Maurizio Lambardi

Application of Tissue Culture to the Germplasm Conservation of Temperate Broad-Leaf Trees

The recovery and conservation of a broad genetic pool of wild and domesticated species are essential for the maintenance of plant biodiversity. Traditionalex-situconservation systems (i.e., seed and field collections), alongsidein-situconservation, make a fundamental contribution to the preservation of plant genetic resources. Up to now these approaches have been the only reliable option for the long-term germplasm preservation of woody species, although they have several drawbacks. Numerous forest Angiosperms (e.g.,Acerspp.,Quercusspp., chestnut, horsechestnut, many tropical species) have non-orthodox (recalcitrant) seeds that quickly loose their viability or are subject to a rapid decrease in germinability during storage (Pence, 1995). Moreover, recalcitrant seeds do not tolerate the level of dessication that is commonly required to prolong conservation (Roberts, 1973). As regards temperate fruit trees, the majority of the species belonging to this group are reproduced by vegetative propagation, thus requiring the conservation in clonal orchards of huge numbers of accessions, including old and newly selected cultivars, local varieties and wild material. Table 1, for instance, gives a clear idea of the efforts required today for the preservation of the EuropeanPrunusgermplasm, kept in the field repositories of 21 countries.

MICROPROPAGATION OF OLIVE (Olea europaea L.)

The olive (Olea europaea L.), together with the fig and the date, is one of the oldest fruit trees in the Mediterranean area (Zohary and Spiegel-Roy, 1975). The species, originating in the eastern part of the Mediterranean basin, belongs to the genus Olea, which is one of over 30 genera of the Oleaceae family. The basic chromosome number of the genus Olea is 2n=46 (x=23). Although the genus comprises some 60 species distributed over most of Africa, the warmest zones of Europe, South Asia, Central America, South Australia and New Zealand, the O. europaea is typical of the Mediterranean area. Although several different classifications of Olea europaea are reported, it is generally accepted that the Euro-Mediterranean domesticated olive includes a wild type (O. europaea subsp. oleaster) and a cultivated form (O. europaea subsp. saliva Hoffin. et Link = O. europaea subsp. europaea). The oleaster is a shrub producing small bitter-tasting fruit with low oil content. In addition to olive and oleaster, the term “olevaster” is also widely used to refer to domesticated escapes (Lavee, 1996).

Protocols for Micropropagation of Selected Economically-Important Horticultural Plants

Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on 1⁄2 strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 m M N 6 benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1–4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.

In vitro conservation and cryopreservation of ornamental plants.

Today, the conservation of ornamental germplasm can take advantage of innovative techniques which allow preservation in vitro (slow growth storage) or in liquid nitrogen (cryopreservation) of plant material. Slow growth storage refers to the techniques enabling the in vitro conservation of shoot cultures in aseptic conditions by reducing markedly the frequency of periodic subculturing, without affecting the viability and regrowth of shoot cultures. Cryopreservation refers to the storage of explants from tissue culture at ultra-low temperature (-196 degrees C). At such temperature, all the biological reactions within the cells are hampered, hence the technique makes available the storage of plant material for theoretically unlimited periods of time. An exhaustive review of papers dealing with the slow growth storage and the cryopreservation of ornamental species is reported here. Step-by-step protocols for the slow growth storage of rose germplasm, the production of synthetic seeds for the in vitro conservation of ornamentals, and the cryopreservation of Chrysanthemum morifolium are included.

Seed storage in polyethylene bags of a recalcitrant species (Quercus ilex): analysis of some bio-energetic and oxidative parameters

Some technological and physiological aspects were analysed during seed storage of holm oak (Quercus ilex L.), a typical Mediterranean recalcitrant species. Considering the mild dormancy and desiccation sensitivity of these seeds, the influence of the storage environment on viability was examined. Acorns were maintained at low temperature and at high moisture content either inside thin polyethylene bags or mixed with peat in a bin. Storage in polyethylene bags, if compared with peat treatment, maintained optimal seed quality parameters. The effects of the two treatments on some physiological parameters were analysed during 1-year seed storage. Acorns stored in a bin with peat started to germinate early and exhibited a more rapid seed deterioration caused by metabolism-linked oxidative damage. The latter phenomenon was linked to a higher respiration and H2O2 level, induction of catalase activity, as well as lowered glutathione pool and ATP content. In addition, a more oxidized redox poise was observed. On the contrary, the plastic film of polyethylene, limiting gaseous exchanges, maintained acorn metabolic activity at low levels and allowed the accumulation of ethylene inside the storage atmosphere. These factors, inducing a dormant-like state, could have played a crucial role in prolonging seed storage.

Cryopreservation of Germplasm of Populus (Poplar) Species

Trees and shrubs of the family Salicaceae (poplars, aspens and willows) provide a wide range of industrial construction woods (sawtimber, peeler logs, pulpwood), fuel wood, poles, light packing material, plywood, matches, as well as being important in biomass and feedstock production. Furthermore, they are vital for protection of soil, crops, livestock and dwellings, and, as ornamental plants, they are used extensively in parks, gardens, streets and open spaces (Fig. 1). The species are dioecious, with male and female flowers on separate trees. Between the two genera forming the family, i e., Salix and Populus, the latter is by far the more important from an economic point of view and includes both poplars and aspens. The genus Populus is characterized by a chromosome number of 2n=38, although triploid (2n=57) and tetraploid (2n=76) individuals have occasionally been recorded. The 34 species of the genus are botanically divided into five sections (Turanga, Leuce, Aigeiros, Tacamahaca and Leucoides) and these have an extremely wide geographical distribution throughout the Mediterranean, temperate, and colder regions of the northern hemisphere. While two sections (Turanga and Leucoides) do not include species of commercial value, the other three include several species of great economic importance.

