CN104067821A - Preparation method of virus-free seedlings of sweet potato - Google Patents

Abstract

The invention provides a preparation method of virus-free sweet potato seedlings. The potato pieces are sun-dried and placed together with mature bananas for 3-5 days, and then the potato pieces are planted in sandy soil and germinated in an incubator, and a higher temperature is set. . Take the top 1-2cm of the sprouts, cut off the leaves visible to the naked eye, disinfect the surface and rinse with sterile water. The shoot apex with 1-2 leaf primordia was stripped and inoculated on the corresponding medium to induce the differentiation of adventitious buds and somatic embryogenesis respectively. Virus detection is carried out on the regenerated plants. After subculture, seedling hardening, and direct planting in plastic greenhouses, the virus-free seedlings are flooded with water. In the first 2 weeks of transplanting, prevent direct sunlight at noon, and after 3 weeks of transplanting, sprinkle urea and water immediately to obtain virus-free seedlings. The invention can obviously improve the survival rate by directly planting the detoxified test-tube seedlings in the plastic greenhouse after domestication, reduces the operation procedure, does not need to go through the process of domestication in the domestication room, and saves manpower, material resources and financial resources.

Description

A kind of preparation method of virus-free sweet potato seedlings

technical field

The invention belongs to the technical field of sweet potato seedling cultivation, and in particular relates to a method for preparing virus-free sweet potato seedlings.

Background technique

Sweet potato is one of the four major food crops in my country. Sweet potato is a vegetatively propagated crop and is susceptible to virus infection during cultivation. Once infected, it is difficult to remove. The accumulation of viruses can cause the degeneration of varieties, the reduction of quality and yield, and even the loss of commodity value, causing serious harm to sweet potato production. So far, there are no practical sweet potato varieties with high resistance to viral diseases at home and abroad, and there are no efficient pesticides for preventing and treating viral diseases. The distribution of viruses in plants is uneven, the closer to the root tip and shoot tip, the lower the virus density; conversely, the higher.

The meristem is generally free of virus infection. The reason why the shoot tip (and root tip) meristem does not contain virus is as follows. First, the transmission route of the virus in the plant is through the vascular system, and the vascular system has not yet formed in the meristem; the second is through plasmodesmata, but the virus moves very slowly through this pathway, and it is difficult to catch up with actively growing meristems. Second, in the vigorously dividing meristem, metabolic activity is high, making virus replication impossible. Third, there are high levels of endogenous hormones in the shoot apex, which can inhibit the proliferation of viruses. Fourth, there is a "virus inactivation system" in plants, and its activity should be the highest in the meristematic tissue, so that the meristem tissue is not infected. Therefore, the shoot apical meristem can be used to culture and produce virus-free seedlings. However, if only the meristem is taken for culture, it is often not easy to survive.

Utilize the sweet potato stem tip to cultivate and produce the existing research and report of virus-free seedlings, but in the past, the stem tip of the field or greenhouse cultivation was mostly used to get the material to strip the stem tip; It is used to accelerate germination from potato cubes and take materials to peel off shoot tips for cultivation, but it is also accelerated germination under normal temperature conditions. Select the above-mentioned methods, grow and cultivate under conventional conditions, because the material for detoxification grows slowly, metabolism is not strong, the detoxification effect of stripping the shoot tip is poor, and the shoot tip is not easy to strip (because the leaf primordium is short, Leaf primordia crowded together), and the regeneration rate was low.

In addition, the domestication and transplanting of virus-free test tube seedlings is very important. Generally, peat soil, vermiculite, perlite, etc. are used as transplanting substrates, and they must be domesticated and cultivated in the acclimation room for about 3 weeks before they can be transplanted into plastic greenhouses or greenhouses. The domestication and cultivation of test-tube seedlings consumes a lot of manpower, material and financial resources, and the growth rate is slow, and the survival rate is reduced due to the susceptibility to infection. And need to be transplanted through 2 times, operation is loaded down with trivial details.

