Sericulture germplasm in BACSA countries

PRESENT STATUS FOR UTILIZING OF SERICULTURAL GENETIC RESOURCES IN SOME EASTERN EUROPEAN AND CENTRAL ASIAN COUNTRIES

By

P.I. Tzenov

Sericulture Experiment Station, 24 Mito Orozov Str., Vratza 3000, Bulgaria; e-mail: panomir@yahoo.com

ABSTRACT

Sericulture should be considered as one of the important potential agro-industry in Eastern Europe and central Asia region countries, now that the increasing number of farmers, i.e. approximately 500,000 households are involved in sericulture industry development activities in order to generate their income resources. 14 countries (Albania, Armenia, Azerbaijan, Bulgaria, Georgia, Greece, Kazakhstan, Kyrgyzstan, Romania, Tajikistan ,Turkmenistan, Turkey ,Ukraine, and Uzbekistan) are engaged actively in sericulture development activities in this region. The total number of mulberry accessions preserved in the region is 931 and the number of silkworm accessions is 767. However some accessions are maintained in parallel in more than one country. If compared with the present cocoon /silk production at the regional level those countries maintain a rich collection of both mulberry and silkworm genetic resources. The countries, having the richest sericulture genetic resources in the region are Azerbaijan, Bulgaria, Uzbekistan and Ukraine. Mulberry varieties from 22 countries and silkworm accessions from 20 different countries are presented at the regional sericulture germplasm, including some accessions originated from one of the most sericulturally advanced countries such as Japan and China. Most of mulberry varieties preserved in the region originated from Azerbaijan (229 and 24.70 %), Uzbekistan (169 and 18.15 %), Japan (139 and 14.93 %), Bulgaria (87 and 9.34 %), Ukraine, Georgia and China. The countries, having the highest diversity as regards the mulberry accessions origin are Uzbekistan (from 15 different countries), Azerbaijan (from 12 countries) and Bulgaria (from 11 countries). The countries represented in the highest extent in the regional silkworm germplasm are Bulgaria (166 and 21.64 %), China (100 and 13. 04 %), Ukraine (83 and 10.82 %), Japan (81 and 10.56 %), Azerbaijan (73 and 9.52 %) and Uzbekistan (69 and 9.00 %). The countries, having the highest diversity as regards the silkworm accessions origin are Bulgaria (from 15 different countries), Uzbekistan (from 14 countries), Ukraine (from 10 countries) and Azerbaijan (from 7 countries). Both mulberry and silkworm genetic resources are maintained by correct methodologies. The mulberry leaf yield in the accessions maintained in the region varies from 5.1 to 12.3 t /ha and appears to be lower than in other sericulturally advanced countries from the temperate belt like Japan, China and Korea. However this lower leaf yield could be more attributed to the comparatively dryer summer in this region rather than the genetic potential of the mulberry varieties. It could be concluded that the mulberry accessions preserved in the region manifest comparatively high genetic potential for production of high quality leaves and good productivity per one hectare. The silkworm accessions preserved in the region manifested comparatively high variation of the main productive characters values, such as cocoon weight from 1.603 g to 2.438 g, shell ratio from 12.23 % to 24.90 % and filament length from 791 m to 1581 m. Some of silkworm accessions and pure lines maintained in the region manifest comparatively high productivity. 424 different mulberry varieties and 262 silkworm breeds have been bred in the region. The biggest number of mulberry varieties and silkworm breeds were selected in Azerbaijan, Uzbekistan, Ukraine and Bulgaria. Some advanced methods in mulberry and silkworm selection such as polyploidy and mutation mulberry breeding, breeding sex limited for egg color and larval marking silkworm strains, breeding tolerant to adverse rearing conditions silkworm breeds, breeding partheno/androgenetic lines, and control the sex-balance have been developed in the region. Almost all the commercial hybrids, produced in the region manifested comparatively high productivity, namely single cocoon weight 2.2 – 2.5 g, shell ratio 23-24 %, shell weight 0.500 – 0.600 g, filament length 1300 – 1500 m under laboratory conditions. The exchange of mulberry and silkworm germplasm between those countries will facilitate the initiation, expansion or rehabilitation of silkworm and/or mulberry breeding programmes in the recipient countries. This will enable the promotion of sericulture industries which will contribute to national economic development. Since the region totally has comparatively rich sericulture genetic resources as well as developed breeding science, in the future some of these countries could be chosen as regional centers for germplasm keeping (conservation), supply with parental silkworm eggs, mulberry saplings, training in the field of mulberry and silkworm germplasm collection, maintenance, conservation and breeding techniques. Those countries could also be used as a source for providing technical assistance, mulberry saplings and silkworm eggs to the newly sericulture developing countries from Africa and the Near East.

