Connotation of Biotechnology in Plant Genetic Resources Management
Author: A. K. Trivedi
National Bureau of Plant Genetic Resources
Regional Station - Bhowali - 263132, Nainital (Uttarakhand)



Biotechnology is defined as 'any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use.' In other word biotechnology is a set of biological techniques developed through basic research and now applied to research and product development. In particular, biotechnology refers to the use of recombinant DNA, cell fusion, and new bio processing techniques.

Plant Genetic Resources (PGR) refer to genetic material of plant origin of actual or potential value in the form of seed, vegetative propagule, tissue, cell, pollen, DNA molecule etc. containing the functional unit of heredity that can includes varieties, landraces and wild/ weedy relatives of economically important pant species. In broad sense, the genetic resources of a crop include the varieties developed by farmers in indigenous farming systems and maintained by them for generations often referred to as traditional varieties, landraces or farmers' varieties, related wild species, modern commercial varieties, obsolete varieties, breeding lines and genetic stocks.

Plant genetic resources are among the most vulnerable of all nonrenewable natural resources-once lost, they are lost forever. That is why, for several decades, there have been concerted international efforts to collect and conserve plant genetic resources for food and agriculture in genebanks worldwide. These efforts culminated in June 1996 during the Fourth International Technical Conference on Plant Genetic Resources in the adoption by 150 countries of a Global Plan of Action for the Conservation and Sustainable Utilization of Plant Genetic Resources for Food and Agriculture (FAO 1996). The framework of the plan is to ensure the long-term preservation of genetic resources at regional, national and international levels, as well as the necessary actions to facilitate use of these valuable resources for the benefit of all humans. Biotechnology is recognized as an important component of implementing the global plan.

Biological diversity is defined as 'the variability among living organisms from all sources including, inter alia, terrestrial, marine and other aquatic ecosystems and the ecological complexes of which they are part; this includes diversity within species, between species and of ecosystems.' Agricultural biodiversity, also known as agrobiodiversity, forms a subset of total biodiversity. It refers to biodiversity related to agriculture and can be described as 'the variety and variability amongst living organisms (animals, plants, and microorganisms) that are important to food and agriculture in the broad sense and associated with cultivating crops and rearing animals and the ecological complexes of which they form a part.' It includes the diversity found in farming systems as well as their surroundings to the extent that the latter influences agriculture. Agro-biodiversity occupies a unique place within biodiversity. It recognises that agriculture evolved from bio-prospecting, selection and development of a few species from plant and animal kingdoms to meet human needs of food, fibre and fuel. All biotic factors related to agriculture, such as, plants, animals, fish, reptiles, insects, birds and microbes are components of agro-biodiversity. The conservation, management and sustainable use of these organisms (and their wild progenitors/ relatives) require specific attention.

The Indian gene centre is among the twelve mega diversity regions of the world. About 25 crop species were domesticated here. It is known to have more than 18,000 species of higher plants including 160 major and minor crop species and 325 of their wild relatives. Around 1,500 wild edible plant species are widely exploited by native tribes. These include 145 species of roots and tubers, 521 of leafy vegetables/greens, 101 of buds and flowers, 647 of fruits and 118 of seeds and nuts. In addition, nearly 9,500 plant species of ethnobotanical uses have been reported from the country.

The traditional farming systems of India are relatively stable and in equilibrium. The species complexes in traditional farming systems exemplify co-existence of plants and human tribes, draught animals, friendly birds, beneficial insects, pollinators, earthworms, soil micro-organisms and bio-control agents. Modern farming systems, which evolved in response to the growing needs of the human society to ensure food and nutritional security, have progressively replaced traditional agriculture. More than half of the cultivated area under major crops is now covered by improved varieties and farming practices. Biotic diversity is maintained in modern agricultural systems primarily through cultivation of "mosaic of improved varieties". It is important that diversity is assured while attaining high production levels and profitability. A combination of ex situ and in situ conservation approaches is required for agro- biodiversity conservation strategies in the Indian gene centre. Human tribes, particularly women, have a long tradition of preserving plant species in situ and conserving the agro-ecosystems. There is a need to preserve the traditional practices and learn from the available indigenous technical knowledge (ITK).

