CN101849509B - Root tip detoxification and rapid propagation technology for purple potatoes - Google Patents
Root tip detoxification and rapid propagation technology for purple potatoes Download PDFInfo
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Abstract
The invention discloses root tip detoxification and rapid propagation technology for purple potatoes and belongs to the technical field of plant detoxification and tissue culture rapid propagation. The method comprises the following steps of: (1) preparing a culture medium; (2) performing root tip detoxification and rapid propagation; and (3) performing multi-reverse transcription-polymerase chain reaction (RT-PCR) synchronous and rapid detection on complex virus infection of a purple potato test tube plantlet or a test tube mini-tuber and the like. In the technology, each level of culture medium is adjusted according to the characteristic of the purple potato and conventional stem tip detoxification is changed into root tip detoxification so as to simplify operation process, avoid high pollution rate and improve working efficiency. Moreover, the technology has the advantages of good inoculant differentiation, high test tube plantlet propagation speed, complete detoxification, high mini-tuber growth vigor, monthly mini-tuber reproduction rate of 4 to 6 times and mini-tuber rooting rate and transplanting survival rate of over 98 percent. The technology can be popularized in regions which are suitable for the growth of purple potatoes.
Description
Technical field
The present invention relates to plant toxic and group culturation rapid propagating technology field, be specifically related to tip of a root detoxification and the group culturation rapid propagating technology of a kind of purple potato.
Background technology
Purple-colored potato belongs to the Solanaceae annual crop, originates in Australia.The long ellipse of potato type, epidermis is smooth, and potato meat is atropurpureus, is rich in anthocyan, Vc, carotin etc., has edible, medicinal function concurrently.Anthocyan is the water colo(u)r that is present in the plant; Belonging to flavonoids, is the disease preventing and treating that present scientific circles find, safeguard human health the most directly, the most effective, safest free radical scavenger; Its ability of removing free radical is 20 times of VC, 50 times of VE.Anthocyan has small molecule structure, is that unique ability sees through the material that blood-brain barrier is removed free radical protection brain cell, can reduce some harm that antibiotic is given human body simultaneously.Anthocyan is except that having the inhibitory action carcinogen, and all right enhancing human immune delays senility, and builds up health enhance eyesight.In addition, purple potato pulp content of starch is high, and mouthfeel is better, and quality is splendid, is fit to deep processing.Therefore, purple potato has excellent research exploitation and comprehensive utilization value.
This project team member has introduced 21 purple horse official seal potato kinds from Australia after 2000, has therefrom cultivated 6 strains through organizing subculture, transplanting the earth in 2006; Carry out systematic breeding, the acquisition eye is shallow, epidermis is smooth, yellowish pink is dark purple, and mouthfeel is good; And purple No. 1 new varieties in the river in Zhejiang Province that is fit to processing, and utilize tip of a root detoxification technology to obtain detoxification test tube plantlet, carry out a large amount of fast breedings in the laboratory through tissue culture technique; So far detoxification test tube plantlet 15.1 ten thousand strains have been accomplished in the laboratory; Carried out the test-tube seedling transplanting test simultaneously, transplanting survival rate has reached more than 98%, produces 320,000 of test tube potato breeder's stocks; Mu surplus the breeding original seed 129 has tentatively been found out the culture technique of the suitable purple potato of a cover.
But plant in the process with tissue-culturing rapid propagation in the examination of the field of purple potato, find to have following two problems very soon:
The one, purple potato fields inter-planting strain virus morbidity is serious.Through detecting, purple potato virus disease is that wherein, 9 kinds is the virus that parasitizes potato specially by due to 18 kinds of compound infecting of virus, and 9 kinds is the virus from other host plant in addition.(the Potato Virus Y of marmor upsilon wherein; PVY), corium solani (Potato Leafroll virus, PLRV), potato virus X (Potato Virus X; PVX) and potato virus S (Potato Virus S; PVS), these 4 kinds is the most serious virus of harm potato (comprising purple potato), is the emphasis viral species of the necessary plant quarantine of Ministry of Agriculture's strict regulations; Simultaneously these viruses all have again be difficult to separate, the characteristics of purifying.To these characteristics; This laboratory has been studied and has successfully been adopted multiple RT-PCR detection technique simultaneously and rapidly; Purple potato is implemented highly sensitive virus to be detected; This technology need not to separate, purifying, can be fast whether purple potato plant or test-tube plantlet be implemented with these 4 kinds main viruses and wherein which kind of viral detection of band.