Key words: mulberry, Morus sp., silkworm Bombyx mori L, germplasm, Eastern Europe, Central Asia, selection, preservation.

1. Introduction

The sericulture should be considered as one of the important potential agro-industry in East European and Ex-Soviet union Central Asian countries, situated around Black and Caspian seas region, now that the increasing number of farmers, i.e. approximately 500,000 households are involved in sericulture industry development activities in order to generate their income resources. 14 countries (Albania, Armenia, Azerbaijan, Bulgaria, Georgia, Greece, Kazakhstan, Kyrgyzstan, Romania, Tajikistan, Turkey, Turkmenistan, Ukraine and Uzbekistan) are engaged actively in sericulture development activities in this region. It is a common status of those countries in Black, Caspian seas and Central Asia region that sericulture industry activities have been recently declined to be in a critical stage of disappearing or inactivating to a serious extent, mainly due to the rapid change of political and economic structure with introduction of free-market system and the lack of potential internal and external market development. Although the cocoon/silk production in the countries from this region dropped dramatically in the recent decade, they still have very big potential for sericulture development because of very long tradition and experience in sericulture, availability of more than 250 million mulberry trees in the region as valuable natural resources which are enough for an annual production of about 50,000 ton of fresh cocoon, favourable climatic conditions allowing to produce high quality bivoltine cocoons and raw silk, still comparatively low average monthly salary in most of these countries, comparatively well developed sericulture science, such as rich mulberry and silkworm genetic resources, own production of mulberry saplings and silkworm eggs. If those countries receive the necessary technical and financial support for sericulture revival and development, they could establish upgraded sericulture industry to reach a fresh cocoon production at the regional level of 80-100 000 t/year, raw silk production – 10 000 – 12 000 t/year and about more than 2 million farmer’s households would get income from sericulture and could probably earn approximately EUR 450-500 per crop/family and also expect to have a potential for possible income of more than EUR 1500 - 2000 per hectare and year from mulberry plantations.

One of the big advantages for future sericulture development of the countries in this region is the availability of comparatively rich own mulberry and silkworm genetic resources and their possible development by using the modern methods of genetics and breeding in order to supply the farmers by high quality mulberry saplings and silkworm eggs and the silk textile industry with high grade cocoons/raw silk.

2. Conservation status of sericulture genetic resources in some Eastern European and Central Asian countries.

2.1.Present status of sericulture germplasm maintenance.

The total number and the share of each country in mulberry and silkworm genetic resources in the region are given in Table 1. The total number of mulberry accessions available is 931 and of silkworm accessions is 767. However these genetic resources are not distributed uniformly between the different countries. Regarding the number of mulberry accessions maintained Azerbaijan occupies the first position with 285 numbers and 30.61 %, followed by Uzbekistan (213 and 22.87 %), Bulgaria (140 and 15.04 %) and Ukraine (110 and 11.82 %). The number of mulberry accessions is the smallest in Greece and Turkey. However there are a lot of mulberry trees growing in Greece, a part of them of old, but unknown and comparatively highly productive varieties. These already available mulberry resources in Greece should be further studied and systematized.

With 230 numbers (30.00 %) Bulgaria occupies the first position as regards the number of silkworm accessions maintained, followed by Uzbekistan, having 172 accessions (22.42 %), Ukraine (116 numbers and 15.12 %) and Azerbaijan with 85 silkworm accessions(11.08 %) .

Romania, Georgia and Turkey maintain 65, 56 and 23 accessions each respectively and Greece has 20 silkworm strains.

It could be concluded that if compared with the sericulture genetic resources, maintained in other sericulturally advanced countries, such as China, Japan, Korea and India the total number of mulberry and silkworm accessions preserved at the countries of Eastern Europe and Central Asia is also huge. Moreover there was a good germplasm exchange system established between most of those countries in the near past.

The origin of mulberry and silkworm accessions, preserved at the regional germplasm is presented in Table 2 and Table 3. It is evident from Table 2 that most of mulberry varieties preserved in the region originated from Azerbaijan (229 and 24.70 %), Uzbekistan (169 and 18.15 %), Japan (139 and 14.93 %), Bulgaria (87 and 9.34 %), Ukraine, Georgia and China. Mulberry varieties from 22 different countries are totally presented at the regional germplasm, including some accessions from one of the most sericulturally advanced countries such as Japan and China. However it must be taken into account that one same mulberry varieties are preserved simultaneously at different countries from the region, therefore the real number of mulberry accessions available is less than the total number presented. The countries, having the highest diversity as regards the mulberry accessions origin are Uzbekistan (from 14 different countries), Azerbaijan (from 12 countries) and Bulgaria (from 11 countries).

