Search

Copyright of Revista Foco (Interdisciplinary Studies Journal) is the property of Revista Foco and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Cryopreservation has the potential for long-term germplasm storage. The metabolic pathways and gene regulation involved in cryopreservation procedures are still not well documented. Hence, the genetic expression profile was evaluated using RNA-Seq in zygotic embryos of grapevines subjected to cryopreservation by vitrification. Sequencing was performed on the Illumina NextSeq 500. The average alignment of reads was 96% against the reference genome. The expression profiles showed 229 genes differentially expressed (186 repressed and 46 induced). The main biological processes showing upregulated enrichment were related to nucleosome assembly, while downregulated processes were related to organ growth. The most highly repressed processes were associated with the organization of the cell wall and membrane components. The unnamed protein product and 17.3 kDa class II heat shock protein (HSP) were highly induced, while ATPase subunit 1 and expansin-A1 were repressed. The response to the cooling and warming process during cryopreservation probably indicates that the changes occurring in transcription may be related to epigenetics. In addition, the cell exhibits an increase in the reserve of nutrients while seeking to survive modestly using available energy and pausing the plant's development. Additionally, energy containment occurred to cope with the stress caused by the treatment where deactivation of components of the cell membrane was observed, possibly due to changes in fluidity caused by alterations in temperature. [ABSTRACT FROM AUTHOR]

Peppermint is an important aromatic-medicinal plant species and it has valuable essential oil contents such as menthol, linalool and limonene. The main purpose of the current study was to optimize a protocol for cryopreservation of M. piperita local cultivars (G-74, Candarli and Gomec). The secondary aim was to investigate the genetic stability after cryopreservation using ISSR marker system. Three different single step freezing techniques were compared for longterm preservation of these local cultivars and the optimum regeneration percentages were acquired by using the droplet vitrification technique. The PVS2 treatments of this technique showed succesful long-term preservation of M. piperita cultivars with regeneration percentages of 72% to Candarli cultivar (60 min PVS2), 52% to Gomec cultivar (75 min PVS2) and 62.5% to G-74 cultivar (30 min PVS2) respectively. The ISSR PCR results showed that the genetic stability from cryopreserved M. piperita cultivars were high. While genetic stability percentage was ~99% for the Candarli cultivar, for the Gomec and G-74 cultivars the genetic fidelity was100%. The shoots that come from the cryopreserved shoot tips showed normal and health rooting, and all of them were also easily adapted to greenhouse conditions. [ABSTRACT FROM AUTHOR]

Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP’s cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes. [ABSTRACT FROM AUTHOR]

Numerous plant species around the world suffer from the presence of viruses, which especially in economically important crops, cause irretrievable damage and/or extensive losses. Many biotechnological approaches have been developed, such as meristem culture, chemotherapy, thermotherapy or cryotherapy, to eliminate viruses from infected plants. These have been used alone or in combination. In this work, meristem culture, thermotherapy and cryotherapy were compared for Apple mosaic virus elimination from hazelnut local cultivar “Palaz”. The virus-free plant was also confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) after each treatment and, the best results were obtained by cryotherapy. A one step freezing technique, droplet vitrification, was used for cryotherapy, and the best regeneration percentage was 52%. After cryotherapy, virus-free seedlings of hazelnut local cultivar “Palaz” were confirmed as being virus-free after three subcultured periods.

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots. [ABSTRACT FROM AUTHOR]

Abstract The objective of this study was to evaluate the efficiency of cryoprotective solution (PVS2) combined with phloroglucinol for the cryopreservation of seeds of two orchid species, Encyclia cordigera and Epidendrum ciliare. Seeds of Encyclia cordigera had 91.03% initial viability and 91.99% germination. The treatment of the seeds with PVS2 at 0 °C with 1% phloroglucinol for 60 min returned 93.79% viability and 91.01% germination after recovery from LN, consequently resulting in faster development of protocorms. For Epidendrum ciliare, seed viability was 85.65% and germination was 85.90%. Seed exposure to the PVS2 at 0 °C with 1% phloroglucinol for 180 min showed viability of 39.23% and germination of 37.88%. Despite lower germination, 78.90% of the protocorms reached stage P3 of development, when evaluated 45 days after sowing, not significantly different from the control 1, and showed normal development. These results indicate that PVS2 cryoprotective solution is efficient when combined with phloroglucinol for the cryopreservation and successful recovery of seeds of Encyclia cordigera and Epidendrum ciliare. The present study also indicates that response to cryopreservation and success of recovery after cold storage is species-specific and requires adjustments in exposure time to PVS2 at 0 °C prior to immersion in LN.