The 2nd, purple potato tissue-culturing rapid propagation pollution rate is high.Under the situation that adopts traditional horse official seal potato stem apex detoxify; Have that operation easier is big, inefficiency is (at microscopically; Peel off surplus in the of ten layer continuously with operation tool and be wrapped in the outer stem sheet of stem apex), cause being difficult to thoroughly peel off and sterilize, general tissue culture seedling pollution rate is up to about 60%; It is slower with the detoxification test tube plantlet reproduction speed also to have explant differentiation speed simultaneously, and the micro potato growing way is weak and transplant problems such as the per mu yield of land for growing field crops production potato is on the low side.
To at present, be the technology that explant carries out detoxification and tissue-culturing rapid propagation about adopting the tip of a root with purple potato, not seeing as yet at present has reported in literature.
Summary of the invention
The present invention seeks to; Train the existing complex operation of traditional stem apex detoxify technology, low, the problems such as difficulty is thorough, tissue culture seedling pollution rate height of sterilizing of efficient to horse official seal potato group; Propose a kind of easy and simple to handle, efficient is high, sterilization is prone to thoroughly, the tissue culture seedling pollution rate is low, reproduction speed is fast, seedling matter detoxification of a kind of purple potato tip of a root and fast breeding technique good and that match with virus detection techniques.
Detoxification of a kind of purple potato tip of a root and fast breeding technique, this technology is carried out according to the following steps:
(1) culture medium preparation comprises that component and every liter of contained weight thereof of minimal medium and each stage medium is:
1) minimal medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
350mg/L+KH
2PO
4500mg/L+ sucrose 25g/L+ agar 7-9g/L, pH 5.6-5.8;
2) tip of a root inducing culture: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.5-1mg/L+NAA0.3-0.7mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
3) proliferated culture medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.2-0.5mg/L+NAA0.2-0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
4) subculture growth medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/0.5L+BA0.2-0.5mg/L+NAA0.1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
5) root media: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.1-0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
6) micro potato inducing culture: adopt potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.1-0.5mg/L+6BA0.5-1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
(2) tip of a root detoxification is with numerous soon:
1) material preliminary treatment and tip of a root explant selection, sterilization: purple seed potato is put into the sterilization fine sand heat-treat thoughtful the taking out of 6-12 in 37 soil under 2 ℃ and bear root system; Get the long main root tip of a root of 1-2cm, after 70% alcohol disinfecting 30 seconds, 0.1% mercuric chloride add a soil temperature and shake sterilization 8 minutes and aseptic washing 3-5 time, at microscopically with the tip of a root cutting-out of the long 0.1-0.2mm of front end as explant, subsequent use;
2) tip of a root regeneration induction plant cultivation: explant is seeded in step (1) 2) on the tip of a root inducing culture; Under 25 2 ℃ in soil, light intensity 1000lux, 16 hours/d of illumination, cultivate; The constant light intensity of all the other conditions of back is strengthened to 2000lux extremely; Be strengthened to 4000lux after six weeks, be attenuated to 1500lux after eight weeks and continue down to cultivate until induce the regeneration plant young shoot from the tip of a root, this stage need cultivate 3.5-4.5 month altogether;
