3.I. CONSERVATION STATUS OF SERICULTURE GERMPLASM RESOURCES IN JAPAN

By E. Kosegawa

Laboratory of Insect Genetics, National Institute of Agrobiological Science, Kobuchisawa 6585, Kitakoma-gun, Yamanashi-ken, Japan

Paper contributed to expert consultataion on promotion of global exchange of sericulture germplasm satellite session of XIXth ISC Congress, September 21st-25th Bangkok, Thailand

Food and Agriculture Organization of the United Nations

Rome
2002

1. Introduction

It has been inferred that the utilization of cocoon began in China 6000 or 7000 years ago. Ancient Chinese characters used before 11^th century BC, such as thread and mulberry, show the existence of the sericulture. The sericulture had spread from China to the world. The naturalization person who came from each of Korea and China to Japan brought silkworm and technology of the textile between BC 300 and AD 300. It is uncertain for whether the early races of Japanese silkworm are improved as genetically pure race or not. Though the ancient description describing the existence of Japanese silkworm race have been left in great numbers since 1700s, the most varieties were not pure, and the quality was not stabilized. Japan solved the national isolation in 1859, and the trade was started by opening of Yokohama port. The sericultural industry became the important industry of Japan for acquisition of foreign currencies after that. However, there were many complaints about the irregular quality of Japanese silkworm race especially from USA where was the main destination for export in those days. So the Japanese government prohibited farmers the rearing of non-permitted races between 1937 and 1998. At the present, rearing and distribution of any silkworm have been permitted.

Imported silkworm races had an important role for Japanese sericulture. Since 1859, many silkworm races were imported from foreign countries, and many races were bred in Japan. The newly bred races were mainly contributed to establish pure lines to produce F1 hybrid. Subsequently, main races for cocoon production were shifted to hybrid races at the beginning of 20^th century.

In 19^th century or earlier, the most of cocoons produced in Japan were mainly consumed domestically rather than its export. At the era, major race of domesticated silkworm was Japanese old varieties. Since the government encouraged the sericulture in 1870, the cocoon production increased. At the same time, major silkworm race was shifted to the hybrid races between Japanese race and the pure race bred from imported races. In 1920's the cocoon production reached the record, more over 350 000 ton per year. However, the export decreased sharply at the beginning of the Pacific War in 1941. After the war cocoon production gradually declined and finally decreased to 1 300 tons. To prevent the decline of Japanese sericulture, new silkworm races those had value added to obtain the largest profit was bred.

By 1800's, silkworm breeding was carried out as autonomous activities of the farmers. In Japan, sericulture region had been divided nationwide into two purposes, for the cocoon production and the egg production. From 1859, at the beginning of raw silk export, native Japanese silkworm races were used for sericulture without newly breeding. Since 1937, the government prohibited the rearing of non-approval races except for the race that had high and uniform quality to produce raw silk. By the establishment of the license system for the silkworm-egg production and distribution, farmers were forbidden to breed the silkworm freely, and the most of the breeding activity had shifted to experimental stations and private companies. At that time, the breeding goal was focused on the high yielding and healthiness to enhance the further production of standardized raw silk. Since 1970s, however, the cocoon production of Japan was reduced by the pressure of increased raw-silk import, so the income from cocoon production had become too little to support farmer's life. The change of trade balance caused silkworm breeders to change the breeding goal to characteristic silkworm race.

In the modern period, from 1800s to 1900s, many silkworm races were imported from Italy, France, Korea and China. Thereafter, many races were bred from the hybrids between Japanese native race and imported races. Recently, as the number of silkworm races has been more than 500, import of silkworm races is not necessary only by using such races. As an example for recent international activity by Japanese researchers, technical cooperation of the sericulture by bivoltine race is made between Japan and India to promote new sericulture in India where the polyvoltine race sericulture is carried out.

Recently, the breeding activity of domesticated silkworm has been performed especially in National Institute of Agrobiological Science (NIAS) in Japan. In NIAS, the breeding for the production of the standardized raw silk and additional-value race are going on. The breeding goals are e.g. thicker filament race, thinner filament race, oligophagous race and virus-resistant race. On the other hand, the breeding of silkworm races adapted to factory production of physiological-active substances by the use of the vaculovirus-vecter system are proceeding in NIAS. The races are expected to make the great industry in Japan. Since Tamura (2000) succeeded to make transgenic silkworm in NIAS, new silkworm strains has been increased, and we hope to raise the public demand for utilization of the transgenic silkworm.

