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Genetic Resources of small Fruits, Present andFuture DevelopmentV. Kondakovaa, E. Todorovskaa, R. Boichevab, E. Hristovaa, I. Badjakova, M. Todorovac, D.Domosetovab & A. Atanassova

a AgroBioInstitute, Sofia, Bulgariab Institute of Agriculture, Kyustendil, Bulgariac Institute of Mountainous stock breeding and Agriculture, BulgariaPublished online: 15 Apr 2014.

To cite this article: V. Kondakova, E. Todorovska, R. Boicheva, E. Hristova, I. Badjakov, M. Todorova, D. Domosetova & A.Atanassov (2005) Genetic Resources of small Fruits, Present and Future Development, Biotechnology & BiotechnologicalEquipment, 19:sup3, 4-12, DOI: 10.1080/13102818.2005.10817281

To link to this article: http://dx.doi.org/10.1080/13102818.2005.10817281

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CRITICAL REVIEW ABI 2005

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GENETIC RESOURCES OF SMALL FRUITS, PRESENT AND FUTURE DEVELOPMENT V. Kondakova1, E. Todorovska1, R. Boicheva2, E. Hristova1, I.Badjakov1, M. Todorova3, D. Domosetova2, A. Atanassov1 AgroBioInstitute, Sofia, Bulgaria1 Institute of Agriculture, Kyustendil, Bulgaria2 Institute of Mountainous stock breeding and Agriculture, Bulgaria3

ABSTRACT This review focused on application a new biotechnology and molecular methods for pre-servation, evaluation and utilization of Plant Genetic Resources. The assessment of Bul-garian Small Fruits collections take up very important place in agro-horticultural crops with great significance for breeders. A number of strategies for collection, preservation and sustained use of genetic resources for agricultural productivity and food security were applicable. In vitro techniques offer the opportunity for in vitro collecting, rapid propagation, medium and long-term storage of germplasm and its distribution. The appli-cation of molecular markers will allow to selection of new perspective forms and inclu-ding in future breeding programme. Introduction The provocation of new century is in cor-relation with increasing of globalization, urbanization and population growth. The last statistic data showed that more than 800 million people are hungrily and more than 10 million children starve to death each year. The global changes and prob-lems imposed more requirements to agri-cultural productivity. To make this prob-lems manageable, the production of main crops must be made more efficiently.

Nowadays the main obligation of world science community is directed to protection of habitats and the careful management of genetic resources (67, 71). All agricultural commodities, including modern varieties, descend from a variety of wild and im-proved genetic resources found in allover the world. Agriculture also depends on di-verse genetic resources found in the wild, which help sustain the larger ecosystem in which agriculture operates.

The issues of how and under what condi-tions genetic resources can be accessed and

subsequently used, particularly for indus-trial or commercial purposes, have become central topics of discussion of national and international level (28, 29, 66).

Plant genetic resources in agro-horticul-tural crops and their wild relatives are with immense value for plant breeders. They require reservoir of genetic variation for crop improvement, such as resistance genes for diseases, pests and nematodes or for adaptation to wide ecological amplitudes and stress conditions.

There are a number of crops which are normally propagated vegetatively. In this group, the clonal material carries variable gene combinations which have been main-tained by the avoidance of sexual repro-duction. The serious problems is the vul-nerability of such clones to pests and pathogens or natural disasters to which they are almost continuously exposed. Much of the genetic resources is currently maintained as breeders collections in plan-tations and orchards. These field gene banks don’t represent the entire range of

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genetic variability within crop species (19). A number of strategies for collection,

preservation and sustained use of genetic resources for agricultural productivity and food security were developed. In vitro techniques offer the opportunity for in vitro collecting, rapid propagation, medium and long-term storage of germplasm and its distribution.

Genetic resources of small fruits The group of small fruits, mainly straw-berry and raspberry belonging to the vege-tative propagated species are wide-spread fruits and needed special attentions. Berry fruits are rich sources of bioactive com-pounds, such as phenolics and organic ac-ids, which have antimicrobial activities against human pathogens These comprise flavour compounds such as volatiles and sugars, quality-associated and health-asso-ciated compounds, such as antioxidants, tannins and antifungal. A large variability in these compounds is anticipated, espe-cially in wild species. However currently there is very little insight into the actual variation in these compounds between spe-cies and commercial lines (24, 35, 44, 55).

