth of 82%, followed by encapsulation-dehydration (74%). The D cryo-plate protocol is as follows: pour the alginate solution containing 2% (w/v) sodium alginate in calcium-free ½ MS basal medium with 0.4 M sucrose in the wells of the cryo-plate. For encapsulation-dehydration the protocol is the same as D cryo-plate without using cryo-plate. Place 20 to 50 seeds from 3-month-old selfing capsules in each well. Pour calcium chloride solution containing 0.1 M calcium chloride in ½ MS basal medium with 0.4 M sucrose. Expose to 0.4 M sucrose + 2 M glycerol (loading solution) for 30 min and then dehydrate under a laminar air-flow cabinet for 3 h. Put each cryo-plate in a 2-mL cryotube and plunge into liquid nitrogen for 1 d. Warm in 1.2 M sucrose solution (unloading solution) for 15 min then remove the solution and culture cryopreserved beads on ½ MS agar medium.

Encapsulation-vitrification and encapsulation-dehydration cryopreservation of Cattleya labiata Lindley, a threatened Brazilian orchid

Orchid populations have been decreasing due to habitat loss and over-collection for ornamental use. Consequently, there is an urgent need to conserve germplasm resources. Cryopreservation of the threatened orchid Cattleya labiata L. was successfully achieved using encapsulation-vitrification and encapsulation-dehydration cryopreservation methods. Protocorms, of 0.1 cm in diameter, developed from 2-months-old germinating seeds and encapsulated in sodium alginate-beads, were precultured in half-strength Murashige and Skoog liquid medium containing 0.4 M sucrose on a shaker (110 rpm) at 25°C for 24 h. For encapsulation-dehydration, beads were treated in the loading solution (2 M glycerol and 0.4 M sucrose in half-strength Murashige and Skoog liquid medium) for 20 min and then exposed to a sterile air-flow from the laminar air-flow cabinet for 0, 1, 2, 4 or 6 h. For encapsulation-vitrification, beads were treated in the loading solution for 20 min and then exposed to PVS2 solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethyl sulfoxide in half-strength Murashige and Skoog liquid medium containing 0.4 M sucrose at pH 5.7) for 0, 10, 20, 30 and 40 min. Comparing the two methods tested, it was observed that regeneration of cryopreserved protocorms using encapsulation-dehydration was higher (42%) with 4 h dehydration in laminar air-flow cabinet, whereas using the dehydration-vitrification procedure it was 27% after dehydration for 10 min in PVS2 solution. Seedlings developed from these methods did not show any abnormal characteristics of growth in vitro, and were successfully hardened in greenhouse showing 80% survival after 120 days of acclimatization.

Cryopreservation of somatic embryos from Petiveria alliacea L. by different techniques based on vitrification

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.

Cryopreservation of Date Palm Pro-Embryonic Masses Using the D Cryo-plate Technique.

In this chapter, we describe a cryopreservation (liquid nitrogen, -196°C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171g/L sucrose for 3days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20min with a loading solution containing 184g/L glycerol and 136.8g/L sucrose. They were then dehydrated for 90-120min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15min in an unloading solution containing 410.4g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3days in the dark on posttreatment medium containing 102.6g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.