CN109463067A - A method of improving Potyvirus removal efficiency - Google Patents
A method of improving Potyvirus removal efficiency Download PDFInfo
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- CN109463067A CN109463067A CN201811425149.9A CN201811425149A CN109463067A CN 109463067 A CN109463067 A CN 109463067A CN 201811425149 A CN201811425149 A CN 201811425149A CN 109463067 A CN109463067 A CN 109463067A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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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 present invention provides a kind of method for improving Potyvirus removal efficiency, after including the following steps: that (1) cleans potato wedge, carry out first time processing, it is had an injection with 5 milliliters of syringes in potato wedge bud eye center, potato fruit is firmly aspirated while slowly extraction, then half solution is first slowly injected in same pinprick injection Ribavirin dilution, injection with 1 milliliter of syringe, is further continued for injecting the other half solution after suspending 5min；Second of processing is had an injection after first time handles 1-1.5h at the 0.5-0.7cm of bud eye center, other operations handle with second with the 4th processing for the third time with handling for the first time, avoid pin hole intersection；(2) another set syringe and Ribavirin solution are used, handles umbilical region according to the method for processing bud eye.Method of the invention can remove larger stem apex, shorten the detoxification period, improve removing stem apex planting percent and virus elimination rate.
Description
Technical field
The invention belongs to seed seedling detoxification technology fields, and in particular, to a kind of raising Potyvirus removal efficiency
Method, be mainly used for Potyvirus removing, solve variety deterioration and restore kind a property.
Background technique
Potato is the fourth-largest cereal crops in the world, occupies weight in China's grain, vegetables, feed and the production that processes raw material
Status is wanted, proposes potato staple food grainization strategy in recent years, it is significant in terms of ensureing national food security.Potato is because of poison of falling ill
Variety deterioration caused by disease often results in yield and quality degradation, and by with stem-apex Meristem culture Virusfree
(detoxification of abbreviation Shoot Tip Culture) is that the production of seed stock technology of core is to solve potato degeneration, restore kind of an effective way for property.
Potyvirus removing common method mainly has Shoot Tip Culture detoxification, heat treatment to combine Shoot Tip Culture detoxification, chemistry
The detoxification of chemicals treatment combination Shoot Tip Culture, heat treatment combine Chemical treatment in conjunction with Shoot Tip Culture detoxification, stem apex callus group
Culture detoxification, cryotherapy detoxification etc. are knitted, certain detoxification efficiency is all obtained in production.But there are regeneration rates for these methods
It is low, the period is long, removal efficiency is low, the problems such as morphing, the especially detoxification of stem apex callus tissue culture and cryotherapy detoxification, skill
Art difficulty is big, deficient in stability, and practical application effect is poor.It is applied in Chemical treatment combination shoot tip culture
Chemical agent mainly has Ribavirin (Ribavirin, ribavirin), DHT(5- dihydrouracil) and the bis- second of DA-DHT(
Acyl-dihydro -5-AzU) etc..Ribavirin is initially studied as the drug of human body and internal animal virus and opens
It issues, the synthesis of viral nucleic acid can be prevented, have good therapeutic effect to more than 20 kinds of viruses in humans and animals body.Forefathers
A certain concentration Ribavirin is added in culture medium by trial, combines to improve virus elimination rate with stem-apex Meristem culture.
The Ribavirin of various concentration gradient is attached to by present inventor expands breeding culture medium and Storaged media to carrying disease
After the tissue culture seedlings of potatoes of poison carry out processing in 3 months, then carry out 3 months normally expansion it is numerous, do not taken off through viral diagnosis discovery
Except virus.In addition, the Ribavirin of various concentration gradient is attached in stem tip tissue culture base viruliferous different big to taking
Small potato stem-tip tissue (0.1mm, 0.3mm, 0.5mm, 0.8mm) is handled, the results showed that (1) Ribavirin is to stem apex
Survival rate is affected, and stem apex is smaller, influences bigger.0.1mm stem-tip tissue is only in Ribavirin 15mg/L and following concentration
Just can seedling, 0.3mm stem-tip tissue only Ribavirin 20mg/L and following concentration just can seedling, 0.5mm stem-tip tissue exists
Ribavirin 25mg/L and following concentration just can seedling, 0.8mm stem-tip tissue can Cheng Shengchang under Ribavirin 40mg/L concentration
The excessively poor weak seedling of gesture.(2) through viral diagnosis, Ribavirin processing has detoxification effect to 0.3mm and its processing of following size stem apex
Fruit, wherein the processing of 0.1mm stem apex+15mg/L Ribavirin relatively compare viral removal efficiency improve only 2.0%, 0.3mm stem apex+
The processing of 20mg/L Ribavirin relatively compare viral removal efficiency improve only the processing of 4.2%, 0.5mm stem apex+25mg/L Ribavirin compared with
The viral removal efficiency of control improves only the processing of 1.2%, 0.8mm stem apex+40mg/L Ribavirin and does not obtain detoxic seedling.(3) to above-mentioned
(2) it is shown as negative plant through viral diagnosis in, is expanded numerous 3 months with conventional expansion breeding culture medium and carries out second of viral diagnosis.Inspection
It surveys the results show that 0.3mm and following stem apex source plants all keep negative, and 0.5mm stem apex source plants, positive diseased plant go out
Now rate is up to 97.83%.Our research explanation, adds Ribavirin in fast breeding culture medium, can not Virusfree；It utilizes
Stem apex removing and the Ribavirin that low concentration is added in Shoot Tip Culture base, can be improved virus elimination rate, and stem apex is smaller, survive
Though rate is low, viral removal efficiency is effectively improved, this is consistent with the result of study of domestic and foreign scholars.
