2. Conservation of mulberry genetic resources in China

2.1 Present status of germplasm maintenance

Mulberry genetic resources are the backbone of crop management. Collection, introduction and exchange can enrich existing gene pool and provide breeders a great scope for further improvement.

There are more than 2600 mulberry accessions in the mulberry germplasm maintained in China. These mulberry accessions can be divided as following groups according to their characters and growing districts.

1) Zhejiang and Jiangsu area (Taihu valley type): Taihu valley is located in the low reaches of the yangtse River. The warm temperature and abundant rain falls. The mulberry is budding in the end of March or the beginning of April, opening the leaf in the middle of April, and dropped the leaf until November. The growing duration of mulberry is long and reached 220-240 days annually. There are about 360 copies of mulberry germplasm resources conserved in this area.

2) Guangdong area (Zhujiang valley type): this area is the main sericultural district in southern China with tropical or sub-tropical climate. Average temperature is above 20°C and the growth duration is long to more than 350 days. At present, this area keeps about 500 copies of mulberry genetic resources.

3) Sichun basin area: located in Sichun pain and southern Sichun areas. Waum temperature and sub-tropical climate. The budding of mulberry is from the begging of March, and growth duration reached 240-330 days. There are more than 200 copies of mulberry resources.

4) Yellow River area: this area included Shandong and Hebei provinces located in the down valley of the Yellow River. The budding time is from the middle of April. The main type is Morus multicaulis Perr. More than 160 copies of mulberry were collected and conserved.

5) Yangtse River Middle Valley area: this area included southern Anhui, Hubei and Hunan provinces. Average temperature is between 15-20°C. Annual rain fall comes to 1000-1500 ml. More than 50 copies of mulberry genetics resources is kept.

6) Loess plateau area: this area is inland including Shanxi, shuanxi and Gansu provinces. At present there are more than 160 copies of mulberry conserved.

7) Xinjiang area: this is located in northwest of China, and the main mulberry is belong to Morus alba linn. More than 150 copies of mulberry were conserved.

2.2 Management and utilization of germplasm resources

Mulberry tree is a perennial plant. Because mulberry tree is growing in different ecological environment, it become characteristically different ecotype in morphology, anatomical, reproductive, growth, biochemical, disease etc in different areas. The purpose of conservation of mulberry germplasm resources is keeping the genetic diversity, and using them practically through line selection, breeding, organ culture etc.

2.2.1 Methodology for maintenance of mulberry accessions

Mulberry germplasm resources are the basic materials for line selection and breeding. Expectant effects of breeding depend on the abundance of germplasm resources. So it is important to collect extensively, conserve appropriately of mulberry genetic resources for utilizing practically. Some mulberry materials can be used in practical after studying and arranging, some should be used as parent materials for breeding.

Origin of germplasm resources: In China, there are four ways for collecting the mulberry germplasm resources, i.e.,

1) Local indigenous varieties: most of them are selected or bred through long natural selection and artificial selection. The most characteristics is their high suitability to local condition such as weather, soil and high resistance to pest and disease. The percent of local varieties come to about 65% of total resources in China.

2) Introduced varieties: China mainly introduced varieties from Japan, the former USSR, Korea and some south Asia areas such as India, Thailand, Viet Nam etc. But the percent only comes to below 3%.

3) Artificial-made new type: usually the new type of varieties was made through line selection, breeding, radiation breeding and biotechnology. These varieties have more practical characteristics. This type of varieties comes to about 30% in China.

4) Wild type: the economic value is usually low, but they have some special characteristics or super resistance to pest and diseases. It will be an important material to create high quality new practical varieties.

Collection and preservation: collection of mulberry germplasm resources needs deeply investigation. After that, collected races should be recorded as following: Accession number, race name, variety name, collection place, original environment condition, morphology and other main characters, key points in cultivation etc.

Study of mulberry germplasm: the works of mulberry germplasm resources can be outlined as “collection, conservation, study and utilization”. The procedure is described as following:

Utilization of mulberry germplasm resources: the mulberry germplasm resources provided sericulturists to create the new mulberry varieties through following methods:

• Line selection from local races: it is an important way to create new varieties from investigated local races. The new varieties have usually strong suitability to local environment and can be extended directly to practical use.
  
• Selection of seedling mulberry: the procedure is similar with above. The key point of selection is leaf size and thickness, long growth duration, deep leaf color, less side leaf, dense leaf number, less fruit and high resistance to pest and diseases. The Institute of Sericulture Research in Guangdong and Southern China Agricultural University have selected new varieties from seedling mulberry, such as Lu Jiao 40, Bei Qu No.1, Lun Jiao 109, Sha No.2 etc.
  