Contents of the invention

The invention provides a method for preparing virus-free sweet potato seedlings, which can solve the problems in the prior art that the virus-free effect is poor, the stem tip is not easy to strip, the regeneration rate is low, the operation of acclimating and transplanting the virus-free seedlings is cumbersome, and the survival rate is low. The problem of slow growth.

In order to achieve the purpose of solving the above technical problems, the present invention adopts the following technical solutions to achieve:

A preparation method for virus-free sweet potato seedlings, it comprises the following steps:

(1) Select sweet potato pieces without moths and damage, and expose them to the sun for 2 to 5 days;

(2) Put the sun-exposed potato pieces and ripe bananas together for 3-5 days;

(3) Wash the potato pieces with clean water, cover the potato pieces with sand, put them in the incubator for germination, the temperature is 35~37°C; cultivate in the dark for 1 week before germination, after germination, 13 hours of light per day, 1000-2000lx ;

(4) When the sprout grows to more than 10 cm, take the top 1-2 cm long, cut off the leaves visible to the naked eye, first rinse with clean water, then disinfect the surface, and finally rinse with sterile water 3-5 times;

(5) Peel off shoot tips with 1-2 leaf primordia under a stereo dissecting microscope, inoculate them on a medium supplemented with 0.1-0.2 mg/L NAA and 1.0-2.0 mg/L BAP, and culture at a temperature of 25- 27°C, 13 hours of light per day, 2000-3000lx, culture for 3-4 weeks, induce the differentiation of adventitious buds;

Or inoculate the shoot tip on a medium supplemented with 0.2-2.0mg/L 2,4-D and culture in the dark at a culture temperature of 25-27°C to induce somatic embryogenesis;

(6) Cultivate on a medium supplemented with 0.1-0.2mg/L NAA and 1.0-2.0mg/L BAP. When the regenerated seedlings grow to more than 2cm, cut off from the base and transfer to a medium without adding hormones to obtain Complete virus-free vaccine;

Or after 4-5 weeks of culture on a medium supplemented with 0.2-2.0mg/L 2,4-D, the explants will start to form embryogenic callus and somatic embryos successively, and the explants that form somatic embryos will be transferred to Promote the germination of somatic embryos to form virus-free seedlings in the medium without adding hormones;

(7) Adopt the method of cutting stems and tendrils for rapid propagation to obtain virus-free test-tube seedlings;

(8) Domestication of virus-free test-tube plantlets: Subculture the test-tube plantlets for 1 month, open the bottle for domestication;

(9) Transplanting of virus-free test-tube seedlings: Wash the culture medium of the test-tube seedlings, and plant individual plants directly in the soil of plastic greenhouses, and add insect-proof nets at both ends of the plastic greenhouses;

(10) After the virus-free test tube seedlings are planted in the soil of the greenhouse, water them first, and then flood them with water;

(11) During the first 2 weeks of transplanting the virus-free test tube seedlings, set up a shading net at noon to prevent direct sunlight, remove the shading net after 2 weeks, and let the air out at noon;

(12) After 3 weeks of transplanting, sprinkle urea and water immediately to promote the growth of seedlings;

(13) Propagation of virus-free seedlings in plastic greenhouses: When the stems grow to more than 30cm, cut the stems and plant them in plastic greenhouses for re-propagation of virus-free seedlings. After each cutting, sprinkle urea and immediately water water to obtain virus-free seedlings of sweet potato.

