Seed Propagation Technology and Quality Standards of Zhuoyou 1032

Hua WANG Hailin WANG Yao ZHAO Cong LU Fuwen KE

Abstract The breeders seeds of 56A are produced through individual selection， sib mating in pairs and selection in isolation zone （net） plant line nursery. The foundation seeds are produced by isolated propagation of the breeders seeds， and the sterile line seeds for field production are produced by isolated propagation of the foundation seeds. The breeders seeds of restorer line 8020R are produced by bagging and selfing and selection in isolation zone （net） plant line nursery， and the foundation seeds are produced by re-propagation. The hybrid seed production requires better isolation conditions， which can be realized using natural forests， mountains， rivers， roads （railways， highways）， houses， etc.， and the isolation distance in open areas is equal to or larger than 800 m. It also requires early sowing of the male parent， so that the male parent can make full use of the light and temperature resources before winter to grow vegetatively， forming strong male parent plants that can provide more pollen. The seeds of the male parent are sown on September 8-10， and those of the female seeds are sown on September 12-16. Quality test is conducted during breeding and seed production in accordance with relevant national standards. The purity standards of the parent seeds are as follows： purity≥99%， neatness≥98%， germination rate≥80%， moisture≤9%， glucosinolate content≤20 μmol/g， and erucic acid content≤0.5%; the quality standards for parental field production seeds are as follows： purity≥98%， neatness≥98%， germination rate≥80% and moisture≤9%; and quality standards for hybrid seeds： purity≥85%， neatness≥98%， germination rate≥80% and moisture≤9%.

Key words Zhuoyou 1032; Foundation seed; Seed for field production; Breeding technology; Quality standard

Zhuoyou 1032 is a new double-low hybrid rapeseed （Brassica napus） with high oil content successfully bred from double-low genic male sterile rapeseed line 56A of B. napus （original code： 1040A） and restorer line 8020R （original code： 132-4R） by Guizhou Zhuohao Agricultural Science Co.， Ltd. In the two years of 18-site adaptability evaluation tests， the average yield was 172.31 kg/666.7 m2， with a yield increase of 4.78% compared with Youyan 50 （CK）; its whole growth period was about 202 d， which was 2-3 d earlier than that of Youyan 50 （CK）; and the plant height， branching node height， effective branch number， effective pod number per plant and seed number per pod， 1 000-seed weight and average yield per plant were 197.6 cm， 52.1 cm， 5.55 branches， 179.7 pods， 17.7 seeds， 3.99 g and 11.7 g， respectively. Zhuoyou 1032 has the characteristics of early maturity， high yield， fast cluster formation， high uniformity， compact plant type， strong resistance to lodging， good tolerance to sclerotinia， medium pod length and large 1 000-seed weight. In order to better promote Zhuoyou 1032 and realize the matching of good varieties and good methods[1]， from 2016， research on the breeding techniques of breeders seeds and foundation seeds of 56A and 8020R and on high-yielding seed production techniques of hybrid seeds was conducted， and the Zhuoyou 1032 breeding technical specifications and the seed quality standards implemented by Guizhou Zhuohao Agricultural Technology Co.， Ltd. were formulated according to the general characters in the varieties of the two-line method.

Propagation Techniques and Quality Standards of Sterile Line 56A

Breeding of breeders seeds of 56A

Individual selection

In the breeding area for breeders seeds of the male sterile line 56A with isolation conditions， 150 plants with the typical traits of 56A were selected during the overwintering season and hung with a tag. Screening was further performed in the initial flowering stage to retain certain sterile and fertile plants （30-50 each）. The buds that have opened in the initial flowering stage are removed， and adjacent sterile and fertile plants are covered with a large parchment paper bag or 80 mesh gauze bag[2].

Sib mating in pairs

After the main inflorescences and the upper 2-3 branches of the bagged plants are blooming， the fertile plant in a bag is taken to pollinate the sterile plant in the same bag after 10 oclock on a sunny day to make a sib mating combination， and the pollination is performed once every 3-4 d for more than 5 times in the whole flowering stage. The plants are covered by a paper bag or gauze bag again after each time of pollination. After maturity， the sterile plants and fertile plants are individually harvested according to the combination plant number， and the quality of the seeds （glucosinolate and erucic acid contents） from the sterile plants and fertile plants is tested， respectively. The seeds of the sterile plant and the corresponding fertile plant are retained when both of plants satisfy the required quality indicators （56A： glucosinolate content≤25 μmol/g， erucic acid content≤1.0%）.