Genetic diversity of agricultural crops provides the building for sustainable food, health and livelihood security systems. It is the feedstock for both the biotechnology industry and a climate resilient farming system. In spite of the importance given to the conservation of agro-biodiversity, genetic erosion is progressing in an unabated manner, both globally and nationally. According to the International Union for Conservation of Nature (IUCN) over 47,677 species may disappear soon. Therefore, for conserving plant genetic resources there is a need for launching a Biodiveristy Literacy Movement, so that from childhood onwards everyone is aware of the importance of diversity for the maintenance of food, water, health and livelihood security as well as climate resilience food production system.

National Bureau of Plant Genetic Resources is nodal organization in India for exchange, quarantine, collection, conservation, evaluation and systematic documentation of plant genetic resources. More plant genetic resources are conserved and made available for future use, better the chances of fulfilling future demands. Therefore, bureau is involved in the collection of agro-biodiversity on the basis of prioritization both at species and habitat level. The collection of germplasm of priority gene pool is required due to:

1. Danger of loss of genetic resources leading to erosion.

2. Diversity missing or insufficiently represented in existing ex situ germplasm collection.

3. User's need expressed at national and international levels.

4. Rescue collection of genetic diversity at threat due to natural calamities of habitat loss and where in situ conservation methods are not feasible.

5. Collecting for immediate use by local communities engaged in harvest of traditional medicine.

If we will compare the food grain availability in India, today with that of 50 years ago, the developments may be divided in three steps. The first major development was the Green Revolution in the 1960s and 1970s which resulted in unprecedented increases in food production. The introduction of high-yielding, semi-dwarf wheat and rice varieties doubled production in a short span of five years. 1965-66 to 1971-72. Wheat production increased from 10.4 million tons in 1965-66 to 26.4 million tons in 1971-72. The same period witnessed a doubling of wheat yield from 827 kg/ha to 1380 kg/ha.

The second development was more modest and associated with the introduction of hybrid seeds which replaced open pollinated varieties (OPVs) primarily in selected vegetable crops such as brinjal, bottlegourd, cabbage, capsicum, chilli, okra, onion and tomato and in field crops such as castor, cotton, maize, pearl millet, sorghum, and sunflower in the 1980s and 1990s. Whereas hybrid seeds need to be replaced by farmers every year, they offer an attractive incentive to both large and small farmers because of the significant yield grains from hybrid vigor, and moreover they provide an important technology platform for enhancing productivity in a sustainable manner in the longer term. Hybrids are bred to exploit vigor by improving seeds. The first generation hybrids were developed for open pollinated crops as it was relatively easy to exploit hybrid vigor in those crops. The second generation hybrids were developed in self-pollinating and naturally inbred crops like rice which present more difficulties. Some of the hybrids are based on the heterosis system and others on the cytoplasmic male sterile (CMS) line system, where three lines are required to exploit hybrid vigor.

The third major development was in 2002, which featured the application of biotechnology to crops which led to the approval and commercialization of Bt cotton, the first biotech crop in India. bt cotton hybrids are incorporated with a new insecticidal gene(s) sourced from the common soil bacterium Bacillus thuringiensis that confers resistance to the critically important lepidopteron insect-pests, cotton bollworms.

All these developments are possible if genetic diversity is available for crop improvement program because plant genetic resources for food and agriculture (PGRFA) are the back bone of global food security. They comprise diversity of genetic material available in traditional varieties, modern cultivars, crop wild relatives and other wild species. Available genetic diversity provides the options to develop, new and more productive crops, through selection and breeding, that are resistant to virulent pests and diseases and adapted to changing environments. In this situation we urgently need a climate resilient agriculture which will have to be based on a two pronged strategy - i.e., maximizing farm productivity and production during a normal monsoon period and minimizing the adverse impact of unfavourable weather. Therefore, to fulfil the ever increasing demand of food, better understanding and management of plant genetic resources is essential. Genetically uniform modern varieties are replacing the highly diverse local cultivars and landraces in traditional agro-ecosystems. Changes in land-use pattern are heavily affecting diversity of the wild species. Industrialization, urbanization, globalization, changing life styles and market economies are contributing indirectly to the loss of diversity, particularly of minor and neglected crops.

