A method for ultra-low temperature freezing and detoxification of potatoes

Abstract

The invention provides a method for ultra-low temperature freezing detoxification of potatoes, belonging to the technical field of potato detoxification. The invention firstly adopts variable temperature heat treatment to treat potato tubers, which can not only inactivate viruses, but also ensure a certain humidity in the process to improve the germination rate, and then carries out dark culture and then light culture during seedling cultivation, which shortens the seedling time and improves the detoxification rate and the survival rate.

Description

Technical Field

[0001] The invention belongs to the technical field of potato detoxification, and in particular relates to a method for ultra-low temperature freezing detoxification of potatoes. Background Art

[0002] The yield and quality of potatoes are closely related to the development of agricultural economy and food industry economy. Potatoes are a kind of crop planted by tubers and propagated asexually. Therefore, during the planting process, if the tubers are infected by viruses, the species will be degraded, which will affect the potato's ability to resist diseases, so that it is very susceptible to various diseases such as viral diseases and bacterial wilt during its growth. It can be seen that although potatoes are a high-yield crop, they cannot achieve high yields or even stable production due to the influence of various factors. At the same time, if potatoes are degraded by viruses, the yield will generally be reduced by 20-30%, and in severe cases by more than 80%. The disease will worsen year by year, and finally lose its value for planting.

[0003] Therefore, in order to ensure that the yield meets the market demand and the quality meets the public demand, potatoes need to be detoxified. Currently, the two most commonly used and traditional methods to solve the degeneration of potato seed potatoes are conventional shoot tip culture and heat treatment + shoot tip culture technology, but there are problems such as time-consuming, browning, difficult plant regeneration and poor detoxification effect (Gao Huiqing et al., 2010, Journal of Shanxi Agricultural University, 30(6)528-532). Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a method for ultra-low temperature freezing and detoxification of potatoes.

[0005] To achieve the above object, the present invention provides a method for ultra-low temperature freezing and detoxification of potatoes, comprising the following steps:

[0006] (1) subjecting potato tubers to a heat treatment, spraying water during the treatment, and maintaining the humidity of the potato tubers at 30% to 40%;

[0007] (2) selecting a 3±0.5 cm sprout from the potato tuber in step (1), cutting off a 1.5±0.3 cm upper stem end, washing and peeling off the stem tip;

[0008] (3) placing the stem tip in step (2) in liquid nitrogen for storage for 1 to 3 days; the present invention directly places the stem tip in liquid nitrogen, and the cooling rate can reach (300 to 400)°C / min. The ultra-fast freezing rate can reduce the water in the cells to a safe temperature of -196°C before it has time to form ice crystals, thereby avoiding the risk of freezing in the cells;

[0009] (4) The shoot tip in step (3) is removed from the liquid nitrogen, and after recovery treatment, is placed in a culture medium for cultivation, first in the dark for 1 to 3 days, and then in the light.

[0010] Furthermore, the heat treatment in step (1) is to first expose the potato tubers to light for 12 hours at 28-30°C, and then treat them in the dark for 6 hours at 22-25°C. The heat treatment process of the present invention can not only inactivate the virus, but also ensure a certain humidity during the process to improve the germination rate. If the heat treatment temperature is too high, the young sprouts will be thin and weak, and if the heat treatment temperature is too low, it will be difficult to germinate and will not have the effect of inactivating the virus. Therefore, in order to take into account both the germination and detoxification effects, the present invention adopts a variable temperature heat treatment method for potato tubers.

[0011] Furthermore, in step (1), the humidity of the potato tubers is maintained at 30% to 35%.

[0012] Furthermore, the cleaning in step (2) refers to first soaking the stem tip in Tween 20 for 3 to 5 minutes, then rinsing with running water for 10 to 15 minutes, then disinfecting with alcohol for 10 seconds, disinfecting with NaClO for 5 minutes, and finally washing with sterile water for 3 to 5 times.

[0013] Furthermore, the method of peeling the stem apex in step (2) is: under a dissecting microscope, use a scalpel to cut off the uppermost 0.7±0.2 mm of the stem apex, and then peel off 2 to 3 extra leaflets, leaving only 0.3 to 0.6 mm of the stem apex with 1 to 2 leaf primordia.

[0014] Furthermore, the recovery treatment in step (4) is to place the shoot tip taken out from the liquid nitrogen in a water bath at 25 to 30° C. for 3 to 5 minutes.

[0015] Furthermore, the culture medium in step (4) is MS culture medium supplemented with VC.

[0016] Furthermore, the composition of the culture medium in step (4) is MS + 0.4 mg·L -1 6-BA+0.08mg·L -1 NAA + 0.01~0.03mg·L -1 KH 2 PO 4 +0.01~0.03mg·L -1 (NH 4 ) 2 SO 4 +0.003~0.005mg·L -1 VC+25g·L -1 sucrose.

