Technical Specification for Producing Hybrid Pepper Seed Using Three-line Male Sterility

Xuefeng LI, Chengliang LIANG, Yanqing MA, Xuexiao ZOU

1. Hunan Vegetable Research Institute, Changsha 410125, China;

2. Hunan Xingshu Seed Industry Co. Ltd., Changsha 410125, China;

3. Hunan Research Center for Vegetable Engineering Technology, Changsha 410125, China;

4. Hunan Academy of Agricultural Sciences, Changsha 410125, China

With the accelerated urbanization in China, labor costs has risen sharply, making pepper male sterile line seed production become the development trend of the industry. Using three-line male sterility to produce hybrid pepper seed has the characteristics of simple production, low cost, purity guarantee,and it is also easy to keep secrets between parents, making it hard to lose. At present, pepper research and development institutions and seed companies at home and abroad have made great achievements in this area[1].WEI et al.[2]found that using three-line male sterility as the parent can make the hybrid fruit setting rate and seed numbers higher than those using maintainer line as parent.Through the study on the breeding technology and seed production techniques of three-line pepper, WANG et al.[3]innovated and improved the cross pollination process,which ensured the seed quality, reduced production cost and further improved the market competitiveness of F1 hybrid pepper seed.

Based on the previous achievements,we descried the whole process of three line hybrid pepper seed production in detail,including requirement of the seed production base, parent cultivation, field management, and specified the key operation techniques in seed production, such as parental impurity to preserve pure state, pollen collection, pollination and seed collecting essentials. This specification is of guiding significance for the production of hybrid pepper seed and ensuring the purity of hybrid pepper seed.

Seed Production Base Condition

(1) Environmental quality. The ecological, air and water environment of the pepper seed production base should be in accordance with the requirements of GB/T18407.1-2001. (2)Climate conditions.The climatic conditions in seed production base shouldbe suitable for pepper production.Requirements: average temperature in 25-30 ℃, minimum temperature of above 15 ℃, the highest temperature of less than 35 ℃; l no or few natural disasters throughout the year, less rainfall with sufficient sunlight. (3)Soil conditions. The best soil should be with flat terrain,, fertile soil, even soil productivity, adequate water, convenient irrigation and drainage,no planting of Solanacae crops for more than 3 years,and the soil pH value should be 6.2-7.2. (4) Isolation condition. Safety isolation refers to the method to set a barrier by adjusting time, space or man-made during the seed production process to prevent the biological contamination of different varieties. In addition to the stated parents, all other varieties should be isolated for over 1 000 m within the hybrid seed production region, or using 60-mesh insect gauze to achieve the isolation.(5)Other conditions. The seed production regions should be one with adequate labor resources, where the farmers have some understanding of hybrid seed production, and a certain elite breeding experience preferred[4-7].

Parental Cultivation

Seed sowing

Seeding time In order to provide enough male parent pollen for the maternal plant to produce hybrid seed during the flowering period, it should determine different seeding time according to the maturity of male and female parents, and generally, the male parent should be son 7-15 d prior to the female parent.

Sowing quantity Artificial pollination is preferred to produce seed, and the ratio of planting area or planting numbers of male parent to female parent is about 1:2.5-5.0.The required number of premature female parent seed to sow is 40 g/667 m2, while 30 g/667 m2for late maturing female seeds, and the mating male parent is 10-15 g/667 m2. The male parent varieties can be nearby planted in female varieties in the region. In order to avoid the loss parents can be planted within the regions of female parents. In order to avoid parent loss, male and female parents can be planted separately,but attention should be paid to the isolation and preservation of pure state.

Seeding method The nutritional soil should be evenly spread in the seedbed with the thickness of about 8 cm.The seedbed should be watered thoroughly the day before sowing.Seeds should be sown evenly with 15-20 g/m2,and then cover with a layer of nutritional fine earth with the thickness of 1.0-1.5 cm after sowing, then well watered.When the temperature is low,plastic film is used to cover the seeds to keep moisture and rise temperature,and when the temperature is high,sunshade net is used to decrease temperature and keep moisture.

Seedling stage management

Seeding stage Warming or cooling measures can be taken to keep the temperature at 20-30 ℃,soil humidity of about 80%,and relative air humidity over 80%.

Sprouting stage Illumination should be strengthened, and the temperature and humidity should be controlled to promote the healthy growth of seedlings. The daytime temperature should be controlled at 18-20 ℃, and night temperature at 14-16 ℃;air relative humidity should be controlled within 60%-70%.

Basic vegetative stage Attention should also be paid to enhance light,and the seedbed temperature should be increased appropriately with strict control of humidity. The temperature should be controlled at 16-25 ℃, and humidity is the best if the bed soil show no naked area. The watering quantity should not be too much.