• Breeding: it is a common method to create new varieties. Following procedures should be taken care of: 1. Parent selection from genetic characters such as growth vigor, internode length, budding date and budding rate, disease resistance and leaf characters. It is important to select parents of more fine characters and less poor characters that are complementary each other. The differences of ecological type of parents should be bigger in order to create more heterosis in F1 hydride. For exsample, Sang No.5801 and Yu No. 2 bred by Institute of Sericultural Research, China Agricultural Acedemy were created from Hu Sang 38 × Guangdong Sang, and Hu Sang 39 × Guangdong Sang respectively. 2. breeding ways: according to the breeding purpose, it can be divided to single breeding, multi-breeding, back breeding, and mix-pollination.

Bud mutation: because mulberry can be multiplicated through asexual propagation, the good mutation of bud can be fixed through asexual reproduction and create a new variety.

Mutation breeding: there are many induced mutation factors such as chemical mutagenic agents, radiation rays and other physical factors etc.

Mulberry organ or tissue culture: it is a new method to create new variety, although is still in the laboratory level.

2.2.2 Methodology for mulberry germplasm characterization and evaluation

MULBERRY CLASSIFICATION METHODS (table 4)

Name of variety
Accession number
Donor name
National accession number

1. Morphology

1.1 Branching nature

1.1.1 Curve or straightness of the branch 1.1.2 Color of young shoot 1.1.3 Color of mature shoot 1.1.4 Phyllotaxy 1.1.5 Stipule nature 1.1.6 Stipule duration 1.1.7 Lenticel density/sq.mm

1.2 Leaf nature

1.2.1 Leaf lobation type 1.2.2 Leaf lobation number 1.2.3 Leaf color 1.2.4 Leaf surface 1.2.5 Leaf texture 1.2.6 Leaf apex 1.2.7 Leaf margin 1.2.8 Leaf base 1.2.9 Leaf Shape 1.2.10 Leaf length (cm) 1.2.11 Leaf width (cm) 1.2.12 Petiole length (cm) 1.2.13 Petiole width (cm)

1. Anatomical

2. Reproductive

3.1 Sex

3.1.1 Male inflorescence length (cm) 3.1.2 Female inflorescence length (cm) 3.1.3 Bisexual inflorescence length (cm) 3.1.4 Male inflorescence width (cm) 3.1.5 Female inflorescence width (cm) 3.1.6 Bisexual inflorescence width (cm)

3.2 Flower

3.2.1 No.of flowers/catkin (Male) 3.2.2 No.of flowers/catkin (Female) 3.2.3 No.of flowers/catkin (Bisexual) 3.2.4 Male inflorescence peduncle length 3.2.5 Female inflorescence peduncle length 3.2.6 Bisexual inflorescence peduncle length

3.3 Pollen and Stigma nature

3.3.1 Stamen length (mm) 3.3.2 Anther length (mm) 3.3.3 Pollen diameter (µm) 3.3.4 Pollen viability (%) 3.3.5 Style length (mm) 3.3.6 Stigma length (mm) 3.3.7 Stigma type

3.4 Fruit

3.4.1 Fruit length (cm) 3.4.2 Fruit width (cm) 3.4.3 Fruit weight (gm) 3.4.5 Fruit color 3.4.6 Fruit taste

4. Growth

4.1 Budding

4.1.1 Budding time 4.1.2 Budding rate(%)

4.2 Shoot

4.2.1 No.of branches/plant 4.2.2 Longest shoot length (cm) 4.2.3 Total shoot length (cm) 4.2.4 Total shoot length (cm) 4.2.5 Internodal distance (cm)

4.3 Leaf

4.3.1 Leaf number/kg(spring) 4.3.2 Leaf number/kg(autumn) 4.3.3 Leaf yield(kg)/Mu 4.3.4 Leaf yield(g)/shoot(m) 4.3.5 Leaf yield/plant (kg) 4.3.6 Hundred leaf weight (gm) 4.3.7 Leaf area (sq.cm) 4.3.8 First leaf opening time 4.3.9 Lamina weight (gm) 4.3.10 Petiole weight (gm) 4.3.11 Lamina index (%) 4.3.12 Leaf petiole ratio by length 4.3.13 Leaf petiole ratio by weight 4.3.14 Cocoon yield(kg)/100kg leaf 4.3.15 Leaf shoot ratio 4.3.16 Moisture content (%) 4.3.17 Moisture content after 6 hours (%) 4.3.18 Moisture retention capacity (%)

