Slow Growth Storage Research Articles

In vitro proliferation and minimum growth storage of fraser photinia: Influences of different medium, sugar combinations and culture vessels

Protocols for the in vitro proliferation and storage of fraser photinia were developed by comparing 6-benzyladenine (BA) concentrations (0.5–4 mg/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 1 mg/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 1 mg/L BA supplemented QL medium. Both in vitro propagated and conserved microshoots were rooted (∼75%) on QL medium with 1 mg/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.

Biotechnologies et conservation des ressources phytogénétiques

In vitro techniques have numerous applications in collecting, propagating and conserving plant biodiversity. In vitro collecting techniques make it possible to introduce in vitro explants under field conditions from recalcitrant seeds and vegetatively propagated species. In vitro culture techniques make production and large scale propagation of disease-free material possible. Utilising in vitro culture techniques is of high interest for conserving: 1) genetic resources of recalcitrant seeds and vegetatively propagated species; biotechnology products (elite genotypes, metabolite-producing cell lines, transformed material) and 3) rare and endangered plant species. Medium term conservation is performed by reducing growth of the plant material, thereby increasing intervals between subcultures. Slow growth storage protocols are employed for numerous temperate and tropical species. For long term storage, cryopreservation (liquid nitrogen -196 °C) makes it possible to store plant material without modification or alteration for extended periods, sheltered from contamination and with limited maintenance. Cryopreservation is well advanced for vegetatively propagated plant species. Research is less advanced for recalcitrant species due to certain characteristics including their sensitivity to desiccation, their structural complexity and heterogeneity in terms of developmental stages and their water content at maturity. However, various technical approaches exist in order to establish cryopreservation techniques for a larger number of recalcitrant species. Routine utilisation of cryopreservation is still limited. However, the number of examples for which it is used on a large scale is increasing steadily.

BIOTECHNOLOGIES FOR THE PRESERVATION OF SELECTED RED CHICORY (CICHORIUM INTYBUS L.) LINES

Red chicory (Cichorium intybus L.) is one of the most economically important vegetables produced in the Italian region of Veneto. Here, a programme for the medium-term (by slow-growth storage) and long-term (by cryopreservation) conservation of red chicory selected lines was initiated in 2002. As regards slow growth storage, shoots from the proliferation stage were stored in one of the following conditions: (i) at 4°C, in darkness or under a 8-h photoperiod (with 15 μmol m -2 sec -1 of light intensity), (ii) on a storage medium added of osmotically active compounds (OACs), i.e., NaCl (4.48 g L -1 ) or mannitol (40 g L -1 ), and (iii) at 4°C and in darkness, comparing the storage in gas-tight plastic cylinders, in slow gas-permeable glass jars or in gas-permeable plastic bags ('StarPac'). After 9 months, best storage conditions for the 'Treviso precoce' line in terms of shoot survival (95%) were obtained with the conservation at 4°C, with the shoots maintained in the dark showing a lower relative growth rate (RGR). As for the storage in OACs, a higher shoot survival (70%) and a lower RGR was obtained with the use of mannitol. A general lower adaptability to SGS was evidenced by the 'Chioggia' line, as 100% shoot survival was recorded after 6 months of conservation at 4°C in darkness, but this value dramatically dropped to 45% after 9 months. All the tested containers allowed a very satisfactory conservation of shoots at 4°C in the dark for the whole period (9 months). However, higher RGR values were always recorded from shoots stored in the 'StarPac' gas-permeable plastic bags. As regards cryopreservation, red chicory shoot tips survived and regrew satisfactorily from the storage at -196°C when a procedure of vitrification/one-step freezing, consisting in loading the explants in the PVS2 vitrification solution, was applied.

THE APPLICATION OF BIOTECHNOLOGY IN AN INTEGRATED PROJECT OF CONSERVATION AND UTILIZATION OF PAPAYA AND ITS WILD RELATIVES