Compared with prior art, advantage and positive effect of the present invention are:

1. In the present invention, potato pieces and ripe bananas are put together for 3-5 days, and mature bananas are used to release ethylene to promote early germination of potato pieces, because the newly harvested potato pieces germinate slowly, and the slow germination causes easy rotten potatoes . the

2. The present invention combines high-temperature germination and shoot tip culture to produce sweet potato virus-free seedlings. The germination is set at a higher temperature (35-37°C), based on the fact that viruses are sensitive to high temperatures and sweet potatoes are resistant to high temperatures (because sweet potatoes are plants of tropical origin). This difference chooses the appropriate temperature, which can reduce the virus concentration in the sweet potato body, slow down the transmission speed or lose its activity, and accelerate the cell division of the sweet potato meristem, and accelerate the growth of potato buds. Therefore, the detoxification effect of the stripped shoot tip is good, and because the internodes of the leaf primordium are elongated, the shoot tip is easy to strip (because the internodes of the leaf primordium are elongated, the meristem is easy to see under the dissecting microscope, and the shoot tip is easy to strip), regeneration The rate is high (because of the vigorous growth and metabolism at higher temperatures). Tests have shown that after 8-10 days of accelerating germination in the incubator, the potato pieces begin to germinate, and after 11-13 days, the material can be stripped to get the shoot tip. When peeling off the shoot tip under a dissecting microscope, it is easy to peel off due to the long internodes of the leaf primordia. Through the organogenesis pathway, the frequency of shoot tip regenerated plants was more than 90%.

3. Immediately after harvesting sweet potatoes in autumn in the present invention, germination of potato pieces, stem tip stripping and cultivation are carried out, so that virus-free seedlings can be obtained early. After rapid propagation in test tubes, the second year after acclimatization, the plastic greenhouses are propagated and planted directly in the field, which reduces the time of cultivation in the culture bottle, thereby avoiding the cultivation variation caused by the long cultivation time. Because if the regenerated virus-free seedlings are obtained later, the virus-free seedlings will not be able to plant in the field in the second year, and they will have to wait until the third year to plant in the field. This will prolong the cultivation time in the culture bottle, and gene mutations will easily occur.

4. Directly planting the virus-free test-tube seedlings in plastic greenhouses after domestication can significantly improve the survival rate. In the past, the seedlings were first transplanted into the nutrient matrix and placed in the acclimation room, and then transplanted to plastic greenhouses or glass greenhouses after they survived. This process takes about 3 weeks. And the present invention is that seedlings are directly transplanted in the soil of plastic greenhouse, can avoid being transplanted in the matrix easy to infect bacteria, the difficulty that survival rate is low. Because the microorganisms in the soil are in balance, beneficial bacteria can inhibit the reproduction of harmful bacteria. In the nutrient matrix, because the test-tube plantlets are weak and easy to infect bacteria, thereby reducing the survival rate. The survival rate of the present invention can reach more than 95%.

5. The virus-free test-tube seedlings are directly transplanted into the greenhouse, which reduces the operating procedures and does not need to go through the process of domestication in the domestication room, saving manpower, material and financial resources. And because the procedure is reduced, it grows directly in the soil, reducing the impact of transplanting on seedlings. Because the seedlings are directly planted in the soil, the root system has a large space for expansion, the roots are deep and the leaves are luxuriant, and the root system is developed, which promotes the fast and strong growth of the aboveground part. 9 days after transplanting, the leaves of the seedlings have stretched and new leaves have grown. After 3 weeks, the main vines have begun to elongate and grow side vines. After 5-6 weeks, the vines can grow to more than 30-40cm. Cut vine cuttings for repropagation.

Description of drawings

Fig. 1 is the adventitious buds grown on the callus formed by the shoot tip culture of sweet potato in the present invention, left: Shangshu 19; right: Xushu 22.

Fig. 2 is a complete plant diagram grown from axillary buds in the present invention.

Fig. 3 is the virus-free test-tube plantlet after washing off the culture medium in the present invention.

Fig. 4 is the process of transplanting virus-free seedlings in the present invention.

Fig. 5 is the virus-free seedling just transplanted in the plastic greenhouse soil in the present invention.

Fig. 6 is to take shade net on the plastic greenhouse among the present invention.

Fig. 7 is the virus-free seedling after transplanting 9 days in the present invention.

Fig. 8 is the virus-free seedling transplanted for 3 weeks in the present invention.

Fig. 9 is the virus-free seedling transplanted for 6 weeks in the present invention.

