CN110393154B - Culture method of virus-free tissue culture seedlings of fresh-eating sweet potatoes - Google Patents
Culture method of virus-free tissue culture seedlings of fresh-eating sweet potatoes Download PDFInfo
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- CN110393154B CN110393154B CN201910773868.8A CN201910773868A CN110393154B CN 110393154 B CN110393154 B CN 110393154B CN 201910773868 A CN201910773868 A CN 201910773868A CN 110393154 B CN110393154 B CN 110393154B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a method for culturing virus-free tissue culture seedlings of fresh-eating sweet potatoes, which comprises the following steps: 1) Selecting potato seedlings without virus symptoms to be transplanted, culturing in an incubator at 34 ℃, spraying the potato seedlings with a compound antiviral agent at regular time every two days, and selecting proper stem tips for normal virus-free culture after culturing for 7 days; 2) Culturing and detecting the stem tip of the sweet potato to obtain qualified detoxified tissue culture seedlings; 3) And (3) quickly propagating the detected tissue culture seedlings in a special culture medium for cutting off the anti-pollution sweet potato tissue culture seedlings, and after a certain number of the tissue culture seedlings are obtained, moving out and culturing the tissue culture seedlings into domesticated seedlings for breeding stock. The culture method of the invention improves the detoxification efficiency of the fresh food variety detoxification tissue culture seedling by 40 percent, can carry out industrial production, reduce the cost, expand the application, prevent and control sweet potato virus diseases and ensure the healthy development of the sweet potato industry.
Description
Technical Field
The invention relates to a method for culturing a virus-free tissue culture seedling of a fresh-eating sweet potato.
Background
The sweet potato is used as the seventh food crop of the world and is mainly distributed in Asia, africa, latin America and other regions, and the planting area of China accounts for about 80 percent of the total number of the world. With the enhancement of health care consciousness of people, the edible sweet potatoes become the first choice of vast citizens, the price of fresh edible sweet potatoes is always high and stable, the planting enthusiasm of potato farmers is high, particularly, the sweet potato varieties including 32 common potatoes and 9 Longshu are good in potato shape, good in taste and high in commodity value, and are well loved by the potato farmers and vast consumers, the sweet potatoes have large-area planting in south and north potato areas, account for 30% of the planting of the fresh edible sweet potatoes, are main varieties with high yield and high efficiency in the current production, but the varieties have poor disease resistance and high virus sensitivity, and seriously affect the yield and the quality. The data show that the yield of the strain is reduced by 30 to 50 percent due to the virus.
The sweet potato stem tip tissue virus-free culture is a main measure for preventing and controlling sweet potato virus diseases and a main means for obtaining healthy seedlings, and in recent years, SPVD and leaf curl viruses of sweet potatoes spread rapidly in each sweet potato area, thereby causing serious threat to sweet potato production. The popularization of the virus-free seed potato seedlings can effectively prevent and control the harm of sweet potato virus diseases to production, but several old fresh food seeds are difficult to culture due to the growth characteristics of the fresh food seeds, the sweet potato tissue culture technology is applied for many years, and the industrial operation is difficult to carry out all the time, the main reasons are that the requirements on conditions are high in the tissue culture process, the virus-free efficiency is low, the pollution is easy to generate, once all the work is polluted, the work needs to be started from the beginning, the risk is high, the requirements on operators are high, the sweet potato virus-free tissue culture technology is not industrialized all the time, and the research aims to solve the problems of low virus-free efficiency and high pollution in the tissue culture process.
The method for improving the detoxification efficiency has a plurality of methods, but not all varieties are effective, so that the SPVD and the leaf curl virus of the sweet potatoes which have serious influence in the current production are very important to set different methods for improving the detoxification efficiency aiming at different varieties.