The data presented in Table 3 manifest that the silkworm accessions preserved in the region originated from 20 different countries. The countries represented in the highest extent in the regional silkworm germplasm are Bulgaria (166 and 21.64 %), China (100 and 13. 04 %), Ukraine (83 and 10.82 %), Japan (81 and 10.56 %), Azerbaijan (73 and 9.52 %) and Uzbekistan (69 and 9.00 %). Similarly to mulberry varieties, a part of silkworm accessions are maintained simultaneously at different countries. The countries, having the highest diversity as regards the silkworm accessions origin are Bulgaria (from 15 different countries), Uzbekistan (from 14 countries), Ukraine (from 10 countries) and Azerbaijan (from 7 countries). We could make the conclusion that compared with the present cocoon /silk production at the regional level those countries maintain a rich collection of both mulberry and silkworm genetic resources.

2.2.Management and utilization of sericulture germplasm resources.

2.2.1.Methodology for maintenance of mulberry accessions.

Most of mulberry accessions in those countries were planted in base collections. Each variety is maintained in volume of 6 -10 low (0.6 m) or middle stem (1.2-1.5 m) trees, planted 3/1m or 4/4 m. At present the plantations are maintained as tree form, bottom pruned once in a year, during the spring silkworm rearing season and used as active collection for recording data on morphology, anatomy, reproductive and growth parameters, as well as for collection of cuttings. The recommended cultivation practice is maintained accordingly.

However during the recent decade due to economical problems the necessary agronomy practices in mulberry cultivation were not applied completely in some countries.

2.2.2.Methodology for maintenance of silkworm accessions.

The main purpose in the silkworm accessions maintenance is to conserve their characters closed to the characteristics in the passport, therefore the selection is made only for the qualitative characters, but the main quantitative traits values are also checked. Other important purpose of the maintenance methodology is to avoid inbreeding.

The silkworm accessions from the germpalsm, maintained in this region are reared only once per year, namely during the most favorable spring season-April/May/June.

In Bulgaria, there were some investigations regarding the methods for maintenance of the polyvoltine races (Tzenov, 1998;Tzenov et al., 2001), proved that it’s possible to maintain the polyvoltine race Bonde 517 by only one rearing per year within 4 years in Bulgaria.

Per each accession 25 – 60 batches are produced during the papionage and further preserved under the standard regime up to the next spring. Before putting into incubation from 8 batches (Bulgaria) to 40 batches (Uzbekistan, Georgia), having biggest number of normal eggs are selected and treated by 2 % formaline solution or other suitable chemical for disinfection.

The batches are hatched mixed and the larvae are grown also mixed. After the 2nd molt 200 - 250 larvae in one or two replications are counted per each accession and grown up to the cocooning. Out of the good quality cocoons, produced from each accession 80- 150 cocoons are selected for the qualitative characters, namely cocoon color, shape, hardness and nature of grains. The cocoons selected are put mixed for the papionage and the eggs are produced on cartoons or in paper bags having one mother moth isolated. Time to time a random cocoon sample is taken from each accession in order to investigate the cocoon filament characters by reeling test.

2.2.3.Methodology for silkworm pure lines maintenance

The main purposes in the maintenance of the pure lines, which are parents of the commercial hybrids is to keep the values of the main breeding characters at least at the level of their selection by the breeders and to improve the grainage characters like, moth emergence percentage and fecundity. Also the inbreeding must be avoided obligatory.

When the amount of egg production is comparatively small (less than 2000 boxes of F1/year) the pure lines of the commercial F1 hybrids could be maintained in two categories, namely P3 and P1.In the case of bigger egg production there must be produced P2 category as well.

Each batch is reared separately. After the 2nd moult 200 - 300 larvae are counted from each batch for growing up to cocooning. During the larval period the batches (if any) having not typical for the race larval marking are rejected (separated for production of P1/ F1 or for reeling).

Also the batches having not uniform larval growth or some diseases (NPV) are rejected. After cocoon harvesting, pupation rate, the cocoon yield, average cocoon weight, shell weight and shell percentage are checked for each batch.

Usually from the 55 batches reared, 20-25 batches, having the highest pupation rate are selected for reproduction of P3. After that only 8-12 batches out of them are selected based on the highest cocoon weight, shell weight and shell percentage. The cocoons of the selected 8 batches are assorted for alive pupa, cocoon color, shape, texture and shell hardness manually by experienced researchers in silkworm breeding and after that cut and the pupae are separated by sex. Then all the selected cocoons are measured individually for checking their cocoon weight, shell weight and shell percentage. Only those having the highest cocoon/shell weight and sufficiently high shell ratio are selected for papionage. Practically out of about 180 cocoons produced per a batch only about 50-60 are selected for P3 multiplication. If having 8 selected batches per line, 400-500 cocoons are selected for papionage and about 200 batches of P3 are produced for reproduction. For papionage the female pupae of the first four batches are mixed with the male pupae from the rest 4 batches and vice versa in order to avoid the inbreeding.