The concept of germplasm conservation requires collection methods that initially capture maximum variation and subsequently, conservation and regeneration techniques to minimize losses through time (Astley, 1992). To this effect, plant genetic resources (PGR) conservation activities comprise of collection, conservation identification of potentially valuable material by characterization and evaluation for subsequent use. Recent advances in biotechnology, provide important tools for improved conservation and management of plant genetic resources. Role of Biotechnology in PGR management can be explained under following activities related to PGR management:

1. GERMPLASM COLLECTION

Germplasm collection involves gathering samples of a species from populations in the field or natural habitats for conservation and subsequent use. The unit of collection may be seeds or vegetative propagules, depending on the breeding system of the species. Collection is easy in species producing small seeds in abundance. However, it becomes problematic when seeds are unavailable or non-viable due to damage of plants by grazing or diseases; large and fleshy seeds that are difficult to transport; or where samples are not likely to remain viable during transportation due to remoteness of the collection site from the genebank. Advances in biotechnology provide useful solutions for collection of such problem species (Withers, 1995). For example, in coconut (Cocos nucifera), where the major difficulty for standard seed collection is the large size of the seeds, in vitro techniques have been developed that allow collection of the relatively small zygotic embryos in the field and transporting them back in sterile conditions to the laboratory to inoculate and germinate them on a culture medium (Assy-Bah et al., 1989; Ashburner et al., 1996).

2. GERMPLASM EXCHANGE

Planting material is an international commodity used for exchange of germplasm, infected seeds inadvertently serve as means of introducing plant pathogens into new areas. Therefore, seed health tests and phytosanitary regulations are generally incorporated as part of the genebank activities. The demand to store and exchange seeds of high quality and for greater sensitivity in detecting seed-borne pathogens including viruses and bacteria and short turn around times for seed testing by molecular techniques have led genebanks adopt these technologies. The PCR technique used to amplify small amounts of DNA, also improves the efficiency of seed health tests.

Apical meristem culture has been used to successfully eliminate many viruses from a variety of plant species. Exchange of germplasm as in vitro cultures, offers considerable advantages like reduced volume and weight as well as the improved health status of the cultures. In crops like Musa spp., potato, yam and cassava in vitro germplasm exchange is a routine.

3. GERMPLASM QUARANTINE

Exchange of plant genetic resources (PGR) has contributed significantly towards crop improvement and increased crop production. For safe exchange of germplasm the quarantine related issues which are either legislative or technical need to be addressed. Quarantine is a government endeavour enforced through legislative measures to regulate the introduction of planting materials, plant products, soil, living organisms, etc. in order to prevent inadvertent introduction of pests (including fungi, bacteria, viruses, nematodes, insects and weeds) harmful to the agriculture and if introduced, prevent their establishment and further spread. There are several examples of entry of pests into new areas along with introduced planting material. The introduction of the late blight pathogen (Phytophthora infestans) of potato into Europe from Central America in the middle of 19th century, powdery mildew (Uncinula necator), root eating aphid (Phylloxera vitifolia) and downy mildew (Plasmopara viticola) of grapes into France in mid 19th century from America are a few examples of international spread of important plant diseases. All these introduced pests are causing enormous loss to human health, environment and biodiversity besides direct loss to crop production. Hence, there is a dire need to check the entry of the pests through quarantine measures. In case infestation/ infection is observed in imported material the consignment is tested further using specialized detection methods and the material is salvaged using suitable fumigation or fungicidal/ insecticidal treatments. Advent of biotechnological techniques for detection of pests have contributing for identification as well as remedial measures.

