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Germplasm Conservation
Chapter 10
Germplasm Conservation
10.1. Background The necessity of conservation of plant germplasms is well understood at present, and perhaps needs no explanatory phrases in this book. Our main concern is how to operate it. Yet, we may remember the first alarm on genetic erosion presented by the late Dr. Harry V. Harlan in 1936 (USDA Yearbook): "In the great laboratory of Asia, Europe, and Africa, unguided barley breeding has been going on for thousands of years. Types without number have arisen over an enormous area. The better ones have survived. Many of the surviving types are old... The progenies of these fields with all their surviving variations constitute the world's priceless reservoir of germ plasms. It has waited through long centuries. Unfortunately, from the breeder's standpoint, it is now being imperiled. When new barleys replace those grown by the farmers of Ethiopia or Tibet, the world will have lost something irreplaceable." Efforts to collect and preserve the germplasms of useful plants are being made on international and national levels and by certain research institutions and individual scientists. The International Board for Plant Genetic Resources (IBPGR) was established in 1973 at Rome as an organization attached to the FAO. It aims to build up liaison between national and regional centers dealing with germplasm conservation, to promote exploration activities by those centers, and to hold meetings, symposia, and training cources for personnel engaged in germplasm conservation. There are three major books on plant genetic resources: Frankel and Bennett (eds.), 1970. "Genetic Resources in Plants—Their Exploration and 225
Chapter 10. Germplasm Conservation
226
Conservation" (Blackwell, Oxford); Frankel and Hawkes (eds.), 1975. "Crop Genetic Resources for Today and Tomorrow" (Cambridge Univ. Press); Frankel and Soule, 1981. "Conservation and Evolution" (Cambridge Univ. Press). The central bank of rice germplasms is the International Rice Germplasm Center of the International Rice Research Institute (IRRI). It now maintains over 70,000 accessions. For the outline of the Center, the reader may refer to Chang (1980, 1984a). The routine of the work consists of 1) collection of seed samples from the field, with records on various items, 2) registration and cold storage of the seeds, 3) tests for various traits and seed multiplication in greenhouse or in an experimental field, 4) recording and cold storage of multiplied seeds, and 5) tests for particular characters or genes. 10.2.
Collection from the Field
If the target plant is rare or approaching extinction, the limiting factor will be our capacity to find it. If the plant is commonly distributed and abundant in the area of exploration, the number of plants to be sampled per site and the number of sites to be visited must be considered so as to maximize the efficiency of the work. There are also limits to the size of samples that can be managed effectively in a conservation program. The size of samples to be collected was discussed first by Oka (1969), who proposed a probability formula to estimate the fraction (G) of total genetic variation to be captured by a sampling procedure as: G = l - { ( 1 - P ) + P(l-/>)"}*, where P, proportion of total genetic variation represented by a population, p, proportion of genetic variation per population represented by a plant, N, number of populations sampled, and n, number of plants sampled per population. The values of P and p were suggested for different kinds of populations based on the estimates of variations within and between populations, and the minimum values of N and n to fulfill G>0.95 were computed. There were different combinations of N and n values. To estimate N and n on a more practical basis, Marshall and Brown (1975) proposed: E=N(a+bn), where E is total effort (time) available for collection, a is time necessary for finding a site, and b is time spent for sampling one plant. Thus, the N and n values which would maximize efficiency were investigated, which differed according to the estimates of P and/? (Table 10-1).