Hops (Humulus lupulus L.) is essentially used in the brewing industry as it contributes to flavor, and aroma of beer. However, the genetic diversity of hops is increasingly threatened by diseases, environmental changes, and urbanization. Cryopreservation has emerged as a pivotal strategy for safeguarding and maintaining the genetic diversity of hops. The present work presents a comprehensive study on the cryopreservation of hops, focusing on the development and optimization of a droplet vitrification based cryopreservation protocol. Shoot tips excised from one month old in vitro cultures were precultured on 0.3 M sucrose, dehydrated in a loading solution followed by treatment with PVS2 solution for different durations. Significant effect of PVS2 dehydration was observed on post-thaw survival and regeneration after cryoconservation with maximum 50% post-thaw regeneration observed in shoot tips dehydrated in PVS2 for 30 min. Genetic fidelity of the regenerated plants was confirmed using 30 ISSR markers. Reproducibility of the developed protocol was tested on seven other accessions and post thaw regeneration ranging from 43 to 70% was observed across the accessions. The present study reports a highly efficient protocol for conservation of hops germplasm. The results indicate that droplet vitrification can be used as a reliable and sustainable approach for hop genetic preservation, with high survival rates and minimal genetic alterations observed in cryopreserved samples. To the best of our knowledge, this is the first report on DV based cryopreservation of hops germplasm. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the efficiency of cryoprotective solution (PVS2) combined with phloroglucinol for the cryopreservation of seeds of two orchid species, Encyclia cordigera and Epidendrum ciliare. Seeds of Encyclia cordigera had 91.03% initial viability and 91.99% germination. The treatment of the seeds with PVS2 at 0 °C with 1% phloroglucinol for 60 min returned 93.79% viability and 91.01% germination after recovery from LN, consequently resulting in faster development of protocorms. For Epidendrum ciliare, seed viability was 85.65% and germination was 85.90%. Seed exposure to the PVS2 at 0 °C with 1% phloroglucinol for 180 min showed viability of 39.23% and germination of 37.88%. Despite lower germination, 78.90% of the protocorms reached stage P3 of development, when evaluated 45 days after sowing, not significantly different from the control 1, and showed normal development. These results indicate that PVS2 cryoprotective solution is efficient when combined with phloroglucinol for the cryopreservation and successful recovery of seeds of Encyclia cordigera and Epidendrum ciliare. The present study also indicates that response to cryopreservation and success of recovery after cold storage is species-specific and requires adjustments in exposure time to PVS2 at 0 °C prior to immersion in LN. [ABSTRACT FROM AUTHOR]

Numerous plant species around the world suffer from the presence of viruses, which especially in economically important crops, cause irretrievable damage and/or extensive losses. Many biotechnological approaches have been developed, such as meristem culture, chemotherapy, thermotherapy or cryotherapy, to eliminate viruses from infected plants. These have been used alone or in combination. In this work, meristem culture, thermotherapy and cryotherapy were compared for Apple mosaic virus elimination from hazelnut local cultivar "Palaz". The virus-free plant was also confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) after each treatment and, the best results were obtained by cryotherapy. A one step freezing technique, droplet vitrification, was used for cryotherapy, and the best regeneration percentage was 52%. After cryotherapy, virus-free seedlings of hazelnut local cultivar "Palaz" were confirmed as being virus-free after three subcultured periods. [ABSTRACT FROM AUTHOR]

Cryopreservation combined with in vitro culture offers a safe and cost-effective method to conserve germplasm. Conservation of Persea spp. has been limited to heterozygous somatic embryos that are not true-to-type. A method for shoot-tip cryopreservation is vital to preserve the exact gene pool of interest. For the first time cryopreservation protocols for mature shoot tips of two avocado cultivars (cvs) 'Velvick' and 'Reed', were established. In vitro shoots were subjected to two different optimised pre-treatments; (1) cv 'Velvick'—high sucrose (0.3 M) or (2) cv 'Reed'—low temperature (10 °C) incubation, over a 2-week period prior shoot tip dissection. Two different plant vitrification solutions, plant vitrification solution 2 (PVS2) and vitrification solution L (VSL) were tested at 0 °C for 0, 10, 20, 30 and 40 min. Vitrified shoots were evaluated for survival and regrowth at 2 and 8 weeks after vitrification treatment and either with or without liquid nitrogen exposure. The study revealed that the optimal exposure time for each cultivar varied with the cryoprotectant used. After liquid nitrogen cv 'Velvick' highest regrowth levels were observed with 20 min exposure to either PVS2 or VSL, however, vigorous plants were produced only from VSL treated shoots. In the case of cv 'Reed' highest regrowth levels were observed with 10 min exposure to PVS2 however only morphologically normal plants were recovered from VSL treated shoots. Key message: Cryopreservation of avocado shoot tips was successful using PVS2 and VSL with both recording similar recovery rates for 'Velvick' and 'Reed'; although only vigorous and morphologically normal plants were developed from VSL treatments. [ABSTRACT FROM AUTHOR]