The method is a method for rapidly proliferating the detoxified test-tube plantlets in the detoxified tissue culture of sweet potatoes and is also a step which is most easily polluted in operation, if a substance is added into a culture medium, the method can well prevent pollution and can also be used for directly performing high-pressure disinfection, so that the risk of rapid propagation in the tissue culture is much less, but the addition amount of the antibacterial substance can directly influence the growth of tissue culture plantlets, so that the most suitable prevention and control agent and the amount of the antibacterial substance are researched to achieve the purposes of safety, labor saving and high efficiency, and provide possibility and safety guarantee for the industrialized production of the detoxified tissue culture. The phytofuling is a bactericide used for preventing pollution in a tissue culture experiment, the factory recommended concentration of the phytofuling is 2.5ml/L, but after actual operation, the concentration is found to have certain probability of yellowing, seedling death and the like when the phytofuling is used for tissue culture of sweet potatoes, great risk is caused to the breeding work of tissue culture seedlings, in order to obtain the optimal method and concentration which can be applied to the detoxification tissue culture of the sweet potatoes, a series of tests are carried out, and finally, the special culture medium formula for cutting off the detoxification tissue culture seedlings is determined.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for culturing virus-free tissue culture seedlings of fresh-eating sweet potatoes.
In order to realize the aim, the invention adopts a method for culturing the virus-free tissue culture seedling of the fresh-eating sweet potato, which comprises the following steps:
1) Selecting potato seedlings without virus symptoms to be transplanted, culturing in an incubator at 34 ℃, spraying the potato seedlings with a compound antiviral agent at regular time every two days, and selecting proper stem tips for normal virus-free culture after culturing for 7 days;
2) Culturing and detecting the stem tip of the sweet potato to obtain qualified detoxified tissue culture seedlings;
3) And (3) carrying out rapid propagation on the detected tissue culture seedlings in an efficient anti-pollution culture medium, and after a certain number of tissue culture seedlings are obtained, moving out and culturing the tissue culture seedlings to obtain domesticated seedlings for breeding original seeds.
As an improvement, the compound antiviral agent in the step 1) is prepared by compounding triazole and moroxydine.
As an improvement, the formula of the high-efficiency anti-pollution culture medium in the step 3) comprises MS, phytophthora root rot 1.0ml/L, sucrose 30g/L and agar 7.0g/L.
As an improvement, the yam seedlings in the step 1) adopt Pushu 32 or Longshu 9.
The culture method solves the problems of low detoxification efficiency and high pollution rate during cutting and rapid propagation of the main fresh-eating sweet potato variety Pushu 32 and Longshu 9 in sweet potato detoxification tissue culture at present in China, improves the detoxification efficiency of the fresh-eating sweet potato variety detoxification tissue culture seedlings by 40 percent, can perform industrial production, reduces the cost, expands the application, prevents and controls sweet potato virus diseases, and ensures the healthy development of the sweet potato industry. The invention determines a feasible technical route according to the actual situation in production, starts with the temperature for promoting growth and the medicament screening for inactivating viruses, determines a method for improving the detoxification efficiency, simultaneously screens the medicaments for high-pressure disinfection, determines the optimal concentration after screening the medicaments, finally obtains an efficient anti-pollution formula, and has good application effect in production.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It should be understood, however, that the detailed description herein of specific embodiments is intended to illustrate the invention and not to limit the scope of the invention.