227 TABLE 10-1 Expected Values of Optimal Number of Plants Sampled per Site and Optimal Number of Sites Sampled a Day, for a Range of Genetic Models Po lation opu anon P p alb ratio 25 100 a b 25 1 50 0.5
Improved Primitive cultivars cultivars 0.01 0.05 0.95 0.20 Number of plants per site (ri) 1 10 2 15 Number of sites per day (N) 18 14 10 8
Wild relative 0.10 0.05
Outbreeding species 0.50 0.05
15 39
30 50
12 7
9 6
P, proportion of total genetic variation represented by a population; p, proportion of genetic variation per population represented by a plant; a, time (min) necessary for visiting a site; b, time (min) spent to sample one plant, (from Marshall and Brown, 1975)
The author and colleagues have made a number of exploration trips as mentioned (Chapter 1, p. 7). In the field, we have various limitations. In our experience, field samples are evaluated by their quality and quantity. Quality depends on the adequacy of sampling and other information including records of plant characters and habitat conditions or cultivation method, facts learned from local people and so on. Sampling sites are usually limited to the roadside. To locate a good site depends on luck although careful watching is important while travelling by car. It is necessary that the collector has basic knowledge of the variations of target plants. The distribution of genetic variation is often localized (Brown, 1978). When populations at different sites seem to be differentiated, sampling from as many populations in different environments as possible is recommended even if the sample size per population is thereby reduced (Marshall and Brown, 1975). When the populations are apparently polymorphic, large samples from fewer populations are recommended to capture rare genes. Random sampling is the normal method although sampling of different types to maximize visual diversity is also recommended. Explorations are envisaged with priority given to areas where genetic diversity is rich but is subjected to erosion. Chang (1984b) designated several areas for O. sativa: northeast India, the interior of Bangladesh, northern Burma, northern Indochina, and southwestern China. Unfortunately, it is difficult to visit most of these places because of political or military restrictions.
228
Chapter 10. Germplasm Conservation
10.3. Preservation of Population Samples Rice seed can be kept alive for a long time if stored at low temperature with low moisture content. The half-life of stored dry seed is estimated by: logio p=Kv - Cxm - C2t, where p is the half-life in week, m is seed moisture content in percent, t is temperature in centigrade, and KV9 Cl9 and C2 are constants characteristic of species, genotype, and initial physiological condition of the seed (Roberts, 1960). In rice, it was estimated that Kr=5.3-5.6, (^=0.159 and C 2 =0.069 (Ito, 1965, 1975). It is not difficult to maintain seed viability over 100 years if moisture is below 7% and temperature is below zero. However, a fraction of seeds in storage loses viability much earlier than expected, possibly because of poor initial physiological conditions as in immature or damaged seed. The standard deviations of the above parameter values remain unknown. The seed of O. coarctata is recalcitrant; it does not withstand desiccation like those of Zizania aquatica (American wild rice), some other herbs and trees. The seeds of all other Oryza species can be stored like that of O. sativa, although their relative longevities are not known. In O. sativa, Indica seeds have a longer life than Japonica seeds (Oka and Tsai, 1955). The seeds collected from the field must be germinated for various tests and multiplication. This is a simple procedure when the plants are homogeneous genetically and self-pollinating. With heterogeneous and outcrossed populations like those of the common wild rice, however, special care must be taken for adequate seed multiplication. The seeds collected from a field population will be either in bulk in which seeds from a number of plants are mixed together, or a set of seed lots sampled on a single plant basis. The number of plants sampled will be 10 to 50 per population in many cases. There are two methods of seed multiplication, pedigree and bulk. When the original seed is in bulk, a bulk population is raised in the first generation, and the seed produced is harvested either in bulk or on a single plant basis (pedigree). When the original seed consists of a number of lots each from a single plant, their progeny lines are raised separately (ear-to-row) and the seeds produced are harvested either on a single plant basis (pedigree) or in bulk. Which method is better would depend on the objective of the operation and characteristics of the target populations (Oka, 1983d). When wild-rice populations are grown in the field, particularly when they are grown in bulk, the plants rapidly absorb genes from cultivars grown in the vicinity and change toward a cultivated type in response to the cultivation pressure (Oka