[0017] Furthermore, the composition of the culture medium in step (4) is MS + 0.4 mg·L -1 6-BA+0.08mg·L -1 NAA+0.02mg·L -1 KH 2 PO 4 +0.02mg·L -1 (NH 4 ) 2 SO 4 +0.003mg·L -1 VC+25g·L -1 sucrose.

[0018] Furthermore, in step (4), the culture temperature is 20-25° C., the light intensity is 2000-3000 lx, the light duration is 14-16 hours per day, and the relative humidity is 70%-80%.

[0019] Furthermore, in step (4), the culture temperature is 23±1°C, the light intensity is 2500 lx, the lighting time is 15 h per day, and the relative humidity is 75%.

[0020] The principle of the present invention is as follows: the traditional stem tip detoxification adopts the method of peeling off the stem tip growth point (0.1-0.3mm), transferring it into the meristem culture medium for 3-5 months, and transferring it into the growth culture medium for 15-20 days to form seedlings after the meristem differentiation. The traditional stem tip detoxification needs to be transferred twice from the peeled off growth point to the seedling, which takes nearly half a year. The present invention first uses the variable temperature heat treatment method to treat the potato tubers, which can not only inactivate the virus, but also ensure a certain humidity in the process to improve the germination rate. Moreover, after the variable temperature treatment, the potato tubers generate large buds. The present invention directly cuts the upper stem end of the large bud 1.5±0.3cm, cleans it, separates the stem tip, freezes it with liquid nitrogen, and then cultures it in the dark. Most of the cells are in the mitotic state, and do not need to be cultured through meristem culture. They can be directly cultured into seedlings in the culture medium. It only takes 18-25 days from cutting off the bud tip to seedling formation, which greatly shortens the seedling formation time. Different types of culture media have an important influence on whether the stem tip can be detoxified and survive. The present invention adds K to the MS culture medium. + NH 4 + and VC, which not only play an important role in the growth and development of the stem tip, but also can improve the detoxification effect and the survival rate. During the seedling cultivation, the present invention first performs dark cultivation and then performs light cultivation. The dark cultivation process can promote plant cell mitosis, so that most cells are in a mitotic state, and then performs light cultivation, which can greatly shorten the seedling time. In addition, the virus in the potato stem tip meristem is slow in propagation, which also indirectly improves the survival rate of the virus-free seedlings.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] (1) The virus-free potato plants cultured by the present invention have good growth status, high virus-free rate and high survival rate. The method of the present invention is simple and easy to implement, has a short culture cycle, and can be mass-produced.

[0023] (2) After the ultra-low temperature freezing and detoxification of the present invention, the regenerated plants were tested and no mutations in the DNA genetic sequence were found, indicating that the plants have good genetic stability.

[0024] (3) The present invention only takes 18 to 25 days from cutting off the bud tip to seedling formation, which greatly shortens the seedling formation time. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solution of the present invention is further illustrated by the following embodiments.

[0031] Example 1

[0032] (1) selecting healthy seed potatoes of Longshu No. 17 with large size and standard potato shape as potato tubers, and subjecting them to heat treatment in an artificial climate chamber, wherein the heat treatment comprises first irradiating the potato tubers with light for 12 hours at 28° C., and then subjecting the potato tubers to dark treatment for 6 hours at 22° C., spraying water during the heat treatment, and maintaining the humidity of the potato tubers at 30%;

[0033] (2) Select the 3±0.5 cm tender shoots germinated from the potato tuber in step (1), cut off the 1.5±0.3 cm upper stem tip, first soak the stem tip in Tween 20 for 3 min, then rinse with running water for 10 min, then disinfect with alcohol for 10 s, disinfect with NaClO for 5 min, and finally wash with sterile water 3 times, under a dissecting microscope, cut off the uppermost 0.7±0.2 mm of the stem tip with a scalpel, then peel off the excess 2 to 3 leaflets, and only retain 0.3 to 0.6 mm of the stem tip of 1 to 2 leaf primordia;

[0034] (3) placing the stem tip obtained in step (2) into liquid nitrogen for 1 day;

[0035] (4) After 1 day of storage, the shoot tips were removed from the liquid nitrogen, restored in a 25°C water bath for 5 min, and then cultured in a medium containing MS medium supplemented with VC, containing MS + 0.4 mg·L -1 6-BA+0.08mg·L -1 NAA+0.01mg·L -1 KH 2 PO 4 +0.01mg·L -1 (NH 4 ) 2 SO 4 +0.003mg·L -1 VC+25g·L -1 Sucrose was first cultured in the dark at 20°C for 1 day, and then cultured under light conditions with a light intensity of 2000 lx, a light duration of 16 h per day, and a relative humidity of 70%.