Fig. 10 is the embryogenic callus formed from sweet potato shoot tip culture in the present invention.

Fig. 11 is a somatic embryo formed from embryogenic callus in the present invention.

Figure 12 is a somatic embryo germinated in the present invention.

Fig. 13 is a virus-free test-tube plantlet grown from somatic embryos in the present invention after germination.

Fig. 14 is the potato cubes of No. 14 virus-free seedlings of the middle and high line of the present invention.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the drawings and embodiments.

Example 1

The preparation method of sweet potato virus-free seedling of the present invention specifically comprises the following steps:

1. Treatment of sweet potato pieces before germination:

(1) After harvesting in autumn, select the currently popular sweet potato varieties Xushu 22, Yanshu 25, and Shangshu 19 (commercially available varieties) without moth-eaten and damage-free potato pieces, and expose them to the sun for 2-3 days (No. One has a bactericidal effect; the second can promote the germination of potato pieces).

(2) Prepare ripe bananas, put the sun-exposed potato pieces and ripe bananas together in a plastic storage box, close the lid, and store them in the laboratory for 3-5 days (at room temperature not lower than 13°C) (Use mature Bananas release ethylene, which promotes the germination of the potato cubes. Because the newly harvested potato cubes germinate slowly, resulting in rotten potatoes).

2. Germination:

Wash the above-mentioned potato pieces with clear water, put them into sand, cover the potato pieces with sand with a thickness of 1-2 cm, and put them into an incubator for germination. The temperature setting is 35~37°C. (According to the virus is sensitive to high temperature, because sweet potato is a plant of tropical origin, so sweet potato is a high temperature resistant plant, using this difference, the present invention selects an appropriate high temperature through experiments, which can reduce the virus concentration in the sweet potato and slow down the transmission speed, while sweet potato Meristem cell division is accelerated, and potato bud growth is accelerated. Therefore, the detoxification effect of the stripped shoot tip is good, and because the internodes of the leaf primordia are elongated, the shoot tip is easy to strip and the regeneration rate is high). Cultivate in the dark for 1 week before germination, and after germination, light 13 hours a day, 1000-2000lx. Spray water in an appropriate amount according to the humidity of the sand. The water content in the sand should not be too large, otherwise the seeds will rot easily. After being cultivated in the incubator for 8-10 days, the potato pieces begin to germinate, and after 11-13 days, some sprouts have reached 10cm in length.

3. Shoot tip stripping and cultivation

(1) Disinfection of materials: When the sprout grows to more than 10 cm, take the top 1-2 cm long, cut off the leaves visible to the naked eye, rinse it with clean water, and then soak it in 70% alcohol in an ultra-quiet workbench for 10- For 20 seconds, soak in 2% sodium hypochlorite solution for 5-7 minutes for surface disinfection, and finally rinse with sterile water 3-5 times.

(2) Shoot apex stripping and culture: The shoot tip with 1-2 leaf primordia was stripped under a stereo dissecting microscope, and inoculated on MS medium supplemented with 0.2 mg/L NAA and 2.0 mg/L BAP. After 1 week of culture, the shoot tip began to form callus, and after 3-4 weeks, adventitious buds began to form from the callus wound, as shown in Figure 1. The frequency of adventitious bud formation is listed in Table 1. The frequencies of adventitious bud formation at the shoot tips of the three tested varieties were all higher than 90%.

Table 1 Regeneration rate of shoot tip cultured plants of different sweet potato varieties

Variety Xu Shu No. 22 Smoked Potatoes 25 Shangshu 19 Regeneration rate (%) 95.6 92.3 93.8

After being cultured for one month on the medium supplemented with 0.2mg/L NAA and 2.0mg/L BAP, when the regenerated seedlings grow to more than 2cm, cut off from the base, transfer to the MS medium without adding hormones, and grow into complete Virus-free vaccines.

The culture conditions are 25-27°C, 13 hours of light per day, 2000-3000lx.