Once per 3 years random samples for making silk reeling test are taken from each pure line.

For control of pebrine disease after hatching, the egg shells of each batch are microscoped, samples of silkworm skins after each molt, early spun cocoons etc. are also taken for microscope examination.

The cocoons of the rest batches, produced are assorted and used for production of P2, P1 or directly for F1 if it is a single hybrid.

The maximum amount of silkworm eggs which could be produced following this scheme is as follows:

P3: 200 batches per pure sub-line (or pure line in the parents of four - way hybrids )

P2: 35 boxes per pure sub-line

P1: 1200 boxes per pure line (or 2400 boxes P1)

F1: 96 000 boxes

Therefore, countries like Uzbekistan, producing big amount of silkworm eggs normally produce more number of P3 batches.

3 .Characterization and evaluation of sericulture genetic resources in some countries in Eastern Europe and Central Asia.

Petkov (1998) detected that the leaf dry matter in some mulberry varieties from the region, grown in Bulgaria was from 27.12 % to 30.27 %, crude protein content – from 23.57 % to 26.25 %, the crude fats from 3.05 to 4.77 % and the crude fibers percentage was from 10.59 to 12.40 %.

It was detected that there were clearly expressed variety differences regarding the mulberry productivity. The average leaf and shoot yield per one tree obtained was 6.21 kg and it varied from 4.54 kg in Kairio Ichinose to 8.11 kg in Emin tut variety. In most of the accessions studied the leaf and shoot yield per one tree is from 6 to 7 kg (40%). Other important character influencing the mulberry productivity is the leaves percentage from the whole vegetation yield. Petkov(1998) detected that the leaves percentage was higher than the mean in the varieties Tbilisuri, Georgia, Husan 1, Husan 39, Saniish 5, Shinso 2 and Siozisu. The most of mulberry varieties studied have leaf percentage higher than 50 %. The data regarding the leaf annual yield per 1 ha rain fed mulberry plantation manifested that 16 accessions had comparatively lower productivity – less than 10 t/ha. Other 7 accessions showed much higher productivity. Most of the accessions manifested a leaf yield per 1 ha from 10.50 t to 12.00 t (62%).

The data about the productivity of some Georgian and Azerbaijani mulberry varieties tested in the respective countries manifest that the leaf yield per one hectare varies from 6.5 t to 12.00 t in the Georgian varieties and from 5.1 t to 12.3 t in the Azerbaijani varieties.

The leaf yield in those countries from East European and Central Asian region is lower if compared with the mulberry leaf yield per 1 ha in other sericulturally advanced countries from the temperate belt like Japan, China and Korea where the leaf yield is 17-20 t/ha. However this lower leaf yield could be more attributed to the comparatively dryer summer in this region rather than the genetic potential of the mulberry varieties.

It could be concluded that the mulberry accessions preserved in the region manifest comparatively high genetic potential for production of high quality leaves and good productivity per one hectare.

In many tests, held in different region countries the silkworm accessions preserved in the region manifested comparatively high variation of the main productive characters, such as cocoon weight from 1.603 g to 2.438 g, shell ratio from 12.23 % to 24.90 % and filament length from 791 m to 1581 m. Some of silkworm accessions and pure lines manifest comparatively high productivity. There also are some accessions, having particular characters like sex-limitation, male progeny, tolerance to adverse rearing conditions, parthenogenesis ability etc. which are very precious not only for the current silkworm breeding process, but also for the use of silkworm as organism for basic research in genetics, phisiology etc.

Taking into account the information collected for sericulture genetic resources in East European and Central Asian region, the countries could be divided into the following 3 groups:

I. Countries having comparatively rich sericulture genetic resources (Azerbaijan, Bulgaria, Ukraine and Uzbekistan).

II. Countries, having sericulture genetic resources, enough as a basis for own production of mulberry saplings and commercial silkworm eggs (Georgia, Romania and Turkey).

III. Countries, which have too small number of sericulture genetic resources, maintained systematically (Greece) and from now on should start establishing their mulberry and silkworm gene banks.