3) young shoot enrichment culture: young shoot is seeded in step (1) 3) on the proliferated culture medium, in 2 ℃ in 25 soil, light intensity 2000-3000lux, 16 hours/d of illumination cultivate 1 month to growing up to the long seedling of 8-10cm down;
4) subculture grown cultures: seedling is cut into the long segment of 2-2.5cm is seeded in step (1) 4) on the subculture growth medium,, be cultured under light intensity 2000-3000lux, the 16 hours/d of illumination and grow up to the long seedling of 8-10cm in 2 ℃ in 25 soil; It is numerous that every later on separated 3-4 week is carried out a subculture expansion by the same technology, counts requirement up to satisfying seedling;
5) culture of rootage: the subculture seedling that increases is inoculated into step (1) 5) on the root media, in 2 ℃ in 25 soil, light intensity 2000-3000lux, 16 hours/d of illumination cultivate down, and sending out roots after 1 week to be tied to form is complete test-tube plantlet;
6) cultivation of test tube micro potato: will grow tall to the test-tube plantlet of 8-10cm, and be cut into the stem section that has 1-2 blade and axillalry bud, and be seeded to the step (1) 6 that is loaded in the triangular flask by every bottle of 5-6 stem section) on the micro potato inducing culture; Under 22 ℃, dark condition, cultivating 10-15 days beginnings forms micro potato, and the same terms continues to cultivate 6-8 down can gather in the crops micro potato after week;
(3) virus of purple potato test-tube plantlet or test tube micro potato detects:
1) preparation of purple Potyvirus ssRNA: choose purple potato marmor upsilon (Potato Virus Y; PVY), corium solani (Potato Leafroll virus; PLRV), potato virus X (Potato Virus X; PVX) and potato virus S (Potato Virus S, blade PVS), leaf stalk, stem or potato tubers are material, put into 100mL0.5Triton X-100 0.4%Na
2SO
3Viral leaching liquor in; Put 37 ℃ of lixiviate 20min, get supernatant behind the room temperature lixiviate 1h; Through 12, behind the centrifugal 10min of 000r/min centrifuge, get supernatant and be stored in-20 ℃, subsequent use;
2) cDNA is synthetic: design potato virus X (Potato Virus X according to Potyvirus CP gene order; PVX) marmor upsilon (Potato Virus Y; PVY) corium solani (Potato Leafroll virus, PLRV) and the Auele Specific Primer of potato virus S (Potato Virus S) right; Synthetic each the reaction cumulative volume of cDNA is 10 μ L, and reaction reagent comprises virus total RNA 2.5 μ L, and 1 * MMLV contains 3mmol/L MgCl
2Buffer solution, 1.5mmol/L dNTPs, 2.5U RNA enzyme inhibitor, 50U MMLV reverse transcriptase MMLVRT, 0.5 μ mol/L antisense primer; Response procedures is 25 ℃ of incubation 10mim, 42 ℃ of reverse transcription 45mim, 95 ℃ of deactivation 2mim;
Described Potyvirus Auele Specific Primer is to being:
PVS upstream primer P1:5 '-ATGCCGCCCAAACCGGATCCGTCA-3 '
PVS downstream primer P2:5 '-TCATTGGGTGATTGCATTACGGTG-3 '
PVY upstream primer P3:5 '-GCAAATGACACAATCGATGCAGGA-3 '
PVY downstream primer P4:5 '-TGGTGTTCGGTGATGTGACCT-3 '
PVX upstream primer P5:5 '-GAGCACAACACAGACCACAG-3 '
PVX downstream primer P6:5 '-GACTAGTTATGGTGGTGGGAGAG-3 '
PLRV upstream primer P7:5 '-CCCTTCGCAGGCGCGCTAACA-3 '
PLRV downstream primer P8:5 '-GGGGTCCAACTCATAAGCGAT-3 ';
3) pcr amplification: PCR reaction cumulative volume is 25 μ L, gets the synthetic cDNA of 4 μ L reverse transcriptions as the PCR reaction template; The reaction system of multiplex PCR comprises: 1 * PCR reaction buffer, and 3.5mmol/L dNTPs, Taq archaeal dna polymerase 2U, the 0.5mmol/L Auele Specific Primer is right; Amplification program is 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, and 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 10min; The volume ratio that wherein adds primer in the PCR reaction is PVX: PVY: PLRV: PVS=0.4: 0.5: 0.9: 1.0;
4) observe electrophoretic band: have with electrophoretic band contrast, the purple potato test-tube plantlet of establishment or test tube micro potato and do not infect corresponding virus.
