CN102017895B - Method for producing potato virus A (PVA)-free seedling by utilizing variable temperature detoxification technology - Google Patents
Method for producing potato virus A (PVA)-free seedling by utilizing variable temperature detoxification technology Download PDFInfo
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- CN102017895B CN102017895B CN2010105100493A CN201010510049A CN102017895B CN 102017895 B CN102017895 B CN 102017895B CN 2010105100493 A CN2010105100493 A CN 2010105100493A CN 201010510049 A CN201010510049 A CN 201010510049A CN 102017895 B CN102017895 B CN 102017895B
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Abstract
The invention discloses a method for producing a potato virus A (PVA)-free seedling by utilizing variable temperature virus elimination technology, which belongs to a part of a superior potato breeding system. The method for producing the PVA-free seedling by utilizing the variable temperature virus elimination technology comprises the following steps of: (1) obtaining a tissue culture seedling; (2) performing variable temperature treatment; (3) performing subculture and continuous variable temperature treatment; and (4) performing conventional propagation. In the method, the PVA-free seedling can be obtained by performing the variable temperature test treatment totally for 42 days. The method ensures high PVA-free seedling production efficiency, saves time, labor and energy sources, and promotes the health and sustainable development of potato industry, and is implemented by a simple technical procedure.
Description
Technical field
The present invention relates to the detoxification technology in a kind of germchit of potato of taking off poison breeding of method, especially relate to a kind of detoxification technology that adopts the alternating temperature detoxification technology to produce potato PVA detoxic seedling.
Background technology
Potato is one of the fourth-largest cereal crops of China, and it infects multiple virus and viroids in the course of cultivation easily, and phenotype produces complicated symptoms such as leaf roll, floral leaf, yellow, plant lotus throneization.In agricultural production, potato mainly enlarges production through vegetative propagation, and the viral level in the plant body can cause the degeneration of viral germ plasm resource by the generation accumulation, and underproduction amplitude reaches 40～70%.Because the global interchange of Content Germplasm Resources in Potato enlarges day by day; Caused the worldwide propagation of Potyvirus; What China was comparatively general has 7 kinds more than, and the perhaps compound separately perhaps physiological various symptoms that cause various phenotypes of multiple virus have had a strong impact on yield of potato and quality.
Potato PVA virus is that a few can cause that the potato main breed shows one of viral species of flower leaf paresthesia.Potato PVA virus is contaminated separately and is caused slight floral leaf or non-evident sympton; But it is often and other viruses, as potato virus X (Potato virus X, PVX), marmor upsilon (potato virus Y; PVY), marmor angliae (Potato virusM; PVM), (Potato virus S, PVS) etc. virus is compound infects potato virus S, leads to great drop in production.PVA can propagate with potato seed, and has at least 7 kinds of aphids to pass poison with the mode of perishability, and these aphids are at China's ubiquity, and is therefore should virus very big in China's diffusion possibility, has higher popular risk.
Utilizing virus-elimination seedlings to produce virus-free seed potato is the important means that improves potato yield and quality.The acquisition of traditional virus-elimination seedlings mainly is to adopt the mode of Shoot Tip Culture to cultivate virus-elimination seedlings, and then bears virus-free stem tuber, forms a complete detoxifying fast breeding system.The method that adopts tissue culture can not be removed potato PVA virus through the method that potato tubers or plant is changed into aseptic seedling, and " research of potato PVA test-tube plantlet preservation effect " delivered like plant protection journal in 2002 shows that the stem apex that cuts potato test-tube plantlet 0.6～1cm has identical infecting potential with the conventional PVA that cultivates preservation.Research through adopting Shoot Tip Culture and Shoot Tip Culture to combine the mode of thermal treatment, drug treating to obtain potato virus-free plantlet has had a lot; As: calendar year 2001 the Master's thesis " efficient the detoxification of potato stem apex, fast numerous and micro potato raising technology research " of Xibei Univ. of Agricultural & Forest Science & Technology show to handle for 34 ℃ and handle aseptic seedling 3 weeks and handled for 3 weeks with 38 ℃ and be with the bud potato tuberss better; Stripping stem apex after the thermal treatment is 80～82.5% to the PVY removal efficiency, is between 50～65% to the PVX removal efficiency; Be published in the medium optimization of the potato stem apex seedling differentiation " research " of Agricultural University Of