Recent statistics on world production of this species (38) indicate that they are the most important soft fruit worldwide. Ap-proximately 25% of world production is concentrated in North America (36) with 85 % of this in California, USA. In Euro-pean Union, Spain and Italy account for approximately 50% of production. Japan also has a sub stained small fruits industry, which is second only to that of the USA (59).

For the first time in Europe, under the framework of COST Action 836 an initial strawberry inventory was undertaken, re-sulting in the identification of 900 straw-berry cultivars in the main European col-lections and about 400 wild species acces-sions are preserved. (66). The main objec-tive was to use strawberry to create a model applicable to another berries in order to

improve fundamental knowledge. By the new project COST Action 863, started this year, the inventory of another small fruits will be continue (53).

In the first project the cultivars were clas-sified in four categories :old cultivars of genetic interest designated for preservation in the repository; old cultivars designated for eventual preservation after additional characterization; cultivars with less than 20 years under protection rights and old culti-vars with little known value and of minor interest (20).

It is important that these European col-lections are not only maintained but also utilized by the different European breeding programs. However, for this to be done effectively the germplasm must be well characterized (71).

Genetic resources of small fruits in Bulgaria Bulgaria is traditionally an important pro-ducer of soft fruits. The climate is very suitable for cultivation and a large genetic variation in wild and cultivated species is available. Over the last decades, this diver-sity has only been exploited to a limited extent. Most effort has been invested in selection of disease resistant lines and in improving productivity. Other properties, more focused on consumer benefits (fruit quality, flavor and healthiness), have been largely ignored. A large genetic reservoir providing tremendous variation in quality aspects has thankfully been maintained and one of the largest European collections re-sides at Institute of Agriculture, Kyustendil (the experimental station–Kostinbrod) and AgroBioInstitute (ABI), Sofia.

The present number of strawberry culti-vars in Bulgaria is 190 accessions, and for raspberry 125 accessions and 205 elites, maintained on field conditions (9, 10, 11, 12). The collection, classification and pres-ervation of this collections is one of the most important tasks of the both Institutes. The establishment of Modern Gene Bank

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for vegetative propagated soft fruits in ABI is an excellent source for high quality planting material The accessions are sub-jected to physiological, cytogenetically and molecular studies in order to evaluate the possibilities for their improvement using up-to date biotechnology methods. The in vitro techniques for the conservation of plant genetic resources include in vitro col-lecting, quarantine, disease indexing (ELISA, PCR and grafting), propagation, charac-terization, evaluation, storage by slow growth and criopreservation. (6, 13, 70, 73).

In vitro collecting and micropropagation The potential of plant biotechnology is based on the totipotency of plant cells re-generation of complete plants from cul-tured cells, and the production of genetic variants with useful characters. In vitro collecting techniques are effective for a large number of species, and have been developed to allow collecting at any time, to reduce bulk for transport, to maintain viability of tissue during transport and to provide uncontaminated germplasm which can facilitate their protection, characteriza-tion and utilization (65).

It offers not only means for mass propa-gation, but also plays an important role to conserve of elite plants (14). Micropropa-gation can be used also to accelerate the breeding process by in vitro selection of genotypes for the traits of interest, selection for salt tolerance has been evaluated by germinating seedlings in vitro on various concentrations of sodium chloride In vitro testing for resistance to V.dahliae and Ph.cactorum has also been reported (23).

Strawberry. Micropropagation of straw-berry has been used for commercial propa-gation of elite selections. They are estab-lished in vitro from meristem and axillary buds after surface sterilized and explanted on MS basal medium with supplementation of growth regulations-0.49 µM indolebu-tyric acid (IBA) and 8.9 µ M benzyladenin (BA). Shoot elongation and rooting of in vitro plants are in progress on MS medium

without growth regulators It is a very clear demonstration of the conviction that en-dogenous level of cytokinin in strawberry plants is enough for maintain of regenera-tion process. Among the researchers exist a big discussion about the yield and quality of in vitro and ex vitro rooted plant mate-rial. After a 4 weeks rooting period, plantlets that are rooted ex vitro have a larger root system. During subsequent growth, differences in development occur, e.g. more than twice as many runners are formed by ex vitro than in vitro-rooted plants (20, 21, 22, 32, 42).