229 and Morishima, 1971). Therefore, the pedigree method is preferred and bagging before flowering is needed. The populations of the common wild rice as well as of land races are highly polymorphic, as mentioned (Chapter 4). In the common wild rice, the average gene diversity per population varied from nearly zero to about 0.4 (Table 4-10), and it was correlated with observed heterozygosity, average number of alleles per locus and proportion of polymorphic loci per population (cf. p. 72). The number of potential allelic variants is innumerable, but one plant can represent an appreciable portion of the alleles. In a relatively small population, most alleles are expected to be either very common or rare, showing a U-shaped curve of distribution (Kimura and Crow, 1964; Marshall and Brown, 1975). Rare genes (/?<0.05) cannot be the object of conservation, if not isolated (Marshall and Brown, 1975). When the frequency of the most frequent allele is less than 0.95, the locus may be considered polymorphic operationally (Frankel and Soule, 1981, p.42). When it is projected to maintain 95% of the genes (gametes) distributed in a population with a 5% frequency, 59 or more plants are needed (Oka, 1969, estimated as the probability of at least one success). If it is projected to maintain 95% of genes with 5% or higher frequencies (5-50%) whose distribution is logarithmic (U-shaped), the plants needed will be about 20 (Oka, 1983d). In this context, Yonezawa (1986) pointed out theoretically that the single-seed-descent method (pedigree) is most efficient in reducing sampling variance in the rejuvenation of populations with mixed selfing and outcrossing, although he did not deal with population size. The collected and rejuvenated population is much smaller in size than the mother population in the field. This may be compared with the bottleneck effect in evolutionary theory. The loss of genes due to a finite effective population size (N) is shown by: (1 —^ΤΓ] > w n e r e t stands for generation number. The number of alleles remaining after a bottleneck is: E(n)=m— Σ (1 — Pi)2N9 where m is the number of alleles prior to the bottleneck and pt is the frequency of z-th allele (Frankel and Soule, 1981, p.35). When m=2, p=0.05, and N=20, ls(n) = 1.87. It is expected that the loss of genes due to a bottleneck is not very serious even though rare genes are lost, if the population is increased in size in the next generation. A large number of genetic stocks of wild Oryza species and land races of O. sativa and O. glaberrima are being preserved in the Plant Germplasm Laboratory, National Institute of Genetics, Japan. Dr. Y. Sano is presently in charge of this operation. The seeds collected from field populations, put in many small envelopes, are registered with collector's records and are kept at — 10°C with desiccating material. A part of the seeds are germinated in
230
Chapter 10. Germplasm Conservation
the season following collection, and the plants are grown for seed multiplication and research. As the plants are grown in concrete beds with automatic shortday control, the space available for this work is limited. If not used for further research, the multiplied seeds are kept at 0°C with desiccating material for 10 years or more. At present, of about 1,200 accessions of wild Oryza species, 52% are of O. perennis complex and 29% O. breviligulata, the remaining 19% belonging to 15 other wild species (Rice Genetics Newsl., 2, pp.26-28). Many of these are population samples. In about 50 accessions, an accession consists of more than 20 lines each representing a plant in the mother population and consisting of 4 to 5 plants. Sixty other accessions are each represented by more than 10 lines. In the remainder, each is represented by 4 to 5 lines. Thus, the total number of lines exceeds 12,000. For the greater part of these genetic stocks, duplicate seed lots are being kept in the International Rice Germplasm Center of the IRRI. Various technical problems are encountered in this operation. The first is bagging before flowering for self-pollination and after flowering (internode elongation) to keep the seeds from shedding. Each bag is tied to a bamboo stick, and this is a labor-consuming procedure. Second, highly perennial plants have a low seed productivity. They are kept alive in a greenhouse, each in a pot, yet not all of them persist for many years and rejuvenation procedures are needed. In the germplasm conservation programs, wild relatives are considered most important. Wild germplasms and indigenous land races arising from the wild progenitors are the most essential part of the conservation of biological diversity (Walsh, 1981). They are expected to carry a greater gene pool than cultivars, as their isoenzymic diversity suggests, even though not yet fully exploited. However, the conservation of wild germplasms requires much more labor than that of self-pollinated cultivars. Efforts for germplasm conservation are now being made throughout the world. However, genetic erosion proceeds rapidly as the result of land exploitation and lumbering. It is not known to what extent the present system of germplasm conservation can achieve its general aim to carry over the gene pools necessary for crop evolution to our future generations. In addition to the preservation of seeds in laboratories, even more important is the protection of areas rich in diversity from destruction and conservation of the environment.