Southwest's journal natural science edition in 2005,40 ℃ of (4h)+25 of Agricultural University Of Southwest's Master's thesis " research of potato stem tip detoxification system optimization " report in 2005 ℃ (20h) alternating temperature is handled the methods in 2 weeks and is heat-treated being with the sprout tuber stem; To the PLRV removal efficiency is 100%; To the PVY removal efficiency is 96.87%; To the PVX removal efficiency is 89.33%; To the PVS removal efficiency be 87.93%, 2006 year Xinjiang Agricultural Univ's Master's thesis " stem apex detoxify of potato, rejuvenation and RT-PCR detect ", be published in 2007 the Shanxi agricultural science " the potato stem apex is cultivated the influence factor test of virus elimination rate ", be published in " research of potato stem apex tissue culture detoxicating " of Jilin agricultural science etc. in 2007; And 40 ℃ of (4h)+16 of the Master's thesis of Shihezi Univ in 2007 " technical research of specialized potato high-efficiency detoxicating " report ℃ (20h) alternating temperature is handled 8 all band bud potato tuberss; Its PVX removal efficiency is 62%, is 78% to the PVY removal efficiency.The weak point of above potato detoxicating technology is: the one, and stem apex detoxify specification requirement technical merit is high, is skilled in technique; The 2nd, different potato kinds or strain require special-purpose prescription, and adaptability is not extensive; The 3rd, the cycle of producing virus-elimination seedlings is long, and the virus-elimination seedlings technology of disposable acquisition is little, is unfavorable for the large batch of production of short time.
Summary of the invention
The purpose of this invention is to provide a kind of on the basis that changes into test-tube plantlet or existing test-tube plantlet; Adopt the alternating temperature detoxification technology; Can reach reduce production costs, the saving of labor, save time, energy-conservation, simplify the production technology program; Produce the detoxification technology of potato PVA detoxic seedling efficiently, can adapt to the production of batch production.
The objective of the invention is to realize that through following technical scheme concrete steps are following:
(1) obtains tissue cultivating seedling;
(2) alternating temperature is handled: get the tissue cultivating seedling that above-mentioned steps obtains, it was handled 12 hours down at 36 ℃; Handled 12 hours down for 20 ℃, the photoperiod is 12h/12h, and intensity of illumination is 2000～4000 luxs; Handled for 4 weeks continuously;
(3) subculture and alternating temperature processing continuously: the tissue cultivating seedling of getting respectively after above-mentioned alternating temperature is handled saves position stem section according to the stem fragment position from first segment position, the second joint position and the 3rd of morphology upper end; Classification is inoculated on the MS medium of no hormone additive; Continue alternating temperature and handled for 2 weeks; The same step of alternating temperature treatment conditions (2), intensity of illumination are 2000～4000 luxs, and the caloric test processing that amounts to 42 days can obtain potato PVA detoxic seedling.
(4) conventional expansion is numerous: get the tissue cultivating seedling that amounts to 6 weeks of processing continuously, routine is cultivated behind the subculture, and cultivation temperature is 25 ℃, and illumination is 2000～4000 luxs, 10 every bottle; Photoperiod is illumination in 14 hours, 10 hours dark.
Be preferably in above-mentioned (4) step, increase to the tissue cultivating seedling after continuous 6 weeks of processing sampling and carry out virus and detect, as detect confirm virus-free after, then conventional cultivation behind the subculture again.
The method that obtains tissue cultivating seedling is the method for knowing in the prior art.But the survival rate of heat treatment period and tissue cultivating seedling has relation very significantly among the present invention.The combination of different temperature and time is influential to the growth of test-tube plantlet, because have only suitable temperature combinations, could suppress duplicating of virus, and the continued growth of potato tissue cultivating seedling, thereby take off virus.
In the article of existing report, there was similar alternating temperature detoxification treatment technology to come toxicity-removing white potato virus.Like potato leaf roll virus, potato PVY virus, potato PVX virus, potato PVS virus etc.But what the present invention and other relevant reports were significantly different is, the present invention is the detoxification material with the potato tissue cultivating seedling, need not strip the stem apex material and cultivate, and range of application is that research material is more extensive than other research reports to handle potato tubers; Next is that the research of alternating temperature processing parameter of the present invention and other reports differs greatly, and most importantly to potato PVA virus.