4. Virus detection of virus-free vaccines

Virus detection was carried out on the virus-free seedlings, and the detection results are shown in Table 2. The virus-free rates of the three varieties all reached 100%.

Table 2 Virus-free rate of regenerated plants of different sweet potato varieties

Variety Xu Shu No. 22 Smoked Potatoes 25 Shangshu 19 Detoxification rate (%) 100% 100% 100%

5. Aseptic Rapid Propagation of Virus-free Vaccines

The propagation of the virus-free seedlings adopts the method of cutting stems and tendrils. The 1-2 leaf nodes of the virus-free seedlings are divided into 1 section, and the cuttings are inserted into the MS medium without adding hormones. plants, as shown in Figure 2. In this way, the subculture is continuously cut into sections, and the propagation speed increases geometrically, and a large number of virus-free seedlings can be propagated in the culture bottle. The culture conditions are 25-27°C, 13 hours of light per day, 2000-3000lx.

6. Domestication and transplanting of virus-free test-tube plantlets:

(1) Subculture the test-tube seedlings for 1 month, open the mouth of the bottle for domestication, open the mouth a little on the first day, and then open a little more on the second day, so that the seedlings are hardened continuously for 2-3 days, until the mouth of the bottle is half opened ( Because the hardening time is short, it is easy to lose water and wilt if they are all opened).

(2) Clean the culture medium of the test-tube seedlings, and plant individual plants directly in the soil of plastic greenhouses with a spacing of 20-25 cm, as shown in Figures 3, 4, and 5. Be careful not to damage the roots during cleaning and transplanting, which can shorten the time for slowing down seedlings and improve the survival rate and growth rate of seedlings. Add insect-proof nets at both ends of the plastic greenhouse (to prevent virus re-infection, because the virus is mainly transmitted by insects, adding insect-proof nets can prevent insects from entering the plastic greenhouse).

(3) After the virus-free test-tube seedlings are planted in the soil of the greenhouse, water is first poured (to prevent the seedlings from being washed up), and then flooded with water. The function of flood irrigation can firstly provide the water needed by the seedlings, and secondly, it can provide guarantee for maintaining the air humidity in the plastic greenhouse. Because the air humidity of the test tube seedlings in the test tube is very high, a certain air humidity should be maintained in the early stage of transplanting, otherwise it is easy to lose water and wilt.

7. Management of virus-free seedlings in plastic greenhouses:

(1) In the first two weeks after transplanting the virus-free test-tube seedlings, set up a shade net at noon (that is, from 10:00 am to 3:00 pm) to prevent direct sunlight, as shown in Figure 6. And after the shading net is set up, the temperature in the plastic greenhouse will not be too high if the wind is not released at noon, and the air humidity in the plastic greenhouse can be maintained. After 2 weeks, remove the shading net and release the wind at noon. Water according to soil moisture.

(2) Three weeks after transplanting, sprinkle urea at a rate of 8-10 catties per mu, and then water immediately to promote the growth of seedlings. In the invention, since the seedlings are directly planted in the soil, the root system has a large space for extending, the roots are deep and the leaves are lush, and the root system is developed, so that the aboveground part grows fast and strong. Observe after transplanting 9 days, seedling blade stretches, and grows new blade, as shown in Figure 7, statistical survival rate is in table 3, and 3 test varieties survival rates are all more than 95%. Three weeks after transplanting, the main tendrils have begun to elongate, and some plants have grown side tendrils, as shown in Figure 8. After 5-6 weeks of transplanting, the vines have grown to 30-40cm, as shown in Figure 9.

Table 3 Statistical survival rate of virus-free seedlings 9 days after transplanting

Variety Number of transplanted seedlings Number of surviving seedlings Survival rate (%) Xu Shu 22 240 231 96.25 Smoked Potatoes 25 113 109 96.46% Shangshu 19 148 142 95.95%

8. Propagation of virus-free seedlings in plastic greenhouses:

When the vine grows to more than 30cm, cut the vine and cut it into the soil of the plastic greenhouse for re-propagation of the virus-free seedlings. The distance between the planting plants is 20-25cm. 8 to 10 catties per mu, and then watered immediately, a large number of robust virus-free seedlings can be obtained.

