CN116267601A - Method for detoxification of potato stem tip by variable temperature heat treatment - Google Patents

Abstract

The invention provides a potato variable-temperature heat treatment stem tip detoxification method, which comprises the following treatment steps: carrying out photothermal variable-temperature heat treatment on the buds of the potatoes, and then peeling off the stem tips to obtain detoxified potato micro stem tips; the variable temperature heat treatment of different periods of light and heat refers to the cyclic variable temperature heat treatment according to the conditions of low-temperature illumination and high-temperature darkness in the process of culturing the buds of the potatoes. The invention sets the high-temperature treatment stage process as a dark period and the normal-temperature stage as a photoperiod so as to ensure that the growth state of the buds of the potatoes under illumination is good, and the vacuum range of the stem tip viruses is enlarged through pretreatment. The invention combines the variable temperature heat treatment with the stem tip culture, can achieve higher detoxification rate, enlarges the stripped stem tip, improves the stripping speed, increases the survival rate and the detoxification rate, expands the application range of the potato by simple heat treatment detoxification, improves the detoxification perfection technology of the potato stem tip, and is more beneficial to production practice.

Description

A kind of detoxification method of potato stem tip with variable temperature heat treatment

technical field

The invention relates to a potato stem tip detoxification method, and belongs to the technical field of crop virus disease detoxification.

Background technique

Potato shoot apex meristem culture detoxification technology is the earliest potato detoxification technology that has been studied and applied. It can obtain good detoxification effect and is still widely used so far. This technique is a method for obtaining virus-free plants by peeling off tiny shoot tips from the plants for tissue culture.

The shoot tip detoxification technology is basically mature, with good detoxification effect and fast speed, but it still has significant defects: peeling off the shoot tip under the microscope exposes it to the air, which increases the risk of contamination; the shoot tip that needs to be cut is tiny , Observation and operation are more difficult, and the efficiency of peeling off the shoot tip is low. Therefore, some studies on the combination of shoot tip detoxification technology and other detoxification methods have begun to appear. At present, the research direction at home and abroad mainly includes the optimization of shoot apex detoxification technology, exploring the influence of different factors on shoot apex detoxification technology and the influence of different factors on shoot apex detoxification technology. Combination of technology with other detoxification methods.

The method of stem tip stripping combined with chemical agents has lower requirements on the length of the cut shoot tip than the simple shoot tip culture detoxification technology, and the stripped shoot tip can be larger than 1mm, which improves the efficiency of shoot tip stripping and reduces the excessive length of the shoot tip in the past. The risk of browning and vitrification caused by shortness increases the seedling rate.

Shoot tip culture combined with heat treatment detoxification method is currently a relatively recognized method in the industry and is suitable for large-scale operations. Potato capsid protein is partially inactivated or completely inactivated due to deformation under high temperature conditions. Under this condition, the plant grows and develops normally, so that the growth rate of the cells near the growth point of the stem or the plant is faster than the speed of virus reproduction, so that the growth point is non-toxic. In the process of shoot tip stripping, sometimes due to the longer shoot tip that is stripped, virus will remain at the tail of the shoot tip. There are also some viruses that are too close to the growth point, even if the cut tip is very short, it is difficult to get rid of it. However, heat treatment combined with shoot tip culture detoxification can inactivate viruses, thereby promoting the removal of these viruses and improving the detoxification rate of potato plants.

High-temperature heat treatment can enhance the detoxification effect of shoot tip tissue culture, which is determined by the treatment time, temperature and location. However, long-term continuous high temperature treatment will inactivate the virus, but it will also slow down the growth rate of shoot tip differentiation, hinder the growth of shoot tip, and cause the decline of shoot tip vitality and seedling rate. In addition, there are few studies on heat treatment time, temperature, and the interaction between light intensity and high temperature during the treatment process. There is no unified conclusion, and the system is still immature, which brings a lot of inconvenience to operators. To sum up, determining a shoot tip detoxification technology with strong operability and high detoxification rate is still an urgent problem to be solved in potato detoxification.

Contents of the invention

The object of the present invention is to provide a method for detoxification of potato stem tips by heat treatment at variable temperatures in view of the technical problems in the background technology.