In vitro grown strawberry plants demon-strated changes in the large and small subunit of ribulose 1,5-biphosphate car-boxylase (Rubisco), increased leaf chloro-phyll concentration and C : N ratios (31) Although micropropagated strawberries produce more flowers than conventionally propagated plants, they also produce more runners, which can lead to smaller fruits (74). Strawberry improvement by genetic engineering is already done in AgroBioIn-stitute. Two cultivars - “Selva “ and “Elsanta” were successfully transformed with LBA 4404 Agrobacterium lines harboring two dif-ferent reporter gene -uidA gene and agpB1.

Raspberry. The first report for prolifera-tion of Rubus shoots under in vitro condi-tions was reported from Broome and Zim-merman (1978) and Harper (1978).

The effective protocols for rooting and acclimatization of in vitro plants have also been described (18, 25, 30, 42, 46, 50, 72). The adaptation of protocols for micro propagation and rooting of raspberry de-pends in grate degree of genotypes. Disease indexing The production of virus-free planting mate-rial from strawberry and raspberry is rou-tine procedure for many laboratories in the world. The diagnostic by different methods for virus test (biological, serological and electron microscopy) is an obligatory step. The control of the health status of the strawberry mother plants has been mainly concerned with virus transmitted by aphids-

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Strawberry mottle virus (SMV) Strawberry crinkle virus (SCV) and Strawberry yellow edge virus (SYEV) where exist a great various strains with different pathogenicity. By this way there are explanation with various symptoms observed when testing plants by grafting on the susceptible indi-cators as clones of Fragaria vesca. (50). The extended exchange of planting mate-rial make us be especially careful in the field of an other group of viruses – nema-tode transmitted viruses (NEPO) (75, 76). The diseases caused by viruses of the NEPO group constitute a potential threat for the raspberry plantations. It is related to the raspberry monocultures growing (48).

In vitro culture allows maintenance of genetic resources in an environment free from pathogens, including fungal and bac-terial pathogens. Endogenous pathogens, particularly viruses can be eliminated using meristem-tip culture, thermotherapy and chemotherapy (47, 51).

In vitro culture and virus- elimination techniques need to be more widely applied for species where disease problems exist and are not restricted to clonally propa-gated species or species with recalcitrant seed (7, 58, 46, 49, 78).

Disease like grey mould (Botrytis cine-rea), powdery mildew (Sphaerotheca macularis), leaf spot (Mycosphaerella fra-gariae), verticillium wilt (Verticillium dahliae), crown and leather rot (Phy-tophthora cactorum) are very harmfull for soft fruits production.

Severity of diseases depends on weather conditions, susceptibility of cultivars and presence of infection source (31, 37, 43, 54, 55, 56).

Virus elimination With in vitro techniques it is possible to provide a germplasm storage procedure which uniquely combines the possibilities of disease elimination and rapid clonal propagation (44, 45). There is not defini-tive explanation can be given to understand

this virus eradication. Various explanations have been given: absence of plasmodesm in the meristematic domes, competition between synthesis of nucleoproteins for cellular division and viral replication, in-hibitor substances, absence of enzymes replication, inhibitor substances by exci-sion of small meristematic domes (79).

Endogenous pathogens, particularly vi-ruses can be eliminated using meristem-tip culture, thermotherapy and chemotherapy (45, 52). Two mainly approaches are ap-propriate for elimination of virus according Upadhya (1988), some viroids and viruses particularly are not necessarily eliminated or even detected and can readily multiply in tissue culture. The protocol involves of virus infected plants at relatively high tem-perature (36-38 ºC) for approximately 3-4 weeks. At the end of this period, meristem tips are excised and explanted on the suit-able medium until plantlets are large enough to be indexed by immunosorbent assay (ELISA) and PCR. By another ap-proach the virus detection fellow the clonal selection of mother plants with exclusive characters in means of yield, quality and genetic stability of cultivars. This mother plants can be used as sources for micro-propagation for more long time after pro-tect under the appropriate conditions. The success in virus elimination depends on the choice of the explants and the virus. This is the main approach for study of genetic re-sources in AgroBioInstitute, directed for evaluation of the health status of small fruits accessions. Samples of evaluated plants are stored under in-vitro conditions in growth chambers The part of valuable cultivars interesting for private farmers and customers are propagated and commer-cially distributed.

