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Euonymus koopmannii is a rare and protected species in Kazakhstan, valued for its ecological role in soil stabilization and its ornamental properties. This study presents the first use of micropropagation and phylogenetic analysis for the endemic plant E. koopmannii. Seedlings of E. koopmannii proved to be more effective than internodes as primary explants for plant micropropagation of in vitro culture, with a multiplication coefficient of 28.5 from seedlings and 6.1 from internodes. On MSR I medium supplemented with 0.5 mg/L IBA and 0.05 mg/L IAA, a higher success rate of 67% was achieved for root formation of test tube-grown E. koopmannii plants. Using mannitol as an osmotic agent at a concentration of 8 mg/L prolonged the storage time of E. koopmannii under slow growth conditions when compared to CCC and abscisic acid. Phylogenetic relationships and species identification were analyzed using four DNA-barcoding markers, comparing E. koopmannii with species from NCBI. All candidate barcoding markers showed sufficient levels of interspecific genetic variation among Euonymus species. In addition, ITS region and rbcL gene sequences effectively distinguished E. koopmannii from other species. These results provide fundamental information that will be valuable for future biotechnological and molecular studies. [ABSTRACT FROM AUTHOR]

Euonymus koopmannii is a rare and protected species in Kazakhstan, valued for its ecological role in soil stabilization and its ornamental properties. This study presents the first use of micropropagation and phylogenetic analysis for the endemic plant E. koopmannii. Seedlings of E. koopmannii proved to be more effective than internodes as primary explants for plant micropropagation of in vitro culture, with a multiplication coefficient of 28.5 from seedlings and 6.1 from internodes. On MSR I medium supplemented with 0.5 mg/L IBA and 0.05 mg/L IAA, a higher success rate of 67% was achieved for root formation of test tube-grown E. koopmannii plants. Using mannitol as an osmotic agent at a concentration of 8 mg/L prolonged the storage time of E. koopmannii under slow growth conditions when compared to CCC and abscisic acid. Phylogenetic relationships and species identification were analyzed using four DNA-barcoding markers, comparing E. koopmannii with species from NCBI. All candidate barcoding markers showed sufficient levels of interspecific genetic variation among Euonymus species. In addition, ITS region and rbcL gene sequences effectively distinguished E. koopmannii from other species. These results provide fundamental information that will be valuable for future biotechnological and molecular studies.

ElecTIS is a new single container bioreactor which does not require forced air blowing, instead making the culture material mobile and the liquid medium stationary. The timed up-and-down movement of the basket containing the shoot culture ensures periodic contact with the liquid medium positioned at the base of the container. In this study we tested for the first time its use in the recovery of blackberry shoot cultures (Rubus fruticosus L., cvs Thornfree and Chester), coming from 5 months of slow growth storage (SGS), at 4 °C and in the dark. The shoot recovery at standard culture conditions was performed on two different types of ElecTIS, i.e., one with a smaller basket (ElecTISS, 234 cm2 of culture area), and one with a large basket (ElecTISL, 336 cm2), comparing the culture in TIS (cycle of 8 min every 6 h, equal to 32 min/day) with the traditional one in a gelled medium in glass jars (500 cc). After each one of the three 4-week subcultures, the shoot growth parameters and the relative growth rate highlighted a clear superiority of ElecTIS in promoting the recovery of shoot cultures coming from SGS. The analyses of chlorophyll content and stoma functionality confirmed the superior quality of shoots cultured in the ElecTIS bioreactor, and these shoots were afterwards easily rooted and acclimatized ex vitro. [ABSTRACT FROM AUTHOR]

ElecTIS is a new single container bioreactor which does not require forced air blowing, instead making the culture material mobile and the liquid medium stationary. The timed up-and-down movement of the basket containing the shoot culture ensures periodic contact with the liquid medium positioned at the base of the container. In this study we tested for the first time its use in the recovery of blackberry shoot cultures (Rubus fruticosus L., cvs Thornfree and Chester), coming from 5 months of slow growth storage (SGS), at 4 °C and in the dark. The shoot recovery at standard culture conditions was performed on two different types of ElecTIS, i.e., one with a smaller basket (ElecTISS, 234 cm2 of culture area), and one with a large basket (ElecTISL, 336 cm2), comparing the culture in TIS (cycle of 8 min every 6 h, equal to 32 min/day) with the traditional one in a gelled medium in glass jars (500 cc). After each one of the three 4-week subcultures, the shoot growth parameters and the relative growth rate highlighted a clear superiority of ElecTIS in promoting the recovery of shoot cultures coming from SGS. The analyses of chlorophyll content and stoma functionality confirmed the superior quality of shoots cultured in the ElecTIS bioreactor, and these shoots were afterwards easily rooted and acclimatized ex vitro.