Transplanting and its management

The seedlings can be transplanted after sprouting 2 -3 main leaves,and the transplanted seed spacing should be (5-10) cm×10 cm. It also can be directly transplanted in the nutrition bowl and nutrition dish.

Rejuvenation stage After transplanting, the seedlings should be in close plastic shed for 2-3 d, and it should keep the temperature and humidity within 5-7 d after the transplanting to promote the survival of seedling roots.The daytime temperature should be controlled at 25-30 ℃, night temperature at above 20 ℃,and the relative humidity of air should be over 9%.

Vigorous growth stage The daytime temperature should be controlled at 20-25 ℃, and night temperature at 15-16 ℃. Attention should be paid to the ventilation to make the seedlings exposed to light,and proper amount of water and dressing fertilizer should be applied. When the seedbed is dry,0.2% of nitrogen phosphorus and potassium fertilizer (15%of N,15%of P2O5, 15% of K2O) or 10% of dilute compost pig slurry can be sprayed at noon in sunny days, and the soil can be loosed based on the practical situations.

Training seedling stage One week before the field planting, attention should be paid to control water and fertilization, and the covering should be removed gradually to increase the adaptive capacity of pepper seedlings.

Nutrient and water management

According to the growth of pepper,topdressing should be applied in 3 different periods of survive to pre-flowering, pollination, after-pollination. The period of survive to pre-flowering: light application of fertilizer for seed bed(10% of dilute compost pig slurry or 0.1% -0.2% of compound fertilizer)should be preferred every 4-6 d.Pollination period:steady application of fertilizers for potted flowers is preferred.When the four cross-bar pepper set well,compound fertilizer should be applied through topdressing or hole fertilization once combined with watering,and the application amount should be 10-15 kg/667 m2.After-pollination:fruit fertilizers should be applied repeatedly,and the amount of compound fertilizer for each hold fertilization or spraying should be 7-10 kg/667 m2with 5-7 kg/667 m2of potassium sulfate. And the fertilizations should be carried out every 10 d until the fruits begin to coloring.In different growth period,attention should be paid to the pruning and ventilation, and the yellow leaves should be picked out and buried or burnt out of the field.

Pest control

Pest control should be based on the principle of prevention, and it should enhance the resistance to disease and insects of plant through reasonable layout of the field,no continuous cropping of eggplant crops, reinforcing field management,and reasonable fertilization and other measure.Specific measures are as follows:first,it should weep out the fields, carry out deep tillage to eradicate overwintered pathogen, larvae and pupae, and reduce the cardinal number of diseases and pests; second, it should remove the serously sick plants, pick out the diseased leaves and fruits, which should be burnt or buried outside the field.

In the meantime,chemical means should be taken to timely prevent insect disease. The 1 500 times dilution of 10% imidacloprid wettable powder can be sprayed to control and aphids,thrips. In sunny days, 600 times dilution of 75%chlorothalonil,500 times of dilution of 64% sandofan, 500 times dilution of 70% mancozeb can be sprayed to control and prevent daming-off and epidemic disease.During the early period of viral disease,500 times dilution of 20% of virus A,50% of dioctyl divinyl triamino glycine wettable powder or 1 000 times dilution of 1.5% plant disease emulsion,Dufei can be sprayed every 6-10 d for 3 -4 continuous time to control the disease.During the early period of anthracnose and scab,200 mg/L of agricultural streptomycin and new phytomycin or 700 times dilution of 80% of zineb wettable powder or Bordeaux mixture and be sprayed every 7-10 d for 2-3 continuous time to control the disease. When the bacterial wilt appears, the diseased or infected plants should be removed immediately, and the diseased holes should be poured with 2% of formalin or 20% of whitewash to sterilize the holes to prevent the further proliferation of germs.

Seed Production Techniques

Parent propagation

Sterile line propagation is to propagate seeds by pollinating the pollen of maintainer line to the stilus of sterile line, and both the maintainer line and restorer line are self-fruit to propagate parents. Parent propagation requires to be bred in the greenhouse covered with 60-mesh insect prevention guaze to prevent biological contamination.

Parent configuration

Parent configuration refers to the planting proportion of male parent to female parent in hybrid seed production. Generally, the ration of female parent to male parent is 3:1,which can be adjusted according to the number of plant flowers of male and female parents and the amount of pollen.