After virus detects, carry out illumination cultivation, such purpose is in order to enlarge the production of virus-free potato set.And main be that expanding numerous again after detecting through virus is in order to get rid of the potato tissue cultivating seedling that not detoxification is fallen.The light treatment of back does not belong to the scope of detoxification, but the multiplication technique of normal potato tissue cultivating seedling.
In embodiments of the present invention, explained and before carrying out alternating temperature detoxification step, to have detected earlier, seen whether the tissue cultivating seedling that is obtained is with PVA virus.And then carry out the alternating temperature detoxification.
Handle malicious in spite of illness potato tissue cultivating seedling with alternating temperature poison-removing method provided by the invention, can obtain potato PVA virus detoxic seedling, efficient is high, save time, laborsaving, energy savings, technical program are simple, promotes health, the sustainable development of Potato Industry.
Description of drawings
Fig. 1 is the present invention's design flow diagram;
Fig. 2 is the RT-PCR electrophoretogram of potato fields thinning, tissue cultivating seedling in the embodiment of the invention, wherein, and M:Mark; 1-2: negative control; 3-4: tissue cultivating seedling; 5-6: potted plant seedling; 7-8: field seedling; 9: positive control;
Fig. 3 is a RT-PCR electrophoretogram after the potato tissue cultivating seedling alternating temperature detoxification treatment in the embodiment of the invention, wherein, and 1: negative control; 2: alternating temperature is handled 4 all first segments position seedlings; 3: alternating temperature was handled for 4 weeks second and is saved the position seedling; 4: alternating temperature was handled for 4 weeks the 3rd and is saved the position seedling; 5: alternating temperature is handled 6 all first segments position seedlings; 6: alternating temperature was handled for 6 weeks second and is saved the position seedling; 7: alternating temperature was handled for 6 weeks the 3rd and is saved the position seedling; 8: positive control; M:Mark;
Fig. 4 is a RT-PCR electrophoretogram after the potato tissue cultivating seedling alternating temperature detoxification treatment in the embodiment of the invention, wherein, and 1: negative control; 2-16: alternating temperature is handled 6 all follow-up band tissue cultivating seedling; 17: positive control; M:Mark.
Embodiment
1) conversion of tissue cultivating seedling: the potato kind of the normal temperature of learning from else's experience storage is the stem tuber of middle stockaded village Calusena lansium; Be to handle after 7 days under 25 ℃ the dark condition to obtain tender shoots in temperature; Get tender shoots through 0.1% mercuric chloride sterilization 10 minutes, behind the aseptic water washing 4 times, 25 ℃ of cultivations on the MS of no hormone additive medium; Intensity of illumination is 2000～4000 luxs, obtains aseptic seedling after 5 days.
2) virus detects: the asexual seedling offspring to the same potato tubers of originating carries out the virus detection; Rely on the RT-PCR technology, come the band poison situation of test tubes seedling with PVA primer sequence Sense:5 '-TGCCCAGGTATGGTCTTCAACG-3 ' and Reverse:5 '-ACTTCGGTTACACCCCCTTCAC-3 '.Method for detecting virus carries out (X.Nie by the method for X.NIE; R.P.Sing.2001; A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids; Leaves, and tubers.Journal of Virological Methods 91 37-49) carries out.Testing result is seen Fig. 2.Fig. 2 shows that the handled test material of the present invention all is viruliferous before detoxification.
3) expansion of basic seedling is numerous: the tissue cultivating seedling expansion to detecting band PVA virus is numerous, and medium is the MS medium of no hormone additive, and cultivation temperature is 25 ℃, and illumination is 2000～4000 luxs, 10 every bottle; The group culture container is footpath, end 6cm, the vial of height 9cm.
4) alternating temperature is handled: get 10 bottles of alternating temperature processing in illumination box of 10 days tissue cultivating seedling of growth, and 36 ℃ of processing in 12 hours, 20 ℃ of processing in 12 hours, the photoperiod is 2000～4000 luxs for the 12h/12h intensity of illumination.Alternating temperature handled for 4 weeks continuously.
5) continuously alternating temperature is handled: get continuous alternating temperature respectively and handle 4 all tissue cultivating seedling and handled for 2 weeks from first segment position, the second joint position and the 3rd joint position stem section of morphology upper end according to the MS medium continuation alternating temperature that the classification of stem fragment position is inoculated in no hormone additive, intensity of illumination is 2000～4000 luxs.