The technical scheme that the present invention takes is as follows:

A potato shoot tip detoxification method with variable temperature heat treatment, comprising the following processing steps: after the potato buds are subjected to variable temperature heat treatment at different stages of light and heat, and then the shoot tip is peeled off to obtain a virus-free potato shoot tip;

The variable temperature heat treatment with different photothermal periods refers to the cycle variable temperature heat treatment under the conditions of low-temperature light and high-temperature darkness during the process of cultivating potato buds.

Variable temperature heat treatment partially or completely inactivates many viruses in plant tissues. This treatment has little damage to the host tissue and makes the shoot tip vigorously differentiate into seedlings; alternate light and heat treatment improves the plant vitality and prevents the shoot tip from being burned due to high temperature. ; Therefore, the present invention adopts variable temperature heat treatment in combination with shoot tip culture to achieve a higher detoxification rate. Variable temperature heat treatment makes the critical part of virus distribution move back, the peeled shoot tip becomes larger, the peeling speed is increased, the survival rate and virus-free rate are increased, and the application range of potato detoxification through simple heat treatment is expanded, and the potato shoot tip is detoxified. Perfect technology More perfect, more conducive to production practice.

High temperature under light is a conventional treatment method for detoxification. Considering the double stress of strong light and high temperature on plant growth, the present invention improves this by setting the high temperature treatment stage as a dark cycle, and the normal temperature stage as a photoperiod to ensure that the potato The buds grow well under the light. After the pretreatment of the method, the vacuum range of the shoot apex virus is expanded, thereby laying a foundation for obtaining a higher virus-free rate by taking a shoot tip of 0.5-1 mm.

Further preferably, the conditions of the above variable temperature heat treatment are: 18-20°C, 10-14h light; 36-39°C, 10-14h dark. The time and temperature of high temperature treatment have an important impact on the survival rate and seedling rate. Through experiments, it is found that the comprehensive treatment method of variable temperature heat treatment combined with shoot tip culture within this range can greatly increase the peeling rate and detoxification rate of the shoot tip. .

Further preferably, the light intensity of the above variable temperature heat treatment is 2000-3000LUX. Under different light intensities, the seedling rate and detoxification rate of tissue cultured seedlings will be different. Under the light intensity of 2000-3000LUX, the test tube seedlings grow well, and their fresh weight and dry matter are higher than those treated with low light density. Illumination is not only an energy source for photosynthesis, but also an important signaling molecule, regulating gene expression, affecting enzyme activity and morphogenesis, etc. Illumination can promote plants to better adapt to the external environment, but high light intensity and high temperature dual treatment is easy. The combined stress caused poor growth of in vitro culture materials. Therefore, appropriate light intensity combined with variable temperature treatment can achieve better detoxification effect.

Further preferably, the above-mentioned temperature-variable heat treatment with different phases of light and heat has a culture treatment time of one month or more.

Further preferably, when the above-mentioned temperature-varying heat treatment is performed on the potato sprouts, the sprouts selected are the sprouts of potato tissue cultured seedlings.

Further preferably, the above-mentioned potato tissue-cultured plantlets are virus-infected tissue-cultured plantlets obtained by cutting off buds from germinated potato tubers carrying viruses and culturing them with medium.

Traditional tuber germination micro shoots can only be sampled at a specific time point in a specific season every year, and the amount is small; the present invention changes the traditional tuber germination micro shoots into in vitro cultured micro shoots by taking materials from the shoot tips, so that it is not limited by seasons, Get a lot of raw materials.

Further preferably, the above-mentioned culturing using the culture medium includes sequentially performing tissue culture and subculture;

The tissue culture needs to first cut off the buds on the germinated potato tubers carrying PVY virus, wash and sterilize them, and then cultivate and grow regenerated buds in MS medium; , Tissue culture under the condition of full light 3000-4000LUX light intensity;

The subculture expansion refers to cutting 1-1.5 cm long stems with a leaf and transferring them to other MS medium for subgeneration expansion to obtain multiple batches of tissue culture seedlings when the plants with leaves are obtained through tissue culture.

Through the pre-treatment method for tissue culture seedlings of the present invention, it is more convenient to obtain materials for in vitro cultured micro shoot tips that were difficult to obtain, and at the same time, due to in vitro propagation, the source of micro shoot tip materials is no longer limited.