Based on this classification we can recommend some strategies about mulberry and silkworm breeding, propagation and egg production in order to use in a maximum extent the available genetic resources in the region. For example Azerbaijan, Bulgaria, Georgia, Romania, Ukraine and Uzbekistan could exchange between each other some mulberry and silkworm accessions and pure lines in order to use them in their future breeding programmes. Simultaneously Bulgaria and Ukraine can sell mulberry saplings and F 1 silkworm eggs to Uzbekistan, Greece and Turkey. In the future Bulgaria can also provide Greece with technical assistance and some parental silkworm line eggs in order to start gradually a local egg production.

As a main conclusion, since the region has totally comparatively rich sericulture genetic resources, one of these countries could be chosen in the future as a regional center for germplasm keeping (conservation), supply with parental and commercial silkworm eggs, mulberry saplings, training in the field of mulberry and silkworm germplasm collection, maintenance, conservation and breeding techniques.

The countries from East European/West/Central Asian region could also be used as a source for providing technical assistance, mulberry saplings and silkworm eggs to the newly sericulture developing countries from Africa and Near East .

4. Development of sericulture genetic resources in East Europe and West/Central Asia.

The following main methods have been implemented in mulberry breeding:

• Collection of different mulberry forms obtained by natural open pollination.
• Artificial hybridization.
• Polyploidy breeding
• Mutation breeding

Using the above mentioned methods both single or in combinations, 424 different mulberry varieties have been bred in the region. The biggest number of mulberry varieties were selected in Azerbaijan and Uzbekistan, followed by Bulgaria and Ukraine. Each candidate-variety is planted in 3 replicates, having 10-12 trees and the testing is made after the 5th year.

In the silkworm breeding the following different methods have been used:

• Segregation from foreign F1 hybrids.
• Separation from foreign F1 hybrids.
• Using populations of Japanese races.
• By making initial hybrid populations for further breeding.
• Breeding sex limited for egg color and larval marking strains: By this method as initial material sex limited silkworm lines, crossed with other strains, having plain larvae or yellow eggs have been used. After that the hybrid population was maintained by batch rearing for 4 generations and with inside batch mating (inbreeding). On the 5th generation an inter batch crossing was performed, followed again by inbreeding in the 6th and 7th generations. At the 8th generation the two inbred lines were crossed and the new breed was created. Using this method in Bulgaria Petkov (1995) selected 2 sex limited for egg color lines (T 15/4 and HT-215/38) and 5 sex limited for larval marking lines (B2/6, BTV-2/64, TBV-2/24, HB-2/22 and TV-3/2.)
• Method of breeding sex limited for larval marking lines as analogues of pure lines of already approved commercial hybrids: In the recent time the efforts of silkworm breeding in Bulgaria were directed to create lines/hybrids having higher tolerance to adverse rearing conditions. The reasons were the necessity to intensify the cocoon production in Bulgaria by adoption of summer and autumn rearing as well as the possibilities to export silkworm eggs to some tropical countries.

As a result a new hardy silkworm four-way commercial hybrid (Hesa 1x KK) x (Gergana2xVesletz2) was selected, authorized by the government in 1999 and widely adopted in the field during the period 2000-2003.

In order to improve the silkworm egg production technology we (Tzenov and Guzman, 2004) came to the idea to use the parental pure lines of the already created hybrid for breeding new sex-limited for larval markings pure lines. The breeding process had been started since the spring season of 1998 and 3 generations were reared in each year for four years. The breeding strategy aimed to use the existing pure lines, parents of the hardy commercial hybrid (Hesa 1xKK) x (Gergana2xVesletz2) as male parents as well as four introduced recently from Japan sex-limited for larval markings breeds as female parents in order to create new sex-limited lines, having similar qualitative characteristics and quantitative characters values to those of the parental pure lines of the commercial hybrid. In all the cases the sex-limited females were crossed with ordinary lines males, having same larval markings like the males of the sex-limited race. For example the Japanese sex – limited race Ze Aojuku had zebra marked females and normal marked males and the zebra marked females were crossed with males of the line Hesa 1, having also normal larval markings like the males of Ze Aojuku. By using 11 backcrosses, combined with proper selection methods 4 new silkworm sex-limited for larval markings pure lines, having similar qualitative and quantitative characters values to those in the original pure lines were created. Practically the selection was made only with the female individuals of newly selected sex-limited lines, using selected males of the ordinary lines. In this case the basic pure lines were maintained in bigger volume, allowing to make the necessary selection and the sex-limited analogues were maintained in lower volume (8-10 batches during rearing).

• Method of breeding tolerant to adverse rearing conditions silkworm breeds: In Bulgaria, 3 breeds (SB1, VB1, HB 2) were selected by this method in the recent years by using crosses between the uni-bivoltine pure lines Super 1 and Hesa 2 and the polyvoltine race Bonde 517.

