Section I Propagation of virus-free seedlings

1. Strawberry virus-free seedlings

Strawberry virus-free seedlings are those that have completely removed strawberry virus. Strawberry virus disease is a disease caused by strawberry infection, which shows two symptom types in cultivation: yellowing type and shrinking leaf type. Strawberry virus disease has a wide range of hazards. According to incomplete statistics, there are as many as 62 kinds of strawberry disease viruses, among which Strawberry mottle virus (SmoV), Strawberry mild yellow edge virus (SMTEV), Strawberry vein-band virus (SVBV) and Strawberry crinkle virus (sCrV) are four main viruses infecting strawberries in China, with a total infection rate of 80.2%, The infection rate of single virus is 41.6%, and the combined infection rate of two or more viruses is 38.6% (Ma Chongjian, 2004). Strawberries infected with virus disease grow slowly, their leaves shrink, their fruits become smaller, deformed, and their quality decreases year by year. At present, there is no medicine to control the virus disease. The use of virus-free cultivation seedlings such as strawberry stem tip is an effective way to control the virus disease, improve the quality of strawberry and improve the yield of strawberry. Strawberry virus-free seedlings have the characteristics of fast growth, vigorous growth, thick stems and leaves, strong resistance to disease, high temperature and cold, many flowers, high fruit setting rate, large fruit, neat and uniform, bright color, no abnormal fruit, high yield and good economic benefits, and are generally welcomed by fruit farmers. However, virus-free strawberry seedlings will be re-infected with the virus under field production conditions, and the infection rate is generally 10%~20% per year. Therefore, the majority of fruit farmers can continue to ensure high economic benefits by replacing the virus-free seedlings after 2 to 3 years of application.

Qin Lanying et al. (1988) showed that stem tip culture had different degrees of virus removal. The smaller the stem tip, the greater the chance of virus removal. The higher the virus removal rate of the stem tip below 0.3 mm, and the tissue culture seedlings did not carry virus; Only 20% of the stems above 0.5mm were virus-free. After heat treatment, the effect of virus-free was significantly increased by taking stem meristem for culture, which was proved to be more reliable by electron microscope observation.

The stem tip tissue culture of strawberry virus-free seedlings goes through the steps of explant selection, induction culture, proliferation culture, rooting culture, etc. Generally, from June to August, select middle-aged, disease-free, pure and healthy plants with fine weather, and cut 2~3cm stolon segments with growth points from the new shoots as explants, and wash them with running water. Place the surface cleaned explants on the ultra-clean workbench, disinfect the surface with 70% ethanol for 1 minute, discard the ethanol, add 0.1% mercuric chloride and 1 drop of Tween-20 to disinfect for 8 to 10 minutes, and shake it constantly, then wash it with sterile water for 5 to 8 times, use sterile filter paper to absorb the water, remove the scales, and use the scalpel to pick up 0.2 to 0.3 mm of the stem tip under the dissecting microscope, and inoculate it in the stem tip induction medium. Stem tip culture conditions: daytime temperature 25 ℃ soil 2 ℃, light intensity 30~60 umol/(m2. s); The temperature at night is 18 ℃, the soil temperature is 2 ℃, the culture is dark, and the daily illumination is 12-16 hours. After induction and culture for 2 to 3 months, when the cluster adventitious buds are 1.5 to 2.0 cm, the test-tube plantlets that have passed the virus test are inoculated on the proliferation medium for multiplication. The multiplication culture is subcultured every 20 to 30 days (the total number of subcultures is not more than 8), and the seedlings with a height of 2 to 3 cm are selected to be transferred to the rooting medium for rooting culture. After 20~30 days of induced rooting culture, tissue culture seedlings of complete plants can be formed.

2. Application of growth regulators in strawberry virus-free seedling propagation

In the process of stem tip tissue induction culture and proliferation culture, growth regulators have significant effects on improving shoot tip germination rate, subculture proliferation and promoting rooting.