Further preferably, before the above-mentioned variable temperature heat treatment is performed on the tissue cultured seedlings, it is also necessary to pre-cultivate the tissue cultured seedlings under full light, and then perform subsequent treatment after the tissue cultured seedlings take root.

The tissue cultured seedlings after rooting are more resistant to high temperature, so they can grow under high temperature treatment; otherwise, the survival rate of tissue cultured seedlings will be greatly reduced if they enter high temperature treatment without rooting. This is because the unrooted tissue culture seedlings lack the root system to absorb nutrients, the survival rate is low, and the shoot tips are easy to burn and die under heat treatment, so they cannot be used for subsequent shoot tip detoxification operations.

Further preferably, the above-mentioned full-light pre-cultivation is to pre-cultivate the tissue-cultured seedlings for 5-7 days under the condition of 18-20° C. and full light of 3000-4000 LUX light intensity.

Further preferably, it also includes the following steps: after undergoing different periods of heat treatment with light and heat, strip off the 0.5-1mm long micro-shoot tips of the tissue cultured seedlings under a microscope, and follow the conditions of 18-20°C and full light of 2000-3000LUX. conditions, use MS medium to culture the micro-shoot tips for 2 months, and after the regenerated shoots grow, cut off the regenerated shoots and carry out cultivation and propagation in a full light environment of 3000-4000LUX to obtain virus-free regenerated tissue culture seedlings.

In order to obtain a virus-free rate of more than 50% for ordinary micro shoot tips, the length of the shoot tip must be less than 0.5 mm, or even less than 0.2 mm. The operation of stripping the micro shoot tips is difficult, and the survival rate of the culture of too small micro shoot tips is low. The pretreatment method of the present invention greatly reduces the amount of virus in the range of 0.5-1 mm, and expands the range of virus-free micro shoot tips, so that the operator can strip off longer micro shoot tips, so that the survival rate of micro shoot tips can be improved. , to obtain a higher detoxification rate. The pretreatment method of the present invention makes the shoot tip vigor better, and the ordinary MS medium without hormone addition is used in the culture process of the stripped micro shoot tip, so as to reduce the culture cost and ensure the genetic stability. Then through the regeneration bud culture and propagation, enough virus-free regeneration tissue culture seedlings for production can be obtained.

Compared with prior art, the beneficial effect of the present invention is:

Variable temperature heat treatment partially or completely inactivates many viruses in plant tissues. This treatment has little damage to the host tissue and makes the shoot tip vigorously differentiate into seedlings; alternate light and heat treatment improves the plant vitality and prevents the shoot tip from being burned due to high temperature. ; Therefore, the present invention adopts variable temperature heat treatment in combination with micro-shoot tip culture to achieve a higher detoxification rate. The variable temperature heat treatment makes the critical part of the virus distribution move back, the peeled shoot tip becomes larger, and the survival rate and virus-free rate increase, which expands the application range of potato detoxification through simple heat treatment, and makes the perfect potato micro-shoot tip virus-free technology more perfect. It is more conducive to production practice. High temperature under light is a conventional treatment method for detoxification. Considering the double stress of strong light and high temperature on plant growth, the present invention improves this by setting the high temperature treatment stage as a dark cycle, and the normal temperature stage as a photoperiod to ensure that the potato The buds grow well under the light. After the pretreatment of the method, the vacuum range of the shoot apex virus is expanded, thereby laying a foundation for obtaining a higher virus-free rate by taking a shoot tip of 0.5-1 mm.

The invention adopts temperature-variable pretreatment and shoot tip culture combined detoxification on the virus-infected potato tissue culture seedlings, which is easy to operate, not limited by seasons, and has a large base of materials that can be used for detoxification. Through the pre-treatment method for tissue culture seedlings of the present invention, it is more convenient to obtain materials for in vitro cultured micro shoot tips that were difficult to obtain, and at the same time, due to in vitro propagation, the source of micro shoot tip materials is no longer limited. Different temperature treatment tissue culture seedlings are not limited by season and space like seedlings. Tissue culture seedlings can be inoculated and cultivated at any time to obtain the required materials at any time, with high operability.