Currently used Potato Shoot-tips tissue culture detoxicating method, stem apex size requirements are less than 0.3mm, and only 10% is left
Right survival rate and 50% or so viral removal efficiency, in real work, testing crew is for disposable detoxification success, every kind
A large amount of stem apex is often shelled to alternative, labor intensive, time and increases cost.In addition, harvesting from potato potato wedge to stem apex
Tissue seedling at least last 6 wheat harvesting periods (200d), if front detoxification is unsuccessful, repeat shell stem apex last 8 months (240d) with
On.The applicant once to a Potato Clones time-consuming 3 years, just removed main several Potyvirus.
Summary of the invention
In order to solve the problems in the existing technology, the higher poison-removing method of saving of work and time, efficiency, the applicant are sought
By using Ribavirin being passivated processing to virus from initial phase is sprouted to taking viruliferous potato potato wedge, discovery has
Processing bud no longer carry virus through viral diagnosis, enlightenment is established and is directly located using Ribavirin to potato wedge bud eye for the first time
Reason, in conjunction with the innovation poison-removing method of big Shoot-tip Culture (0.3-0.5mm).Using this poison-removing method, removed in Potato Shoot-tips
In the larger situation of size (0.3-0.5 mm), a variety of main viruses of removing potato are easier to, and conventional removing stem apex is big
Small requirement is less than 0.3 mm.This method overcomes that stem apex is too small to be easy to browning, vitrifying and the low technical problem of survival rate, mentions
High Shoot-tip Culture shoot survival percent, while shortening the Shoot-tip Culture detoxification period, improves work efficiency, be it is a kind of it is simple and practical,
At low cost, high-efficient Potyvirus removal methods.
The present invention provides a kind of method for improving Potyvirus removal efficiency, includes the following steps:
(1) potato wedge bud eye is handled: after potato wedge is cleaned, first time processing is carried out, with 5 milliliters of syringes in potato wedge bud eye center
It has an injection, depth 1.0-1.5cm firmly aspirates potato fruit, then with 1 milliliter of syringe same while slowly extraction
Pinprick injects 0.2ml-0.3ml Ribavirin dilution, first slow 1/2 bulk solution of injection when injection, suspend after 5min followed by
It is continuous to inject remaining 1/2 bulk solution；Second of processing is after first time handles 1h-1.5h, apart from bud eye center 0.5cm-
It has an injection at 0.7cm, adjustment syringe and potato wedge section angle are 25 ° -35 °, and with handling for the first time, third time is handled for other operations
It is handled with the 4th processing with second, position of accomplishing to have an injection does not repeat, and avoids pin hole intersection；
(2) umbilical region is handled: while processing bud eye, with another set syringe and Ribavirin solution, according to processing bud eye
Method handles umbilical region, wherein processing depth is 2.0cm-3.0cm.
Preferably, the concentration of the Ribavirin dilution is 5g/L-10g/L in step (1).
Preferably, the concentration of the Ribavirin solution is 0.1g/ml in step (2).
Preferably, the potato wedge removing stem apex size is 0.3 mm-0.5mm.
The present invention provides a kind of potato virus-free plantlet, is prepared using method described in any of the above embodiments.
Advantages of the present invention and good effect are as follows:
The present invention has the characteristic of passivation virus using Ribavirin, sprouts starting to explant formation stages to disease from potato
Poison is passivated processing, be the larger stem apex of removing 0.3mm-0.5mm in subsequent detoxification program, 84% or more stem apex survival rate and
63.8% virus elimination rate lays the foundation.
Explant is reduced in the present invention and expands numerous number, can save 30 days or so time, while increasing removing stem apex size
To 0.3mm-0.5mm, the seedling time shortens to 30d-40d, the short detoxification period 60d-70d of two cocondensations by conventional 60d-70d.