The invention has the beneficial effects as follows:
One, the present invention is improved to tip of a root detoxification with traditional stem apex detoxify, some excellent effect below having produced in purple potato group training work:
1, processing ease, efficient significantly improve: because of " stem apex " of horse official seal potato is made up of the stem apex at layer stem sheet parcel center surplus peripheral ten, in the past at microscopically, divesting surplus in the of ten a layer stem sheet continuously with operation tool is the big and very low work of efficient of a difficulty; And outside " tip of a root " of horse official seal potato be directly exposed to, the tip that cuts its tip of a root at microscopically just seemed that very easily, efficient can improve tens times to hundreds of times;
2, sterilization is thorough, and pollution rate is low: stem apex detoxify is because its peripheral stem sheet is difficult for Ex-all, and sterilization also just is difficult to thoroughly cause the tissue culture seedling pollution rate often up to about 60%; And tip of a root detoxification sees Table 1 because its peripheral no any parcel is sterilized easily and thoroughly so its tissue culture seedling pollution rate can drop to 13.3%();
3, improved the quality of group training seedling: the present invention is that explant has not only successfully been cultivated purple potato group training seedling with the tip of a root; And clear and definite by the test back, tip of a root group training seedling breaks up on speed, plant regeneration speed, test-tube plantlet reproduction speed and the micro potato output be improved (seeing Table 1) than stem apex group training seedling at explant;
4, clear and definite after the field planting test, its rooting rate of tip of a root tissue cultural seedlings of free and transplanting survival rate are up to more than 98%, and potato output also slightly improves than the stem apex detoxify tissue cultivating seedling.
Two, the present invention is directed to the especially characteristics of its tip of a root of purple potato, medium at different levels have been done adaptation and adjustment, guaranteed the success of tip of a root group training.
Three, the present invention to tip of a root explant completely disinfectant measure detect virus technology simultaneously and rapidly with unique multiple RT-PCR and combine; Guaranteed zero band poison of purple potato tissue cultivating seedling, thereby laid a good foundation for the high-quality of purple potato tissue cultivating seedling, safety, high yield and large tracts of land popularization.
Embodiment
Through following examples the present invention is done further detailed description, but content of the present invention is not limited thereto.
Embodiment 1: (detoxification of a kind of purple potato tip of a root and fast breeding technique 1)
(1) culture medium preparation comprises that component and every liter of contained weight thereof of minimal medium and each stage medium is:
1) minimal medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
350mg/L+KH
2PO
4500mg/L+ sucrose 25g/L+ agar 7g/L, pH 5.8;
2) tip of a root inducing culture: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.5mg/L+NAA0.7mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
3) proliferated culture medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.5mg/L+NAA0.2mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
4) subculture growth medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/0.5L+BA0.2mg/L+NAA0.1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
5) root media: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
6) micro potato inducing culture: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.1mg/L+6BA0.75mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
Described potato-based basal culture medium is: potato 200g/L+ sugarcane 10g/L+ agar 20g/L;
(2) tip of a root detoxification is with numerous soon:
1) material preliminary treatment and tip of a root explant selection, sterilization: with purple seed potato put into the sterilization fine sand in 37 soil heat-treat under 2 ℃ 6-12 thoughtful take out to take root be; Get the long main root tip of a root of 1-2cm, after 70% alcohol disinfecting 30 seconds, 0.1% mercuric chloride add a soil temperature and shake sterilization 8 minutes and aseptic washing 3-5 time, at microscopically with the cutting-out of the front end 0.1-0.2mm tip of a root as explant, subsequent use;