4. GERMPLASM CONSERVATION

Plant Genetic Resources can be conserved by two methods, namely in situ and ex situ. In situ conservation involves maintaining genetic resources in the natural habitats where they occur. In this method wild and uncultivated plant communities are conserved in their natural habitat and crop cultivars in farmers' fields as in the traditional agricultural systems. Ex situ conservation involves conservation outside the native habitat. It is usually used to safeguard populations in danger of destruction, replacement or deterioration. Approaches to ex situ conservation include methods like seed storage, field genebanks and botanical gardens. DNA and pollen storage also contribute indirectly to ex situ conservation of PGR. Among the various ex situ conservation methods, seed storage is the most convenient for long-term conservation of plant genetic resources. This involves desiccation of seeds to low moisture contents and storage at low temperatures. However, there are a large number of important tropical and sub-tropical tree species which produce recalcitrant seeds that quickly lose viability and do not survive desiccation, hence conventional seed storage strategies are not possible (Roberts, 1973). There are also a number of other important crop species that are sterile or do not easily produce seeds or seed is highly heterozygous and clonal propagation is preferred to conserve elite genotypes such as banana, sweet potato, sugarcane etc. These species are usually conserved in field genebanks. Although field genebanks provide easy access to conserved material for use, they run the risk of destruction by natural calamities, pests and diseases. For this reason, safety duplicates of the living collections are established using alternate strategies of conservation and it is in this area that biotechnology contributed significantly by providing complementary in vitro conservation options through tissue culture techniques. In vitro conservation also offers other distinct advantages. For example, the material can be maintained in a pathogen-tested state, thereby facilitating safer distribution. Further, the cultures are not subjected to environmental disturbances (Withers and Engelmann, 1997). In vitro conservation may be through (i) slow growth procedures, where germplasm accessions are kept as sterile plant tissues or plantlets on nutrient gels; and (ii) cryopreservation where plant material is stored in liquid nitrogen. Slow growth procedures provide short- and medium-term storage options, while cryopreservation enables long-term storage of the plant material.

Cryopreservation technique is well established for vegetatively propagated species. However, in case of recalcitrant seed species it is not much useful due to some of their characteristics, including their very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity.

5. GERMPLASM CHARACTERIZATION AND EVALUATION

Identification of genetic variability of collected material is essential for its management and further utilization. Phenotypic traits such as plant height, growth habit, flower colour, fruit size, pod size and agronomic traits like yield potential, stress tolerance, etc., are easy characters for assessing genotypic diversity. But genetic variability assessed by phenotypic characters has some limitations and quantitative traits are influenced by environmental factors.

In the second stage variability may be assessed by biochemical traits such as differences between seed protein and enzyme electrophoresis. Biochemical characterization removes the effects of environmental factors. However, these methods have some inherent problems; these can not detect low level of variation.

Furthermore, molecular characterization can analyse the variation at the DNA level itself, excluding all environmental influences. The analysis can be performed at any growth stage using any plant part and it requires only small amounts of material. Several methods have been developed for analysis of genetic diversity. These methods differ with respect to technical requirements, level of polymorphism detected, reproducibility and cost (Karp et al., 1997; Karp, 2002). Molecular methods used for detecting DNA sequence variation are generally based on the use of restriction enzymes that recognize and cut specific short sequences of DNA (e.g., Restriction Fragment Length Polymorphism, RFLP) or polymerase chain reaction (PCR), which involves amplification of target DNA sequences using short oligonucleotide primers. PCR based techniques such as Random Amplified Polymorphic DNAs (RAPDs), Amplified Fragment Length Polymorphisms (AFLPs) and Simple Sequence Repeats (SSRs, microsatellites) have proved especially important in diversity studies. Newer and more powerful molecular techniques that detect variation at specific gene loci, which can be automated for high throughput of samples, are becoming available (Sicard et al., 1999), permitting precise and versatile analyses of genetic variation. Molecular methods are increasingly playing an important role in conservation and management of plant genetic resources.

Specific areas in which molecular marker techniques have been used are developing sampling strategies and identification of gaps in the collections to plan for future collecting and acquisition and managing conserved germplasm - including identification of redundancies, development of core collections, fingerprinting, identification of genetic contamination and quantification of genetic drifts/shifts.

6. GERMPLASM USE

Challenges for effective utilization and enhancement of plant genetic resources have been minimized by biotechnological interventions. Such as, a number of techniques have been developed to overcome problems of sexual incompatibility that lead to hybrid sterility or lack of genetic recombination in wide crosses involving distant wild relatives and cultivated species.

a. Molecular markers in sampling strategies

Molecular markers have been applied to study genetic diversity from natural populations and formulate efficient sampling strategies to capture maximum variation for genetic resources conservation. For example, the substantially higher level of RFLP variation observed in self-incompatible, as compared with self-compatible species of Lycopersicon was used to recommend predominant sampling of self-incompatible species for germplasm acquisition (Miller and Tanksley, 1990).