Cryopreservation (also called as cryobanking) of gel-embedded shoot tips on the cryo-plate dehydrated by vitrification solution (V cryo-plate method) was examined for saving plant resources of hybrid aspen. Cold-hardened shoot tips of in vitro grown hybrid aspen were precultured for 1 day at 25℃ on solidified 1/2 Murashige and Skoog (MS) medium containing 0.3 M sucrose. Pretreated shoot tips were placed in elliptical wells on an aluminium cryo-plate and embedded in calcium alginate gel. Osmoprotection treatment was performed in a loading solution containing 2.0 M glycerol and 1.0 M sucrose for 30 min at 25℃. After the shoot tip samples on the plates were treated with the plant vitrification solution 2 for 20 min at 25℃, the samples on the plates were directly plunged into liquid nitrogen. For rewarming, shoot tip samples on the cryo-plates were immersed in 1.0 M sucrose solution at 25℃. Then, shoot tips in the gels were regrown at 25℃ on solidified 1/2 MS medium. The regrowth rate of cryopreserved shoot tips was almost 100%, and normal shoot growth was observed without callus formation. This protocol appears to be a promising technique for cryobanking of hybrid aspen genetic resources. [ABSTRACT FROM AUTHOR]

Main conclusion: This paper reviews the cryopreservation of the ornamental, carnation (Dianthus caryophyllus L.), as an important method for the long-term preservation of this plant’s germplasm. Carnation (Dianthus caryophyllus L.) is an important ornamental plant that is used as a potted plant as well as a cut flower. Important Dianthus germplasm would benefit from long-term strategies such as cryopreservation. Unlike the in vitro tissue culture literature of this ornamental, which has been studied in considerable detail, and with several genetic transformation protocols, surprisingly, the literature on its cryopreservation is still fairly scant, with barely two dozen or so studies, mostly having employed shoot tips. Early (< 2007) and more recent (2007–2020) cryopreservation techniques for carnation, including ultra-rapid cooling, encapsulation-vitrification, and encapsulation-dehydration, efficiently replaced programmed slow cooling processes used in early studies in the 1980s. Two large gaps (1997–2006, and 2016–2020) in which no carnation cryopreservation studies were published, requires future studies to cover new knowledge to fill gaps in information. Carnation cryopreservation research would benefit from testing a wide range of in vitro explants, new techniques such as the cryo-mesh, improved regeneration protocols for post-cryopreserved material, and the use of low-temperature storage as a mid- to long-term complementary germplasm storage strategy. This mini-review provides details of what has been achieved thus far and future objectives that could fortify cryopreservation research of this ornamental, as well as provide a robust long-term germplasm repository. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine the optimum parameters for high regrowth following cryostorage (− 196 °C) of seven endemic and endangered Dianthus species. A cryopreservation approach based on a droplet-vitrification protocol was successfully applied using explants (shoot apices and axillary buds) from a collection of in vitro grown Dianthus species. The plants were micropropagated for two years prior using them as explant donors. Osmoprotection in different sucrose concentrations and various dehydration durations in the plant vitrification solution (PVS2) were tested to assess survival and regrowth following cryostorage. The regrowth rates after cryostorage ranged between 63% (D. glacialis ssp. gelidus) and 73% (D. nardiformis) and were achieved after osmoprotection in 0.5 M sucrose and 30 min dehydration in PVS2 for D. glacialis ssp. gelidus and osmoprotection in 0.25 M sucrose and 30 min dehydration in PVS2 for D. nardiformis. The morphogenetic response to liquid nitrogen storage was direct multiple shoot formation for both non-cryopreserved and cryopreserved explants for all species. This biotechnological approach can be efficiently applied for the ex situ conservation of endemic and endangered Dianthus species to ensure the long-term conservation of biodiversity. [ABSTRACT FROM AUTHOR]

The objective of this research was to investigate the combination effects of phosphate and sodium chloride (NaCl) on the quality of frozen Nile tilapia fillets (control and treated with sodium tripolyphosphate 1.4 % STPP + 2.7 % NaCl) during storage at -18 °C for up to 8 months. Results showed that moisture content decreased slightly (P = 0.05), while pH gradual decreased, total volatile base nitrogen (TVB-N) increased, and hardness and gumminess decreased with increasing time (P = 0.05). Thiobarbituric acid-reactive substances (TBARS) values were low (0.01 - 0.03 mg malonaldehyde/kg) and phosphate content ranged from 3350 - 3900 mg/kg. There was no significant difference (P > 0.05) in drip loss during storage. The control had higher cooking losses and the L* value increased with increasing storage time, while a*, b*, C*, and h* values were not significantly different (P > 0.05). Appearance and texture acceptability scores of treated fish were significantly higher than the control throughout storage (P = 0.05). Total aerobic psychrophilic and mesophilic bacteria were relatively unchanged at about 4 log CFU/g. [ABSTRACT FROM AUTHOR]