Stem apex planting percent is removed in the present invention and is increased to 84% or more by conventional 14.9%, and viral removal efficiency is by routine
39.1% is increased to 63.8%, compared with the conventional method, carries out detoxification to a kind, it is not necessary to prepare stripping tens even up to a hundred
Explant it is spare, material resources and human resources can be saved, to reduce cost.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
Embodiment 1
A kind of method of raising Potyvirus removal efficiency of the invention is specific as follows:
(1) to the potato plant of the listed mark of selection, full ripe stage, harvests potato wedge ahead of time, takes back at laboratory immediately after harvest
Reason.
(2) potato wedge is rinsed well under tap water, is contained in sterilized plate, each potato wedge marking pen is in bud
Eye center position draws "+" and identifies 5 bud eyes, and number consecutively is 1#-5# since terminal bud, draws in potato wedge umbilical region center position
"+" mark, number 6#.Each potato wedge selects 5 milliliters and 1 milliliter of syringe each 6, each syringe on tube wall successively
Identify 1#-6#, to the purpose that bud eye and syringe carry out reference numeral be required in subsequent processes syringe and bud eye or
Umbilical region corresponds in number, is unable to Confusion Cross use.
There are several bud eyes in each potato wedge, because potato wedge situation difference especially kind is different, bud eye 5-15 are differed.
(3) dilution and 2 kinds of stoste processing is arranged in Ribavirin (specification is 1ml=0.1g) treatment fluid, and dilution is to use nothing
Bacterium distilled water is diluted the solution of 20 times -10 times (concentration 5g/L-10g/L) to stoste by volumetric ratio.
(4) bud eye is handled: being handled, is had an injection with 5 milliliters of syringes of 1# in potato wedge 1# bud eye "+" center for the first time, is injected
Device and potato wedge section angle are 90 °, depth 1.0cm-1.5cm, and potato fruit is firmly aspirated while slowly extraction, is then used
1 milliliter of syringe of 1# first slowly injects 1/2 volume in same pinprick injection 0.2ml-0.3ml Ribavirin dilution, injection
Solution is further continued for injecting remaining 1/2 bulk solution after suspending 5min；2# bud eye is handled according to the method described above with 2 kinds of 2# syringes,
And so on 5 bud eyes successively handle；Second of processing, after first time handles 1h-1.5h, apart from bud eye "+" center
It has an injection at 0.5cm-0.7cm, adjustment syringe and potato wedge section angle are 25 ° -35 °, other operation sequences are the same as processing for the first time；
Third time processing and the 4th processing are handled with second, and position of accomplishing to have an injection does not repeat, and by choose have an injection position and
Adjustment syringe is avoided pin hole with potato wedge section angle and is intersected.
(5) umbilical region is handled: while processing bud eye, with corresponding 6# syringe, with Ribavirin stoste, according to processing bud
Eye handles umbilical region to the 4th step for the first time, handles depth 2.0cm-3.0cm.
Umbilical region is stolon inserted part, and it is by stolon skeleton that virus, which passes to stem tuber (potato wedge) by plant,
Bast passes to the bast of potato wedge skeleton.The same aerial stem of plant of potato tubers skeleton, stolon internode vascular bundle,
Belong to bicollateral bundle.Potato wedge is derived from complicated crop field environment, and umbilical region is had an injection, and is the virus that incoming potato wedge is possible to based on passivation
Assuming that.In addition potato wedge perimedullary region is dispersed with the solid netted branch with the contact of entire potato wedge conducting system.
The potato wedge vanning of above-mentioned processing is placed in room temperature or greenhouse germinates.Subsequent detoxification program removes size except stem apex
0.3mm-0.5mm is changed to by conventional 0.1mm-0.3mm and explant culture medium adds outside 25mg/L Ribavirin, other foundation
Explant, Shoot Tip Culture, method for detecting virus are the same as conventional.
Certainly method of the invention is also applied for the case where stem apex removing size is 0.1mm-0.3mm, only same operation
Under, stem apex is smaller, and planting percent is lower.In the case where reaching same detoxification purpose, with larger stem apex, not only removes stem apex and be easy
Operation, planting percent is also high, without preparing more potato wedge, without setting up a large amount of explant, without shelling more stem apex.
Embodiment 2
It tests and is tried in the spring -2013 years autumns in 2012 in potato seed raising technology research department, potato research institute, Gansu Academy of Agricultural Science
Test room progress.
Respectively in the red Gu in Lanzhou, Lanzhou Yuzhong, Dingxi Weiyuan, Dingxi be stable and level-one potato seed, the commodity potato of Lanzhou peacefulness
It produces in field and Germplasm Resources, chooses Favorita, gram new No. 1, No. 3 potato wedges of Gansu Province potato, LK99 and Gansu Province No. a kind Ma Ling of coloured silk
Potato, specifying information are shown in Table 1.