Example 2

The preparation method of sweet potato virus-free seedling described in the present embodiment specifically comprises the steps:

1. Treatment of sweet potato pieces before germination:

(1) After harvesting in autumn, select sweet potato varieties Gaoxi No. 14 (commercially available variety) without insects and damage, and expose them to the sun for 2-3 days (the first has a bactericidal effect; the second can promote the growth of the potato pieces). bud).

(2) Prepare ripe bananas, put the sun-exposed potato pieces and ripe bananas together in a plastic storage box, close the lid, and store them in the laboratory for 3-5 days (at room temperature not lower than 13°C) (Use mature Bananas release ethylene, which promotes the germination of the potato cubes. Because the newly harvested potato cubes germinate slowly, resulting in rotten potatoes).

2. Germination:

Wash the above-mentioned potato pieces with clean water, put them into sand, cover the potato pieces with about 1-2cm of sand, and put them into an incubator for germination. The temperature setting is 35~37°C. (According to the fact that the virus is sensitive to high temperature, because sweet potato is a plant of tropical origin, sweet potato is a high temperature resistant plant. Using this difference, choosing an appropriate high temperature can reduce the virus concentration in sweet potato and slow down the transmission speed, while sweet potato meristematic cells The division is accelerated, and the growth of potato buds is accelerated. Therefore, the detoxification effect of the stripped shoot tips is good, and because the internodes of the leaf primordia are elongated, the shoot tips are easy to strip, and the regeneration rate is high.) Before sprouting, culture in the dark for 1 week, after sprouting, daily 13 hours light, 1000-2000lx. Spray water in an appropriate amount according to the humidity of the sand. The water content in the sand should not be too large, otherwise the seeds will rot easily. After being cultivated in the incubator for 10 days, the potato pieces began to sprout, and after 13 days, the length of some sprouts had reached 10cm.

3. Shoot tip stripping and cultivation

(1) Disinfection of materials: When the sprout grows to about 10 cm, take the top 1-2 cm long, cut off the leaves visible to the naked eye, wash it with clean water first, and then soak it in 70% alcohol in an ultra-quiet workbench for 10- For 20 seconds, soak in 2% sodium hypochlorite solution for 5-7 minutes for surface disinfection, and finally rinse with sterile water 3-5 times.

(2) Shoot apex stripping and culture: Strip the shoot tip with 1-2 leaf primordia under a stereo dissecting microscope, and inoculate it on MS medium supplemented with 0.2-2.0 mg/L 2,4-D for culture ( In this case, the concentration of 2,4-D is 2.0mg/L). After 3 weeks of culture, embryogenic callus began to form, as shown in Figure 10, the frequency of embryogenic callus at the shoot tip was 76.7%. After 4-5 weeks of culture, somatic embryos were formed from the embryogenic callus, as shown in Figure 11, and the culture was carried out at 25-27°C under dark conditions.

The explants that formed somatic embryos were transferred to the MS medium without adding hormones, and the somatic embryos germinated into seedlings, as shown in FIG. 12 . When the regenerated seedling grows to more than 2 cm, it is cut off and transferred to a new MS medium to grow into a complete plant, as shown in Figure 13. The culture conditions are 25-27°C, 13 hours of light per day, 2000-3000lx.

4. Aseptic Rapid Propagation of Virus-free Vaccines

The propagation of the virus-free seedlings adopts the method of cutting stems and tendrils. The 1-2 leaf nodes of the virus-free seedlings are divided into 1 section, and the cuttings are inserted into the MS medium without adding hormones. Virus-free seedlings, as shown in Figure 2. In this way, the multiplication and cultivation by continuous cutting, the propagation speed increases geometrically, and a large number of virus-free seedlings can be propagated in the culture bottle. The culture conditions are 25-27°C, 13 hours of light per day, 2000-3000lx.