Using AFLP markers to study distribution of genetic diversity in coconut populations it has been found that emphasis should be placed on collecting relatively large numbers of plants from few populations since most of the diversity is within populations rather than between populations (Perera et al., 1998). Genetic structure across natural distribution areas of marginal pawpaw (Asimina triloba) populations was established by RAPD analysis (Huang et al., 2000) and used for collection.

b. Molecular markers in managing genetic resources

Molecular techniques proved useful in a number of ways to improve the conservation and management of PGR. In particular, genetic diversity data provides information on gaps in terms of coverage in gene pools as well as redundancies, i.e., material with similar characteristics that wastes resources through increased cost of management. Molecular markers have been used to identify groups from which core collection accessions can be selected or to monitor the effectiveness of one or the other strategy in capturing genetic diversity found in the whole collection. Molecular markers have been employed for fingerprinting, verification of accession identity and genetic contamination. Molecular markers are being increasingly used to resolve problems of taxonomy and phylogenetic relationships, as a good knowledge of genomic homologies helps in devising suitable breeding strategies for appropriate conservation as well as transfer of genes from one species to another.

c. Molecular markers in in situ conservation

Molecular genetic markers made it easy to discriminate between wanted and unwanted genes of agronomic importance in segregating populations. If linkages are established between a heritable agronomic trait and a genetic marker/ markers can be used to identify the location of genes, such linkages allow direct selection for the trait using marker assisted selection in a backcrossing programme. Molecular markers which densely cover an entire crop genome can be applied to develop a molecular map for a crop, which could be used to determine linkage between a specific molecular marker and a strongly heritable trait.

d. Embryo rescue

In embryo rescue, an otherwise non-viable hybrid embryo is transferred to a culture medium where viable plants may be regenerated and backcrossed to the cultivated species to introduce the desired genetic trait. Such as: interspecific hybrids between Lycopersicon esculentum and L. peruvianum (Thomas and Pratt, 1981).

A combination of ovary culture and embryo rescue was used to develop fertile hybrid plants from the intergeneric cross between Brassica napus and Sinapsis alba, which has many desirable traits like resistance or tolerance to all major insect pests of brassica crops, tolerance to high temperatures and drought besides being shatter resistant (Brown et al., 1997; Momotaz et al. 1998).

f. Somatic hybridization

Protoplast fusion and somatic hybridization provide an alternative way for transfer of traits between distantly related species. It has been particularly useful in breeding programs to transfer beneficial characteristics from wild and weedy plants to the cultivated crop species, breaking the barrier for gene transfer. Protoplast surfaces bear strong negative charges, and intact protoplasts in suspension repel each other. Hence fusion is accomplished by addition of calcium ions or polyethylene glycol (PEG) or using electric fields. Successful gene transfer via protoplasm fusion depends on the ability to regenerate a mature plant from the fusion product. Somatic hybrids were produced and late blight resistance was successfully transferred from Solanum nigrum into S. tuberosum (Horsman et al., 1997; Zimnoch-Guzowska, 2003).

CONCLUSION

Biotechnology has made significant contributions in PGR management. The rapid progress made in in vitro culture technology has helped in improving the conservation of genetic resources especially of problem species. In vitro collection, slow growth and cryopreservation has made important contribution. By facilitating better understanding of genetic diversity, both in extent and structure, molecular marker techniques are proving extremely useful in identification of redundancies in collections, in testing accession stability and integrity, and in supporting the development of effective management strategies both for ex situ and in situ conservation. Molecular genetic studies are being increasingly used to support improved use of plant genetic resources. The sequence data that are becoming available for increasing numbers of genes as a result of the rapid advances in DNA technology have stimulated the development of novel molecular technologies which allow the screening of germplasm for functional diversity and identify variation at an early developmental stage without the need for performing the time-consuming evaluation tests

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