Somatic embryogenesis in cypress (Cupressus sempervirens L.)

The genus Cupressus, native to warm temperate climates of the Northern hemisphere, can be found around the Mediterranean, in North America and in Asia. Twenty-five taxa have been identified in the genus and described as species (Ducrey et al., 1999), all generically named “cypress”. Here, we refer to the species Cupressus sempervirens L., also called “common”, “Mediterranean” or “Italian” cypress, by far the most important and widespread cypress in the Mediterranean basin. The species is native to northern Persia, as well as Syria, Turkey, Cyprus and several Greek islands. However, during the Roman Empire it was introduced into all the Mediterranean countries, where it can now be considered naturalised. The cypress grows up to 30 m in height, it is monoecious, and bears male and female strobili (cones) separately at the end of short branchlets. Depending on the crown branch habit, the species is divided into two varieties, i.e.: C. sempervirens var. horizontalis, the most common in natural areas, characterized by spreading branches and a broad conical crown; C. sempervirens var. pyramidalis (= var. fastigiata), the most popular for ornamental use because of its erect branches, parallel to the trunk, which give the tree its typical columnar shape, resembling a flame (Fig. 1).

Impact of two arbuscular mycorrhizal fungi on Arundo donax L. response to salt stress

AbstractMain conclusionAM symbiosis did not strongly affectArundo donax performances under salt stress, although differences in the plants inoculated with two different fungi were recorded. The mechanisms at the basis of the improved tolerance to abiotic stresses by arbuscular mycorrhizal (AM) fungi have been investigated mainly focusing on food crops. In this work, the potential impact of AM symbiosis on the performance of a bioenergy crop, Arundo donax, under saline conditions was considered. Specifically, we tried to understand whether AM symbiosis helps this fast-growing plant, often widespread in marginal soils, withstand salt. A combined approach, involving eco-physiological, morphometric and biochemical measurements, was used and the effects of two different AM fungal species (Funneliformis mosseae and Rhizophagus irregularis) were compared. Results indicate that potted A. donax plants do not suffer permanent damage induced by salt stress, but photosynthesis and growth are considerably reduced. Since A. donax is a high-yield biomass crop, reduction of biomass might be a serious agronomical problem in saline conditions. At least under the presently experienced growth conditions, and plant–AM combinations, the negative effect of salt on plant performance was not rescued by AM fungal colonization. However, some changes in plant metabolisms were observed following AM-inoculation, including a significant increase in proline accumulation and a trend toward higher isoprene emission and higher H2O2, especially in plants colonized by R. irregularis. This suggests that AM fungal symbiosis influences plant metabolism, and plant–AM fungus combination is an important factor for improving plant performance and productivity, in presence or absence of stress conditions.

Cryobiotechnology of forest trees: recent advances and future prospects

Globally, forests are of great economic importance and play a vital role in maintaining friendly ecological environments, sustainability of eco-systems, and biodiversity. Harsh environments, human activities and climate warming have long threatened the diversity of forest genetic resources. Among all conservation strategies, cryopreservation is at present time considered an ideal means for long-term conservation of plant genetic resources. To date, studies on cryopreservation of forest trees have been far behind agricultural and horticultural crops. The present review provides a comprehensive and update information on recent advances in cryopreservation of shoot tips, somatic embryogenic callus and seeds of forest trees. Assessments of genetic stability in the regenerants following cryopreservation were also analyzed and addressed. Further studies on cryopreservation of forest trees are proposed and needed. By doing so, we expect to re-evoke research interests and promote further developments in forest tree cryobiotechnology, thus assisting to ensure maintenance of biodiversity of genetic resources of forest trees.

Somatic plant regeneration from selected common cypress ( Cupressus sempervirens L.) clones resistant to the bark canker disease

The effectiveness of a protocol for somatic embryogenesis in conifers requires both the proliferation of embryonal masses and their conversion into somatic plants. Despite several successful protocols developed for Pinaceae, species belonging to Cuperessaceae family are often characterized by a problematic and unsatisfactory maturation of somatic embryos. Hence, the main goal of this study was to overcome the problem of embryo maturation and plant regeneration in Cupressus sempervirens, a Mediterranean species widely used for its ornamental value, timber production and interest in reforestation programmes. Embryogenic lines were produced from selected canker-resistant genotypes of common cypress; the effect of polyethylene glycol (PEG), desiccation period, medium composition and culturing period on the somatic embryo maturation and conversion, were evaluated. Despite significant variations observed among genotypes, the PEG based medium was the most effective for somatic embryo maturation. Germination and conversion of mature somatic embryos took place after three months of culture in a low-sucrose LP medium with activated charcoal. A short desiccation period failed to improve the germination rate of the mature somatic embryos. To our knowledge this is the first protocol reporting on somatic plant regeneration from somatic embryos of C. sempervirens.