Ex Situ Conservation Methods

Ex situ conservation is a form of conservation of forest genetic resources outside their natural habitat (out of site). It may include conservation of single individuals, groups of individuals or just conserving their individual parts. Preserving individu-als as individual conservation units is performed in the botanical gardens, arboreta or living archives. Conservation of the smaller or larger groups of individuals is per-formed by establishing seed orchards, clonal archives, progeny tests or provenance trials. Preservation, and conservation of certain parts of an individual, is realized in gene banks. As classical methods of static ex situ conservation can be considered only seed banks, pollen banks, DNA banks, in vitro explants, cryopreservation and clonal archives in which are, due to genetic uniformity of individuals and vegeta-tive turnover of generations, almost eliminated the effects of natural selection. Any other method of ex situ conservation, primarily, can be regarded as dynamic.

7.3.2.1 Arboreta and Botanical Gardens

Botanical Garden represents a collection of living plants, which is mainly used for improvement and dissemination of botanical knowledge (Potočić 1980). As forerun-ner of today’s botanical gardens can be considered private gardens where wealthy people, in being and nature lovers, collected and cultivated different plants. The first public botanical garden was founded by the Venetian Republic in 1545, in Padua as a scientific institution of the university of that time. Modeled on the botanical gardens in Padua, Pisa and Bologna, botanical gardens were founded throughout Europe. Nowdays, there are being kept unique specimens of various plant species, some of which a long time ago disappeared from the natural habitat. An arboretum (lat. arbor-tree) is a separate space or part of the botanical gardens, in which are grown trees and shrubs in the scientific, ornamental and breeding purposes (Potočić 1980). The oldest such collection of trees and shrubs was mentioned in botanical garden in Tokyo, which is reportedly over 800 years old.

7.3.2.2 Seed Orchads

Establishment of ex situ populations requires considerable financial resources. The economic justifiability of these populations appears only in cases when achieve double aim, such as simultaneously collecting seeds for commercial purposes and long-term conservation of genetic diversity of seed sources, which is best achieved by establishing seed orchards. Seed orchards and clonal archives are examples of static ex situ conservation units, because there are no dynamic changes in their ge-netic structures. However, those are important pilot objects in the program of testing the genetic potential of initial population, because effects of simultaneous natural and artificial selection processes may be observed. The purpose of establishing these

orchards is to establish populations that will maintain the original genetic variability to the maximum extent and allow long-term adaptation to local conditions, where planting was done. In addition to preserving the original genetic variability, seed orchards are used as sources of reproductive material for commercial forestry.

Seed orchard means specialized, artificial culture for long-lasting production of genetic quality seeds of economically significant tree species (Tucović 1990). Seed orchards are established from phenotypic and genotypic best individuals of one, or various, species, at area which is supposed to be spatially isolated from physically mature trees of the same species, in order to prevent uncontrolled pollination of in-dividuals that come out of the seed orchard. According to the character of planting material, plantations can be vegetative, formed of clones or generative, formed of plants originating from the half-sib or full-sib lines of selected genotypes.

7.3.2.3 Progeny Tests

Progeny tests represent an opportunity for exploring the genetic potential of certain species, provenances, populations or the genotype. The basic principle of the es-tablishment of such experiments is to create uniform conditions for the cultivation of plants that are being tested, so that mutually appeared differences are reflection of various genotypes, not various environmental conditions. Progeny tests may be established of full-sib lines, which represent offspring of controlled hybridization (both mother and father are known), or half-sib lines, where the mother is known, and the father is unknown (free pollination). Tests can be performed in the phyto-trons, greenhouses, nursery or field conditions. According to duration of experi-ment, there are early tests, short-term and long-term tests. A particular form of ge-netic potential assessment, which are constantly gaining in importance are early tests. Research conducted in the earliest stages of ontogenesis in Serbian spruce have pointed to the importance of the same for exploring the variability of species, its taxonomy, genetic potential, cultivation, breeding and conservation direction (Šijačić-Nikolić and Milovanović 2010). The assessment of adaptive and produc-tion potentials of different lines of half-sibs in progeny tests is used for the selecproduc-tion of elite trees, which represent a basis for seed collection and seedling production, aimed at spreading the population of some species and preservation of genetic vari-ability (Nonić et al. 2012).

The age variability is the result of natural selection, which is expressed at each stage of the life cycle. Therefore, the variability of plants in the juvenile develop-ment phase can be considered as products of the interaction of hereditary basis and selection. Genetic variability of the mature population is significantly reduced, compared to the variability of baseline levels seedlings, 1 year or 2 years old plants, considering that selection was the highest in the germination stage, after which it was reduced, but continuous. Wherein the larger dimensions of the cotyledons, hy-pocotyls and epicotyls characteristics, good root system characteristics of, and simi-lar, indicate the potential superiority of adult individuals in comparison to the aver-age. Every geneticist, breeder or nurseryman, who knows spontaneous variability,

already after the formation of cotyledons, can observe possible deviations from the usual form, in particular in the haploids, polyploids or polysomics, which provides high speciation (Tucović 1990). The results of the early tests should be considered approximate they need to be checked in the following years of research.