Optimized cryopreservation protocols are essential for the safe, cost-effective and long-term conservation of germplasm of great economic interest, such as Guinea (Petiveria alliacea L.), a medicinal species that synthesizes a great diversity of bioactive substances, among them polysulfides with antitumor activity. Somatic embryos (SEs) produced from in vitro roots were cryopreserved using the V-cryoplate technique. Their viability was measured using the triphenyltetrazolium test and their recovery by counting the number of secondary SEs produced per cryopreserved SE 90 days after liquid nitrogen (LN) exposure. Structural alterations were evaluated qualitatively and quantitatively during the successive stages of the protocol. SEs were dehydrated in 0.5 M sucrose, attached to cryoplates using sodium alginate solution (3%) and treated with PVS2 for different periods. After immersion in LN, SEs were rewarmed in unloading solution (1.2 M sucrose) at room temperature (25 °C) for 20 min. Viability based on the triphenyltetrazolium test was 100% after 15 min of treatment with PVS2 and LN exposure, and recovery, based on multiplication rate, was 21 somatic embryos produced per cryopreserved somatic embryo after 90 days of culture. The histological analysis of cryopreserved somatic embryos showed plasmolysis in the different cell types after treatment with PVS2, with meristematic and parenchymatic cells presenting a higher plasmolysis level. However, after rewarming, the level of plasmolysis decreased over cultivation time, reaching only 2% in meristematic cells after 30 days, indicating the good ability of Petiveria alliacea SEs to develop cryotolerance under the experimental conditions tested. [ABSTRACT FROM AUTHOR]

This study evaluated the effect of vitrification solutions and exposure time on the cryopreservation of Brazilian green dwarf coconut plumules (BGD) using the droplet vitrification technique. Explants were excised from BGD mature fruits from the Active Germplasm Bank of Embrapa Tabuleiros Costeiros, Sergipe, Brazil. Firstly, embryos were disinfected, and after excision, plumules were pre-cultivated for 72 hours in Y3 + 0.6 M sucrose + 2.2 g L-1 Gelrite® culture medium. Plumules were exposed to PVS2 and PVS3 solutions for 15 and 30 minutes and rapidly immersed in liquid nitrogen (-196 °C). After cryopreservation, they were thawed in culture medium solution (Y3 + 1.2 Msucrose) and cultured in regeneration medium. The experimental design was completely randomized in a 2x2 factorial scheme (vitrification solutions per exposure times), with five replicates per treatment. Data were compared by the Tukey 's test at 5% probability. Significant differences were observed in the callogenesis percentage for the solutions x exposure time interaction for non-cryopreserved cultures (-NL) and for exposure time after cryopreservation (+NL). PVS2 and PVS3 combined with 15 minutes of exposure promoted the highest callus formation (70 and 100%, respectively) in control cultures. The exposure time of 30 min, regardless of vitrification solution, resulted in 30% embryogenic callus formation after cryopreservation. These results contributed to the long-term conservation of coconut palm. [ABSTRACT FROM AUTHOR]

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.

Cryopreservation is one of the most appropriate and safest tool to preserve plant germplasm species including plants seeds. In this temperature, physiologic activities and metabolic processes of the seeds and plant members are almost held up and seeds are able to survive at unlimited time periods. In this research, in order to preserve seeds of medicinal plant Foeniculum vulgare L.(Apiaceae) in cryopreservation condition, we used three pre-treatments: Glycerol 30 %, and Plant vitrification solution 2 and Desiccation. After a week all of treated seeds were extracted from the liquid Nitrogen(LN) and were melted in Bain-Marie heater with 42°C. Then seeds were transferred to a germinator in 24°C and 16/8H optical period. Results analyzed statistically through laboratory exploration. Results show, Foeniculum vulgare L. seeds' survival in LN are maintained in all three treatments among which Glycerol 30 % treatment has the highest survival percentage 65%, then Desiccation() and PVS2 with 53% and 15% have germinated, comparing the control 83%. Thus, Glycerol 30 % is recommended as appropriate alternative to preserve this plant seeds for long periods of time in preserving centers of plant germplasm preservation.

ABSTRACT: This study evaluated the effect of vitrification solutions and exposure time on the cryopreservation of Brazilian green dwarf coconut plumules (BGD) using the droplet vitrification technique. Explants were excised from BGD mature fruits from the Active Germplasm Bank of Embrapa Tabuleiros Costeiros, Sergipe, Brazil. Firstly, embryos were disinfected, and after excision, plumules were pre-cultivated for 72 hours in Y3 + 0.6 M sucrose + 2.2 g L-1 Gelrite® culture medium. Plumules were exposed to PVS2 and PVS3 solutions for 15 and 30 minutes and rapidly immersed in liquid nitrogen (-196 ºC). After cryopreservation, they were thawed in culture medium solution (Y3 + 1.2 M sucrose) and cultured in regeneration medium. The experimental design was completely randomized in a 2x2 factorial scheme (vitrification solutions per exposure times), with five replicates per treatment. Data were compared by the Tukey’s test at 5% probability. Significant differences were observed in the callogenesis percentage for the solutions x exposure time interaction for non-cryopreserved cultures (-NL) and for exposure time after cryopreservation (+NL). PVS2 and PVS3 combined with 15 minutes of exposure promoted the highest callus formation (70 and 100%, respectively) in control cultures. The exposure time of 30 min, regardless of vitrification solution, resulted in 30% embryogenic callus formation after cryopreservation. These results contributed to the long-term conservation of coconut palm.