5. Biochemical

5.1 Dried material (%)
5.2 Water content (%)
5.3 Crude lipid (%)
5.4 Chlorophyll

5.4.1 Chlorophyll-a
5.4.2 Chlorophyll-b
5.4.3 Total Chlorophyll
5.4.4 Chlorophyll-a/b

5.5 Protein

5.5.1 Crude protein content (%)
5.5.2 Protein soluble (dry weight)
5.5.3 Protein soluble (dry weight)

5.6 Carbohydrate

5.6.1 Crude cellulose (%)
5.6.2 Carbohydrate soluble (fresh weight)
5.6.3 Carbohydrate soluble (dry weight)

5.7 C/N

6. Disease

6.1 Powdery mildew
6.2 Leaf spot
6.3 Bacterial blight

7. Propagation

7.1 Survival %

7.2 leaf

7.2.1 No.of Leaves/saplings 7.2.2 Leaf length (cm) 7.2.3 Leaf width (cm) 7.2.4 Fresh leaf weight/sapling (gm) 7.2.5 Dry leaf weight/sapling (gm)

7.3 shoot

7.3.1 Shoot diameter (cm) 7.3.2 Shoot length (cm) 7.3.3 Fresh shoot weight/sapling (gm) 7.3.4 Dry shoot weight/sapling (gm)

7.4 biomass:

7.4.1 Total fresh biomass/sapling (gm) 7.4.2 Total dry biomass/sapling (gm)

7.5 root

7.5.1 No.of roots/sapling 7.5.2 Fresh root weight/sapling (gm) 7.5.3 Dry root weight/sapling (gm) 7.5.4 Longest root length/sapling (cm) 7.5.5 Root volume/sapling (ml)

SPECIFIC INVESTIGATING METHODS FOR MULBERRY

1. Investigation of silkworm rearing, seed preparation, reeling of mulberry (Table-5)

1.1 Rearing results(spring, autumn)

1.1.1 Rate of survival (%) 1.1.2 Died larva in cocooning (%) 1.1.3 Cocoon weight (kg/10000 larvae) 1.1.4 Cocoon number per kg 1.1.5 Cocoon weight(g) 1.1.6 Shell weight(g) 1.1.7 Shell ratio(%) 1.1.8 Moultism ratio

1.2 Egg-production results(spring, autumn)

1.2.1 Number of healthy/fertilized eggs 1.2.2 Number of unhealthy or unfertilizrd eggs 1.2.3 Total egg number 1.2.4 Egg color uniform 1.2.5 Egg weight (g/10000eggs)

1.3 Reeling results

1.3.1 Cocoon weight (g) 1.3.2 Shell weight (g) 1.3.3 Filament length (m) 1.3.4 Reelable filament length (M) 1.3.5 Reelability(%) 1.3.6 Filament size (d) 1.3.7 Size deviation

2. Investigation of morphology of mulberry germplasm (Table-6)

Mulberry variety:
Tree years:
Cut type:
Origin:

2.1 Outline

2.1.1 Height 2.1.2 Tree type: 2.1.2.1 Type 2.1.2.2 Shoot number 2.1.2.3 Shoot length 2.1.3 stem: 2.1.3.1 Curve or straight 2.1.3.2 Color 2.1.3.3 Tree skin

2.2. Shoot

2.2.1 Type: 2.2.1.1 Curve or straight 2.2.1.2 Stand or bending 2.2.2 Size 2.2.3 Length 2.2.4 side shoot number 2.2.5 Internode 2.2.5.1 Length 2.2.5.2 Curve or straight 2.2.6 Skin color 2.2.7 Skin veins 2.2.8 Stoma ostiole 2.2.8.1 morpha 2.2.8.2 Size 2.2.8.3 Protruding 2.2.8.3 Distribution 2.2.8.4 Number

2.3 Winter bud

2.3.1 Morpha 2.3.2 Growth character 2.3.3 Size 2.3.4 Color 2.3.5 Bud scale 2.3.5.1 Number 2.3.5.2 Layers 2.3.6 Accessory bud number 2.3.7 Bud bract 2.3.8 Leaf rank 2.3.9 Leaf scar 2.3.9.1 morpha 2.3.9.2 Rhombic 2.3.9.3 Size