Description of drawings

Fig. 1 is the morphological characterization comparison figure after the tissue culture seedling precultivation 7 days+variation temperature treatment in embodiment 1 and comparative example 1,2 one month;

Fig. 2 is the morphological feature dyeing contrast figure of each part after the tissue culture seedling variable temperature treatment in embodiment 1 and comparative example 1,2;

Fig. 3 is the TTC dyeing situation contrast figure of tissue culture plantlet variable temperature treatment 14 days in embodiment 1 and comparative example;

Fig. 4 is the physical figure of the forming process of micro-shoot tip culture in embodiment 1;

Fig. 5 is the detection situation diagram of PVY virus RT-PCR in embodiment 1 and comparative example 1,2;

Fig. 6 is the detection situation diagram of PVS virus RT-PCR in embodiment 1 and comparative example 1,2;

Fig. 7 is a comparison chart of the survival rate of tissue-cultured seedlings in Example 1 and Comparative Example 2 without rooting treatment and after rooting treatment.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully and in detail below, but the protection scope of the present invention is not limited to the following specific examples.

Unless otherwise defined, all technical terms used hereinafter have the same meanings as commonly understood by those skilled in the art. The terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the protection scope of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

Embodiment 1: (label is T2 in the accompanying drawing)

A method for detoxifying shoot tips of potato tissue cultured seedlings with variable temperature heat treatment, the steps are as follows:

(1) Take the germinated tuber material of Hunan Potato No. 1 carrying PVY virus, cut the buds on the tuber into about 0.5cm long, wash it under running water for at least 2 hours, and place the stem section in 75% alcohol in an ultra-clean workbench Disinfect in the solution for 30s; then put it in 0.1% HgCI ₂ solution for about 4min, rinse the sterilized buds with sterile water at least 3 times, about 15s each time; blot the residual water with sterilized filter paper and transfer to 3% In MS medium, cut off the regenerated buds when they grow, and conduct tissue culture at 18°C under full light of 3000-4000LUX.

(2) When the regenerated buds obtained through tissue culture are used to obtain plants with leaves, cut the stem section with a leaf about 1 cm long, and transfer to 3% MS medium for subgeneration and propagation to obtain enough virus-infected tissue culture seedlings, First place the propagated tissue cultured seedlings at 18°C under the condition of full light of 3000-4000LUX for pre-cultivation for 7 days, and then carry out subsequent treatment after the tissue cultured seedlings take root.

(3) Place the tissue-cultured seedlings under the conditions of light intensity 2000-3000LUX, daily normal temperature 18°C/12h, and night high temperature 38°C/12h for 1 month.

(4) Peel off 0.5-1mm micro-shoot tips under a stereomicroscope and transfer them to ordinary 3% MS medium, cultivate them under the conditions of light intensity 2000-3000LUX, normal day and night temperature (18°C) for 12 hours, and dark culture for 12 hours. After one month, the regenerated shoots were cut off and propagated for 1 month in a full-light environment of 3000-4000 LUX to obtain virus-free regenerated tissue culture seedlings.

The morphological characteristics of the tissue culture seedlings after 7 days of pre-cultivation + variable temperature treatment for one month are shown in Figure 1; The dyeing situation is shown in Figure 3; the physical picture of the formation process of the micro shoot tip culture is shown in Figure 4; the statistical comparison of the survival rate and the seedling rate is shown in Table 1.

Take 0.1 g of the regenerated tissue culture seedlings obtained through (4), extract the total RNA by liquid nitrogen quick-frozen grinding, and detect whether the virus is carried by PCR after reverse transcription. The PVY virus detection results are shown in Table 2 and Figure 5.

Example 2:

In this embodiment, other implementations are the same as in Example 1, only step (1) "get the germinated tuber material of No. 1 Hunan Potato carrying PVY virus" in Example 1 and change it into "Take No. 1 Hunan Potato and germinate carrying PVS virus tuber material". After obtaining the regenerated tissue culture seedlings, the same method was adopted to detect whether the virus was carried, and the PVS virus detection results are shown in Table 2 and Figure 6.

Example 3:

In this embodiment, other implementations are the same as in Example 1, only step (1) "get the germinated tuber material of Hunan potato No. 1 carrying PVY virus" into "take Hunan potato No. 1 carrying PVY+PVS virus" Germinated tuber material". After obtaining the regenerated tissue culture seedlings, the same method was adopted to detect whether the virus was carried, and the PVY+PVS virus detection results are shown in Table 2.