The method is following:

1. Super 1 x Bonde 517 F1

2. Super 1 x Bonde 517 F2. There was segregation in colored and white cocoons with different shape and texture. Seven male cocoons having white color, elongated shape, similar to those of the uni-bivoltine line texture out of 32 kg cocoons produced were selected and mated with selected females of the line Vratza 35.

3. Inbreeding for 6 generations, batch rearing, selection for cocoon color, shape, texture and the main quantitative characters.

4. Outbreeding for 5 generations, batch rearing, selection for cocoon color, shape, texture and the main quantitative characters.

5. New breed VB 1

The newly selected breeds manifested a higher tolerance to adverse rearing conditions, but their productivity was comparatively low, therefore the further idea was to use them as components of four-way hybrids in combination with highly productive, but sensitive to bad environment pure lines.

* Silkworm breeding by using the help of computer programs: The following breeding scheme was performed by Greiss (2003):

1 Tested optimal rearing inputs for the silkworm Bombyx mori L. during 1995-1996 in mixed batches.

2 Reared 4-6 sib-matted inbred lines in these optimal inputs in 4 generations to increase genetic homozygosity during 1997-1998.

3 Checked the best combiner inbred lines versus a top cross of the same breed reared in a mixed batch rearing and randomly mated .

4 Crossed the best two general combiner lines (GRAND PARENTS) to produce the PARENTS egg batches.

5 Selection by “relative culling method”, having the following selection parameters:

5.1 Choosing the individuals that are within the top 30 individuals from 100 population per sex in two or more characters from each sex.

5.2 The minimum number of selected individuals should be four or more from each sex.

5.3 Priority was given to shell ratio and the cocoon weight character, if not possible the shell weight character was selected for, instead of cocoon weight.

Computer programs on Microsoft Excel base were specially designed to undergo selection according to the criteria given according to the Skew and Kurt parameters of each of the male and female populations, which dictated the selection criteria in the case of the experiments.

* *Breeding partheno/androgenetic lines, and control the sex-balance: There are some methods developed to control the sex balance of silkworm under the joint effort of scientists from different countries such as androgenesis, parthenogenesis and balanced lethal mutation.

Ameiotic parthenogenesis: The method is based on treatment by pure water with temperature 46 o C for 18 min of silkworm eggs extracted from the ovariole. The reductional ovarian separation is prevented by this treatment and the diploid chromosome set is preserved in the nucleus. In the same time the ovaries are stimulated for development and as a result only female genetical copies of the mother moth are obtained. This method was successfully used in Russia (Strunnikov et al., 1986), Georgia, Ukraine and Uzbekistan for breeding special partheno- lines. The Sericulture institutes in Georgia and Uzbekistan had a good cooperation with the Laboratory of Cytogenetics and Sex Regulation at the Institute of Developing Biology under the Russian Academy of Sciences in the field of selection new parthenogenesis lines and F1 hybrids between them and ordinary lines. The estimated advantages of the parthenogenesis line x ordinary breed F1 hybrids over the other hybrids are as follows:

• the percentage of seed cocoons used is much higher(85 % while 40 % in the ordinary ).
• all the seed cocoons from the maternal breed give female moths who lay eggs.
• the uniformity in F 1 is better.
• in the maternal breed there is no any sex discrimination, because all the individuals are female.
• the F1 hybrid purity is 100 %.

This new breeding method was further developed in Uzbekistan by creation of breeds, having Z lethal genes and giving male progeny only. In this case the female parent of the hybrid is parthenogenetic line, having only females and the male line is “Z lethal gene male progeny”, having only males. By this scientific achievement the problem with sex separation in the silkworm egg production was solved completely.

*Meiotic parthenogenesis: This method was developed for the first time in the Soviet Union (Russia ) by Strunnikov (1987). The silkworm ovaries are allowed to reductive separation and the nucleus has haploid number of chromosomes. If such kind of ovaries are preliminarily stimulated by high (46 o C ) or low(-11 o C) temperature they separate into two sister’s cells which fuse without meeting any spermatozoa. In this fusion, the two cells having haploid chromosome set each, make a cell with diploid chromosome set in the nucleus. In the case of meiotic parthenogenesis both the cells are heterogametic and in their fusion the possible combinations are WW and ZZ. From the eggs having ZZ chromosomes male individuals develop, but the eggs having WW chromosomes die.

*Androgenesis: Newly laid eggs are first treated by X-rays (12000 – 15000 R) and then they are treated by high temperature (40 o C) water for 1 h. The X – rays destroy the ovarian nucleus, so the female nucleus become incapable to fuse with spermatozoa and the development starts after fusion of two spermatozoids nucleuses with the participance of maternal cytoplasm. Only the father’s genotype is inherited (ZZ) and only male individuals are obtained. Since both spermatozoids are from one same father the progeny is with low viability.