2) tip of a root regeneration induction plant cultivation: explant is seeded in step (1) 2) on the tip of a root inducing culture; Under 25 2 ℃ in soil, light intensity 1000lux, 16 hours/d of illumination, cultivate; The constant light intensity of all the other conditions of back is strengthened to 2000lux extremely; Be strengthened to 4000lux after six weeks, be attenuated to 1500lux after eight weeks and continue down to cultivate until induce the regeneration plant young shoot from the tip of a root, this stage need cultivate 3.5-4.5 month altogether;
3) young shoot enrichment culture: the regeneration plant young shoot is seeded in step (1) 3) on the proliferated culture medium, in 2 ℃ in 25 soil, light intensity 2000-3000lux, 16 hours/d of illumination cultivate 1 month to growing up to the long seedling of 8-10cm down;
4) subculture grown cultures: seedling is cut into the long segment of 2-2.5cm is seeded in step (1) 4) on the subculture growth medium,, be cultured to the long seedling of 8-10cm under light intensity 2000-3000lux, the 16 hours/d of illumination in 2 ℃ in 25 soil; It is numerous that every later on separated 3-4 week is carried out a subculture expansion by the same technology, counts requirement up to satisfying seedling;
5) culture of rootage: subculture is expanded numerous seedling be inoculated into step (1) 5) on the root media, in 2 ℃ in 25 soil, light intensity 2000-3000lux, 16 hours/d of illumination cultivate down, and sending out roots after 1 week to be tied to form is complete test-tube plantlet;
6) cultivation of test tube micro potato: will grow tall to the test-tube plantlet of 8-10cm; Be cut into the stem section that has 1-2 blade and axillalry bud; Be seeded to the step (1) 6 that is loaded in the triangular flask by every bottle of 5-6 stem section) on the micro potato inducing culture; Under 22 ℃, dark condition of culture, cultivate 10 days to 15 days beginnings formation micro potato; The same terms continues to cultivate 6-8 down can gather in the crops micro potato after week;
(3) virus of purple potato test-tube plantlet detects:
1) preparation of purple Potyvirus ssRNA: choose purple potato marmor upsilon (Potato Virus Y; PVY), corium solani (Potato Leafroll virus; PLRV), potato virus X (Potato Virus X; PVX) and potato virus S (Potato Virus S, blade PVS), leaf stalk, stem or potato tubers are material, put into 100mL0.5Triton X-100 0.4%Na
2SO
3Viral leaching liquor in; Put 37 ℃ of lixiviate 20min, get supernatant behind the room temperature lixiviate 1h; Through 12, behind the centrifugal 10min of 000r/min centrifuge, get supernatant and be stored in-20 ℃, subsequent use;
2) cDNA is synthetic: design potato virus X (Potato Virus X according to Potyvirus CP gene order; PVX) marmor upsilon (Potato Virus Y; PVY) corium solani (Potato Leafroll virus, PLRV) and the Auele Specific Primer of potato virus S (Potato Virus S) right; Synthetic each the reaction cumulative volume of cDNA is 10 μ L, and reaction reagent comprises virus total RNA 2.5 μ L, and 1 * MMLV contains 3mmol/L MgCl
2Buffer solution, 1.5mmol/L dNTPs, 2.5U RNA enzyme inhibitor, 50U MMLV reverse transcriptase MMLVRT, 0.5 μ mol/L antisense primer; Response procedures is 25 ℃ of incubation 10mim, 42 ℃ of reverse transcription 45mim, 95 ℃ of deactivation 2mim;
Described Potyvirus (PVX, PVY, PLRV, PVS) Auele Specific Primer is to being:
3) pcr amplification: PCR reaction cumulative volume is 25 μ L, gets the synthetic cDNA of 4 μ L reverse transcriptions as the PCR reaction template; The reaction system of multiplex PCR comprises: 1 * PCR reaction buffer, and 3.5mmol/L dNTPs, Taq archaeal dna polymerase 2U, the 0.5mmol/L Auele Specific Primer is right; Amplification program is 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, and 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 10min; The volume ratio that wherein adds primer in the PCR reaction is PVX: PVY: PLRV: PVS=0.4: 0.5: 0.9: 1.0;
4) observe electrophoretic band: with the electrophoretic band contrast, establish this routine purple potato test-tube plantlet and do not infect corresponding virus.