Comparative example 1: (marked as CK in the attached drawing)

Other implementations are the same as in Example 1 in this comparative example, only the conditions of the variable temperature heat treatment of step (3) tissue culture seedlings in Example 1 are changed to: light intensity 3000-4000LUX, day and night normal temperature (18 ℃) light cultivation 12h , cultivated in dark for 12h. After obtaining the regenerated tissue culture seedlings, the same statistical method was adopted to compare the survival rate and seedling rate.

The morphological characteristics of the tissue culture seedlings after 7 days of pre-cultivation + variable temperature treatment for one month are shown in Figure 1; The dyeing situation is shown in Figure 3; the statistical comparison of the survival rate and seedling rate is shown in Table 1.

0.1 g of the obtained regenerated tissue culture seedlings were quick-frozen and ground in liquid nitrogen to extract total RNA. After reverse transcription, PCR was used to detect whether the virus was carried. The PVY virus detection results are shown in Table 2.

Comparative example 2: (labeled as T1 in the attached drawing)

Other implementations are the same as Example 1 in this comparative example, only change the condition of the temperature-variable heat treatment of step (3) tissue culture seedling in Example 1 into: light intensity 2000-3000LUX, day high temperature (38 ℃) cultivate 12h, night Incubate at normal temperature (18°C) for 12 hours. After obtaining the regenerated tissue culture seedlings, the same statistical method was adopted to compare the survival rate and seedling rate.

The morphological characteristics of the tissue culture seedlings after 7 days of pre-cultivation + variable temperature treatment for one month are shown in Figure 1; The dyeing situation is shown in Figure 3; the statistical comparison of the survival rate and seedling rate is shown in Table 1.

0.1 g of the obtained regenerated tissue culture seedlings were quick-frozen and ground in liquid nitrogen to extract total RNA. After reverse transcription, PCR was used to detect whether the virus was carried. The PVY virus detection results are shown in Table 2.

Comparative example 3: (marked as T3 in the attached drawing)

Other implementations are the same as Example 1 in this comparative example, only change the condition of the temperature-variable heat treatment of step (3) tissue culture seedling among the Example 1 into: light intensity 2000-3000LUX, day and night high temperature (38 ℃) light culture 12h, Incubate in dark for 12h. After obtaining the regenerated tissue culture seedlings, the same statistical method was adopted to compare the survival rate and seedling rate. T3 is continuous high temperature treatment, a commonly used method for traditional potted seedlings. Experiments have found that continuous high temperature is completely infeasible for tissue cultured seedlings. Apoptosis begins after 1 week of treatment, and all die within 2 weeks. Therefore, the subsequent survival rate is 0, and the detoxification rate is 0. 0.

The present invention also aims at whether to perform follow-up treatment after taking root during the pre-cultivation of the tissue cultured seedlings, and has done a relevant small comparative test on the basis of T1 and T2, and the results are as shown in Figure 7.

The specific statistical analysis of situations such as the regeneration tissue culture seedling survival rate of embodiment and comparative example and seedling rate:

Measure the plant height, stem diameter and root length of the tissue cultured seedlings, then cut the stems of each group of tissue cultured seedlings, and stain them with TTC. It can be seen from Fig. 1 and Fig. 2 that the stems and petioles of T2 are the most Slender, with the lightest leaf color, the smallest leaves and stem tips, but with high viability; the stem cell activity of T2 in Figure 3 is good.

The stem tips of the tissue cultured seedlings were stripped and cultured for 3 months, and the survival rate and seedling rate were counted. It can be seen from Table 1 that the survival rate of T2 heat treatment is 69.23%, and the seedling rate is 53.85%. The seedling rate of variable temperature treatment is far lower than that of constant temperature treatment, and there is a significant difference. The seedling rate was the same at night high temperature and daily high temperature, and the detoxification effect was the result of the joint action of time, temperature and location of high temperature treatment. The consistent seedling rate may be the accidental result caused by the small number of samples. Although the shoot tip survival rate of T2 was lower than that of constant temperature treatment, it was higher than that of T1, so night high temperature combined with shoot tip culture detoxification method was better than day high temperature.