(1) Paclobutrazol: In strawberry tissue culture, with the increase of differentiation times and subculture times, the test-tube seedlings degenerated. Such as petiole thinning, leaf yellowing, and reduced differentiation rate. In addition, the survival rate of transplanting is one of the key factors for the success of rapid propagation of strawberry virus-free seedlings, and the survival rate of transplanting of test-tube seedlings is directly related to the rooting of test-tube seedlings. In the rooting stage, the plantlets with thin and weak yellowing have poor rooting effect, and the survival rate is low after the bottle is out. At present, the application of paclobutrazol in strawberry tissue culture has solved this problem well.

Zhang Xitai et al. (1997) used strawberry tube seedlings that have been subcultured for five times and have grown thin, thin and yellow as the test material, added 0.2~1.0mg/L15% paclobutrazol and 0.25mg/L6-BA to MS medium for subculture, and added 0.2~0.8mg/L15% paclobutrazol and 0.2mg/L6-BA for rooting culture. The results showed that paclobutrazol had obvious dwarfing effect on strawberry tube seedlings in subculture. With the increase of the concentration of paclobutrazol, the rhizome of the plantlets regenerated from the test-tube plantlets gradually became thick, the petiole was short and thick, the leaves were thick, and the chlorophyll content was high. Compared with the control, the rhizome thickness increased by 31.6%~174.4%, the petiole length shortened by 7.3%~71.0%, and the differentiation multiple increased by 14.3%~221.4%. When the concentration of paclobutrazol was increased to 0.6mg/L, the differentiation times of plantlets in vitro began to decline. In strawberry rooting culture, paclobutrazol and 6-BA significantly promoted the root system growth of test-tube seedlings. Compared with the control, the number of roots increased by 24.3%~543.0%, and the effect of adding 0.4mg/L15% paclobutrazol and 0.2mg/L6-BA to 1/2MS medium was better.

According to Ruan Long et al. (2002), adding 0.2~0.6mg/L paclobutrazol to 1/2MS Fengxiang strawberry virus-free seedling rooting medium can promote the increase of rooting number, root thickening and growth of strawberry virus-free seedlings. Compared with the rooting medium without paclobutrazol, the rooting number of Fengxiang test-tube seedlings increased by 10.9~15.7, the root diameter increased by 0.24~0.39 mm, and the root length increased by 0.27~0.70 cm, among which the treatment effect of adding 0.4mg/L paclobutrazol was better.

Section II Regulate the growth of stolon and promote the elongation of the top inflorescence

1. Strawberry stolon and top inflorescence growth

Strawberry stolon is a vegetative reproductive organ on the ground of strawberry, which is sprouted from the axillary buds of new stems. Generally, in the late fruit setting and after harvest, the sunlight in early summer is enhanced, the temperature is increased, and the stolons sprout from the dormant axillary buds formed last autumn. A bract and axillary bud are formed on the first node of the stolon, and the axillary bud remains dormant. The growth point on the second node differentiates the leaf primordium. When 2 to 3 leaves are exposed, adventitious roots are produced, which are embedded in the soil, forming a first-level stolon seedling. At the same time as the first stage stolon seedlings are pregnant and differentiated, the axillary buds between the leaf axils produce new meristematic stolons. The axillary buds on the first node also remain dormant, and the growth points on the second node continue to differentiate leaf primordia. According to this rule, the stolon forms the 1st, 2nd, 3rd and 4th level stolon seedlings and meristematic stolon seedlings on the 2nd, 4th, 6th and 8th even-numbered nodes, and carries out multi-level network meristematic, resulting in a large number of stolon seedlings. The closer these stolons are to the mother plant, the better they will grow. Most of their top buds can form flower buds in the current year and blossom and bear fruit in the next year.

Using stolons is a common method of seedling propagation in strawberry production. The number of stolons varies with varieties. Some varieties can sprout stolons many times in a year, and the number of stolons is more, while some varieties are not. Generally, cold varieties with high demand for low temperature, such as All-Star, Hani, etc., have less stolons; The varieties of warm ground that require short low temperature period, such as Baojiao Zaosheng, Fengxiang, Nufeng, etc., have more stolons.