Flower synchronization

Flower synchronization refers to the synchronized flowering of male and female parent or the male parent flowers during the flowering period of female parent in hybrid seed production. The methods to solve the problem of flowering asynchronism are as follows: first is to sow and transplant male parent in advance according to the early flowering season and pollen amount of male parent,and ensure the supply of pollen timely and enough;second is to use new technology to preserve male parent pollen for long time to provide pollen anytime any where; third is to use agricultural technology to completely clear the male parent plant or put the plant under drought stress to make it flowering in advance; fourth is to use chemical technology by spraying growth regulator like gibberellin to male parent to promote its flowering, but since it is hard to control the growth regulator,this method is not suggested in production.

Parent impurity removal and preservation of pure state

Parent impurity and preservation of pure state refers to the removal of abnormal plant in the male and female parents during the artificial pre-pollination stage and pollination stage, fruiting stage. Before pollination, the opened flowers and set fruits on the female parents should be removed.According to the features and characteristics of male and female parent plants, the abnormal plants should be removed before pollination, and regular check should be carried out during the pollination, one found, removed immediately. The fruits on the female parent plant should also be removed.

Pollen collection

At 7:00-8:00 in the morning and 16:00-17:00 in the afternoon, the big flower buds that have turned white but not sprayed pollen,which can be flowered on that day, can be picked out,and the anther can be collected by tweezers or hand and dried in the air or in the container with calcined lime at the bottom to make the pollen expand naturally.And then pollen mesh can be used to screen out the pollen, which then is dried and than poured into the pollination tube before pollination. Under room temperature, the pollen viability of pepper can last for 1-2 d, and therefore, it should screen the fresh pollen on that day.

Pollination

Artificial pollination refers to the artificial method to pollinate the male parent pollen to the stigmas of female parent flower, and therefore make the female parent fruit and produce quality hybrid F1 seed. First, the pollen is put in the pollination tube,which is to pollinate the pollen to the stigmas of sterile female parent which has been fully open. And 2-3 petals can be picked out to make a mark to avoid repeated pollination. The optimal cross pollination time is 6:00-11:00 in the morning and 16:00-19:00 in the afternoon every day, and it should avoid high temperature pollination to ensure high fruit-setting rate. In general, the pollination period is 20-30 d.

Seed collection

The physiological matured fruits are picked and placed in the room for after-ripening for 2-3 d, and special person is assigned to collect seeds.And special machinery can also be used to smash the fruit, but clean water is needed to wash the fruit to prevent machinery contamination, and then all fruits are labeled. After extraction, the seeds should be spread on the straw mat or gauze to make them dry, and it should not be exposed to the sunlight on high-temperature cement.After dried (moisture content of 7% ), all seeds are cooled down in room temperature, and then put in sealed preservation in the plastic bag with drying agent.

Seed Production Record

First,production operation record,including land preparation, sowing,field planting, fertilization, irrigation,pest control and other farming operation, which should be itemized and recorded truthfully. Second, seed breeding record, which mainly records impurity removal,pollination date,personnel and pollination quality, and the file is kept for 3-5 a.Third,seed quali-ty record, which mainly records the harvest date, batch and quality of the seed,and this file is kept for 3-5 a.

[1]LI XF(李雪峰),LIANG CL(梁成亮).Seed production technique of hybrid pepper and its application status(雜交辣椒制種技術(shù)研究及應(yīng)用現(xiàn)狀)[J]. Hunan Agricultural Sciences,2012,(10):20-22.

[2]WEI BQ (魏兵強),WANG LL (王蘭蘭),CHEN LZ(陳靈芝),et al.Comparison of relevant characters of hybrid seeds production between hot pepper cytoplasmic male sterile line and fertile line(辣椒不育系制種與可育系制種相關(guān)性狀的比較研究)[J]. Journal of Changjiang Vegetables,2011,(24):10-11.

[3]WANG LL (王蘭蘭),WEI BQ (魏兵強),CHEN LZ (陳靈芝),et al. Research on seed production technique with cytoplasmic male sterile line and its maintainer line and restorer line in pepper(辣椒胞質(zhì)雄性不育三系配套制種技術(shù)研究)[J]. Northern Horticulture, 2013,(8):53-54.

[4]GB T18407.1-2001. safety qualification for agricultural product environmental requirements for origin of non-environmental pollution vegetable(農(nóng)產(chǎn)品安全質(zhì)量無公害蔬菜產(chǎn)地環(huán)境要求)[S].

[5]DB43/115.1-1998.Hybrid Pepper Seeds雜交辣椒種子[S].

[6]DB43/T115.2-1998.Technical regulations for the production of hybrid pepper parent(雜交辣椒親本原種生產(chǎn)技術(shù)規(guī)程)[S].

[7]GB16715.3-1999. National Standard for the Seeds Quality of Solanaceous Vegetable[S].