An experiment for strawberry Fragaria ananassa cv. Festival in vitro conservation of cultures for nine months was studied. Three MS medium strength (full strength MS, ¾ MS, ½ MS, and ¼ MS) and the storage period (3, 6, and 9 months) in slowing down or reducing growth. Results showed a significant effect of the medium salt strength, as the full strength treatment was higher by achieved the highest percentage of viability (survival %) and storage characteristics of cultures (N%, P%, carbohydrates%, protein %) and regeneration ability characteristics (shoots and roots number and length). Three-month storage period achieved the highest rate compared to the nine-month storage period, which recorded the lowest average for the studied characteristics. The full strength treatment for three months storage period realized the highest rate compared to treatment ¼ strength for the storage period of nine months, which recorded the lowest rate of the studied traits. The application of minimal strength for in vitro conservation allows strawberry cultures to remain viable with a slow growth rate and a significant increase in the storage life of the tissues to nine months.

An experiment for strawberry Fragaria ananassa cv. Festival in vitro conservation of cultures for nine months was studied. Three MS medium strength (full strength MS, ¾ MS, ½ MS, and ¼ MS) and the storage period (3, 6, and 9 months) in slowing down or reducing growth. Results showed a significant effect of the medium salt strength, as the full strength treatment was higher by achieved the highest percentage of viability (survival %) and storage characteristics of cultures (N%, P%, carbohydrates%, protein %) and regeneration ability characteristics (shoots and roots number and length). Three-month storage period achieved the highest rate compared to the nine-month storage period, which recorded the lowest average for the studied characteristics. The full strength treatment for three months storage period realized the highest rate compared to treatment ¼ strength for the storage period of nine months, which recorded the lowest rate of the studied traits. The application of minimal strength for in vitro conservation allows strawberry cultures to remain viable with a slow growth rate and a significant increase in the storage life of the tissues to nine months. [ABSTRACT FROM AUTHOR]

The high endemism, the natural habitat degradation, and the over-collection for ornamental purposes have led some species such as Melocactus paucispinus and Melocactus glaucescens to be threatened with extinction. The use of in vitro conservation techniques, such as slow growth storage, promotes the preservation of genetic diversity with integrity. The goal of this study was to establish a strategy for in vitro conservation of apical segments of the cladode of M. paucispinus and M. glaucescens and evaluate the genetic diversity of individuals from in vitro germinated plants. For such purpose, different concentrations of the plant regulator ancymidol and the osmotic agent sucrose on the inhibition of the in vitro growth were tested, and the genetic diversity of M. paucispinus and M. glaucescens individuals stored in vitro was evaluated. Sucrose showed higher efficiency in the reduction of growth than ancymidol for both species. However, due to the reduction in survival percentage, the use of sucrose over 75 g L−1 in the in vitro conservation of both species for 360 days is not recommended. In the genetic diversity analysis, 76.92% of polymorphic loci (P), expected heterozygosity (He) = 0.276 and Shannon index (S) = 0.414 were observed for M. paucispinus. For M. glaucescens, the observed values were P = 95.38%, He = 0.228 and S = 0.369. These values observed here were higher than those previously found for the natural populations of these species, which demonstrated that this in vitro collection showed genetic diversity and can be used in management and reintroduction programs of these species. [ABSTRACT FROM AUTHOR]

Today, application of in vitro culture by means of slow growth storage of shoot cultures and cryopreservation of organs, tissues and cells in liquid nitrogen presents a remarkable strategic tool to support medium- and long-term conservation of plant genetic resources. Over the last 30 years, considerable progresses have been made in the development of both methods that are currently considered as ex situ conservation strategies, complementary to traditional seed banks and in-field clonal collections. Efficient protocols were developed for the conservation of a large number of crops, including strategically-important vegetables, such as garlic, artichoke, asparagus, cassava, Jerusalem artichoke, mint, potato, sweet potato, chicory, taro, thyme and yam. As a consequence, more than 45,000 accessions of vegetable crops are maintained in 22 genetic resources conservation centers (biobanks), located in 16 countries and 6 continents (Europe, Asia, Africa, Oceania, North and South America). Approximately 4/5 of these accessions are maintained in vitro by means of slow growth storage of shoot cultures, but cryopreservation is also constantly growing, with almost 8300 vegetable accessions being stored in liquid nitrogen at − 196 °C. [ABSTRACT FROM AUTHOR]

Copyright of Italus Hortus is the property of Societa di Ortoflorofrutticoltura Italiana 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.)