6) virus detects with expansion numerous: get the tissue cultivating seedling of handling for 6 weeks altogether continuously, take a sample and carry out the virus detection, detection method is with the detection method in the 2nd step, and testing result is seen Fig. 3,4.The 2-4 bar swimming lane of Fig. 3 shows the present invention after handling through the continuous alternating temperature in 4 weeks, and potato PVA virus is not take off fully; And the 5-7 swimming lane shows that handling the potato material through continuous 6 all alternating temperatures takes off potato PVA virus.The difference of taking from continuous 6 weeks of processing that shows Fig. 4 saves the potato tissue cultivating seedling of position and has taken off virus fully through the virus detection.
The conventional cultivation behind the subculture, cultivation temperature is 25 ℃, illumination is 2000～4000 luxs, 10 every bottle; Photoperiod is illumination in 14 hours, 10 hours dark; The caloric test processing that amounts to 42 days can obtain potato PVA detoxic seedling.
Claims (2)
1. a method of utilizing the alternating temperature detoxification technology to produce potato PVA detoxic seedling comprises the steps:
(1) obtains tissue cultivating seedling;
(2) alternating temperature is handled: get the tissue cultivating seedling that above-mentioned steps obtains, it was handled 12 hours down at 36 ℃; Handled 12 hours down for 20 ℃, the photoperiod is 12h/12h, and intensity of illumination is 2000～4000 luxs; Handled for 4 weeks continuously;
(3) subculture and alternating temperature processing continuously: the tissue cultivating seedling of getting respectively after above-mentioned alternating temperature is handled saves position stem section according to the stem fragment position from first segment position, the second joint position and the 3rd of morphology upper end; Classification is inoculated on the MS medium of no hormone additive; Continue alternating temperature and handled for 2 weeks; The same step of alternating temperature treatment conditions (2), intensity of illumination are 2000～4000 luxs;
(4) conventional expansion is numerous: get the tissue cultivating seedling that amounts to 6 weeks of processing continuously, routine is cultivated behind the subculture, and cultivation temperature is 25 ℃, and illumination is 2000～4000 luxs, 10 every bottle; Photoperiod is illumination in 14 hours, 10 hours dark; The caloric test processing that amounts to 42 days can obtain potato PVA detoxic seedling.
2. the method for utilizing the alternating temperature detoxification technology to produce potato PVA detoxic seedling according to claim 1; It is characterized in that in above-mentioned (4) step; The tissue cultivating seedling sampling of increase after to continuous 6 weeks of processing carried out virus and detected, as detect confirm virus-free after, then conventional cultivation behind the subculture again.
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| CN2010105100493A CN102017895B (en) | 2010-07-01 | 2010-10-18 | Method for producing potato virus A (PVA)-free seedling by utilizing variable temperature detoxification technology |
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| CN102217544A (en) * | 2011-05-24 | 2011-10-19 | 崔刚 | Open tissue culture and factory fast propagation method for potatoes |
| 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 |
| CN103598097B (en) * | 2013-11-18 | 2015-08-12 | 绍兴市农业科学研究院 | The cultural method of virus-free snake gourd plantlet in vitro |
| CN105191793B (en) * | 2015-09-10 | 2017-12-19 | 宁波市农业科学研究院 | The efficient breeding method of purple tissue culture seedlings of potatoes |
| CN115281089B (en) * | 2022-08-08 | 2023-04-18 | 镇江金豆豆种业有限公司 | Method for improving potato virus disease removal efficiency |
| CN116267601A (en) * | 2022-12-19 | 2023-06-23 | 湖南农业大学 | Method for detoxification of potato stem tip by variable temperature heat treatment |
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| CN1709027A (en) * | 2005-06-16 | 2005-12-21 | 中国农业大学 | Potato seed breeding method |
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| CN1709027A (en) * | 2005-06-16 | 2005-12-21 | 中国农业大学 | Potato seed breeding method |
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| 张岳莉等.马铃薯脱毒种薯生产技术.《新疆农业科技》.2008,(第6期),第31-32页. * |
| 赵佐敏,艾勇.马铃薯组培脱毒试管苗繁育技术.《中国马铃薯》.2003,第17卷(第5期),第301-304页. * |
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