Germplasm Conservation
10.1. Background The necessity of conservation of plant germplasms is well understood at present, and perhaps needs no explanatory phrases in this book. Our main concern is how to operate it. Yet, we may remember the first alarm on genetic erosion presented by the late Dr. Harry V. Harlan in 1936 (USDA Yearbook): "In the great laboratory of Asia, Europe, and Africa, unguided barley breeding has been going on for thousands of years. Types without number have arisen over an enormous area. The better ones have survived. Many of the surviving types are old... The progenies of these fields with all their surviving variations constitute the world's priceless reservoir of germ plasms. It has waited through long centuries. Unfortunately, from the breeder's standpoint, it is now being imperiled. When new barleys replace those grown by the farmers of Ethiopia or Tibet, the world will have lost something irreplaceable." Efforts to collect and preserve the germplasms of useful plants are being made on international and national levels and by certain research institutions and individual scientists. The International Board for Plant Genetic Resources (IBPGR) was established in 1973 at Rome as an organization attached to the FAO. It aims to build up liaison between national and regional centers dealing with germplasm conservation, to promote exploration activities by those centers, and to hold meetings, symposia, and training cources for personnel engaged in germplasm conservation. There are three major books on plant genetic resources: Frankel and Bennett (eds.), 1970. "Genetic Resources in Plants—Their Exploration and 225
Chapter 10. Germplasm Conservation
226
Conservation" (Blackwell, Oxford); Frankel and Hawkes (eds.), 1975. "Crop Genetic Resources for Today and Tomorrow" (Cambridge Univ. Press); Frankel and Soule, 1981. "Conservation and Evolution" (Cambridge Univ. Press). The central bank of rice germplasms is the International Rice Germplasm Center of the International Rice Research Institute (IRRI). It now maintains over 70,000 accessions. For the outline of the Center, the reader may refer to Chang (1980, 1984a). The routine of the work consists of 1) collection of seed samples from the field, with records on various items, 2) registration and cold storage of the seeds, 3) tests for various traits and seed multiplication in greenhouse or in an experimental field, 4) recording and cold storage of multiplied seeds, and 5) tests for particular characters or genes. 10.2.
Collection from the Field
If the target plant is rare or approaching extinction, the limiting factor will be our capacity to find it. If the plant is commonly distributed and abundant in the area of exploration, the number of plants to be sampled per site and the number of sites to be visited must be considered so as to maximize the efficiency of the work. There are also limits to the size of samples that can be managed effectively in a conservation program. The size of samples to be collected was discussed first by Oka (1969), who proposed a probability formula to estimate the fraction (G) of total genetic variation to be captured by a sampling procedure as: G = l - { ( 1 - P ) + P(l-/>)"}*, where P, proportion of total genetic variation represented by a population, p, proportion of genetic variation per population represented by a plant, N, number of populations sampled, and n, number of plants sampled per population. The values of P and p were suggested for different kinds of populations based on the estimates of variations within and between populations, and the minimum values of N and n to fulfill G>0.95 were computed. There were different combinations of N and n values. To estimate N and n on a more practical basis, Marshall and Brown (1975) proposed: E=N(a+bn), where E is total effort (time) available for collection, a is time necessary for finding a site, and b is time spent for sampling one plant. Thus, the N and n values which would maximize efficiency were investigated, which differed according to the estimates of P and/? (Table 10-1).