BACKGROUND: Vaccinium myrtillus L. is a valuable wild fruit species of conservation concern in the Tuscan Apennines (Italy). OBJECTIVE: The development of a micropropagation protocol for the conservation of this species in in vitro-banks for the year-round mass production of specific selected superior bilberry genotypes, maintaining the genetic variability of natural populations, avoiding genetic erosion. METHODS: Nodal segments from seedlings were cultured on mMS medium with zeatin or 2iP for multiplication and elongation stage, afterwards shoots were rooting in vitro and in vivo. The RAPD analysis was applied to lines obtained and slow growth storage (4°C – darkness) was evaluated for the conservation. RESULTS: Zeatin concentration 9.1 μM was the most favorable for shoot proliferation. The activated charcoal was essential for in vitro rooting. Both in vitro and ex vitro methods were successfully used to root and acclimatize bilberry shoots. The RAPD analysis indicated a large genetic diversity among the micropropagated genotypes. Shoots were successfully in vitro preserved for up to 6 months with no apparent deterioration effect. CONCLUSIONS: In vitro propagation and conservation are fundamental tools for exploiting the whole potential of this wild crop species for breeding and genetic improvement purposes. [ABSTRACT FROM AUTHOR]

The market of flowers and ornamental plants is dependent on the diversification of species and the availability of high quality propagation materials. Actually, in vitro culture techniques performance a prominent role in the multiplication and maintenance of commercially propagated ornamental plant species, and are promising for the production of thousands of high quality plants in relatively short term. In addition, when market demand for a particular species is low or zero in a specific period of the year, in vitro culture techniques allow the conservation of cultures under aseptic conditions, by Slow Growth Storage (SGS), from a few weeks to one year (or more), without affecting their viability and potential regrowth. This can be achieved by modifying the constitution of the culture medium and the maintenance conditions of in vitro cultures. Obviously, the success of the technique depends on greatly on the physiological characteristics of the species to be conserved, as well. Once a SGS protocol is optimized, the expenses labor, the possibility of contamination and the probability of somaclonal variation can be reduced markedly.

Ziziphora tenuior is a medicinal plant which has been used to treat many diseases due to the presence of many valuable medicinal ingredients such as, pulegone. This valuable plant has become extinct from its natural environment due to illegal collection, urbanization, overgrazing and drought. In this study, in vitro slow growth storage was applied to study the possibility of conserving Ziziphora tenuior L. microshoots using elevated levels of different carbon sources ( sucrose, sorbitol and mannitol) at (0.1, 0.2, 0.3 and 0.4 M) or ABA at different concentrations (0, 3.8, 7.6, 11.4 μM). Data were recorded after two periods (6 and 12 weeks) of cultivation for growth parameters, survival and recovery percentages. Recovery percentage was used as a survival indicator for shoots after 12 weeks of conservation. Furthermore, effect of slow growth storage on the total oil content and pulegone concentration of the microshoots was investigated after 3 months conservation using Gas chromatographic method (GC/MS) for the identification and quantification of the marker compounds pulegone in Ziziphora tenuior. The current study revealed that, microshoots prestored in MS media + (0.2 M) sucrose or ABA were able to reduce the all measured growth parameters and to maintain recovery after storage. On the other hand, using sorbitol or mannitol had a strong negative impact on recovery after storage. Meanwhile, there were differences between the amounts of total oil yield and pulegone extracted from the wild type plants and the in vitro prestored microshoots. Maximum total oil yield content (5%) and pulegone (0.0312 M) were obtained from the wild type plants. On the other hand, the amount of total oil yield extracted from the in vitro stored microshoots were one third the amounts of oil obtained from the wild type plants. Also, the concentration of pulegone found in the dried in vitro stored samples (0.0041-0.0046 M) was about the same in all in vitro prestored microshoots but much lower than those obtained from the wild plants samples. [ABSTRACT FROM AUTHOR]

Copyright of Italus Hortus is the property of Societa di Ortoflorofrutticoltura Italiana 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.)