2. Conservation of silkworm genetic resources in Japan

In Japan, silkworm races are consists of native race, improved race and mutants. The silkworm races owned by NIAS are grouped into three major categories: silkworm genetic stock, breeding resources and currently popularized race. The later two categories were excluded from the genetic stock because their characters are varied by annual selection depending on the change of public needs, and the distribution of the eggs are left for the breeder to keep their title to an race. Such races will become prospective the genetic stock. In the case of mutants, the largest stock is conserved in Kyushu University while NIAS stock is only 172 lines. By the way, the most popularized race has been bred and distributed by private companies. Additionally, Gunma Prifecutural Experimantal Station of Sericulture and some other experimental stations own original races for districted sericulture too. The genetic stocks of NIAS are mentioned in the following paragraphs.

2.1 In Present status of silkworm germplasm maintenance in NIAS

Six hundreds and forty five lines of domesticated silkworm have been preserved as the genetic stock of NIAS (Table 1). The genetic stock is greatly classified into four types: native race, improved race, mutant and intermediate stock. The native races are classified by geographical origin, like the tropics, Japanese, China and Europe. The improved variety is classified into three categories, Japanese, Chinese and European races. Although, the detailed description is omitted, mutants are preserved mainly for the experimental purpose. The breeding middle mother plant has not been completed as a variety, but it recognized that the line is a characteristic compared with common commercial races.

The breeding section of NIAS possess many lines, those are not yet registered to the genetic stock. It could be guessed that the number of such lines reach 50 - 200 per laboratory. Those lines are improved and newly established in each rearing period, so we cannot count the accurate number.

NIAS is managed by the budget from Ministry of Agriculture, Forestry and Fisheries and expected to perform the contribution to the Japanese agriculture. NIAS established Gene Bank Project in relation to many genetic resources: crops, domestic animals, insects including silkworms. In the project, great number of the genetic stock has been preserved and distributed. The half of the budget necessary for the maintenance of the genetic stock has been supplied from the gene bank project. It manages the remainder by the research budget of NIAS. Previously, over 2000 lines of silkworm were maintained in Japan, and 1000 lines of the genetic stock had been preserved in Sericultural Experimental Station, it's the former of NIAS. However, the preservation system is reduced with the decrease of farmers engaging to sericulture, consequently the number of the genetic stock had decreased. Recently, the importance of the genetic stock was well understood by the nation, so the decrease in the number has been stopped. On the contrary, present NIAS became possible to accept the additional preservation of silkworm germplasm. As the gene-bank project have the plane to evaluate its outcome in every five years, the person, who have the charge to preserve the genetic stock, has to make efforts to obtain the budget in each period.

2.2 Management and utilization of silkworm germplasm resources

Except for the breeding resources now on use, the genetic stock is preserved in the laboratory of insect genetics in NIAS. The breeding resources have been preserved in Laboratory of Sericultural Science and Laboratory of Insect Molecular Evolution. The present state in the laboratory of insect genetics is described in the following paragraphs.

2.2.1 Methodology for maintenance of silkworm germplasm resources

The silkworm races were bred to make homozygote for important characters in relation to cocoon filament and to maintain heterozygosity in other characters, like disease tolerance. It is concerned that inbreeding makes the silkworm race wrong. Therefore the silkworm breeders always select the excellent individuals or lines from the silkworm race in each generation to maintain the average performance owned by itself. It has been suggested historically that the maintainance of excellent character of race is difficult because of less number of silkworm race reared continuously more than ten years in Japan.

The laboratory of insect genetics is preserving 470 lines of the genetic stock by the hands of five staffs and seven part-time workers. The number of persons is too small to perform the selection in all races. Therefore, the laboratory gave up a part of selection step like weighing the individual cocoon and reeling test. In other words, the laboratory abandoned the definition that silkworm race always express its own phenotypes, while concerned that a race is the pool of the excellent genes. If we are able to avoid the loss of excellent genes without the selection in a race on every generation, the race will revived again by re-selection in future.