Aosiraci Carica papaya is an important crop species throughout tropical and subtropical regions of the world, including northern regions of Australia. Biotechnology is relatively advanced in papaya including techniques for micropropagation, embryogenesis, embryo rescue, transformation and the application of molecular markers. Plant improvement projects are underpinned by such technologies but also require a range of germplasm as a source of new and useful agronomic traits. A collection of valuable germplasm in the form of superior cropping genotypes of papaya and wild relatives of papaya, the Vasconcella spp., are being used in a range of plant improvement projects in our laboratories. The Vasconcella spp. are a particularly rich source of agronomically important traits, including resistance to papaya ringspot virus type P (PRSV-P), phytophthora, phytoplasma and blackspot. Intergeneric hybrids between wild relatives and papaya are now segregating for PVSP-P and molecular mapping and marker technologies are assisting with further selection of useful hybrid plants. The long-term conservation and availability of this range of germplasm is critical to sustainable utilization. Tropical fruits are typically difficult to store as seed and we are currently involved in international collaborations to develop alternative conservation technologies for tropical fruits, including papaya. This paper reports on advances in seed storage technologies, slow-growth storage and cryopreservation of both shoot tips and seeds as alternatives for use across a range of papaya genotypes for long-term ex situ conservation.

IN VITRO SLOW GROWTH STORAGE OF FRUIT ROOTSTOCKS INSIDE GAS-TIGHT OR GAS-PERMEABLE CONTAINERS

The maintenance of stock cultures in darkness and at low temperatures is by far the most common slow growth storage technique used in commercial laboratories. This study was carried out with in vitro shoot cultures of three fruit rootstocks: cherry rootstock ('Gisela 5®'), apple rootstock ('M26'), and pear rootstock ('A74'). The shoot cultures were stored at 4°C in darkness inside 500-ml glass jars or StarPac plastic bags. Moreover, with 'Gisela 5®' rootstock, three different sucrose concentrations (10, 20, and 30 g L -1 ) were tested in the storage medium. Very high levels of CO 2 were detected inside the gas-tight containers, particularly at the highest sucrose concentration which determined over 130,000 ppm accumulation after just 8 weeks of conservation. Differently, the gas-permeable bags proved to be effective in maintaining low levels of CO 2 accumulation. In both containers, ethylene accumulation never exceeded 10 ppm. After 9 months of 'Gisela 5®' conservation in glass jars, a lower growth rate was recorded from shoot cultures stored in the lowest sucrose concentration. However, with both containers, shoots stored this way were highly affected by hyperidricity and decay, and had to be discarded in great quantity. Best conditions of conservation and regrowth were obtained when shoot cultures were stored in a medium containing 30 g L -1 sucrose. As regard 'M26' apple and 'A74' pear rootstocks, the shoot cultures maintained a good standard of quality for the whole period of storage at 4°C in darkness, regrowing easily when moved again to proliferative conditions. Differently, the use of mannitol as an osmotically active compound allowed a satisfactory conservation only for the pear rootstock.

Non-destructive evaluation of in vitro-stored plants: A comparison of visual and image analysis

In vitro plants, in slow-growth storage require routine evaluation for assessment of viability and need for repropagation. Determination of plantlet health by visual assessment is subjective and varies by genus due to variations in growth pattern and plant structure. Developing a standardized plant evaluation system would improve the efficiency of in vitro storage. This study was initiated to develop digital image analysis techniques for plantlets during slow-growth cold storage and to compare that system with visual examinations. Pear (Pyrus communis L) cultivars were chosen for this initial trial because they have an open structure and clear internode position for image composition. Pear shoots stored at 4°C in tissue culture bags were evaluated monthly by standard visual examination and by digital image analysis. Digital images were evaluated for red, green, blue, modified normalized differences of vegetation index (MNDVI), green/red ratio (G/R), intensity, hue, and saturation at the first two nodes of each, plantlet. At 6 mo., the visual ratings had declined steadily for P. communis ‘Luscious’ and ‘Bartlett—Swiss’, while ‘Belle Lucrative’ and ‘Louse Bonne de Jersey’ ratings did not show significant declines until 9 mo. Correlations between visual ratings and G/R and MNDVI values were significant (r2>=0.5) for all cultivars. Regression analysis indicated that the MNDVI and G/R ratios changed significantly over the 15-mo. rating period for most cultivars. Intensity, hue and saturation values were not consistently significant and did not correlate with visual ratings. These results will assist in the development of digital imaging as an alternative technique for evaluation of stored in vitro plantlets.