• An efficient, sustainable and genotype-wide cryopreservation protocol developed for dahlia germplasm conservation. • The protocol was tested for its sustainability for 6 years of cryobanking. • This is the first report on cryopreservation of dahlia germplasm. Dahlias are the one amongst the tuberous herbaceous plants, valued for their attractive colorful flowers with varied size cultivated due to its ornamental value as a potted plant or cut flower in many countries. Dahlia germplasm was being conserved in the in vitro Genebank of ICAR- NBPGR, New Delhi since 2005–06 under normal growth and slow growth with a regular subculturing in every 6 months and 1 year respectively, which is laborious and time-consuming. Hence, in the current study we attempted to develop an efficient droplet-vitrification cryopreservation protocol for safe and long-term conservation of dahlia germplasm. Significant effect of type and position of explants, plant vitrification solution-2 (PVS2) dehydration duration, pregrowth period and loading solution treatment duration on post-thaw regrowth after cryoconservation was observed. Shoot tips, each being 1.0 mm in length and 0.5 mm in width positioned at the tip of in vitro shoots with a similar developmental stage (similar shoot length) were excised from in vitro cultures pregrown for 6 wks on B5 basal medium supplemented with 0.5 mg/l BAP, 30 mg/l silver nitrate and 3% sucrose. The excised shoot tips were precultured on MS + 0.3 M sucrose for overnight followed by loading solution (2.0 M glycerol and 0.4 M sucrose in liquid MS medium) treatment for 80 min, PVS2 dehydration for 30 min and then transferred onto PVS2 droplets on aluminum foil strips and cryostored in liquid nitrogen (LN). An average of 44.44% post-thaw regrowth was obtained in six accessions tested and no significant difference among the accessions was observed. There was no significant loss in regrowth of cryostored shoot tips after 1 year and 6 years of cryobanking. This justifies the genotype-wide and sustainability of the protocol for cryoconservation of Dahlia germplasm using droplet-vitrification cryopreservation procedure in the cryobanks. To the best of our knowledge, this is the first report on cryoconservation of Dahlia germplasm. [ABSTRACT FROM AUTHOR]

Common cypress (Cupressus sempervirens L.) is one of the most widespread species in the Mediterranean area. It has been traditionally cultivated for its ornamental value, becoming a typical feature of urban and rural landscapes, and high timber quality. In the last 30 years, cypress has been subjected to important breeding programmes, aimed to select clones tolerant to the widespread canker caused by the pathogenic fungus Seiridium cardinale, leading to various patented varieties today, available on the market, as well as for genotypes producing null or low amount of allergenic pollen. Somatic embryogenesis is a suitable in vitro regeneration method for fast cloning of conifer trees, and the cryopreservation of embryogenic callus is a significant tool for the safe long-term conservation of valuable cell lines. Recently, a complete protocol for the production of cypress plants from somatic embryogenesis was developed for the patented clone 'Mediterraneo'. Here, the coupling of somatic embryogenesis and cryopreservation may offer a superior tool to propagate and maintain superior genotypes of cypress by overcoming repetitive subculturing of selected embryogenic callus lines. For the above, this study aimed to compare different cryopreservation techniques (PVS2-based vitrification and slow cooling) with the 'Mediterraneo' embryogenic callus line. Best results were obtained after the optimization of a slow cooling procedure, based on the 30-min treatment of embryogenic masses with a cryoprotective solution containing 180 g l-1 sucrose and 7.5% DMSO, followed by the reduction of the temperature at a rate of -1 °C min-1 up to -40 °C and the subsequent immersion in liquid nitrogen ("two-step freezing"). [ABSTRACT FROM AUTHOR]

The cryopreservation has revolutionized the field of biotechnology. Frozen in liquid nitrogen (LN) preserves long living cells. In this sense, in this paper were evaluated three conditions of cryopreservation based on vitrification of buds of grapevine. The buds were subjected to the PVS2, PVS3 and glycerol for 0-420 min, and submerged in LN for one hour. After the incubation time electrolyte leakage was determined as a viability measurement, and tissue damage was evaluated through stereoscopic observation. Based on the viability percentage the best preservation method was using PVS3 solution (30%) followed by glycerol (25%) and PVS2 (<10%). The images of the buds exposed to PVS3 shows no tissue damage unlike to PVS2 and glycerol, were not sufficient to preserve buds tissue. The results shown here suggest that using PVS3 as protocol can be considered for buds grapevine germplasm preservation [ABSTRACT FROM AUTHOR]

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [ Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% ( v/v) dimethyl sulfoxide (DMSO), 15% ( v/v) ethylene glycol, 30% ( v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% ( v/v) glycerol, 50% ( w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% ( w/v) PEG 8000, 10% ( w/v) glucose, 10% ( v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% ( v/v) DMSO or 15% ( v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures. [ABSTRACT FROM AUTHOR]

The objective of this work was to evaluate the phytotoxicity of a plant vitrification solution (PVS2), and the survival of shoot tips of the sugarcane variety SP716949, after cryopreservation by droplet-vitrification. Shoot tips were precultured for 24 hours in MS medium containing 0.3 mol L-1 sucrose, and exposed to PVS2 for 0, 20 or 30 min. Shoot tips were then immersed in liquid nitrogen. Thawing was fast in concentrated sucrose solution (1.2 mol L-1). PVS2 is a nontoxic to shoot tips, which in turn are sensitive to liquid nitrogen. The best results occurred when shoot tips were maintained for up to 20 min in PVS2 solution, before freezing, with 20% survival.