At present, strawberries are cultivated in plastic greenhouse in Jiangnan area. Due to the lack of heating equipment in the plastic greenhouse and the low temperature in winter, the growth of the top inflorescence is shorter, and the fruit shrinks under the plant. In addition, the poor ventilation and light transmission conditions in the greenhouse are easy to cause rotten fruit and cause economic losses. In order to promote the elongation of the top inflorescence of strawberry, make the fruit hang at the edge of the ditch, and reduce the rotten fruit, gibberellin was applied in production to promote the elongation of the top inflorescence, which has obvious effect.

2. Regulation measures for the growth of stolon and inflorescence of grape

(1) Promote stolon growth:

In order to promote the occurrence of strawberry stolons, especially for varieties with few stolons, gibberellin can be sprayed. The method is to spray 50 mg/L gibberellin 1 to 2 times after the mother plant survives and grows three new leaves, and spray 5 to 10 mL per plant, which can effectively promote the occurrence of strawberry stolons and expand the propagation coefficient of seedlings.

Yu Gengxu et al; The total number of secondary stolons was 18.7, which was 59.8% higher than that of water control; The secondary stolon occurred one week earlier than the control. In addition, GA3 can also relieve the inhibition of PP333 on strawberry, increase the number of stolons and petiole length of plants treated with PP333, and eliminate the inhibition of axillary bud development, but has no sustained effect on plant growth (Yang Huirong et al., 1993).

(2) Inhibition of stolon growth:

① PP333 sprayed with 250mg/L PP333 has obvious inhibitory effect on the length of stolon, plant height and petiole length of strawberry, and has an effect on increasing the yield of strawberry. It is worth noting that the inhibition effect of PP333 is too large. For example, after spraying with 500mg/L PP333 solution, it not only has extremely strong inhibitory effect on strawberry, but also can cause yield reduction. At the same time, until the fruit development period in May of the next year, there was still a large inhibitory effect, and the photosynthetic area was seriously reduced. Gibberellin has the effect of relieving the inhibition of paclobutrazol on strawberry. After spraying 20mg/L gibberellin solution on the leaves, it can take effect in about a week. After treatment with gibberellin, the strawberry seriously inhibited by paclobutrazol for a long time, the plant height was significantly increased, the petiole length was also significantly increased, and the growth potential was strengthened.

(3) Promote terminal inflorescence elongation

Zhou Huayue (1997) experiment showed that after planting in the middle of September in the Fengxiang strawberry garden cultivated in the protected area, spraying 10mg/kg gibberellin once at the beginning of the bud emergence of the top inflorescence, and spraying once again 10 days after spraying, a total of two times, can significantly promote the elongation of the top inflorescence of Fengxiang strawberry, mature early, improve the early yield, and reduce rotten fruit. Compared with the control, the average length of the top inflorescence increased by 7.9 cm, the initial harvest date of fresh fruit was 13 days earlier, and the weight of fresh strawberry fruit in the early stage before the end of February increased by 44.6%. The number of fresh and rotten strawberries in the early stage (before the end of February) was only 8.5% of the control, with significant economic benefits.

Section 3 Break dormancy and promote flower bud differentiation

1. Strawberry dormancy and flowering physiology

After the flower bud differentiation of strawberry, under the conditions of lower temperature and shorter sunshine in late autumn and early winter, the plant enters into dormancy, with dwarfing morphology, small new leaves and short petioles. Although there are flowers and fruits, there are no stolons. The dormancy period of strawberry starts from a period of time after flower bud differentiation and gradually deepens, and generally reaches a deeper dormancy period in the middle and late November. After dormancy, different varieties need to experience different low temperature hours to break dormancy. The northern varieties have deep dormancy and need a long time of low temperature; the southern varieties have shallow dormancy or no dormancy and need a few hours of low temperature. Too many low temperature hours will lead to vigorous growth. Too many stolons occur and the result is bad. Varieties with low demand for low temperature are suitable for facilitating cultivation, and vice versa are suitable for semi-facilitating or open field cultivation. Long sunshine, high temperature or spraying gibberellin can break its dormancy. The above treatment can prevent it from entering dormancy and continue to blossom and bear fruit when it is about to enter dormancy; In the late stage of dormancy, the dormancy can be broken early and the growth can be resumed in flowering and fruiting.