Embodiment 2: (detoxification of a kind of purple potato tip of a root and fast breeding technique 2)
(1) culture medium preparation comprises that component and every liter of contained weight thereof of minimal medium and each stage medium is:
1) minimal medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
350mg/L KH
2PO
4500mg/L+ sucrose 25g/L+ agar 8g/L, pH 5.7;
2) tip of a root inducing culture: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.8mg/L+NAA0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
3) proliferated culture medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.35mg/L+NAA0.35mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
4) subculture growth medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/0.5L+BA0.35mg/L+NAA0.1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
5) root media: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.3mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
6) micro potato inducing culture: adopt potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.3mg/L+6BA0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
The tip of a root detoxification of step (2) with fast numerous in, its step, technology are with embodiment 1;
The virus of step (3) is material with purple potato test tube micro potato in detecting, and all the other steps, technology are with embodiment 1, and the clear and definite purple potato test tube micro potato of testing result does not infect corresponding virus.
Embodiment 3: (detoxification of a kind of purple potato tip of a root and fast breeding technique 3)
(1) culture medium preparation comprises that component and every liter of contained weight thereof of minimal medium and each stage medium is:
1) minimal medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
350mg/L KH
2PO
4500mg/L+ sucrose 25g/L+ agar 9g/L, pH 5.6;
2) tip of a root inducing culture: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA1.0mg/L+NAA0.3mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
3) proliferated culture medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.2mg/L+NAA0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
4) subculture growth medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/0.5L+BA0.5mg/L+NAA0.1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
5) root media: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
6) micro potato inducing culture: adopt potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.5mg/L+6BA1.0mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
The tip of a root detoxification of step (2) with fast numerous in, its step, technology are with embodiment 1;
The virus of step (3) is material with purple potato test-tube plantlet in detecting, and all the other steps, technology are with embodiment 1, and the clear and definite purple potato test-tube plantlet of testing result does not infect corresponding virus.
Test Example: (the effect comparison test of purple potato stem apex detoxifying fast breeding and tip of a root detoxifying fast breeding)
This test between academy of agricultural sciences, Zhejiang Province virology and biotechnology research institute satellite car-academy of agricultural sciences, Ningbo City, Zhejiang Province tissue culture in intracardiac carrying out.
On July 11st, 2005 was put into the sterilization fine sand with the purple No. 1 purple seed potato in river in Zhejiang Province, under 2 ℃ in 37 soil, heat-treated thoughtful the taking out of 6-12 and bore root system; Get 158 at the long main root tip of a root of 1-2cm, add a soil temperature through 70% alcohol disinfecting 30 seconds, 0.1% mercuric chloride and shake after sterilization 8 minutes and aseptic washing accomplish sterilization 5 times, at microscopically the tip of a root of its front end 0.1-0.2mm is downcut as the tip of a root group training and use explant; Other gets 518 of the purple No. 1 purple potato stem apexs in river in Zhejiang Province; Press with quadrat method after sterilization treatment; Microscopically in desinfection chamber; Divest continuously with operation tool one by one and be wrapped in it surplus outer ten behind layer stem sheet, extract the stem apex of long 0.1～0.3mm in it, and select 158 comparatively complete persons of structure as the stem apex group training use explant;
With embodiment 1 each stage medium as medium; And by this routine step, technology; The above-mentioned tip of a root and the training of stem apex group are inoculated, managed and cultivate with explant, at last the micro potato kind is gone into to isolate the solarium and produce potato production, the comparing result of various test stages is seen table 1.
Visible by table 1, the tip of a root pollutes 21, and pollution rate is 13.3%, and stem apex pollutes 94 pollution rates up to 59.5%; The average callus of the tip of a root is 27 days, and stem apex is 28 days; The tip of a root forms regeneration plant average out to 123 days, and the stem apex regeneration plant is 128 days; The monthly average reproduction speed of tip of a root seedling is 4.8 times, and the monthly average reproduction speed of stem sharp has only 4.3 times; The average micro potato of tip of a root tissue cultivating seedling is 3.1/strain, and the average micro potato of stem apex tissue cultivating seedling is 2.4/strain; Tip of a root seedling land for growing field crops product per mu reaches 3088 jin, and stem sharp product per mu is 2995 jin.This shows that tip of a root tissue cultivating seedling all increases in tissue cultivating seedling and the land for growing field crops output that explant breaks up speed, plant regeneration speed, test-tube plantlet reproduction speed and micro potato than stem apex tissue cultivating seedling.