[0036] The new leaves of the virus-free potato plants of Longshu No. 17 of this embodiment are in good development state, with a survival rate of 95%. It only takes 25 days from cutting off the stem tip to seedling, which greatly shortens the seedling time. According to the RT-PCR test of the virus, the average virus-free rate (including potato leaf roll virus, potato virus Y, potato virus X, potato virus S, potato virus M and potato virus A) is about 94%. The regenerated plants after the ultra-low temperature freezing and virus-freezing of the present invention have no mutation in the DNA genetic sequence after testing, and have good genetic stability.

[0037] Example 2

[0038] (1) selecting healthy seed potatoes of Longshu No. 17 with large size and standard potato shape as potato tubers, and subjecting them to heat treatment in an artificial climate chamber, wherein the heat treatment comprises first subjecting the potato tubers to light at 30° C. for 12 h, and then subjecting them to dark treatment at 25° C. for 6 h, spraying water during the heat treatment, and maintaining the humidity of the potato tubers at 35%;

[0039] (2) Select the 3±0.5 cm tender shoots germinated from the potato tuber in step (1), cut off the 1.5±0.3 cm upper stem tip, soak the stem tip in Tween 20 for 3 min, rinse with running water for 12 min, disinfect with alcohol for 10 s, disinfect with NaClO for 5 min, and finally wash with sterile water 3 times. Under a dissecting microscope, cut off the uppermost 0.7±0.2 mm of the stem tip with a scalpel, then peel off the excess 2 to 3 leaflets, and only retain 0.3 to 0.6 mm of the stem tip of 1 to 2 leaf primordia;

[0040] (3) placing the stem tip obtained in step (2) into liquid nitrogen for 2 days;

[0041] (4) After 2 days of storage, the shoot tips were removed from the liquid nitrogen, restored in a 28°C water bath for 3 min, and then cultured in a medium containing MS medium supplemented with VC (MS + 0.4 mg L -1 6-BA+0.08mg·L -1 NAA+0.02mg·L -1 KH 2 PO 4 +0.02mg·L -1 (NH 4 ) 2 SO 4 +0.003mg·L -1 VC+25g·L -1 Sucrose was first cultured in the dark at 23±1℃ for 2 days, and then cultured under light conditions with a light intensity of 2500lx, a light duration of 15h per day, and a relative humidity of 75%.

[0042] The new leaves of the virus-free potato plants of Longshu No. 17 of this embodiment are in good development state, with a survival rate of 98%. It only takes 18 days from cutting off the stem tip to seedling, which greatly shortens the seedling time. According to the RT-PCR test of the virus, the average virus-free rate (including potato leaf roll virus, potato virus Y, potato virus X, potato virus S, potato virus M and potato virus A) is about 98%. The regenerated plants after the ultra-low temperature freezing and virus-freezing of the present invention have no mutation in the DNA genetic sequence after testing, and have good genetic stability.

[0043] Example 3

[0044] (1) selecting healthy seed potatoes of Longshu No. 17 with large size and standard potato shape as potato tubers, and subjecting them to heat treatment in an artificial climate chamber, wherein the heat treatment comprises first irradiating the potato tubers with light at 30° C. for 12 h, and then subjecting the potato tubers to dark treatment at 22° C. for 6 h, spraying water during the heat treatment, and maintaining the humidity of the potato tubers at 40%;

[0045] (2) Select the 3±0.5 cm tender shoots germinated from the potato tuber in step (1), cut off the 1.5±0.3 cm upper stem tip, soak the stem tip in Tween 20 for 5 min, rinse with running water for 15 min, disinfect with alcohol for 10 s, disinfect with NaClO for 5 min, and finally wash with sterile water 5 times. Under a dissecting microscope, cut off the uppermost 0.7±0.2 mm of the stem tip with a scalpel, then peel off the excess 2 to 3 leaflets, and only retain 0.3 to 0.6 mm of the stem tip of 1 to 2 leaf primordia;

[0046] (3) placing the stem tip obtained in step (2) in liquid nitrogen for 3 days;

[0047] (4) After 3 days of storage, the shoot tips were removed from liquid nitrogen, restored in a 30°C water bath for 5 min, and then cultured in a medium containing MS medium supplemented with VC, containing MS + 0.4 mg·L -1 6-BA+0.08mg·L -1 NAA+0.03mg·L -1 KH 2 PO 4 +0.03mg·L -1 (NH 4 ) 2 SO 4 +0.005mg·L -1 VC+25g·L -1 Sucrose was first cultured in the dark at 23±1℃ for 2 days, and then cultured under light conditions with a light intensity of 3000lx, a light duration of 14h per day, and a relative humidity of 80%.