5. Domestication and transplanting of virus-free test-tube plantlets:

(1) Subculture the test-tube seedlings for 1 month, open the mouth of the bottle for domestication, open a little on the first day, and then open a little on the next day, so that the seedlings will be hardened continuously for 2-3 days, until the bottle mouth is half opened ( Because the hardening time is short, it is easy to lose water and wilt if they are all opened).

(2) Clean the culture medium of the test-tube seedlings, and plant individual plants directly in the soil of plastic greenhouses with a spacing of 20-25 cm, as shown in Figures 3, 4, and 5. Be careful not to damage the roots during cleaning and transplanting, which can shorten the time for slowing down seedlings and improve the survival rate and growth rate of seedlings. Add insect-proof nets at both ends of the plastic greenhouse (to prevent virus re-infection, because the virus is mainly transmitted by insects, adding insect-proof nets can prevent insects from entering the plastic greenhouse).

(3) After the virus-free test-tube seedlings are planted in the soil of the greenhouse, water is first poured (to prevent the seedlings from being washed up), and then flooded with water. The function of flood irrigation can firstly provide the water needed by the seedlings, and secondly, it can provide guarantee for maintaining the air humidity in the plastic greenhouse. Because the air humidity of the test tube seedlings in the test tube is very high, a certain air humidity should be maintained in the early stage of transplanting, otherwise it is easy to lose water and wilt.

6. Management of virus-free seedlings in plastic greenhouses:

(1) In the first two weeks after transplanting the virus-free test-tube seedlings, set up a shade net at noon (that is, from 10:00 am to 3:00 pm) to prevent direct sunlight, as shown in Figure 6. And after the shading net is set up, the temperature in the plastic greenhouse will not be too high if the wind is not released at noon, and the air humidity in the plastic greenhouse can be maintained. After 2 weeks, remove the shading net and release the wind at noon. Water according to soil moisture.

(2) 3 weeks after transplanting, sprinkle urea with a fertilizer rate of 8-10 catties per mu, and then water immediately to promote the growth of seedlings.

Observed after 9 days of transplanting, the leaves of the seedlings have stretched and new leaves have grown, as shown in Figure 7, and the statistical survival rate is 96%. Three weeks after transplanting, the main tendrils have begun to elongate, and some plants have grown side tendrils, as shown in Figure 8. After 5-6 weeks of transplanting, the vines have grown to 30-40cm, as shown in Figure 9.

7. Propagation of virus-free seedlings in plastic greenhouses:

When the vine grows to more than 30cm, cut the vine and cut it in the soil of the plastic greenhouse for re-propagation of the virus-free seedlings. The distance between the plants is 20-25cm. Mu 8 ~ 10 catties, then watered, you can get a lot of robust virus-free seedlings.

After the detoxified sweet potato seedlings in the plastic greenhouse were transplanted into the field, the conventional cultivation and management were carried out. The potato seedlings grew robustly and vigorously, and the leaves were stretched. No mutant plants were observed in the 4 varieties tested in the present invention. Observation after harvest in autumn, the skin of the potato pieces is smooth and beautiful, which increases the commodity value, as shown in Figure 14.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still understand the foregoing embodiments. Modifications are made to the technical solutions described, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

Claims (8)

1. a preparation method for Sweetpotato Viruses Elimination seedling, is characterized in that, it comprises the following steps:

(1) choose the undamaged sweet potato potato of free from insect pests piece, under the sun, be exposed to the sun 2 ~ 5 days;

(2) the potato piece after being exposed to the sun and ripe banana are put together 3-5 days;

(3) potato piece is cleaned with clear water, on potato piece, covered sand, put into incubator and carry out vernalization, 35 ~ 37 ℃ of temperature; Before sprouting, dark culturing is 1 week, after sprouting, and illumination 13 hours every days, 1000-2000lx;