227 TABLE 10-1 Expected Values of Optimal Number of Plants Sampled per Site and Optimal Number of Sites Sampled a Day, for a Range of Genetic Models Po lation opu anon P p alb ratio 25 100 a b 25 1 50 0.5
Improved Primitive cultivars cultivars 0.01 0.05 0.95 0.20 Number of plants per site (ri) 1 10 2 15 Number of sites per day (N) 18 14 10 8
Wild relative 0.10 0.05
Outbreeding species 0.50 0.05
15 39
30 50
12 7
9 6
P, proportion of total genetic variation represented by a population; p, proportion of genetic variation per population represented by a plant; a, time (min) necessary for visiting a site; b, time (min) spent to sample one plant, (from Marshall and Brown, 1975)
The author and colleagues have made a number of exploration trips as mentioned (Chapter 1, p. 7). In the field, we have various limitations. In our experience, field samples are evaluated by their quality and quantity. Quality depends on the adequacy of sampling and other information including records of plant characters and habitat conditions or cultivation method, facts learned from local people and so on. Sampling sites are usually limited to the roadside. To locate a good site depends on luck although careful watching is important while travelling by car. It is necessary that the collector has basic knowledge of the variations of target plants. The distribution of genetic variation is often localized (Brown, 1978). When populations at different sites seem to be differentiated, sampling from as many populations in different environments as possible is recommended even if the sample size per population is thereby reduced (Marshall and Brown, 1975). When the populations are apparently polymorphic, large samples from fewer populations are recommended to capture rare genes. Random sampling is the normal method although sampling of different types to maximize visual diversity is also recommended. Explorations are envisaged with priority given to areas where genetic diversity is rich but is subjected to erosion. Chang (1984b) designated several areas for O. sativa: northeast India, the interior of Bangladesh, northern Burma, northern Indochina, and southwestern China. Unfortunately, it is difficult to visit most of these places because of political or military restrictions.
228
Chapter 10. Germplasm Conservation
10.3. Preservation of Population Samples Rice seed can be kept alive for a long time if stored at low temperature with low moisture content. The half-life of stored dry seed is estimated by: logio p=Kv - Cxm - C2t, where p is the half-life in week, m is seed moisture content in percent, t is temperature in centigrade, and KV9 Cl9 and C2 are constants characteristic of species, genotype, and initial physiological condition of the seed (Roberts, 1960). In rice, it was estimated that Kr=5.3-5.6, (^=0.159 and C 2 =0.069 (Ito, 1965, 1975). It is not difficult to maintain seed viability over 100 years if moisture is below 7% and temperature is below zero. However, a fraction of seeds in storage loses viability much earlier than expected, possibly because of poor initial physiological conditions as in immature or damaged seed. The standard deviations of the above parameter values remain unknown. The seed of O. coarctata is recalcitrant; it does not withstand desiccation like those of Zizania aquatica (American wild rice), some other herbs and trees. The seeds of all other Oryza species can be stored like that of O. sativa, although their relative longevities are not known. In O. sativa, Indica seeds have a longer life than Japonica seeds (Oka and Tsai, 1955). The seeds collected from the field must be germinated for various tests and multiplication. This is a simple procedure when the plants are homogeneous genetically and self-pollinating. With heterogeneous and outcrossed populations like those of the common wild rice, however, special care must be taken for adequate seed multiplication. The seeds collected from a field population will be either in bulk in which seeds from a number of plants are mixed together, or a set of seed lots sampled on a single plant basis. The number of plants sampled will be 10 to 50 per population in many cases. There are two methods of seed multiplication, pedigree and bulk. When the original seed is in bulk, a bulk population is raised in the first generation, and the seed produced is harvested either in bulk or on a single plant basis (pedigree). When the original seed consists of a number of lots each from a single plant, their progeny lines are raised separately (ear-to-row) and the seeds produced are harvested either on a single plant basis (pedigree) or in bulk. Which method is better would depend on the objective of the operation and characteristics of the target populations (Oka, 1983d). When wild-rice populations are grown in the field, particularly when they are grown in bulk, the plants rapidly absorb genes from cultivars grown in the vicinity and change toward a cultivated type in response to the cultivation pressure (Oka