[0048] The new leaves of the virus-free potato plants of Longshu No. 17 of this embodiment are in good development state, with a survival rate of 97%. It only takes 21 days from cutting off the stem tip to seedling, which greatly shortens the seedling time. According to the RT-PCR test of the virus, the average virus-free rate (including potato leaf roll virus, potato virus Y, potato virus X, potato virus S, potato virus M and potato virus A) is about 96%. The regenerated plants after the ultra-low temperature freezing and virus-freezing of the present invention have no mutation in the DNA genetic sequence after testing, and have good genetic stability.

[0049] Comparative Example 1

[0050] Same as Example 2, except that the conventional shoot tip detoxification method was used, the shoot tip growth point (0.1-0.3 mm) was peeled off, and the meristem culture medium (MS + 0.5 M chitosan oligosaccharide) was used for culture, and then the growth medium (MS + 0.4 mg·L -1 6-BA+0.05mg·L -1 NAA+0.15mg·L -1 GA 3 +8g·L -1 Agar + 30 g L -1 Sucrose) was cultured into seedlings.

[0051] The new leaves of the virus-free potato plants of this comparative example were in good development state, with a survival rate of 90%, and it took 5 months from cutting off the stem tip to seedling. The average virus-free rate (including potato leaf roll virus, potato virus Y, potato virus X, potato virus S, potato virus M and potato virus A) was about 85% through RT-PCR detection of viruses.

[0052] Comparative Example 2

[0053] Same as Example 2, except that KH was not added to the culture medium in step (4). 2 PO 4 NH 4 ) 2 SO 4 and VC.

[0054] The new leaves of the virus-free potato plants of this comparative example were in good development state, with a survival rate of 88%, and it took 30 days from cutting off the stem tip to seedling. The average virus-free rate (including potato leaf roll virus, potato virus Y, potato virus S and potato virus M) was about 80% through RT-PCR detection of viruses.

[0055] Comparative Example 3

[0056] The same as Example 2, except that in step (4), dark culture is not performed, but light culture is performed directly.

[0057] The new leaves of the virus-free potato plants of this comparative example were in good development state, with a survival rate of 75%, and it took 50 days from cutting off the stem tip to seedling. The average virus-free rate (including potato leaf roll virus, potato virus Y, potato virus X, potato virus S, potato virus M and potato virus A) was about 88% through RT-PCR detection of viruses.

[0058] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for ultra-low temperature freezing and detoxification of potatoes, characterized in that: The following steps are involved: (1) heat-treating potato tubers, spraying water during the heat treatment, and maintaining the humidity of the potato tubers at 30% to 40%; (2) Select the 3±0.5 cm sprouts from the potato tuber in step (1), cut off the 1.5±0.3 cm upper stem tip, wash it and peel off the stem tip; (3) Place the stem tip obtained in step (2) in liquid nitrogen and store for 1 to 3 days; (4) taking out the shoot tip from the liquid nitrogen in step (3), performing a recovery treatment, and then placing it in a culture medium for culturing, first in the dark for 1 to 3 days, and then in the light; The potato tubers are Longshu No. 17 potato tubers; The heat treatment in step (1) is to first expose the potato tubers to light at 28-30°C for 12 hours and then to dark treatment at 22-25°C for 6 hours; The composition of the culture medium in step (4) is MS + 0.4 mg·L -1 6-BA+0.08mg·L -1 NAA + 0.01~0.03mg·L -1 KH2PO4+0.01~0.03mg·L -1 (NH4)2SO4+0.003~0.005mg·L -1 VC+25g·L -1 sucrose; The cleaning in step (2) refers to first soaking the stem tip in Tween 20 for 3-5 minutes, then washing with running water for 10-15 minutes, then disinfecting with alcohol for 10 seconds, NaClO for 5 minutes, and finally washing with sterile water for 3-5 times; The recovery treatment in step (4) is to place the stem tip taken out of liquid nitrogen in a 25-30°C water bath for 3-5 minutes; The temperature during cultivation in step (4) is 20-25° C., the light intensity is 2000-3000 lx, the light duration is 14-16 h per day, and the relative humidity is 70%-80%.

2. The method for ultra-low temperature freezing and detoxification of potatoes according to claim 1, characterized in that: In step (1), the humidity of the potato pieces is maintained at 30% to 35%.

3. The method for ultra-low temperature freezing and detoxification of potatoes according to claim 1, characterized in that: The culture temperature in step (4) is 23±1°C, the light intensity is 2500 lx, the light duration is 15 h per day, and the relative humidity is 75%.