Plant line sorting and selection

In an isolation net room or a parent sorting area with isolation conditions， qualified seeds harvested and tested to be qualified in the previous year are planted into plant lines according to combinations， and 15-20 rows are transplanted for each plant line， including a total of about 200-400 plants. The plant characteristics are checked at the seedling stage， and atypical plants are marked. Investigate fertility ratio at flowering period. The fertility segregation ratio （sterile∶fertile） is investigated in the flowering stage， and 3-4 plant lines with a fertility segregation ratio at 1∶1 having typical characteristics of 56A were retained， while other plant lines were cut off. For each of the remaining plane line， sickly and off-type plants are removed strictly. Sterile plants are marked by tagging， tying with red rope or smearing red paint during the flowering stage. The fertile plants， which are early flowering， late flowering or have few flowers， are removed. Then， bees that have been cleaned for more than 3 d are released into the net room， or artificial pollination is performed. The marked sterile plants are screened in the maturation stage， the seeds of the selected sterile plants were harvested in a mixed manner， obtaining the breeders seeds of 56A， which are sun-dried and stored in a low-temperature storehouse under dry conditions.

Breeding of foundation seeds

According to the hybrid seed production plan， an appropriate amount of the breeders seeds in the low-temperature storehouse are taken and transplanted in the propagation area with an isolation distance of more than 1 500 m that has not been planted with rapeseed for 2-3 years. Meanwhile， it is forbidden to cultivate Cruciferae plants such as rapeseed and cabbage in the isolation area， and bee colony stocking is also strictly prohibited. Sickly and off-type plants are strictly removed at the seedling stage， bolting stage， and early flowering stage. During the flowering stage， the fertility is checked plant by plant， and sterile plants are marked by tagging， tying with red rope or smearing red paint. The marked sterile plants are screened in the maturation stage， and the seeds of the selected sterile plants are harvested in a mixed manner， i.e.， the foundation seeds of 56A[3].

Propagation of seeds for field seed production

The seeds produced from the foundation seeds of 56A satisfying the quality standards through isolated propagation isolation are the seeds for large-area hybrid seed production. The propagation method is the same as that of the foundation seeds， while the the isolation distance can be reduced to 1 000 m.

Propagation Techniques and Quality Standards of the Restorer Line

Breeding of breeders seeds of the restorer line

Individual selection

The seeds of restorer line 8020R individuals are planted in a parent sorting field or an isolation net room， 150-200 plants with typical traits of the parent are selected at the seedling stage and hung with a tag. The number of bagged plants is determined in the initial flowering stage according to the quantity of the parental seeds desired. Generally， 80-100 plants are retained.  The individuals are numbered， and harvested， threshed and bagged， separately. The seed quality of each individual is analyzed （restorer line 8020R， glucosinolate content≤25μmol/g， erucic acid content≤0.5%）. Finally， 20-30 excellent individuals are retained.

Plant line selection

In an isolation net room or a parent sorting area with isolation conditions， qualified seeds harvested and tested to be qualified in the previous year are planted into plant lines according to plant lines， and 15-20 rows are transplanted for each plant line， including a total of about 160-250 plants. The plant characteristics are checked at the seedling stage， and 1-2 typical plant lines are retained， while other plant lines were cut off. For the remaining lines， sickly and off-type plants are removed strictly. The， the remaining plants are screened in the maturation stage， and the seeds of the selected plants were harvested in a mixed manner， obtaining the breeders seeds of restorer line 8020R， which are dried and stored in a low-temperature storehouse under dry conditions after drying.