Example 1
A method for culturing the virus-free tissue culture seedlings of fresh-eating sweet potatoes comprises the following steps:
because the tissue culture is performed by using the principle that the virus content of the plant growing points is low for virus-free culture, but some varieties grow slowly, the virus conduction is fast, and the virus-free efficiency is low, the invention uses high temperature to promote the growth points to grow rapidly, and then uses the screened antiviral agent to passivate the virus in the plant body, so as to limit the virus conduction speed and achieve the purpose of improving the virus-free efficiency;
specifically, selecting potato seedlings without virus symptoms to be pointed, culturing at high temperature in an incubator at 34 ℃, spraying screened special compound antiviral agents at regular time, and culturing for 7 days, and then selecting proper stem tips for normal virus-free culture;
the optimal temperature and antiviral agent screening process comprises the following steps:
1) Materials and methods
1.1 Design temperature and reference agents: setting the temperature at 32 deg.C, 34 deg.C, 36 deg.C, 38 deg.C, and the control at 28 deg.C;
the reference medicament comprises: the moroxydine hydrochloride, the triazole and the moroxydine are compounded (the most suitable concentration of each medicament is compounded according to the proportion of 1;
1.2 Test materials): 100 seedlings of No. 9 Ipomoea batatas and Ipomoea batatas 32 in field;
1.3 ) the specific steps:
a. planting potato seedlings with the length of 15cm (to heart leaves) in flowerpots respectively, planting 5 potato seedlings in each flowerpot, and planting 20 potato seedlings in each variety for later use;
b. adjusting an illumination incubator to 28 ℃, 32 ℃,34 ℃, 36 ℃ and 38 ℃ respectively, placing 5 pots at each temperature, spraying the prepared antiviral agent for 1 time each day (the spraying amount is based on the fact that the leaf surface of each plant is moist but not dropping liquid), observing the growth potential and the plant performance of the antiviral agent, investigating the plant height of the plant on the 8 th day, observing the growth point condition under a microscope, and determining the optimal temperature;
c. under the condition of the determined optimal temperature, carrying out virus passivation spraying on the potato seedlings for 7 days, taking plant stem tips treated by different medicaments, and disinfecting the stem tips on a superclean bench (a disinfection method comprises the steps of shearing 2-3 cm of terminal buds, shearing off outer leaves, washing with tap water for 20-30 min, soaking in 75% ethanol for 40s in the superclean bench, then soaking in 2% sodium hypochlorite solution for 5min, and washing with sterile water for 4-5 times);
d. peeling and inoculating stem tips (in a clean bench, peeling the treated stem tips under a 40-fold binocular stereovision dissecting mirror, using forceps and a dissecting needle or a scalpel to peel the stem tips until semicircular smooth growing points are exposed, using the scalpel or the dissecting needle to cut micro stem tips only containing 1-2 leaf primordia from a position of 0.1-0.3 mm, quickly inoculating the micro stem tips on a stem tip culture medium, carrying out in vitro culture, baking container mouths with an alcohol lamp and sealing, and marking numbers, variety names and inoculation time on the containers); culturing seedlings (the optimum temperature of the sweet potato tissue culture chamber is kept at 26-30 ℃, the relative humidity is less than or equal to 70 percent, the illumination time is 12-16 h/d, and the illumination intensity is 2000lx-3000 lx); and (4) detecting virus molecules (referring to agricultural industry standards). The test results are shown in table 1 below.
TABLE 1 influence of different temperatures on growth and seedling rate of sweet potato variety
Analysis table 1 shows that under different temperature culture conditions, the potato seedlings cultured at 34-36 ℃ have the best growth potential, and the stem tips are most easy to tip and culture into seedlings; the growth is fast at 38 ℃, but the effect of tip pulling of the stem tip is not ideal, and the seedling rate is low; the growth is slow below 34 ℃, and the purpose of growth promotion can not be achieved.
1.4 Moroxydine hydrochloride, the compound of triazole and moroxydine, the compound of 4 percent of pyritinomycin, the compound of moroxydine and albendazole, four medicaments are respectively sprayed on potato seedlings, and virus detection is carried out after stem tip culture, as shown in the following table 2.
TABLE 2 influence of different agents on growth and seedling rate of sweet potato variety
The results in Table 2 are combined to show that: the four medicaments have certain effects, but in terms of the growth vigor of potato seedlings, the stem tip seedling rate and the virus detection rate, the sterilization effect of the compound spraying of triazole and moroxydine (the compound is prepared according to the optimal concentration of each medicament and the proportion of 1.