The study focused on an economically-important ornamental outdoor shrub, Nandina domestica, with the aims to (i) optimize an effective in vitro conservation method, and (ii) develop a cryopreservation protocol for shoot tips by the PVS2 vitrification and droplet-vitrification techniques. For in vitro conservation of shoot cultures, the tested parameters were sucrose content in the storage medium (30, 45, 60 g/L) and storage temperature (4 °C or 8 °C). Cryopreservation was performed by applying the PVS2 vitrification solution, in 2-ml cryovials or in drops over aluminum foil strips, for 15, 30, 60 or 90 min at 0 °C, followed by the direct immersion in liquid nitrogen of shoot tips. Results show that N. domestica shoots can be conserved successfully for 6 months at both the temperatures tested, especially when 60 g/L sucrose is used in the storage medium. However, conservation at 4 °C showed to be more appropriate, as hyperhydricity was observed in post-conservation of shoots coming from storage at 8 °C. As for cryopreservation, a daily gradual increase of sucrose concentration (from 0.25 to 1.0 M) produced better protection to the samples that were stored in liquid nitrogen. Indeed, with this sucrose treatment method, a 30-min PVS2 incubation time was enough to produce, 60 days after thawing, the best recovery (47% and 50%) of shoot tips, cryopreserved with PVS2 vitrification and droplet-vitrification, respectively. [ABSTRACT FROM AUTHOR]

As genetic erosion of pistachio ( Pistacia vera L.) has been occurring in the Mediterranean, Central and West Asia and North Africa, experiments were conducted to conserve two cultivars ('Atlı' and 'Siirt') of mature pistachio germplasm by assessing both medium- and long-term conservation techniques. In medium-term conservation, our results showed that it was feasible to conserve both cultivars in the form of either microshoots or encapsulated shoot apices up to 12 months at 4°C in the dark. As regards long-term conservation, encapsulation-dehydration and droplet-vitrification techniques were assessed for cryopreservation of cold-hardened and osmoprotected shoot apices of mature 'Atlı' cultivar. Among the methods tested, 13.6% of regrowth was achieved with incubation of explants in the droplets of vitrification solution for 150 min at 0°C followed by direct immersion in liquid nitrogen (LN), rapidly thawed and then cultured on Murashige and Skoog's (MS) medium containing 1 mg L BA and 0.5 mg L GA The developed droplet-vitrification technique appeared as a promising procedure for long-term preservation of shoot apices of mature pistachio germplasm. Moreover, assesment of genetic fidelity by Random Amplified Polymorphic DNA analysis (RAPD) revealed out high levels of genetic stability between donor plant and cryopreserved plants (similarity indexes between 0.959 and 0.973) after they were subcultured for at least 3 months. The detected low level of genetic instability could be due to the toxic effect of PVS2 and regeneration phase. The optimized conservation techniques, especially slow growth storage, could be applied to preserve other Pistacia species. [ABSTRACT FROM AUTHOR]

In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, −196°C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species. [ABSTRACT FROM AUTHOR]

Abstract: Protocols for the in vitro proliferation and storage of fraser photinia were developed by comparing 6-benzyladenine (BA) concentrations (0.5–4mg/L) together with different media formulations [Murashige and Skoog (MS) media and Quoirin and Lepoivre (QL) media], sugar combinations (sucrose and mannitol), culture vessels (baby food jars and vitrovents) and methods (synthetic seed technology and slow growth storage). The best responses in terms of both proliferation percentage and multiple shoot formation were obtained in QL medium containing 1mg/L BA. Synthetic seed production was optimized by encapsulating shoot apices in 3% sodium alginate. Encapsulated shoot apices could be maintained up to 6 months at 4°C in dark with 91.6% sprouting in MS medium. Microshoots were stored at 4°C up to 15 months on sucrose and mannitol containing QL medium in both baby food jars and vitrovents without subculture. The stored material could be recovered and multiplied normally in 1mg/L BA supplemented QL medium. Both in vitro propagated and conserved microshoots were rooted (∼75%) on QL medium with 1mg/L indole butyric acid (IBA). Optimized synthetic seed and slow growth storage system can be used for short and medium-term storage of fraser photinia germplasm. [ABSTRACT FROM AUTHOR]

Abstract: Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22±2°C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15gl−1 each of sucrose and mannitol in sealed culture vessels at 22±2°C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0mgl−1 BA and 0.5mgl −1 IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources. [Copyright &y& Elsevier]