In this research a new method for long term conservation of Medicago polymorpha seeds was developed by using cryopreservation. Two year cold acclimated seeds, collected from Iran rangelands, were desiccated using silica gel for four days and then cryopreserved by vitrification method. Seeds were loaded by loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then inserted into cryo-vials containing 0.5 ml PVS2 in 0 °C prior to direct immersed into liquid nitrogen. Seeds were kept in liquid nitrogen for a week then thawed in 37 °C water bath for 2 min. The seeds were rehydrated and detoxified in a solution of MS medium salts containing 1.2 M sucrose respectively for 5, 10 and 15 min in 22 °C. At the final stage of post-treatment, three different procedures were used: (a,b) placing seeds under running water for 30 min and 24 h before being transferred to petri-dishes containing moist filter paper, (c) sterilization of seeds with 2% hypochloride then placed on MS medium containing 0.5 mg/lit GA3. Germination percentage of cryopreserved seeds in all three post-treatments was higher than control (not cryopreserved seeds). This figure was respectively 94.37% and 98.21% for cryopreserved seeds of post-treatments a and b which was different at 0.05 levels with controls. However, germination percentage (76.16%) of cryopreserved seeds in post-treatment c showed no significant difference with control.

As a consequence of the difficulty in conventional coffee seed storage, biotechnological alternatives such as cryopreservation have been investigated. The objective of this study was to develop a protocol for the cryopreservation of Cojfea arabica L. (cv. 'Catuaí Vermelho' - IAC 144) zygotic embryos by vitrification. For the cryopreservation study, the embryos were immersed in Plant Vitrification Solution 2 at different times (0, 10, 25, 50, 100, and 250 min) and two temperatures (0 and 25 °C). Subsequently, the best thawing time was determined in a water bath (1,3, 5 minutes or direcdy in Recovery Solution). An anatomical study was conducted on non-stored and stored embryos, with or without the use of Plant Vitrification Solution 2. The immersion in cryoprotectant solution for 100 min at 0 °C allows embryo cryopreservation. Embryos can be directly thawed in Recovery Solution after storage in liquid nitrogen. It was observed that Plant Vitrification Solution 2 reduced internal water content in the cells, allowing subsequent embryo growth resumption. [ABSTRACT FROM AUTHOR]

Germplasm conservation of pineapple [ Ananas comosus (L.) Mer.] is crucial to preserve the genus' genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, that is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (−196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5-1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes. The technique was highly efficient in cryopreserving meristem shoot tips of different pineapple genotypes, and was also less laborious than techniques previously reported. This is a first report on application of the droplet-vitrification technique to diverse genotypes of cultivated and wild pineapples and the first report on histological changes occurring in cryopreserved Ananas tissue. [ABSTRACT FROM AUTHOR]

Adventitious root cultures of Cleome rosea were established using root segments from proximal and middle root regions of in vitro-propagated plants. The root explants were cultured in liquid MS medium supplemented with the following growth regulators: naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), and N-furfuryladenine (KIN). The highest biomass accumulation was obtained with roots, cultured in media with NAA. The use of 2,4-D resulted in callus formation, while explants cultured in the presence of KIN developed buds. Protocols for long-term maintenance of in vitro roots through cryopreservation by vitrification were also evaluated using plant vitrification solution 2 (PVS2). The use of NAA during pre- and post-culture steps was essential to recovery after exposure to liquid nitrogen. On the other hand, the presence or absence of light during culture maintenance had little effect on the process. The highest recovery frequency after cooling in liquid nitrogen (63.6 ± 14.4%) was reached after exposure to PVS2 for 30 min. The cryopreserved roots showed excellent ability to multiply, which remained stable for up to five subcultures. Moreover, these roots also displayed the capacity for shoot induction when cultivated on solid MS medium supplemented with 0.50 mg/L 6-benzyladenine (BA), reaching regeneration frequencies of 60-82% over five subcultures. [ABSTRACT FROM AUTHOR]

The possibility of streamlining general cryoprotection procedures is investigated using aluminium cryoplates with wells, facilitating fluid handling and drying treatment. Precultured shoot tips of potato cultivar 'Sayaka' were embedded in calcium alginate gel in wells of the aluminium cryo-plates. In the V cryo-plate protocol, dehydration was performed for 30 min at 25 °C in plant vitrification solution after osmoprotection. In the D cryo-plate protocol, dehydration was performed by placing the cryo-plates for 2.0 h under an air current in a laminar flow cabinet after osmoprotection. In both protocols, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, the cryo-plates were immersed in liquid MS medium supplemented with 1.0 M sucrose and diluted for 15 min at room temperature. Under these conditions, regrowth rates of cryopreserved shoot tips in V cryo-plate and D cryo-plate were 96.7 and 93.3 %, respectively. Both protocols will facilitate efficient strategies for preservation, storage, and maintenance of genetic stability of important potato germplasm. [ABSTRACT FROM AUTHOR]