Carrying out rapid propagation on the detected tissue culture seedlings in a special culture medium for cutting off the anti-pollution sweet potato tissue culture seedlings, and after a certain number of the tissue culture seedlings are obtained, moving out the tissue culture seedlings to be cultured into domesticated seedlings for breeding original stocks;
the culture medium screening method comprises the following specific steps:
1) The material and the method are as follows:
1.1 Test agents): the medicines such as the ceromycin, the jinggangmycin, the carbendazim and the like are recommended according to the instructions, and no medicine is added as a control;
1.2 Test materials): 10 bottles of healthy Longshu No. 9 test-tube plantlets;
1.3 ) the specific steps:
a. taking 25 empty culture bottles and 1 250ml beaker for later use;
b. preparing 1.25 liters of MS culture medium, adding 250ml of MS culture medium into a beaker, adding 0.625ml of the carbendazim solvent into the beaker by using a liquid transfer gun, uniformly stirring, subpackaging in 5 culture bottles, and subpackaging the residual liquid into 20 culture bottles according to about 50ml of the residual liquid in each bottle and marking;
c. placing 25 culture bottles filled with culture medium into an autoclave for sterilization, sterilizing for 15-20 minutes when the temperature in the autoclave reaches 121 ℃ under the pressure of 0.1MPa, filtering and sterilizing other medicaments before solidification, and then adding the medicaments into the culture bottles;
d. taking out the experimental seedlings in the tissue culture bottle on a clean bench, cutting the experimental seedlings according to a cutting propagation mode, then carrying out classification treatment, dividing the experimental seedlings into three types, namely young stems, main stems and old stems, implanting the experimental seedlings into a culture medium, planting four plants in each bottle (ensuring that at least one young stem is in each bottle, and the rest three are main stems or old stems), finishing 5 treatments, and culturing under normal culture conditions;
e. on days 3, 7, 14, 21, 28 of the experiment, the growth of each treated shoot was recorded: the pollution condition, the type, death state and quantity of the pollution bacteria and the current situation of the experimental seedlings during recording are briefly described. The specific test results are shown in table 3 below.
TABLE 3 contamination questionnaire after different antiseptics cutting propagation culture
The results in table 3 show that: the addition of different antibacterial agents has certain effects, the cerumen, the jinggangmycin and the carbendazim have certain pollution rates which are respectively 10%, 20% and 25%, only the carbendazim has no pollution plants, and the other three antibacterial agents all need to be subjected to secondary operation on an ultraclean workbench, and only the carbendazim can be subjected to high-pressure sterilization, so the carbendazim can be used for sweet potato tissue culture, but when the recommended concentration of the carbendazim is used, the seedlings are partially dead seedlings although no pollution plants exist, and an optimal concentration test needs to be carried out to find an optimal formula.
Therefore, further optimal concentration screening is required:
1) The material and the method are as follows:
1.1 Drug concentration): the plant-cultivating-agent is used for controlling 5 gradients (0.5 ml/L, 1.0ml/L, 1.5ml/L, 2.0ml/L, 2.5ml/L and the like) (taking the factory-recommended concentration of the agent as the highest point, sequentially reducing 0.5ml/L until 0.5ml/L, setting 5 repetitions for each concentration, and taking 25 bottles in total), and a conventional MS culture medium is used as a control;
1.2 Test materials): 10 bottles of healthy Longshu No. 9, pushu No. 32, shanghai Shu 19 and Shulvyi tissue culture bottle seedlings respectively;
1.3 ) the specific steps:
a. taking 120 empty culture bottles and 24 250ml beakers for later use;
b. preparing 6.0 liters of MS culture medium, taking 20 beakers during preparation, respectively adding 0.125ml, 0.250ml, 0.375ml, 0.500ml and 0.625ml of the carbendazim solvent into 4 beakers respectively by using a liquid transfer gun, then evenly distributing the prepared 6.0 liters of MS culture medium into 24 beakers, each 250ml of the culture medium is distributed into 120 empty culture bottles according to about 50ml of each bottle after being evenly stirred, and marking;
c. wrapping the needed tools with newspaper, putting the wrapped tools and 120 culture bottles filled with culture medium into an autoclave for disinfection;
d. taking out experimental seedlings in bottles on a clean bench, cutting the experimental seedlings according to a cutting propagation mode, then carrying out classification treatment, dividing the experimental seedlings into three types, namely young stems, main stems and old stems, implanting the experimental seedlings into a culture medium, planting four plants in each bottle (ensuring that at least one young stem is in each bottle, and the rest three are main stems or old stems), finishing treatment of 6 varieties, and culturing under normal culture conditions;
e. growth was recorded for each treated shoot on days 3, 7, 14, 21, 28, 60 of the experiment: the survival status, the rooting status and quantity, the sprouting status and quantity, the death status and quantity and the status of the experimental seedlings during recording are briefly described. The results are shown in Table 4 below.