2.2.2 Methodology for silkworm pure lines maintenance

The pure lines, except for the tropical races, are reared one time per year and preserved in the stage of diapause egg by next year. Tropical races are reared at least two times per year because of the difficulty to obtain diapause egg in Japan and to avoid long-term chilling that make the hatchability to go down. Three rearing periods has planned to preserve the genetic stock: The first, second and third period are May-June, July-August and September-October respectively. The genetic stock including 470 lines has been divided into three groups reared in different period each other. The diet for silkworm is mulberry leaf in all cases in order to avoid the unconscious selection by artificial diet. Since the allele of the low frequency would not be lost by the genetic drift, the egg is collected from fifty of female moth even in the minimum for the mating. Then, the larva is derived form all moth batches and supplied in the rearing in the next generation. In third inster, 160 of middle-sized larvae are usually selected from each race. After cocooning, 120 of cocoon that looks like good shape are selected to avoid the accidentally mixing of other races. The cocoons were cut, and pupae are moved into the mating box before the emergence of moth. Consequently, approximately 50 egg batches are obtained for the preservation. The individual selection for the preservation is milder than the case of improving, and is performed simply by the removing of the individual that has incorrect character.

Usually, hybrid F1 is not produced in the laboratory, because of no public demand about old hybrid. If current race is needed, the F1 hybrid is produced in the specialist section of NIAS. The pure lines of popularized hybrid are mainly preserved in Laboratory of Sericultural Science.

2.2.3 Methodology for obtaining the data and calculation of the main breeding characters and values

Various characteristics are investigated whether the race is surely preserved through whole preservation process of the genetic stock. In Gene-Bank Project of NIAS, It is imposed primarily that characteristic data of each race are registered. Character items of the gene bank are divided into three groups: primary character (visual character [Table 2a]), secondary character (quantitative character [Table 2b]) and tertiary character (practical and economical character [Table 2c]). At least the clarify of the primary character is necessary to register silkworm race to the gene bank. Moreover, the primary character is reconfirmed in every generation. Though the secondary character is dispensable to the registration, it is always recorded in the preservation. The tertiary character is investigated in the every generation of currently popularized race, while it is not always for the genetic stock. The investigation of the tertiary character needs rearing the double number of larvae at once. The increase of the number of larvae limits the number of the genetic stock to half because of facility. Therefore, the investigation should have been carried out at least one time per ten years in the preservation of the genetic stock. The last investigation was reported in 1986, and it has not been investigated afterwards.

2.3 Recent Development of silkworm germplasm resources

It is supposed that Japanese-native race was improved empirically by the pure-line selection segregated from hybridization. In 1914, Dr Toyama introduced the usefulness of heterosis to Japanese people. Thereafter the F1 hybrid became the mainstream of cocoon production in Japan. Consequently, the improvement of silkworm had intended to establish the parental pure races for F1 hybrid. As a result, sex-limited races by radiation breeding and oligophagous race that grew by the diet excluding mulberry leaf were established. Recently, the breeding goal has been shifted from the standardized raw-silk production to the extraordinary purpose. The idea is based on the practical use of biological function that is unrestricted by raw-silk production. Here, the races improved in such situation are introduced. But it must be notified that the races were excluded from the genetic stock.

2.3.1 Virus resistant silkworm race

The healthiness of silkworm is an important theme in the breeding. By the screening of the silkworm-genetic stock, the densonucleosis virus (DNV) resistant line was discovered. As a result of the genetic analysis, it is demonstrated that the resistant property is controlled by the single-dominant gene. Therefore, it is possible that the resistance expressed at the F1 hybrid. Consequently, the resistant race "Taisei" was successfully established.

2.3.2 The races that has characteristic silk thread

Generally, thin and fineness silk thread is fitting for the making of the high-grade textile. And it was established that the race, Akebono, which formed 2.2 denier of silk thread. The thread was twisted together with chemical fiber, and the product which is called as Hybrid Silk has been sold. In 1998, Hakugin race was established as the race that had the silk fiber thinner than Akebono. Surprisingly, the thread size is 1.6 denier. It is the first record by the thinner size less than 2 denier in spite of tetramolter. It is expected that the super-thin textile made form the raw silk of this race attracts the attention of the worldwide as a particular product of Japan.

In contrast to the upper paragraph, there is a demand of the thickly and short raw silk for Western clothes, and an race which produces the silk thread of 4 deniers has been improved. A characteristic line, the original line of the race, had been established in 1952, though most researchers were not interested in the line. But the line was remained as a private collection until recently. The fact indicates the importance of genetic resources. Presently it was continuously preserved and registered as a collection of the genetic stock.