Breeding of foundation seeds

An appropriate amount of the breeders seeds in the low-temperature storehouse are planted in the propagation area with an isolation distance of more than 1 500 m that has not been planted with Cruciferae plants such as rapeseed， cabbage and leaf mustard for 2-3 years. Meanwhile， it is forbidden to cultivate Cruciferae plants such as rapeseed， cabbage and leaf mustard in the isolation area， and bee colony stocking is also strictly prohibited. The propagation area is determined according to the quantity of restorer line seeds desired. Sickly and off-type plants are strictly removed at the seedling stage， bolting stage， and early flowering stage. The seeds of the plants are harvested in the maturation stage in a mixed manner， i.e.， the foundation seeds of restorer line 8020R， which are sun-dried and stored in a low-temperature storehouse under dry conditions[4].

Propagation of seeds for field seed production

The foundation seeds of 8020R are used for isolated propagation （isolated distance≥800 m）， according to the method for propagation of foundation seeds.

Seed Production Techniques and Quality Standards of Hybrid Seeds

Requirements for the isolation area

The hybrid seed production of hybrid rapeseed Zhuoyou 1032 requires better isolation conditions. The isolation can be realized using natural forests， mountains， rivers， roads （railways， highways）， houses， etc.， and the isolation distance in open areas is equal to or larger than 800 m. Other rape varieties and cruciferous crops cannot be planted in the seed production area. Meanwhile， the seed production area requires certain traffic conditions， concentrated and connected land， high planting management level and good production conditions. Farmers should be highly motivated， have a certain foundation for seed production， and can accept new technologies and methods easily. Local leaders at all levels should attach great importance to it， and rural cadres and counselors should be competent.

Seedling raising and management

Seedbed selection

Selecting paddy fields in the current season or dry land that has not been planted with rapeseeds and other cruciferous crops for more than 2 years as seedbed can reduce or avoid the occurrence of spontaneous rapeseeds. Meanwhile， the seedbed must be exposed to the sun， fertile， close to water sources and transplanting fields， easy to manage and convenient for seedling transportation. For 667 m2 of seed production field， 80-100 m2 of seedbed is required， of which 15-20 m2 is for the male parent and 65-80 m2 is for the female parent. After plowing the seedbed （depth 10-15 cm）， division should be performed according to the width of 1.0-1.2 m. For every 100 m2 of seedbed， 150-180 kg of fully decomposed human and animal excreta， 3-3.8 kg of superphosphate and 0.8-0.9 kg of potassium chloride are applied. For organic fertilizer， when there is no organic fertilizer， 5-7 kg of Xinyangfeng rapeseed compound fertilizer can be evenly applied to every 100 m2 of seedbed， followed by flattening the surface of the seedbed. The furrows between different sections have a width of 30-35 cm and a depth of 15-20 cm. Surrounding furrows and waist furrows （depth： 40-50 cm） should also be formed.

Proper dates for sowing

The seed production of Zhuoyou 1032 requires early sowing of the male parent， so that the male parent can make full use of the light and temperature resources before winter to grow vegetatively， forming strong male parent plants that can provide more pollen. The seeds of the male parent are sown on September 8-10， and those of the female seeds are sown on September 11-13. If the soil is dry during sowing， the seedbed should be watered and moistened thoroughly in advance. And after the soil absorbs water， the soil should be plowed shallowly before sowing.

Covering of seeds

After sowing， the soil is irrigated with clear water or a thin layer of pig manure water. And the seedbed and surrounding soil are sprayed with the solution of 20-30 ml of 48% chlorpyrifos in 15 kg of water to prevent pests， and finally covered corn， rice and other crop stalks or shade nets. The covering is removed 2-3 d later after the emergence of cotyledons which have not stretched flatly.

Seedbed management

In case of dry weather after sowing， the seedbed should be watered at dusk every day until 3 d after emergence. Then， it should be watered once every two days. If ditch irrigation is performed， the water supply is stopped when the water reaches two thirds of the ditch， and excessive water is drained when the bed surface is completely wet. Attention should be paid to the ditches which should be unblocked. The seedlings are thinned in time to ensure good uniformity at the cotyledon stage of the seedlings， and final singling of the seedlings is performed in time at the stage of 2-3 leaves to retain seedlings at a density of 130-160 plants/m2. During thinning and final singling， we should remove seedlings from the too-dense part and remove large， abnormal and sickly ones and leave small ones. Dressing of urea is performed twice after the final singling and 7 d before transplanting at rates of 750 and 450 g per 100 m2， respectively. Note that 48% chlorpyrifos （15 kg in 15-30 kg of water） is sprayed twice in the seedbed period to prevent and control pests such as aphids， green cabbage worms， Hellula undalis Fabricius and flea beetle.