The detoxification rate was detected by RT-PCR method. As can be seen from Table 2, the detoxification rate of PVY virus after T2 treatment is 46.15%, and the detoxification rate increases by 23.07% compared with the micro shoot tip culture of common tissue culture seedlings, which is higher than the micro shoot tip culture after daily high temperature pretreatment 7.69%, both have significant differences; S virus, S+Y virus removal rate increased by 1 times compared with the control, indicating that night high temperature treatment can greatly improve the virus removal rate of shoot tips and achieve a better effect of virus removal. Therefore, the light intensity 2000-3000LUX daily normal temperature 18 ℃ culture 12h, night high temperature 38 ℃ culture 12h is the most ideal way to detoxify potato shoot tips.

Table 1

group pretreatment conditions Survival rate (%) Seedling rate (%) CK 18℃12h L+18℃12h D 92.31 76.92 T1 38℃12h L+18℃12h D 61.54 53.85 T2 18℃12h L+38℃12h D 69.23 53.85

Table 2

The foregoing are only preferred implementations of the present invention, and the scope of protection of the present invention is not limited to the foregoing examples. For those skilled in the art, improvements and transformations obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for detoxification of potato shoot tips with variable temperature heat treatment, characterized in that, comprising the following processing steps: after the buds of potatoes are subjected to variable temperature heat treatment with different phases of light and heat, then the shoot tip is peeled off to obtain the detoxified potato micro shoot tip; The variable temperature heat treatment with different photothermal periods refers to the cycle variable temperature heat treatment under the conditions of low-temperature light and high-temperature darkness during the process of cultivating potato buds. 2 . The method for detoxifying shoot tips of potatoes with variable temperature heat treatment according to claim 1 , characterized in that, the conditions of the variable temperature heat treatment are: 18-20° C., 10-14 hours of light; 36-39° C., 10-14 hours of darkness. 3. The method for detoxifying shoot tips of potatoes with variable temperature heat treatment according to claim 2, characterized in that, the light intensity of the variable temperature heat treatment is 2000-3000LUX. 4 . The method for detoxifying shoot tips of potatoes with variable temperature heat treatment according to claim 3 , characterized in that, the cultivation treatment time of the variable temperature heat treatment with different phases of light and heat is one month or more. 5. The method according to any one of claims 1-4, wherein the potato shoot tip detoxification method with variable temperature heat treatment is characterized in that, the buds selected when the potato buds are subjected to variable temperature heat treatment are buds of potato tissue cultured seedlings. 6. the method for detoxifying potato stem tips with variable temperature heat treatment according to claim 5, wherein the potato tissue culture seedlings are obtained by cutting off the buds on the potato germinated tubers carrying the virus and cultivating them using the culture medium Virus-infected tissue cultures. 7. the method for detoxification of potato variable temperature heat treatment shoot apex according to claim 6, is characterized in that, described utilize medium to cultivate and comprise the tissue culture that carries out successively and subculture expansion; The tissue culture needs to first cut off the buds on the germinated potato tubers carrying PVY virus, wash and sterilize them, and then cultivate and grow regenerated buds in MS medium; , Tissue culture under the condition of full light 3000-4000LUX light intensity; The subculture expansion refers to cutting 1-1.5 cm long stems with a leaf and transferring them to other MS medium for subgeneration expansion to obtain multiple batches of tissue culture seedlings when the plants with leaves are obtained through tissue culture. 8. according to claim 6 or 7 described potato variable temperature heat treatment stem tip detoxification methods, it is characterized in that, before tissue culture seedlings are carried out variable temperature heat treatment cultivation, it is also necessary to carry out full light pre-cultivation to tissue culture seedlings, to treat tissue culture Subsequent treatment is carried out after the seedlings take root. 9. The method for detoxifying shoot tips of potatoes with variable temperature heat treatment according to claim 8, characterized in that, the full light pre-cultivation is to place the tissue cultured seedlings under conditions of 18-20°C and full light 3000-4000LUX light intensity. Culture for 5-7 days. 10. according to claim 6 or 7 described potato variable temperature heat treatment stem tip detoxification method, it is characterized in that, also comprises the following steps: after the variable temperature heat treatment of light and heat different phases, under the microscope, tissue culture seedling is stripped 0.5. -1mm long micro-shoot tips, according to the conditions of 18-20°C and full light of 2000-3000LUX, culture the micro-shoot tips with MS medium for 2 months, and after the regeneration buds grow, cut off the regeneration shoots in full light Cultivate and multiply in a 3000-4000LUX environment to obtain virus-free regenerated tissue culture seedlings.

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