7.3.2.4 Provenance Trials

Provenance trials, within gene pool conservation, are applied as a method of as-sessing the degree of diversity and potential, both autochthonous and allochthonous trees species. Also, provenance trials may contribute to determining the potential and the degree of divergence of isolated populations, in terms of higher productivity and adaptability, respectively, can be used to determine the differences in genetic variability between and within different provenances.

7.3.2.5 Preservation

The gene banks are formed to preserve the total genetic variability on a scientific basis. The task of gene banks was to collect, determine, document, reproduce and preserve genetic variability at the long term, and made it available for use. A fully and easily accessible information are the primary requirements for the use of ge-netic variability that is stored in gene banks (Penčić et al. 1997). Plant gene banks are formed from the parts of plants that contain germplasm—genetic basis of spe-cies. The most commonly applied method is cryopreservation, which allows long-term storage of plant parts (Withers and Engelmann 1998). This long-term refers to seed storage at extremely low temperatures, typically with the use of liquid nitrogen (− 196 °С). Applied together with the in vitro technique, cryopreservation is often the only reliable, safe and economically viable method of storage of some species.

In cases where the seed is not suitable for cryopreservation, the extract of embryo or the core of the embryo should be applied, at the appropriate stage of develop-ment. Establishment of seed banks, where the seed is stored in refrigerators or under other appropriate conditions, is another form of static ex situ conservation. Seed banks can be used only for species whose seed storage is possible. Most species have seeds with high germination rates sustainable a few years, which is extremely short, compared to the long life span of trees, and therefore the seed supplies must be renewed at regular intervals. It involves germination, seedling production, tree growing to the beginning of fruiting, collection and storage of new seeds.

This “rejuvenilization” leads to the appearance of new genetic recombination and new selection pressures during the propagation and growth. For most species, seed banks have to be a short-term form of conservation. Seeds of endangered pop-ulations can be collected and stored in a bank for a fixed period, until the most suit-able moment for the seeding and seedlings development for the establishment of ex situ conservation habitats.

In addition to seed banks, germplasm conservation can be also realized by es-tablishing pollen banks in which the pollen, dryish to 5 % moisture, is stored on dry surfaces and temperature of 0 °C. However, some species produce pollen whose longer storage is not possible. There are experiences with species whose pollen loses viability and germination after 5 years of storage (Towill 1985). Certainly, pollen has a relatively short period of viability compared to the seed. For these rea-sons, despite the great importance of this technique for germplasm conservation of species with low seed storage capabilities using pollen banks is very limited.

Germplasm conservation can be realized by applying the method of DNA banks, which are increasingly gaining in importance. Nowadays, DNA, isolated from the nucleus, mitochondria, or chloroplast, can be easily immobilized in the nitrocel-lulose fibers, where it can be stored. Reproduction of a specific oligonucleotide, or the entire gene from genomic DNA has become a routine procedure by the de-velopment of PCR techniques. This method has enabled the creation of an interna-tional network of DNA storage (Adams 1997). The advantages of this technique are its efficiency, simplicity and small space requirement. The main disadvantage, besides demanding equipment and facilities, lies in the restrictions during the isola-tion, cloning and gene transfer, i.e. impossibility of regenerating of the whole plant (Maxted et al. 1997).

Another type of static ex situ conservation strategy is in vitro conservation. De-pending on the species and techniques, some genetic changes (such as mutations) can occur during the in vitro growth and storage, and may sometimes be called somaclonal variability (Fourre et al. 1997). In vitro (in glass) method involves the manipulation of the explants in a sterile environment, without the pathogen. It is used for the conservation of species whose seeds cannot be stored for a longer time period, the species that do not provide seed or vegetative material obtained in order to maintain the target genotypes (Engelmann 1997).

Although the first studies on the in vitro technique were conducted only a few decades ago, this technique is already used for multiplication, storage and collec-tion of germplasm of over 1000 species (Bigot 1987). In vitro technique can be ef-fectively used for collection, multiplication and storage of particularly problematic species (Еngelmаnn 1997). The technique was developed in order to produce plants from seed or vegetative material, directly from the field, under aseptic conditions (Withers 1995). Such an approach allows the creation of germplasm collections in the fields of restoration, or in situations where the seeds transport is economically unjustified. However, due to the very high costs of implementation, this method is generally less useful in the forest genetic resources conservation. The static con-servation type includes also clonal archives, which represent a set of vegetatively propagated clones. Grafted or rooted trees will still be able to grow up to a certain age, if the grafting was successful. Naturally, for a certain time, grafting or rooting procedure must be repeated. This static conservation type requires continuous and intensive management by a man.