Strawberry is a short-day plant. Low temperature and short-day can induce strawberry to flower. Under natural conditions, strawberry can induce flower bud differentiation under night temperature of about 17 ℃ and sunshine of less than 12 hours, and flower bud can be formed after 9~16 days. Many studies have been done on the effect of hormones on strawberry flowering, among which there are many studies supporting the theory of "flowering inhibitory substances". The research results of Thompson and Guttridge showed that the higher the concentration of gibberellin, the longer the stem and petiole, the greater the occurrence of stolon, and the deeper the inhibition of flowering process. The effect was similar to that of long sunshine treatment. Ballinger et al. also proved the existence of gibberellin at the top of stolon and crown, and the gibberellin substances decreased at the stage of flower bud differentiation, and began to increase after the elongation of flower stalk. Hou Zhixia et al. (2004) believed that spraying gibberellin when some plants began to differentiate flower buds, while the other plants did not differentiate, and destroying the short sunshine conditions suitable for strawberry flower formation, could reverse the meristem of strawberry stem tip, return to the vegetative growth state, and could not form flower buds; Before that, when gibberellin was sprayed, the growth point could not turn into flowers, and flower buds could not form. The higher the concentration of gibberellin, the deeper the inhibition.

2. Technical measures to break dormancy and promote flower bud differentiation

At present, gibberellin is used in strawberry production to release dormancy, promote development, increase yield and promote stolon occurrence. When gibberellin is used to treat strawberry to promote flower bud differentiation, the treatment period must be well controlled. It is better to treat strawberry at the beginning of growth point hypertrophy, which has no effect too early, and has side effects too late. Generally, the suitable period for treatment is from the germination of new buds to the development of flower buds. If it is too late or the temperature after treatment is too high, the rate of abnormal fruit will be high.

Lu Junxia et al. In addition, spraying 50mg/L gibberellin when the growth point is fat at the early stage of flower bud differentiation can make Futian blossom one week earlier; If it is treated in advance, it will not have this effect, but after treatment, it will produce deformed fruit, or make the fruit stem excessively elongate. Shen Xiaoli et al. (1996) showed that the best time to inhibit strawberry dormancy and promote flowering was after the flower bud differentiation of the second and third order flowers when using gibberellin to promote early ripening of strawberry. Generally, the strawberry is sprayed from the end of October to the beginning of November, with a better concentration of 10mg/L, which can make the strawberry maintain a better plant height of about 20cm at the beginning of harvest, and the harvest period is 20 to 30 days ahead of schedule, and the yield and quality are in a better state.

According to the investigation conducted by Wang Zhonghe (2007) in Shandong, ① in the semi-accelerated cultivation of strawberry, spraying 8~10mg L gibberellin at the early stage of growth has the effect of long sunshine, which can promote the development of flower buds and make the first inflorescence blossom early; It can promote the elongation of petiole and increase the three-dimensional photosynthetic space; It can promote the elongation of flower stalks and facilitate pollination and fruit development. ② In the accelerated cultivation of strawberry, early heat preservation and gibberellin treatment have the effect of inhibiting dormancy, and generally spray gibberellin 5~10mg/L for 1~2 times after the beginning of heat preservation. Spraying for the first time at the stage of 2 undeveloped leaves can promote the growth of young leaves and prevent dormancy; Spraying twice at the bud stage can promote the elongation of flower stalks and facilitate pollination and fertilization. When applying gibberellin on strawberries, we must pay attention to: the concentration should not be too high, the dosage should not be too large, 3~5mL per plant is enough, and the wine should be sprayed on the heart of the seedlings; The concentration varies with the variety. The varieties with shallow dormancy use less than those with deep dormancy, and the cold ground uses less than those with warm ground, and the number of times is also less. The varieties with deep dormancy, such as Dukara and Baojiao Zaosheng, can use 10mg