Slow growth storage and cryopreservation—tools to facilitate germplasm maintenance of vegetatively propagated crops in living plant collections

In living plant collections, vegetatively propagated accessions are outstanding material with respect to vulnerability and labour amount. This is also true for the main vegetative material, held in the IPK, Gatersleben. A survey of the preservation of potato, garlic and other alliums, mint and yam is given. More than 630 accessions are in slow growth conditions. Amongst them, 99 clones of garlic and 35 of shallot have been tested to be virus-free. Cryopreservation is routinely applied for potato using the droplet method. The cryo-collection contains more than 1000 accessions, a part of which has been integrated from another collection, formerly established at Braunschweig. Cryopreservation of garlic has been used to store the accessions of the European core collection. Cryopreservation is successful in three Dioscorea species using a combination of the original vitrification with the droplet method. Investigations about morphogenesis and ultrastructural cell parameters before, during and after cryopreservation were included in these activities.

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4 degrees C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.

Cytological and molecular evaluation of strawberry plants recovered from in vitro conservation by slow-growth

SummaryShoot tips of strawberry (Fragaria vesca F. ananassa) cultivar ‘Heilongjiang No.1’ were successfully preserved in vitro using a slow-growth culture method. All shoot tips survived 1 year of storage and showed an increase of 1.56 ± 0.08 cm in shoot length. In addition, eight single-bud-derived sibling lines were established for genetic analysis. Although cytological examination detected chromosomal variations in plants recovered from slow-growth, their ploidy level was similar to the control. Amplified fragment length polymorphism (AFLP) analysis with 16 primer combinations did not detected any change in AFLP patterns between controls and strawberry plants recovered from slow-growth culture. However, methylation-sensitive amplified polymorphism (MSAP) analyses indicated that slow-growth storage induced changes in DNA methylation and DNA de-methylation patterns, while no de novo methylation was found.

In Vitro Propagation and Storage of Brugmansia versicolor Lagerheim

Brugmansia versicolor Lagerheim of the family Solanaceae was propagated through shoot tip culture and shoots were cold-stored in vitro at 5, 10 or 15°C under light or dark condition. All the shoots died after cold storage for 6 months at 5°C irrespective of light condition. When shoots were stored for 12 months at 15°C under light illumination, the best 100% survival rate was obtained. The plants regenerated from shoots stored for 6 or 12 months retained the ability to accumulate scopolamine as much as the control plants which were raised from shoots maintained under normal culture condition without cold-storage. These findings show that slow growth storage of in vitro B. versicolor shoots at 15°C can be used as a germplasm conservation system for short-or medium-term duration without deterioration of the ability to accumulate the secondary metabolites.

Genetically stable regeneration of apple plants from slow growth

Shoot-tips of apple cultivar `Gala' were stored in vitrousing a low temperature slow-growth culture method. All shoot-tips survived 1-year storage, with a significant height increment over that period. Eight `Gala' single-bud sibling lines were established for genetic analysis. Although cytological examination detected chromosomal variation in plants recovered from slow growth culture, the ploidy remained genetically stable relative to the before-storage cultures. An amplified fragment length polymorphism (AFLP) assay was performed to detect DNA sequence variation. No differences in the DNA fragment patterns were observed using 20 primer combinations between the before-storage and the stored samples. In addition, a methylation sensitive amplified polymorphism (MSAP) assay was performed to investigate the DNA methylation status in both the before-storage and stored samples. It was found that the slow-growth storage resulted in a significant DNA methylation change in the stored shoots compared with the before-storage samples.

CO2 and ethylene evolution in the culture vessels during in vitro conservation of olive (cv. Frantoio) at 4 C.

The evolution of CO 2 and ethylene in air-tight vessels of olive shoot cultures (cv. Frantoio) was studied during eight-month slow growth storage at 4°C in darkness. While the production of ethylene was negligible during the whole storage period, CO 2 increased rapidly and reached very high concentrations (up to 70000 μl.l - 1 ). A correlation between this strong accumulation and the poor shoot regrowth after conservation at 4°C is hypothesized.

In vitro storage of Eucalyptus grandis germplasm under minimal growth conditions.

Slow growth-storage, for up to 10 months, has been achieved for Eucalyptus grandis shoot cultures by either the addition of 10 mg l−1 abscisic acid to the growth medium or by the halving of nutrient supply (half MS) and removal of exogenous plant growth regulators. Reduction of light intensity or the addition of mannitol to the media were less effective in reducing growth rate. Isolated in vitro axillary buds encapsulated in calcium alginate and stored under low temperature and low light intensities survived for up to 3 months without loss in viability. Storage of such encapsulated fresh axillary buds at higher temperature resulted in a loss in viability. These methods have immediate applications to forestry breeding and clonal programs.