In general a high temperature (37 ºC) is effective for virus diseases, but is depres-sive for viroids. The introduction of virus inhibitors (ribavirin) into cultural medium can’t inactivate the viruses, but it prevents their replication. A combination of cytoki-

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nin and ribavirin or a heat treatment in vi-tro, are sometimes used with success to inactivate some viruses difficult to elimi-nate by meristem culture (14, 45).

Maintenance Storage using slow growth and criopreservation Slow growth techniques are now success-fully and routinely applied to a range of species and across a range of genotypes within species. This allows storage of healthy germplasm with extended subcul-ture intervals, thus reducing time and cost for maintains. Optimal methods still need to be developed for many species.

In strawberry, cultures have been suc-cessfully stored for six years (57, 68, 69). In meristem plantlets being kept viable at 4 ºC in dark with renewal of fresh medium, occasionally. Experimenting with 19 strawberry cultivars demonstrated that in cultures stored at 2 ºC for 13 months, sur-vival was 100 percent with one cultivar whereas it was 50 percent for two cultivars stored for 27 months (82).

It has been possible to maintain organo-genic suspension cultures of Fragaria vesca for 2 years in induction medium. Regene-rants have been obtained from cell suspen-sions after the cell mass is decanted on to semi-solid regeneration medium (41).

The effects of pre-freezing treatments, cryoprotectants and cooling rate on sur-vival of strawberry meristem have been studied A criopreservation protocol has also been established for strawberry sus-pension cultures (82).

The extreme diversity and large numbers of species and cultivars associated with Rubus germplasm make it difficult to maintain collections of these lines in either a green house or the field. As alternative method would be to store shoots and mer-istems for long periods of time under in vitro conditions. Reed and Lagerstedt (1987) and Reed (1988) reported methods for cryopreserve of Rubus shoots (62, 64,

65). Chang and Reed (1999) reported studies on the long-term survival of Rubus plants after criopreservation.

Characterization, evaluation and monitoring In november 1987, IPGRI held a meeting on the use of molecular techniques in plant genetic resources conservation and since than has collaborated in a number of pro-jects in which molecular techniques have been used. The potential benefits of using the molecular techniques are clear and in-dividual genetic resources programme are likely to make increasing use of the diffe-rent methodologies.

Somaclonal variation may arise in culture or in field-grown plants, and genetic stabi-lity needs to be carefully monitored to en-sure that desirable traits are retained. Fur-ther research is necessary to establish and compare the extent of, and kinds of, varia-tion arising from field-grown material, from slow growth cultures and after crio-preservation.

These include analysis of diversity for different species, fingerprinting and use of RAPD to assess the stability of in vitro conservation (16, 26, 52, 80). Conservation and increasing utilization of genetic re-sources requires it detailed characteriza-tion.

There are many problems to be addressed before a universal strategies for the wide-spread to be recommended as how quickly the techniques can be adapted to work in any new system.

Restriction fragment length polymor-phism (RFLP) analysis was the first tech-nology developed which enabled the de-tection of polymorphism at the sequence level.

RFLP analysis is used extensively in the construction of genetic map and has been successfully applied to genetic diversity assessment, particularly in cultivated plants (17, 27) but also in populations and wild accessions (8, 50).

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Fig. 1. Genetic relationship between 16 Bulgarian raspberry accessions using UPGMA cluster analysis based on SSR markers.

As a techniques for diversity studies,

there are three important advantages which should be considered – a. RFLP is highly reproducible between laboratories and the diversity profiles generated can be reliably transferred b. RFLPs are co-dominant markers, enabling heterozygotes to be dis-tinguished from homozygotes, c. no se-quence-specific information’s is required and provided suitable probes are available, the approach can be applied immediately for diversity screening in any system. They have also been used for studding within and between population variation, for eco-logical studies (1, 76) and for estimating genetic distances (2).