In Bulgaria Nacheva et al.(1998) used androgenetic male individuals for making series of backcrosses with selected females in order to obtain ordinary bi-sexual lines, demonstrating a higher homozygosity .

*Methods for obtaining only male progeny by sex-limited lethal genes: It was discovered by Russian (Strunnikov, 1987) and Chinese scientists (Chen, 2002) that the sex-linked chocolate gene mutation (sch) was recessive gene which is very sensitive to high temperature during incubation stage. When genotype ZW crossed with Z sch Z sch, the female embryos of their hybrid could be killed during the incubation stage, therefore, only male silkworm will be hatched.

Using some of the above mentioned methods 262 silkworm breeds have been selected totally in the region. As leading countries in silkworm breeding, judging from the number of breeds selected appear to be Bulgaria, Azerbaijan and Uzbekistan.

*Methods for creation commercial silkworm hybrids:

Some of the selected silkworm strains are used as pure lines for commercial hybridization.

The first precondition one strain to be selected as pure line is to cover all the breeding characters criteria, to have low variability of the quantitative characters values and the inheritance of the characters to be stable in the generations.

The chosen(or especially selected) breeds are tested for the combining ability and those one showed the highest combining ability between them are used as pure lines of the commercial hybrid. Then the new hybrid is tested at least for 3 years at the station/institute where it has been selected, and the hybrid is tested for at least 2 years in the system of the State Executive Agency for Varieties Testing after that. During the testing the most widely adopted in the field silkworm hybrid for the last 5 years is used as control. To be recognized as new and original the hybrid must display difference from other hybrids/races at least in 1-2 qualitative characters and the most important quantitative characters values should be higher than the standard.

The most popular in the region are single and four-way F 1 commercial hybrids. The testing of some silkworm hybrids, produced in the region manifested that almost all of them had comparatively high productivity, namely single cocoon weight 2.2 – 2.5 g, shell ratio 23-24 %, shell weight 0.500 – 0.600 g, filament length 1300 – 1500 m under laboratory conditions. This is a proof that the hybrids have high genetically determined productivity potential, if the optimal silkworm rearing technology is followed strictly. However, under field conditions some of the hybrids displayed significantly lower productivity.

Regardless the comparatively high productivity manifested at laboratory conditions, it is necessary to organize a simultaneous testing of all the best hybrids, produced in each country from the region in order to make a real comparative test. In order to compare the productivity of the local silkworm hybrids with those of the hybrids produced in some advanced sericultural countries, some hybrids from Japan, China, South Korea and Italy should also be included in this testing.