2.3.3 Utilization of colored cocoon and double cocoon

Colored-cocoon race has well known in worldwide. The yellow pigment of the cocoon is presently attracted because of the antibacterial activity of flavonoid that is a component of the pigment. Besides, the pigment of the colored cocoon is chemically fixed, and the utilization of colored raw silk has been attempted.

Hand spun silk fabric, which is called Tsumugi, formed from floss silk is very popular and traded expensively in Japan. The double cocoon, as an ingredient of the floss silk, is too scarce to use large amount, because the mounting technique of matured silkworms has been improved. So, double cocoon race has been established.

2.3.4 Transgenic silkworm

Tamura (2000) succeeded in generation of the transgenic silkworm in NIAS. Since exogenous protein gene could be integrated into genome by using of the technique, it will result in an increase in number of the genetic stock of silkworm.

2.3.5 Problem of the retirement from silkworm preservation

Presently due to the reduction of Japanese sericulture, it is difficult for the producer of silkworm egg to make an adequate profit. Some private companies retired from the egg production, and original races were lost before we knew it. Other companies are continuing the production by the moving of the location to foreign countries. In some prefectures, the section of the experimental station in relation to sericulture was abolished owing to the reduction of the farmers. In lucky case, the silkworm races possessed in such experimental stations has been donated to NIAS.

2.3.6 Revival of old race: the Koishimaru phenomenon

Some pieces of silk cloth were excavated from remains that seemed to be of ancient Japan around first or second century. When a copy of the ancient-silk cloths had been reproduced as an activity of archaeology, the utilization of the silkworm race, which was same to ancient race, was aspired. Consequently, Koishimaru race, which has been known very well as the silkworm presently reared by the Empress, was chosen for the project. The news reports of the event made Koishimaru the most famous race in Japan. This race was established about 150 years ago and used by 1900s. Thereafter, the race was took the place of F1 hybrids. The race has a little amount of silk and some defects on the silk filament except that the filament is slightly thinner than recent common race. However, it is owing to the brand image in relation to both the imperial silkworm and high class textile that the race has been the fashion in the nation. Since the people love the texture of ancient silk, further improvement of the race is hated. So the raw-silk producer requests farmers to produce the cocoons of the original race. As a conclusion, old races should be preserved for the demand in future

3. Silkworm germplasm database

The genetic-stock database of NIAS has been constructed and opened to the public by using of world wide web (WWW) on Internet (http://www.nises.affrc.go.jp/nises/db.html). The primary characters which described above and photographs of egg, larva and cocoon were carried in the database. Unfortunately the data is written in Japanese language. Most of research reports which has been become the basic knowledge of Japanese researcher were also described in the Japanese. The problems related to the language, the conversion to electronic data and the linking up each data has caused to take time for the analysis of the data.

3.1 Management and utilization of database

The database was constructed by the data prepared in 1991. By now, some races add and some had been lost, however the database has not been updated. As the additional problem, the database is not a well-known fact for researchers in spite of searchable in the www search engine, for example Yahoo and so on. It is expected that further popularization of Internet will increase the user of the database. As the encouragement of domestic sericulture was major purpose of the sericultural-research section of NIAS, the English presentation had not been active. Presently, NIAS is promoting to make English papers.

3.2 Descriptors/Stock Characterization

3.2.1 Characterization of the silkworm genetic stock

The genetic stock is categorized into four groups, native, improved, mutants and intermediate stock. Native race is a pure line, and the farmars are also able to produce the eggs by themselves. But many improved races are F1 hybrid which is functional only in the generation. The mutants were reared for the preservation of the mutant genes, not of gene pool. The breeding resource is fully improved race that will be registered as a new race in near future.

Native race and improved race are respectively divided into three types, Japanese, Chinese and European. The improvement is performed in order to make the races, Chinese and European, adapted to the climate of Japan. As the races were pure line for the production of hybrid, they were preserved in the condition to keep genetic distance against to Japanese in order to maximize the effect of heterosis. Typical features of the races are shown in Table 3.

3.2.2 Evaluation of silkworm genetic resources

In present Japan, any silkworm race could be reared without legal controls even if the race had problem. By 1998, the controls existed in order to permit the distribution as a commercial race. The details of the requirement to register the race were described below.