The effect comparison of table 1 purple potato stem apex detoxification and tip of a root detoxification
Claims (1)
1. purple potato tip of a root detoxification and fast breeding technique is characterized in that carrying out according to the following steps:
(1) culture medium preparation comprises that component and every liter of contained weight thereof of minimal medium and each stage medium is:
1) minimal medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
350mg/L+KH
2PO
4500mg/L+ sucrose 25g/L+ agar 7-9g/L, pH 5.6-5.8;
2) tip of a root inducing culture: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.5-1mg/L+NAA0.3-0.7mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
3) proliferated culture medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+BA0.2-0.5mg/L+NAA0.2-0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
4) subculture growth medium: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/0.5L+BA0.2-0.5mg/L+NAA0.1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
5) root media: potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.1-0.5mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
6) micro potato inducing culture: adopt potato-based basal culture medium+NH
4NO
3400mg/L+KNO
3500mg/L+KH
2PO
450mg/L+NAA0.1-0.5mg/L+6BA0.5-1mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20g/L;
Described potato-based basal culture medium is: potato 200g/L+ sucrose 10g/L+ agar 20g/L;
(2) tip of a root detoxification is with numerous soon:
1) material preliminary treatment and tip of a root explant selection, sterilization: purple seed potato is put into the sterilization fine sand under 37 ± 2 ℃, heat-treat thoughtful the taking out of 6-12 and bear root system; Get the long main root tip of a root of 1-2cm, after 70% alcohol disinfecting 30 seconds, 0.1% mercuric chloride add a soil temperature and shake sterilization 8 minutes and aseptic washing 3-5 time, at microscopically with the tip of a root cutting-out of the long 0.1-0.2mm of front end as explant, subsequent use;
2) tip of a root regeneration induction plant cultivation: explant is seeded in step (1) 2) on the tip of a root inducing culture; Under 25 ± 2 ℃, light intensity 1000lux, 16 hours/d of illumination, cultivate; The constant light intensity of all the other conditions of back is strengthened to 2000lux extremely; Be strengthened to 4000lux after six weeks, be attenuated to 1500lux after eight weeks and continue down to cultivate until induce the regeneration plant young shoot from the tip of a root, this stage need cultivate 3.5-4.5 month altogether;
3) young shoot enrichment culture: young shoot is seeded in step (1) 3) on the proliferated culture medium, at 25 ± 2 ℃, light intensity 2000-3000lux, 16 hours/d of illumination cultivate 1 month down to growing up to the long seedling of 8-10cm;
4) subculture grown cultures: seedling is cut into the long segment of 2-2.5cm is seeded in step (1) 4) on the subculture growth medium,, be cultured under light intensity 2000-3000lux, the 16 hours/d of illumination and grow up to the long seedling of 8-10cm at 25 ± 2 ℃; It is numerous that every later on separated 3-4 week is carried out a subculture expansion by the same technology, counts requirement up to satisfying seedling;
5) culture of rootage: the subculture seedling that increases is inoculated into step (1) 5) on the root media, at 25 ± 2 ℃, light intensity 2000-3000lux, 16 hours/d of illumination cultivate down, and sending out roots after 1 week to be tied to form is complete test-tube plantlet;
6) cultivation of test tube micro potato: will grow tall to the test-tube plantlet of 8-10cm, and be cut into the stem section that has 1-2 blade and axillalry bud, and be seeded to the step (1) 6 that is loaded in the triangular flask by every bottle of 5-6 stem section) on the micro potato inducing culture; Under 22 ℃, dark condition, cultivating 10-15 days beginnings forms micro potato, and the same terms continues to cultivate 6-8 down can gather in the crops micro potato after week;