The effect of cold and sucrose pretreatment for increasing tolerance to cryopreservation was evaluated with eight diverse genotypes, six cultivars belonging to the cultivated species, Solanum tuberosum spp., S. tuberosum subsp. andigena, S. x juzepczukii and S. x ajanhuiri, and two genotypes from the wild species, S. commersonii. In vitro plantlets were cultured at either 6 or 22 °C in media supplemented with either 0.07 or 0.3 M sucrose prior to droplet PVS2 cryopreservation. The sucrose pretreatment appeared to have no positive effect on post-cryo survival. The cold-hardening pretreatment increased significantly post-cryo recovery in drought and frost tolerant cultivars. When 755 accessions, representing 10 taxa, were cryopreserved after cold-hardening, 96 % responded with at least one shoot recovering and 63 % showed a high recovery rate (40-100 %). Therefore this method is recommended for the long term conservation of diverse accessions of potato germplasm. [ABSTRACT FROM AUTHOR]

Shoot tips from in vitro stock plants of red chicory ‘Rosso di Chioggia’ line were cryopreserved by one-step vitrification. After two days of cold-hardening on hormone-free MS medium and loading for 30 min in a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with PVS2 vitrification solution at 0°C for 60 min and plunged directly into liquid nitrogen. The post-thaw survival of shoot tips was achieved 79% when was cultured on recovery medium containing 0.5 mM BA. Observed regrowth, after six weeks of culture in the same medium composition, was 100%. Rooted cryopreserved microshoots showed good quality when transferred to the greenhouse. Preliminary results proved that the genetic fidelity of the cryopreserved line was maintained. The same vitrification protocol was then applied to three other red chicory lines, ‘Rosso di Treviso precoce’, ‘Rosso di Treviso tardivo’ and ‘Castelfranco’. A simple and effective protocol for the cryopreservation of red chicory shoot tips has been successfully developed as a result of this study.

Axillary buds from greenhouse-grown plants of Rosa chinensis 'Old Blush' were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20-80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 °C gave the best regrowth rates (82-86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration. [ABSTRACT FROM AUTHOR]

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5-1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l N-benzyladenine, 0.5 mg l indole-3-acetic acid, 0.2 mg l zeatin, 20 g l glucose and 6 g l Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %. [ABSTRACT FROM AUTHOR]

This work reports on the cryopreservation of immature zygotic embryonic axes (EA) of petai ( Parkia speciosa Hassk.) for the first time. Two cryopreservation protocols, namely desiccation and vitrification method were tested individually using excised EA. Desiccation of EA to lower moisture content (MC) reduced the survival percentage but a drastic decline in survival percentage (~20 %) was recorded at 16 % MC prior to exposure to LN, rendering the EA to be sensitive to desiccation. Cryopreservation of EA after desiccation, irrespective of the MC, did not result in any survival. On the other hand, post-cryopreservation survival was obtained when the EA were exposed to plant vitrification solution-2 (PVS2) for 75-105 min. The best results were obtained when the EA were exposed to PVS2 for 90 min with an average recovery of 55.5 %. EA recovery into whole plantlets was obtained when the EA were cultured on MS medium supplemented with 2 gl activated charcoal and 0.1 mgl of the plant growth regulators α-naphthalene acetic acid, 6-benzylaminopurine and gibberellin A, each. EA, exposed for less than 75 min and more than 105 min to PVS2, did not show any survival after cryopreservation. The optimization of exposure time is necessary to increase survival. This study has shown that the employment of suitable method is important for conservation using cryopreservation. [ABSTRACT FROM AUTHOR]

Shoot apices of in vitro-grown plantlets of white mulberry, Morus alba L. cv Florio, were cryopreserved using either encapsulation-dehydration or vitrification. For encapsulation-dehydration, alginate beads containing apices were dehydrated for 1, 3, 5 or 7 days in a liquid medium containing various sucrose concentrations (0.5, 0.75, 1.0 or 1.25 M). Bead desiccation was performed using silica gel for either 0, 4, 6, 8, 9 or 14 h. For vitrification, apices were directly immersed for either 5, 15, 30 or 60 min in a vitrification solution (PVS2). Following encapsulation-dehydration, treatment of alginate beads with 0.75 M sucrose was more effective in promoting re-growth of explants after immersion in liquid nitrogen than in the presence of 0.5 M sucrose for either 1 or 3 days. Re-growth of explants was also observed following vitrification and this reached 47% with increasing duration of the PVS2 treatment from 5 to 30 min. Overall, the highest frequency of explant re-growth was obtained when explants were subjected to encapsulation-dehydration in the presence of 0.75 M along with a 3 day sucrose dehydration pre-treatment and followed by desiccation for 9 h in silica gel. [ABSTRACT FROM AUTHOR]