2.4 Young shoot

2.4.1 Size and length 2.4.2 Number 2.4.3 Color 2.4.4 Trichome

2.5 leaf

2.5.1 Morpha 2.5.1.1 Round leaf 2.5.1.2 Lobed leaf 2.5.2 Growth character 2.5.3 Incision 2.5.3.1 Depth 2.5.3.2 Size 2.5.3.3 Lobed leaf number 2.5.4 Leaf apex 2.5.5 Margin of leaf 2.5.6 Leaf base 2.5.7 Leaf vein 2.5.8 Leaf color 2.5.9 Lustre 2.5.10 Coarse leaf 2.5.11 Wrinkle 2.5.12 Size 2.5.12.1 Leaf length 2.5.12.2 Leaf width 2.5.13 Trichome 2.5.13.1 Leaf surface 2.5.13.2 Leaf back 2.5.13.3 Leaf vein 2.5.14 Leaf area 2.5.15 Leaf thickness

2.6 Leaf stalk:

2.6.1 Growth character 2.6.2 Length 2.6.3 Width 2.6.4 Color 2.6.5 Trichome

2.7 Stipule:

2.7.1 Morpha 2.7.2 Color 2.7.3 Size

3. Investigation of flower and fruit of mulberry germplasm (Table-7)

3.1 Monoecious or dioecious

3.2 Female and male for one flower or not

3.3 Unisexual or bisexual flower

3.4 Female flower

3.4.1 Catkin: 3.4.1.1 Morpha 3.4.1.2 Size 3.4.1.3 Number 3.4.1.4 Color 3.4.1.5 Flower number 3.4.1.6 Flower character 3.4.1.7 Flower stalk length

3.4.2 Flower

3.4.2.1 Flower cover: 3.4.2.1.1 Morpha 3.4.2.1.2 Size 3.4.2.1.3 Trichome
3.4.2.2 Style length
3.4.2.3 Stigma 3.4.2.3.1 Morpha 3.4.2.3.2 Length 3.4.2.3.3 Trichome 3.4.2.3.4 Bulge 3.4.2.3.5 Color
3.4.2.4 Ovary 3.4.2.4.1 Morpha 3.4.2.4.2 Size

3.5 Male flower

3.5.1 Catkin: 3.5.1.1 Morpha 3.5.1.2 Size 3.5.1.3 Number 3.5.1.4 Color 3.5.1.5 Flower number 3.5.1.6 Flower character 3.5.1.7 Flower stalk length

3.5.2 Flower 3.5.2.1 Flower cover 3.5.2.1.1 Morpha 3.5.2.1.2 Size 3.5.2.1.3 Trichome 3.5.2.2 Flower filament 3.5.2.2.1 Morpha 3.5.2.2.2 Length3.5.2.2.3 Color 3.5.2.3 Anther 3.5.2.3.1 Morpha 3.5.2.3.2 Length 3.5.2.3.3 Color 3.5.2.4 Ovary 3.4.2.4.1 Morpha 3.4.2.4.2 Size Pollen grain size

3.6 Fruit and sorosis

3.6.1 Fruit 3.6.1.1 Structure 3.6.1.2 Size 3.6.1.3 Fruit stalk 3.6.1.3.1 Color 3.6.1.3.2 Trichome

3.6.2 sorosis 3.6.2.1 Structure 3.6.2.2 Size 3.6.2.2.1 Length 3.6.2.2.2 Diameter 3.6.2.3 Color3.6.2.4 Taste 3.6.2.5 Number 3.6.2.6 Fructification 3.6.2.7 Drop character 3.6.2.8 Mature character

4. Growth duration and economic characters of mulberry germplasm (Table-8)

4.1 growth

4.1.1 Budding time 4.1.2 Leaf growth day 4.1.3 Flower open day

4.1.4 Spring leaf 4.1.4.1 Growth duration 4.1.4.2 Mature time

4.1.5 Autumn leaf 4.1.5.1 Growth duration 4.1.5.2 New shoot stopping day 4.1.5.3 Harding day 4.1.5.4 Leaf yellowed day

4.1.6 Full year growth duration

4.2 Budding ratio (%)

4.3 Ratio of growth bud to growth-ended shoot let

4.3.1 Growth bud 4.3.1.1 Percent 4.3.1.2 Length 4.3.1.3 Size 4.3.1.4 Leaf number 4.3.2 Growth-ended shoot let 4.3.2.1 Percent 4.3.2.2 Leaf number