(3) virus of purple potato test-tube plantlet or test tube micro potato detects:
1) preparation of purple Potyvirus ssRNA: choose purple potato marmor upsilon Potato Virus Y; PVY, corium solani Potato Leafroll virus; PLRV, potato virus X Potato Virus X; PVX and potato virus S Potato Virus S, the blade of PVS, leaf stalk, stem or potato tubers are material, put into 100mL0.5Triton X-100 0.4%Na
2SO
3Viral leaching liquor in; Put 37 ℃ of lixiviate 20min, get supernatant behind the room temperature lixiviate 1h; Through 12, behind the centrifugal 10min of 000r/min centrifuge, get supernatant and be stored in-20 ℃, subsequent use;
2) cDNA is synthetic: design potato virus X Potato VirusX according to Potyvirus CP gene order; PVX, marmor upsilon Potato Virus Y; PVY, corium solani Potato Leafrollvirus, the Auele Specific Primer of PLRV and potato virus S Potato Virus S is right; Synthetic each the reaction cumulative volume of cDNA is 10 μ L, and reaction reagent comprises virus total RNA 2.5 μ L, and 1 * MMLV contains 3mmol/LMgCl
2Buffer solution, 1.5mmol/L dNTPs, 2.5U RNA enzyme inhibitor, 50U MMLV reverse transcriptase MMLVRT, 0.5 μ mol/L antisense primer; Response procedures is 25 ℃ of incubation 10mim, 42 ℃ of reverse transcription 45mim, 95 ℃ of deactivation 2mim;
Described Potyvirus Auele Specific Primer is to being:
PVS upstream primer P1:5 '-ATGCCGCCCAAACCGGATCCGTCA-3 '
PVS downstream primer P2:5 '-TCATTGGGTGATTGCATTACGGTG-3 '
PVY upstream primer P3:5 '-GCAAATGACACAATCGATGCAGGA-3 '
PVY downstream primer P4:5 '-TGGTGTTCGGTGATGTGACCT-3 '
PVX upstream primer P5:5 '-GAGCACAACACAGACCACAG-3 '
PVX downstream primer P6:5 '-GACTAGTTATGGTGGTGGGAGAG-3 '
PLRV upstream primer P7:5 '-CCCTTCGCAGGCGCGCTAACA-3 '
PLRV downstream primer P8:5 '-GGGGTCCAACTCATAAGCGAT-3 ';
3) pcr amplification: PCR reaction cumulative volume is 25 μ L, gets the synthetic cDNA of 4 μ L reverse transcriptions as the PCR reaction template; The reaction system of multiplex PCR comprises: 1 * PCR reaction buffer, and 35mmol/L dNTPs, Taq archaeal dna polymerase 2U, the 0.5mmol/L Auele Specific Primer is right; Amplification program is 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, and 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 10min; The volume ratio that wherein adds primer in the PCR reaction is PVX: PVY: PLRV: PVS=0.4: 0.5: 0.9: 1.0;
4) observe electrophoretic band: have with electrophoretic band contrast, the purple potato test-tube plantlet of establishment or test tube micro potato and do not infect corresponding virus.
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| CN102080132B (en) * | 2010-11-16 | 2012-10-17 | 烟台出入境检验检疫局检验检疫技术中心 | Seminested-RT (reverse transcription)-Realtime PCR (polymerase chain reaction) kit for detecting potato virus X |
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| CN102257959B (en) * | 2011-05-17 | 2013-06-05 | 北方民族大学 | Culture medium for late maturing potato detoxified basic seedling and preparation method thereof |
| CN102405831B (en) * | 2011-08-15 | 2012-12-05 | 山东省农业科学院蔬菜研究所 | Solid culture medium for callus seedlings of stem tips of potatoes |
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| CN102919126B (en) * | 2012-11-12 | 2014-05-07 | 达州市农业科学研究所 | Potato circulating production process by means of detoxified potato test tube |
| CN103329805A (en) * | 2013-06-28 | 2013-10-02 | 江苏开昌农业综合开发科技有限公司 | Simplified potato detoxification method |
| CN104488507A (en) * | 2014-12-03 | 2015-04-08 | 云南省宣威市元宝农产品开发有限公司 | Breeding method of purple potatoes |
| 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 |
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| CN105724019A (en) * | 2016-02-24 | 2016-07-06 | 丽江市农业科学研究所 | Potato virus-free test-tube seedling direct transplanting method |
| CN105900838A (en) * | 2016-04-25 | 2016-08-31 | 吉林师范大学 | Purple sweet potato detoxification method |
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