5.Main findings, conclusions and recommendations

• The sericulture should be considered as one of the important potential agro-industry in Eastern Europe and Central Asia region countries, now that the increasing number of farmers, i.e. approximately 500,000 households are involved in order to generate their income resources. 14 countries (Albania, Armenia, Azerbaijan, Bulgaria, Georgia, Greece, Kazakhstan, Kyrgyzstan, Romania, Tajikistan, Turkey, Turkmenistan, Ukraine, and Uzbekistan) are engaged actively in sericulture development activities in this region.
• The total number of mulberry accessions preserved in the region is 931 and the number of silkworm accessions is 767. Compared with the present cocoon /silk production at the regional level those countries maintain a rich collection of both mulberry and silkworm genetic resources.
• The countries, having the richest sericulture genetic resources in the region are Azerbaijan, Bulgaria, Uzbekistan and Ukraine.
• Mulberry varieties from 22 countries and silkworm accessions from 20 different countries are totally presented at the regional sericultural germplasm, including some accessions from one of the most sericulturally advanced countries such as Japan and China.
• Most of mulberry varieties preserved in the region originated from Azerbaijan (229 and 24.70 %), Uzbekistan (169 and 18.15 %), Japan (139 and 14.93 %), Bulgaria (87 and 9.34 %), Ukraine, Georgia and China. The countries, having the highest diversity as regards the mulberry accessions origin are Uzbekistan (from 15 different countries), Azerbaijan (from 12 countries) and Bulgaria (from 11 countries).
• The countries represented in the highest extent in the regional silkworm germplasm are Bulgaria (166 and 21.64 %), China (100 and 13. 04 %), Ukraine (83 and 10.82 %), Japan (81 and 10.56 %), Azerbaijan (73 and 9.52 %) and Uzbekistan (69 and 9.00 %). The countries, having the highest diversity as regards the silkworm accessions origin are Bulgaria (from 15 different countries), Uzbekistan (from 14 countries), Ukraine (from 10 countries) and Azerbaijan (from 7 countries).
• Both mulberry and silkworm genetic resources are maintained by correct methodologies. However, due to the economical problems, the number of accessions preserved has been decreased to some extent during the recent decade in some region countries.
• The mulberry leaf yield in the accessions maintained varies from 5.1 to 12.3 t /ha and appears to be lower than in other sericulturally advanced countries from the temperate belt like Japan, China and Korea. However this lower leaf yield could be more attributed to the comparatively dryer summer in this region rather than the genetic potential of the mulberry varieties. It could be concluded that the mulberry accessions preserved in the region manifest comparatively high genetic potential for production of high quality leaves and good productivity per one hectare.
• The silkworm accessions preserved in the region manifested comparatively high variation of the main productive characters, such as cocoon weight from 1.603 g to 2.438 g, shell ratio from 12.23 % to 24.90 % and filament length from 791 m to 1581 m. Some of silkworm accessions and pure lines maintained in the region manifest comparatively high productivity.
• 424 different mulberry varieties and 262 silkworm breeds have been bred in the region. The biggest number of mulberry varieties and silkworm breeds were selected in Azerbaijan, Uzbekistan, Ukraine and Bulgaria.
• Some advanced methods in mulberry and silkworm selection such as polyploidy and mutation mulberry breeding, breeding sex limited for egg color and larval marking silkworm strains, breeding tolerant to adverse rearing conditions silkworm breeds, breeding parthenogenetic/androgenetic lines, and control the sex-balance have been developed in the region.
• Almost all the commercial hybrids, produced in the region manifested comparatively high productivity, namely single cocoon weight 2.2 – 2.5 g, shell ratio 23-24 %, shell weight 0.500 – 0.600 g, filament length 1300 – 1500 m under laboratory conditions.
• The exchange of mulberry and silkworm germplasm between those countries will facilitate the initiation, expansion or rehabilitation of silkworm and/or mulberry breeding programmes in the recipient countries. This will enable the promotion of sericulture industries which will contribute to national economic development.
• Since the region has comparatively rich sericulture genetic resources and advanced science in the field of mulberry and silkworm breeding, in the future some of these countries could be chosen as a regional centers for germplasm keeping (conservation), supply with parental silkworm eggs, mulberry saplings of chosen varieties, training in the field of mulberry and silkworm germplasm collection, maintenance, conservation and breeding techniques. Those countries could also be used as a source for providing technical expertise, mulberry saplings and silkworm eggs to the newly sericulture developing countries from Africa and Near East.

6. References

Chen Y. (2002) Conservation status of silkworm genetic resources in China. In: ”Expert consultation on promotion of global exchange of sericulture germplasm resources”, Satellite session of XIX th ISC Congress,21st -25th , September 2002,Bangkok,Thailand.

Greiss H. (2003) Genetic-selection studies on silkworm Bombyx mori L. under different environmental conditions. Abstract of the dissertation for conferment of educational and scientific degree PhD, Sofia, 63 pp.

Nacheva Y.,Petkov N.,Malinova K. (1998) Possibilities for silkworm,Bombyx mori L. bisexual androgenetic lines selection, Animal sciences,supplement,82-85. (in Bulgarian)

Petkov Z. (1998) Studies on some mulberry species and varieties (Morus sp.) and influence of some of them on silkworm(Bombyx mori L.) development and productivity., PHD thesis. Sofia. (in Bulgarian)

Petkov N. (1995) Selection and genetic studies and results of the selection of breeds, lines and hybrids of the silkworm,Bombyx mori L., Dr of Sc thesis, Sofia. (In Bulgarian)

Strunnikov V., Gulamova L., Karimova Sh., Jakubov A., Alimova R., Kurbanov (1986) Results of investigations on studying the possibilities for creation of genetical copies of female and male silkworms from the commercial breeds. Sbornik “Nauchnie osnovi razvitija shelkovodstva “, Tashkent, 14-27. (in Russian)

Strunnikov V. (1987) Genetical methods for silkworm breeding and sex regulation, Agropromizdat,Moskow,7-103 (in Russian)

Tzenov P. (1998) Study on possibilities for keeping and maintenance of tropical multivoltine race at Bulgarian climatic condition by rearing of 1 generation yearly, Animal science, supplement, 113-116. (In Bulgarian)

Tzenov P.,Nacheva Y.,Lazarov J. (2001) Study on the preservation terms of nondiapausing silkworm, Bombyx mori L. eggs at low temperature, Bulgarian Journal of Agricultural Science, 7, 467-470.

Tzenov P., Z.I.De Guzman (2004) Breeding the new Bulgarian sex-limited for larval markings silkworm Bombyx mori L. commercial hybrid Ze/4 and study on its performance in the Philippines and Bulgaria, Sericologia, 297-312. (France)