Under cryopreservation conditions or at -196°C, because of extreme reduction of metabolic and physiologic processes, the seed sample or plant organ can survive for a long time. Chinese arbor-vitae (Biota orientalis (L.) Endl.) is one of the endangered forest species in Iran. To preserve B. orientalis seeds under cryogenic conditions, three cryopreservation treatments, including plant vitrification solution 2 (PVS2), 30% glycerol, and seed desiccation, were applied before transferring the seeds into liquid nitrogen. In desiccation, the moisture content of seeds dropped from 8.95% to 5.56% in silica gel at +4°C. All of the treated seeds except control were transferred into liquid nitrogen (-196°C), and preserved for periods of one week, one month, and one year. After removal of the seeds from liquid nitrogen, they were exposed to a heat shock (+42°C), washed, and evaluated by standard germination tests. Furthermore, the second samples of the same seeds were sown in pots and grown in greenhouse (22 ± 4 °C temperature) for one year. Seed germination, root and shoot lengths, germination speed, root/shoot length ratio, and seed vigor index (VI) were recorded and statistically analyzed. Seed germination percentages in PVS2 treatment for one-week, one-month, and one-year storage in liquid nitrogen were 95.94, 94.55, and 94.36, respectively, which statistically were not significant. High level of recovery from liquid nitrogen (-196°C), seed germination, and other attributes, in either laboratory or greenhouse conditions, revealed the cryogenic (-196°C) tolerance of this endangered species. The one-year old plants originated from cryopreserved seeds and grown under greenhouse conditions grew normally and did not show any abnormality when compared with the control plants. Keeping B. orientalis seeds under cryopreservation conditions (liquid nitrogen) is a reliable and long-term method of preserving them. Moreover, with the use of this technology, the seeds of this endangered species can be collected from different habitats and preserved for a long period. To save a species from becoming extinct, the seeds can be recovered and replanted in the disintegrated habitats with the aim of afforestation. [ABSTRACT FROM AUTHOR]

Preservation methods were evaluated for jerusalem artichokes (Helianthus tuberosus L.) to facilitate the back-up of field collections. In vitro plant cultures were established from field-harvested tubers for nine jerusalem artichoke cultivars obtained from the Plant Gene Resources of Canada, Saskatoon Research Center, Saskatoon, Canada. Most cultivars could be maintained at 5 °C or under 25 °C growth room conditions for at least 6 months. Excised shoot tips from in vitro cultures were cryopreserved using plant vitrification solution 2 (PVS2; 15% w/v ethylene glycol, 15% w/v dimethyl sulfoxide, 30% w/v glycerol, and 13.7% w/v sucrose in half strength media salts) as a cryoprotectant. PVS2 exposures of 15 or 30 min at 0 °C resulted in an average regrowth of 34% across five jerusalem artichoke cultivars after liquid nitrogen exposure. Jerusalem artichokes can be successfully maintained using both reduced temperature and cryopreservation approaches. [ABSTRACT FROM AUTHOR]

Abstract: Cryopreservation—the storage of plant germplasm in liquid nitrogen—provides a modern tool for the conservation of forest genetic resources. It is especially applicable for species in which their micropropagation can be initiated from mature tree buds, e.g., silver birch (Betula pendula Roth), thus enabling the conservation of specific genotypes: endangered elite trees and trees expressing rare, valuable or interesting characteristics. The aim of the present study was to develop a vitrification protocol applicable for the cryostorage of silver birch that avoids the use of expensive sophisticated freezers. The average recovery of vitrified axillary silver birch buds was 71% using a protocol that started with four-week cold hardening of bud-bearing in vitro donor shoots on modified medium under short day conditions. After cold hardening, the excised axillary buds were precultivated on medium containing 0.7M sucrose for 24h under the same conditions as during the cold hardening period. Following preculture, the buds were treated with loading solution containing 2M glycerol and 0.4M sucrose for 20min at room temperature. Finally, the buds were dehydrated with PVS2 cryoprotectant for 120min followed by direct immersion in liquid nitrogen. According to the morphology and the RAPD profiles of regenerated plants in the greenhouse, the genetic fidelity of the vitrified birch material seems to have remained unchanged. [Copyright &y& Elsevier]

Axillary shoot tips of in vitro grape ( Vitis vinifera L. cv. Cabernet Sauvignon) were successfully cryopreserved by vitrification. Axillary shoot tips were excised from 4- and 5-month old plantlets were cultured on solidified 1/2 MS medium with 1 mg l-1 BA at 25 °C. Shoot tips were precultured on solidified medium supplemented with 0.3 M sucrose for 3 days and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 20 min at 25 °C. They were then dehydrated with PVS2 for 80 min at 0 °C before being plunged into LN for 1 hr. Samples were then warmed rapidly in water at 40 °C. The recovery of the shoot tips amounted to approximately 60%. To enhance further recovery, a two-step dehydration procedure by vitrification was examined. Osmo-protected shoot tips were first dehydrated with a 50% PVS2 (half strength of the solution) for 30 min, followed by PVS2 for 50 min at 0 °C. This revised dehydration procedure improved shoot recovery from 60 to 80%. This procedure by vitrification was applied to ten other species/cultivars of Vitis with an average recovery of 64%. This method incorporates a simple and reliable approach for providing high levels of growth recovery. It also appears promising for the cryopreservation of Vitis germplasm. [ABSTRACT FROM AUTHOR]