229 and Morishima, 1971). Therefore, the pedigree method is preferred and bagging before flowering is needed. The populations of the common wild rice as well as of land races are highly polymorphic, as mentioned (Chapter 4). In the common wild rice, the average gene diversity per population varied from nearly zero to about 0.4 (Table 4-10), and it was correlated with observed heterozygosity, average number of alleles per locus and proportion of polymorphic loci per population (cf. p. 72). The number of potential allelic variants is innumerable, but one plant can represent an appreciable portion of the alleles. In a relatively small population, most alleles are expected to be either very common or rare, showing a U-shaped curve of distribution (Kimura and Crow, 1964; Marshall and Brown, 1975). Rare genes (/?<0.05) cannot be the object of conservation, if not isolated (Marshall and Brown, 1975). When the frequency of the most frequent allele is less than 0.95, the locus may be considered polymorphic operationally (Frankel and Soule, 1981, p.42). When it is projected to maintain 95% of the genes (gametes) distributed in a population with a 5% frequency, 59 or more plants are needed (Oka, 1969, estimated as the probability of at least one success). If it is projected to maintain 95% of genes with 5% or higher frequencies (5-50%) whose distribution is logarithmic (U-shaped), the plants needed will be about 20 (Oka, 1983d). In this context, Yonezawa (1986) pointed out theoretically that the single-seed-descent method (pedigree) is most efficient in reducing sampling variance in the rejuvenation of populations with mixed selfing and outcrossing, although he did not deal with population size. The collected and rejuvenated population is much smaller in size than the mother population in the field. This may be compared with the bottleneck effect in evolutionary theory. The loss of genes due to a finite effective population size (N) is shown by: (1 —^ΤΓ] > w n e r e t stands for generation number. The number of alleles remaining after a bottleneck is: E(n)=m— Σ (1 — Pi)2N9 where m is the number of alleles prior to the bottleneck and pt is the frequency of z-th allele (Frankel and Soule, 1981, p.35). When m=2, p=0.05, and N=20, ls(n) = 1.87. It is expected that the loss of genes due to a bottleneck is not very serious even though rare genes are lost, if the population is increased in size in the next generation. A large number of genetic stocks of wild Oryza species and land races of O. sativa and O. glaberrima are being preserved in the Plant Germplasm Laboratory, National Institute of Genetics, Japan. Dr. Y. Sano is presently in charge of this operation. The seeds collected from field populations, put in many small envelopes, are registered with collector's records and are kept at — 10°C with desiccating material. A part of the seeds are germinated in
230
Chapter 10. Germplasm Conservation
the season following collection, and the plants are grown for seed multiplication and research. As the plants are grown in concrete beds with automatic shortday control, the space available for this work is limited. If not used for further research, the multiplied seeds are kept at 0°C with desiccating material for 10 years or more. At present, of about 1,200 accessions of wild Oryza species, 52% are of O. perennis complex and 29% O. breviligulata, the remaining 19% belonging to 15 other wild species (Rice Genetics Newsl., 2, pp.26-28). Many of these are population samples. In about 50 accessions, an accession consists of more than 20 lines each representing a plant in the mother population and consisting of 4 to 5 plants. Sixty other accessions are each represented by more than 10 lines. In the remainder, each is represented by 4 to 5 lines. Thus, the total number of lines exceeds 12,000. For the greater part of these genetic stocks, duplicate seed lots are being kept in the International Rice Germplasm Center of the IRRI. Various technical problems are encountered in this operation. The first is bagging before flowering for self-pollination and after flowering (internode elongation) to keep the seeds from shedding. Each bag is tied to a bamboo stick, and this is a labor-consuming procedure. Second, highly perennial plants have a low seed productivity. They are kept alive in a greenhouse, each in a pot, yet not all of them persist for many years and rejuvenation procedures are needed. In the germplasm conservation programs, wild relatives are considered most important. Wild germplasms and indigenous land races arising from the wild progenitors are the most essential part of the conservation of biological diversity (Walsh, 1981). They are expected to carry a greater gene pool than cultivars, as their isoenzymic diversity suggests, even though not yet fully exploited. However, the conservation of wild germplasms requires much more labor than that of self-pollinated cultivars. Efforts for germplasm conservation are now being made throughout the world. However, genetic erosion proceeds rapidly as the result of land exploitation and lumbering. It is not known to what extent the present system of germplasm conservation can achieve its general aim to carry over the gene pools necessary for crop evolution to our future generations. In addition to the preservation of seeds in laboratories, even more important is the protection of areas rich in diversity from destruction and conservation of the environment.