Healthiness: The race is able to be tolerant to the climate high-temperature and humidity in summer season. The mean value of the ratio of pupal number to larval number has to be higher than both control race and the mean value of 10 races those have been registered most recently.

Amount of Cocoon harvested: The mean value of the amount of cocoon harvested have to be higher than both control race and the mean value of 10 races, that has registered most recently..

Cocoon shell weight: It is desirable to be abundant, because the quantity percentage of raw silk is proportional.

Silk fiber size: It is better, as it is thinner.

Silk thread length: It is better, as it is longer.

Reelability: It has to be higher than 73 percent.

Neatness defects: It is better, as it is little. It has to be over 93 points on the defects.

Degumming loss of cocoon shell: It is better, as it is little. It has to be lower than 26 percent.

Number of eggs laid: It is desirable to be both abundant in the number of eggs and high ratio of the number to fresh cocoon weight.

At present, new race can be provided to sericulture, even if it is worse in any characters. However, the race patent has been acquired, since the system that protects the right of the breeder.

3.3 Promotion of global sharing of silkworm germplasm resources

3.3.1 Sharing Information about the silkworm genetic resources

The global sharing of the information about the genetic stock has been promoted in NIAS. The database contains the items described in Table 2. Of course the translation of the data from Japanese to English is also in progress. It is necessary to unify the standard on quantitative character and technical terms, when the information of the genetic stock is exchanged.

3.3.2 Sharing Silkworm genetic resources

Previously, Japan had provided the silkworm races for India and so on. The races were exchanged equivalently with same number of silkworm races. It seems hard to provide silkworm race one-sidedly. Since NIAS make Japanese sericulture to have competitive power, the unique races will not be exported to any other countries. That is depending on the sericultural trend in Japan.

Anybody considers obtaining silk fabric at low price even in Japan. And, the profit increases more for the silk reeling industry, when the raw material cocoon has gotten at low price, too. Though the quality is very high on the cocoon produced in Japan, it is very expensive. Therefore, it is advantageous of that the cocoon is imported from foreign countries, if it intends to produce high-quality silk fabric at low price. Sometimes the quality of the foreign cocoon has been bad to weave fine clothes Japanese traditional. So some private companies has chosen the method that they rear the Japanese silkworm in foreign countries and import the cocoon at low price.

If the genetic stock is reared in foreign country, the silkworms may not adapt to the foreign climate. Therefore, the developing country without the breeding technology may not be able to utilize effectively the genetic stock. In the meantime, the one-sided acceptance of silkworm race to Japan is seemed to be possible, but it is selfish. It is possible to share the maintenance of silkworm races owned by foreign country. When the necessity was occurred, then the country depositing the race could be allotted the eggs. If the races could be also used in Japan according to the contract with the country, it would benefit NIAS.

Recently, the accumulation of waste which does not decay in the natural environment becomes a problem. The raw silk is convenient fiber which never remains due to decay easily in the environment, from the standpoint. And, it is the only long filament got from the nature. Therefore, it is possible to consider that it is useful to keep the environmental. Accordingly, people may prefer such race rather than the race which produce highly heavy cocoon in spite of being often illness as some races in Japan. For the developing countries, the race which is healthy and tolerant to the change of environmental condition may be recommended. The stable production of cocoon is important than the pursuing of cocoon productivity.

Table 1. The number of geographical races of domestic silkworm stocked in National Institute of Agrobiological Science

Table 2a. Primary characters needed to authorize silkworm race as genetic stock of NIAS

Table 2b. Secondary characters needed to authorize silkworm race as genetic resource of NIAS

Table 2c.Tertiary characters needed to authorize silkworm race as a genetic stock of NIAS

Table 3. Characteristics of geographic races of silkworm genetic resources conserved in NIAS

References

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Kosegawa, E., Kikawada, T., Shimizu, K. & Okajima, T. 1995. Evaluation and preservation of silkworm genetic resources in Japan. In MAFF, ed. Animal genetic resources: efficient conservation and effective use, pp. 153-162. Tsukuba, Japan, AFFRCS.

Kosegawa, E. Reddy, G. V., Shimizu, K., & Okajima, T. 2000. Induction of non-diapause egg by dark and low temperature incubation in local variety of the silkworm, Bombyx mori (in Japanese language, and Tables in English). J. Sericult. Sci. Jpn., 69(6): pp 369-376.

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