Hua WANG et al. Seed Propagation Technology and Quality Standards of Zhuoyou 1032

Field transplantation

Fertilization

Before transplanting， 1 000-1 500 kg of decomposed pig and cow waste， 20-25 kg of calcium superphosphate， 7-8 kg of potassium chloride and 0.5-1.0 kg of boron fertilizer are applied to the seed production field. If there is no organic fertilizer， 30-40 kg of compound fertilizer for rape or 30-40 kg of 35%-45% Xinyangfeng Manshanhong compound fertilizer plus 1 kg of boron fertilizer can be applied as the base fertilizer， followed by ploughing the land.

Transplanting

Transplanting is performed when the seedlings are more than 25 days old at the stage of 5-6 leaves. The male parent is planted first， and the female parent is planted later. It must be ensured that the transplanting of the male parent ends in mid October and the transplanting of the female parent ends in late October. The row ratio is better to be 1∶（3-4） （male parent：female parent）. The line spacing between the male and female parents is 50 cm， and the line spacing between the female parent is 40 cm. The male and female parents are both transplanted individually. The plant spacing for the male parent is 20-25 cm， and that for the female parent is 15-18 cm. The row direction should be perpendicular to the perennial wind direction in the area. Meanwhile， it is better to plant pure male parent in some fields in the seed production area， and male parent isolation belts can be planted within 50 m around the seed production area.

Field management

Seedling checking and filling vacant areas

The phenomenon of lacking seedlings should be checked 5-7 d after transplanting in a timely manner line by line. The male parent is first checked， followed by the female parent. After filling the gaps with seedlings， water added with a small amount of urea or diluted clear dung water is applied to fix the roots， so as to ensure that the seedlings filled in the gaps can survive and grow vigorously.

Chemical weeding

After the rape seedlings are transplanted and survive， the soil surface is sprayed uniformly with alachlor at a rate of 100-120 g/667 m2 in 30-45 kg of water to kill weeds. In the later stage， weed control can be carried out once again according to the occurrence of weeds. In paddy fields， there are mainly gramineous weeds， which can be controlled with haloxytop-r-mithyl or quizalofop-p-ethyl. Drylands are dominated by broad-leaved weeds， which can be controlled with Galtak or Haoshiduo.

Intertillage and fertilzation

Urea is applied to the parents as the seedling fertilizer at a rate of 10 kg/667 m2， 15-20 d after the end of transplanting in the farming field at the same time. It can be applied directly on a light rainy day， or in clear water or liquid dung on sunny days. The wax fertilizer is applied at the beginning of January. Specifically， 20 kg of Xinyangfeng special compound fertilizer for rapeseed is mixed with 5-7 kg of potassium chloride or mixed with organic fertilizer and applied per 667 m2 between the lines. Intertillage is also performed to cover the fertilizer. Before bolting， 100-150 g of borax is applied in 75 kg of water per 667 m2 by spraying， to improve the seed setting rate of the female parent[5].

Adjustment of flowering season

Rape has infinite inflorescences and a long flowering period. The middle inflorescences can meet well during seed production， but in order to obtain higher yields， the flowering time of the male parent must be extended. According to the fertility characteristics in the parents of Zhuoyou 1032， top pruning of the male parent can effectively extend its flowering period by 5-10 d. The top pruning is to cutting 5-10 cm off the main stems of 5-10 plants every 5-10 plants when the bolts of male parent are 10-15 cm high. The male parent plants are supplemented with urea at a rate of 2.0-3.0 kg/667 m2 after the top pruning[6].