Slow-growth storage of single node shoots of apple genotypes

A landrace (‘Moscatella’) and a commercial cultivar (‘Starkspur Red’) of Malus pumila Mill. were maintained and proliferated in vitro for ≥ four years. A factorial experiment, planned to evaluate the response of the two cultivars in conservation in determined slow growth conditions, was carried out for eighteen months. Single node cuttings were stored in 4 different media, at 4 °C, in dark conditions, in microvessels to assess the feasibility of reducing space in in vitro gemplasm banks. Culture viability after storage was evaluated after 6, 8,12 and 18 months of storage. Both varieties showed high survival percentages for up to a year of conservation, but the landrace's capability to resume growth dropped dramatically afterwards. On the contrary, ‘Starkspur Red’ maintained substantially unchanged capacity of resuming vigorous growth after 18 months of conservation. Overall, microvessels appeared to be suitable for storing single node cuttings under slow growth conditions up to a year at least. The described techniques could be useful for in vitro germplasm collections where frequent subculturing enhances the risk of genetic changes and personnel, energy and materials costs limit the amount of genotypes that can be managed.

IN VITRO GERMPLASM CONSERVATION

In vitro techniques have great potential for collecting, exchange and conservation of: i) genetic resources of recalcitrant-seed and vegetatively propagated species as well as of endangered species; ii) elite genotypes which are multiplied on a large scale in production laboratories; iii) cultures with special attributes, eg, metabolite-producing cell lines and genetically engineered material. In vitro collecting techniques, which consist of introducing embryos or vegetative tissues in vitro under field conditions, have been developed for collection of germplasm of various problem species. In vitro cultures are routinely used for exchange of plant genetic resources of a number of species, due to their advantages in terms of phytosanitary status and reduced cost. Slow growth techniques have been developed for medium-term conservation of numerous species but their routine use is still restricted to a limited number of crop species. Routine use of cryopreservation is mostly restricted to conservation of cell lines in research laboratories. However, simple and efficient freezing protocols have been developed recently for apices and embryos, and can be considered operational for an increasing number of species. Current research priorities include: i) the development of in vitro collecting techniques for additional species; ii) demonstration of the flexibility, simplicity and practicality of slow growth storage to increase its utilisation; iii) experimentation of existing cryopreservation techniques on a large scale in a genebank context and the development of cryopreservation protocols for additional species.

Storage of potato microplants in vitro in the presence of acetylsalicylic acid

Acetylsalicylic acid was investigated as an alternative medium supplement to mannitol for slow-growth in vitro storage of potato microplants. At 8°C, culture in the presence of either 100 μM acetylsalicylic acid or 4% mannitolretarded microplant stem growth, and required intervals between subcultures ranged from 8 to over 12 months, depending on the genotype. Several media were tested for recovery of clones from slow-growth storage, with similar efficiences of recovery achieved for microplants from either mannitol or acetylsalicylic acid media. Prolonged culture on acetylsalicylic acid had no adverse effects on the yield of minitubers on plantlets transferred to glasshouse cultivation. At 18°C also, intervals between subcultures could be prolonged to 5.5–6 months by culture on either mannitol or acetylsalicylic acid media. Microplants cultured on acetylsalicylic acid did not exhibit the frequent phenotypic abnormalities found in microplants on mannitol medium.

Towards integrated conservation of Australian endangered plants—the Western Australian model

Four percent of the Australian flora is rare and endangered with over 100 taxa presumed extinct. Western Australia contains a large proportion of the endangered flora of Australia with 238 taxa in a critical state of conservation and 70 species presumed extinct. Kings Park and Botanic Garden in south-west Australia is responsible for developing specialized collections of rare and endangered indigenous flora. Macro-and micropropagation procedures are used including conventional cutting and seed propagation, grafting and in thein vitro programme whole seeds (asymbiotic and symbiotic germination), excised seed embryos, shoot apices and inflorescence sections. Wherever possible explants are collected from major provenances of the species and a wide cross section of a species population. Although many of the rare flora of Western Australia are now in theex situ collection maintained by Kings Park and Botanic Garden attempts are being made to develop slow growth storage forin vitro cultures and cryostorage. Trial recovery programmes have commenced with a number of species including the rare and endangered Purdie's donkey orchid (Diuris purdiei). Results of these recovery programmes will guide future efforts in conserving and recovering rare Australian species.