4.4 Shoot

4.4.1 Single tree 4.4.1.1 Shoot number 4.4.1.2 Shoot length for one tree 4.4.2 Single shoot 4.4.2.1 Shoot length 4.4.2.2 Shoot diameter

4.5 Ratio of shoot to leaf to fruit

4.6 Yield quantity per leaf

4.6.1 Spring 4.6.1.1 Leaf number per kg 4.6.1.2 Weight per leaf 4.6.2 Autumn 4.6.2.1 Leaf number per kg 4.6.2.2 Weight per leaf

4.7 Leaf quantity

4.7.1 Spring 4.7.1.1 Shoot let length per meter 4.7.1.2 Yield for one tree 4.7.1.3 Yield for one hectare

4.7.2 Autumn 4.7.2.1 Shoot let length per meter 4.7.2.2 Yield for one tree 4.7.2.3 Yield for one hectare

4.8 Water content and wilting speed

4.8.1 Water content 4.8.1.1 Spring leaf 4.8.1.2 Autumn leaf

4.8.2 Water Evaporation rate 4.8.2.1 After 2 hrs 4.8.2.2 After 4 hrs 4.8.2.3 After 6 hrs 4.8.2.4 After 8 hrs 4.8.2.5 After 10 hrs 4.8.2.6 After 12 hrs

5. Resistance to disease and pest of mulberry germplasm (Table-9)

5.1 Diseases (1)Infection time (2)Disease ratio

5.1.1 Dwarf disease 5.1.2 Twig blight 5.1.3 Dogare blight 5.1.4 Bacterial blight 5.1.5 Powdery mildew 5.1.6 Rust aecidium mori 5.1.7 White root rot 5.1.8 Violet root rot

5.2 Pest (1)Infestation time (2)Harmful degree

5.2.1 Mulberry small weevil 5.2.2 Mulberry shoot dall midge 5.2.3 Mulberry leaf roller 5.2.4 Mulberry pyralid 5.2.5 Mulberry sucker 5.2.6 Fall webworm 5.2.7 Mulberry yellow tail moth 5.2.8 Mulberry looper 5.2.9 Mulberry borer 5.2.10 Mulberry thrip 5.2.11 Mites

5.3 Natural disasters (1)Occurrence time (2)Harmful degree

5.3.1 Frost 5.3.2 Cool 5.3.3 Drought 5.3.4 Flooding 5.3.5 Wind

2.3 Development of germplasm resources

The studies on mulberry germplasm are focused on following aspects:

1) The ecological types suitable to different districts, such as plain, mountain, hilly area, riverbed areas etc.

(1) Ecological types of mulberry under different factors including average temperature, leaf growth duration, rain fall, relative humidity, photo-period, and wind speed etc. Among them, the temperature and humidity come to first places. Following is the sample of plain area (Hang-Jia-Hu sericultural area) in Zhejiang Province.

(2) Effects of meteorological factors on the growth and development of mulberry.

a. Budding in spring and leaf maturation. In plain area such as Hang- Jia-Hu area, mulberry of tong xiang qing, he ye bai, bai tiao sang wu sang etc are budding around March 12 with average temperature of 8.8°C, in the middle of April with the stable average temperature of 12°C, 5 leaves are opened. Usually, it takes about 32 days from budding to leaf maturation with slight difference of 3 to 5 days. But in hilly area, the temperature is higher than plain about 1.2-1.4°C. So the budding time is 3-5 days earlier than that of plain area.

b. the growth of mulberry shoots and leaves in summer and autumn(Table 10).

(3) Effects of meteorological factors on the structure and quality of mulberry

a. leaf area: During spring an average length of leaf 14.7 cm, width 12 cm and the summer-autumn leaf is 24.5 cm and 17.6 cm under suitable temperature, rain fall and sunlight(Table 11). The average shoot number is 30 per mulberry tree with 70% of shoots of 150-200 cm.

b. thickness and leaf quality (Table 12)

2) Analysis of major nutrients and fluoride content in the mulberry leaf.

Mulberry Leaf is the only nutrition for the silkworm. The sericulture institute of Zhejiang province investigated the major nutrients and fluoride content of mulberry preserved in germplasm during 1989-1990. The results were as following:

Table 13 the crude protein content of mulberry

Table 14 the soluble carbohydrate content of mulberry

Table 15 the fluoride content of mulberry

3) Biological investigation

4) Evaluation of economic characters, including leaf quantity, leaf quality, resistance to diseases and pests etc.

5) Application of computer science in mulberry germplasm conservation

6) Genetic analysis of factors affecting mulberry quantity and quality