TABLE 4 survey results of different species and concentrations of carbendazim on growth
The results in table 4 show that: the sensitivity of different cultivars to carbendazim is different, the different concentrations of the same cultivar are different, the contamination rate is increased when the carbendazim concentration is only 0.5ml/L, and the yellow seedling rate and the death rate of tissue culture seedlings are increased when the carbendazim concentration reaches 2.5ml/L, and from the results, all cultivars are better at 1.0ml/L or 1.5ml/L, so that the selection of carbendazim at 1.0-1.5ml/L is most effective.
The research result shows that: when sweet potato virus-free tissue culture is cut off and rapidly propagated, a certain amount of carbendazim is added to achieve normal growth, and the formula of the culture medium which can achieve zero pollution is as follows: MS + carbendazim 1.0-1.5ml/L + sucrose 30g/L + agar 7.0g/L.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (1)
1. A method for culturing the virus-free tissue culture seedlings of fresh-eating sweet potatoes is characterized by comprising the following steps:
1) Selecting a potato to be pointed to be 32 or 9 without virus symptoms, culturing in an incubator at 34 ℃, spraying an antiviral agent compounded by triazole and moroxydine at regular time every two days, and selecting a proper stem tip for normal detoxification culture after culturing for 7 days;
2) Culturing and detecting the stem tip of the sweet potato to obtain qualified detoxified tissue culture seedlings;
3) Carrying out rapid propagation on the detected tissue culture seedlings in an efficient anti-pollution culture medium, and after a certain number of tissue culture seedlings are obtained, moving out and culturing the tissue culture seedlings to obtain domesticated seedlings for breeding original seeds; the formula of the high-efficiency anti-pollution culture medium comprises MS, carbendazim 1.0ml/L, sucrose 30g/L and agar 7.0g/L.
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| KR100604186B1 (en) * | 2004-08-25 | 2006-07-25 | 고려대학교 산학협력단 | DNA nucleotide sequence for directing high level expression in plant storage root plasmid vector using the same and transient assay method in plant storage root using the same vector |
| CN101584301B (en) * | 2009-06-05 | 2011-09-28 | 海南省农业科学院粮食作物研究所 | Method for culturing detoxified seedling by sweet potato stem tip |
| CN102599057A (en) * | 2012-03-22 | 2012-07-25 | 华中农业大学 | Method for removing potato virus by virazole |
| CN103651112B (en) * | 2012-09-06 | 2016-06-15 | 胡荣山 | A kind of potato haulm point peels off the method cultivating detoxic seedling |
| CN106233977A (en) * | 2016-08-03 | 2016-12-21 | 天津丰华裕隆农业发展有限公司 | A kind of cultural method of dragon No. 9 Virus-free Sweetpotatos of potato |
| CN107409669A (en) * | 2017-04-22 | 2017-12-01 | 蚌埠市乔峰农业蔬菜专业合作社 | A kind of sweet potato is heat-treated poison-removing method |
| CN107736380B (en) * | 2017-11-10 | 2020-07-31 | 郑州师范学院 | Composition for preventing and treating sweet potato virus diseases |
| CN108935108A (en) * | 2018-09-29 | 2018-12-07 | 河南云帮农业科技有限公司 | A kind of sweet potato tissue-cultured seedling detoxification tissue culture method |
| CN109392682A (en) * | 2018-12-13 | 2019-03-01 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A kind of sweet potato tissue culture method for transplanting of high-survival rate |
| CN110810247A (en) * | 2019-12-05 | 2020-02-21 | 天津丰华裕隆农业发展股份有限公司 | Sweet potato stem tip detoxification and breeding method |
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