RAPDs markers have also been proved to detect higher levels of polymorphism compared with RFLPs in case where the two techniques have been applied to the same material. RAPDa are dominant mar-kers such that the homozygote conditions are the only genotypes discernible as pre-

sence or absence of the band. RAPDs analysis has been used successfully to identify and differentiate several strawberry cultivars, including closely related ones (34).

The important advantages of choosing SSRs markers for population genetic stu-dies They are usually single loci which, because of their high mutation rate, are often multi-allelic (63), they are co-domi-nant markers and they can be detected by a PCR assay. They are very robust tools that can be exchanged between laboratories and their data is highly informative.

Highly polymorphic molecular markers like RAPD, RFLP and microsatelittes are very suitable for characterizing of the ge-netic resources.

Molecular markers using for characteri-zation of strawberry and Rubus species included isozymes and DNA-based mar-kers such as RFLPs, RAPDs, microsate-lites. These markers have been used for

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cultivar identification, identification of so-maclonal mutations, parentage testing, identification of hybrids, taxonomy studies of genotypic distribution in wild Rubus populations, and marker-assisted selection (3, 40).

The characterization and assessment of genetic diversity in Bulgarian raspberry collection by DNA markers (RAPDs, SSRs) have also been in progress. The ap-plication of SSRs markers should give an answer to breeders for expansion of genetic diversity and could allow selection of new perspective forms in future breeding pro-gramme. The determinate value for genetic variation proved a high genetic diversity (GD) in Bulgarian germplasm collection.

It calculated from SSR data ranged from 0.740 to 0.887 with a mean GD-0.839 The application of co-dominant SSR markers has allowed the evaluation of heterozigos-ity (4).

RAPD analysis proved to be efficient in discrimination of elite raspberry lines with common pedigree. Genetic diversity (GD) was studied in a set of 29 raspberries from Bulgarian collection and separately in the groups representing different geographical regions: Europe, Canada and United States. Mean Genetic Diversity (MGD) in all studied loci was 0.863 Genetic diversity (GD) in the group of Bulgarian varieties was 0.777 compared to European with 0.694 and American - 0.760 respectively. The higher GD observed in the set of Bul-garian raspberry varieties is probably due to more included genotypes (5).

The research activity for DNA charac-terization of small fruits wits DNA markers will be continue for evaluation of all small fruits belong to Bulgarian germplasm col-lection.

Conclusions The collection, preservation and sustained use of genetic resources have become criti-cal for continued agricultural productivity and food security.

The establishment of gene bank for vegetatively propagated plants like small fruits in all over the world and in particular in AgroBioInstitute – Sofia is one of the main priorities. The characterization of accessions is with great potential for them utilization as excellent sources for high quality planting material.

Strawberry improvement will increas-ingly rely on the application of biotechno-logy to develop improved cultivars ac-cording the require of growers and con-sumers. Great and generally unexplored potential exist in the application of muta-tion breeding, perhaps as an alternative to genetic transformation in the short term in Europe.

Somaclonal variation may prove to be a bonus in the application of other micro-propagation techniques. New marker tech-niques such as SSRs and SNPs when de-veloped and applied to strawberry should be useful for development saturated linkage maps for mapping quantitative trait loci (QTLs) (32).

The genus Rubus is taxonomically di-verse and includes plants from many parts of the world. Somaclonal variation, al-though random and undirected, has been demonstrated to have potential for Rubus improvement. Because it is possible to use both sexual and asexual methods to im-prove Rubus species (33).

The AgroBioInstitute strategy for devel-opment and recognization as date base of Bulgaria Small Fruits Collection has made considerable next strategic areas: • Improving and expanding of small fruits

collections. • Molecular, cytogenetical and biochemical

evaluation of existing accessions. • Development and application of in vitro

techniques for the conservation and use of small fruits collections The research activity for the future, ac-

cording plant genetic resources which emerge from the issues above, must be included in the research agenda in all Institutions.

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