(4) more than bud seedling grows to 10 cm, get top 1-2 cm long, cut off macroscopic blade, first with clear water, rinse, then carry out surface sterilization, finally use rinsed with sterile water 3-5 time;

(5) under stereoscopic anatomical lens, strip the stem apex with l-2 leaf primordium, be inoculated on the medium that adds 0.1-0.2mg/L NAA and 1.0-2.0mg/L BAP, cultivation temperature is 25-27 ℃, illumination 13 hours every days, 2000-3000lx, cultivates 3-4 week, the differentiation of evoking adventive bud;

Or described stem apex is inoculated into and adds 0.2-2.0mg/L 2, dark culturing on the medium of 4-D, cultivation temperature is 25-27 ℃, inductor cell stage occurs;

(6) on the medium that adds 0.1-0.2mg/L NAA and 1.0-2.0mg/L BAP, cultivate, when regeneration seedling grows to 2cm when above, from base portion, cut, being transferred in the medium that does not add hormone must complete detoxic seedling;

Or adding 0.2-2.0mg/L 2, on the medium of 4-D, cultivate 4-5 after week, explant successively starts to form embryo callus and body embryo, and the explant of organizator embryo is transferred in the medium that does not add hormone, impels body embryo germination to form detoxic seedling;

(7) adopt the climing segment method of stem Fast-propagation to obtain detoxification test tube plantlet;

(8) domestication of detoxification test tube plantlet: Plantlet subculture is cultivated 1 month, opened bottleneck and tame;

(9) transplanting of detoxification test tube plantlet: test-tube plantlet is cleaned to medium, and a minute individual plant is directly planted in the soil of plastic tunnel, and plastic tunnel two adds fly net;

(10) after detoxification test tube plantlet is planted in the soil of booth, first water-sprinkling, then broad irrigation;

(11) 2 initial Zhou Zhongwu of detoxicating cuvette transplantation of seedlings take shading screen, prevent that the sun directly shines, and after 2 weeks, remove shading screen, and leak informaton noon;

(12) transplant after 3 weeks, spread fertilizer over the fields urea, water immediately, promote growth of seedling;

(13) breeding of detoxic seedling in plastic tunnel: when stem is climing grow to 30cm more than, shear that stem is climing plants in plastic tunnel, carry out breeding again of detoxic seedling, cut at every turn climing after, spread fertilizer over the fields urea, water immediately, obtain Sweetpotato Viruses Elimination seedling.

2. the preparation method of Sweetpotato Viruses Elimination seedling according to claim 1, is characterized in that: the temperature that the potato piece after being exposed to the sun in described step (2) and ripe banana are placed is not less than 13 ℃.

3. the preparation method of Sweetpotato Viruses Elimination seedling according to claim 1, is characterized in that: the thickness that covers sand in described step (3) is 1-2cm.

4. the preparation method of Sweetpotato Viruses Elimination seedling according to claim 1, is characterized in that: in described step (4), with 70% alcohol-pickled 10-20 second, 2% liquor natrii hypochloritis soaks and within 5 minutes-7 minutes, carries out surface sterilization.

5. the preparation method of Sweetpotato Viruses Elimination seedling according to claim 1, is characterized in that: in described step (6) and (7), condition of culture is temperature 25-27 ℃, illumination 13 hours every days, 2000-3000lx.

6. the preparation method of Sweetpotato Viruses Elimination seedling according to claim 1, is characterized in that: in described step (8), the method for domestication is: open gradually bottleneck hardening after 2-3 days, until final bottleneck is opened half.

7. the preparation method of Sweetpotato Viruses Elimination seedling according to claim 1, is characterized in that: the strain spacing of planting in described step (9) and (13) is 20 ~ 25cm.

8. the preparation method of Sweetpotato Viruses Elimination seedling according to claim 1, is characterized in that: the fertilizing amount of spreading fertilizer over the fields urea in described step (12) and (13) is 8 ~ 10 jin every mu.