Removal of abnormal plants

Prompt and thorough removal of about 50% of the fertile plants in the female parent lines is the key to ensuring the quality of seed production. The fertile plants should be removed as early as possible， leaving enough space and time for the sterile plants to grow. The last batch of fertile plants must be cleaned before the female parent blooms. The field flowering inspection is performed every two days during the abnormal plant removal period， and problems are found to be rectified in time. A professional team should be organized to clean up the flowers one by one to ensure that the fertile plant residual rate in the initial flowering stage of the female parent is not higher than 0.2%.

Supplementary pollination at the flowering stage

In order to increase the yield of seed production， we can use the method of releasing bees   in the seed production area after dividing the seed production area into sections， or we can manually shake the male parent plants with a 2 m long bamboo pole at noon every day during the flowering period， so that the parent pollen are scattered above the female parent population， thereby improving the outcrossing rate of the female parent.

Harvesting timely

There is a certain difference in the maturity period in two-line rapeseed seed production fields， and timely harvesting should be carried out according to the maturity of each field. When more than 70% of the pods in the female parent population change from green to light， and the lower pods turn yellow， the female parent must be harvested in time. The entire male parent plants are cut off and transported out of the farming fields， then the female parent plants are cut into small bunches which are evenly placed on the stubbles for air drying when avoiding lying flat on the land. After the female plants are air-dried for 4-6 d， threshing is performed timely when the weather is fine. When the moisture content of the seeds is lower than 8%， wind selection is carried out[5]， so that the neatness is not less than 98.0%， and they are uniformly purchased and quantitatively packed. The packages are provided with tags inside and outside， and are transported to a guarded storage site or temporary warehouse. After the harvesting， they are promptly transferred back to the company for storage.

Quality Requirements

The quality standards of Zhuoyou 1032 seeds are shown in Table 1.

Test Methods

The breeders seed and foundation seed provided by the breeders should be tested by our company for seed quality， and sent to the Quality Inspection and Test Center for Oilseeds Products， Ministry of Agriculture （Wuhan） for erucic acid and glucosinolate content determination. The field purity test is conducted after the removal of abnormal plants in the seed production fields， and the residual rate of fertile plants in the fields should be less than 0.2%. QB/T3543.1-3543.7 standards are implemented for the hybrid sampling and seed purity analysis， germination test， moisture determination， variety authenticity and purity inspection. The purity is indentified by planting in Weining summer and Zunyi regular season field plots. The erucic acid and glucosinolate tests are performed in accordance with NY 414 and ISO 9167-1. The quality classification is performed in accordance with QB 4407.2 and NY 414. The unqualified batches of seeds are weeded out.

Judgment rules

When judging the quality of seeds， the following rules should be met at the same time： If the crop type， variety name and producing area do not match the contents in the seed label， they shall be judged as fake seeds; if any one of the variety purity， neatness， germination rate and moisture detection values does not reach the value specified in the standards， they shall be judged as inferior seeds， and the detection values and the marked values are allowed to be judged according to the allowable error specified in GB/T3543; if any of the quality labeling values of the seed label does not meet the value specified in Table 1， the seeds shall be judged as inferior seeds; and those with quarantine pests stipulated by the state shall be judged as inferior seeds.

References

[1] WANG Hl， JIN Fm， ZHAO Y， et al. Breeding of the new hybrid rape variety Zhuoyou 1032 in Brassica napus L.[J]. Agricultural Biotechnology， 2019， 8（03）： 64-66， 77.

[2] Agricultural Industry Standard of the Peoples Republic of China-NY/T 791-2004. Technical regulations for double-low hybrid rape seed breeding[J]. Agricultural Quality & Standards， 2004（6）： 30-34. （in Chinese）

[3] SU GH， YU XQ， HOU J， et al. Characteristics and high yield seed production technology of hybrid rape Xianyouza No. 2[J]. Seed， 2011， 30（7）： 117-119. （in Chinese）

[4] LIN B， WANG H， WANG HL， et al. The characteristics and high-yielding seed production techniques of Zhuoyou No. 11 in Brassica napus L.[J]. Seed， 2015， 34（10）： 123-125. （in Chinese）

[5] HOU J， HOU Y， HE HP， et al. Hybrid rapeseed Dexinyou 27 species characteristics and seed production techniques[J]. Journal of Anhui Agricultural Sciences， 2013， 41（24）： 9893-9895. （in Chinese）