It is divided into three groups after potato wedge harvest, respectively processing potato wedge and 2 kinds of control potato wedges (CK1 and CK2), handles potato wedge with pressing
Volume ratio dilutes 12.5 times (concentration 8g/L) of Ribavirin dilution processing, and concrete processing procedure carries out by the following method,
It compares potato wedge (CK1) and replaces Ribavirin solution with sterile distilled water, handle in the same way, control potato wedge (CK2) is not appointed
Where reason.
Specific processing method are as follows:
Potato wedge is rinsed well under tap water, is contained in sterilized plate, each potato wedge marking pen is hit exactly in bud eye
Position draws "+" and identifies 5 bud eyes, and number consecutively is 1#-5# since terminal bud, draws "+" mark in potato wedge umbilical region center position,
Number is 6#.Each potato wedge selects 5 milliliters and 1 milliliter of syringe each 6, each syringe successively identifies 1#- on tube wall
6#, the purpose for carrying out reference numeral to bud eye and syringe is that syringe is required to compile in subsequent processes with bud eye or umbilical region
It is corresponded on number, is unable to Confusion Cross use.
Bud eye processing: for the first time handle, had an injection with 1#5 milliliter syringes in potato wedge 1# bud eye "+" center, syringe and
Potato wedge section angle is 90 °, depth 1.2cm, and potato fruit is firmly aspirated while slowly extraction, then with 1#1 milliliters of notes
Emitter first slowly injects 1/2 bulk solution in same pinprick injection 0.2ml Ribavirin dilution, injection, after suspending 5min
It is further continued for injecting remaining 1/2 bulk solution；2# bud eye is handled according to the method described above with 2 kinds of 2# syringes, and so on 5 bud eyes
Successively handle；Second of processing is had an injection after first time handles 1.5h at bud eye "+" center 0.5cm, adjustment note
Emitter and potato wedge section angle are 25 ° -35 °, other operation sequences are the same as processing for the first time；Third time processing and the 4th processing are same
Second is handled, and position of accomplishing to have an injection does not repeat, and by choosing have an injection position and adjustment syringe and potato wedge section angle
Avoid pin hole intersection.
Umbilical region processing: while processing bud eye, with corresponding 6# syringe, with Ribavirin stoste, according to processing bud eye
The step to the 4th time handles umbilical region for the first time, handles depth 3cm.
3 groups of potato wedges are cased respectively and are placed in greenhouse conventionally vernalization.It handles the subsequent detoxification program of potato wedge and removes explant
Body culture medium adds 25mg/L Ribavirin and stem apex removing size is changed to outside 0.3-0.5mm by conventional 0.1-0.3mm, other
Explant, Shoot Tip Culture, method for detecting virus are established with conventional；Control potato wedge (CK1) subsequent detoxification program is same to handle potato wedge
Group；Potato wedge (CK2) subsequent detoxification program is compareed in addition to explant culture medium adds 25mg/L Ribavirin, other programs are the same as conventional
(explant culture medium does not add 25mg/L Ribavirin to poison-removing method in conventional poison-removing method, the reason is that test is proved without de-
Toxic effect fruit, additional purpose is the consistency for condition control in the present embodiment).
It is whether thorough in order to verify detoxification during test, expand after first time viral diagnosis numerous 3 months, carries out second of disease
Poison detection.Special suggestion when operating according to the method for the present invention, need not carry out second of viral diagnosis in concrete practice.
Test result is shown in Table 2, in the present embodiment, using poison-removing method of the invention, reduces explant and expands numerous number, can
30 days or so time is saved, while increasing removing stem apex size is 0.3-0.5mm, the seedling time is contracted by conventional 60d-70d
It is short to 30d-40d, is established from explant to stem apex plumule emergence, the short detoxification period 60d-70d of cocondensation.
In the present embodiment, using poison-removing method of the invention, stem apex planting percent is increased to 84% by conventional 14.9%, virus
Removal efficiency is increased to 63.8% by conventional 39.1%, substantially increases viral removal efficiency.
In the present embodiment, 25 stem apex bud systems are only established to single kind detoxification using poison-removing method of the invention, with
75 comparisons of conventional method, can save material resources and human resources, to reduce cost.It should be noted that this implementation
It is skilled that technician in example shells stem apex, shells stem apex 75, but practical operation to each kind using conventional method (CK2)
In most cases, to the same kind carry out detoxification, it is necessary to prepare eight or nine ten even a explants up to a hundred to stem apex removing with
Culture, work consuming is time-consuming, increases cost.
1 Ribavirin of table handles potato wedge information table
2 Ribavirin of table handles potato wedge detoxification efficiency
Embodiment 3
It tests and is tried in the spring -2014 years autumns in 2013 in potato seed raising technology research department, potato research institute, Gansu Academy of Agricultural Science
Test room progress.
Respectively in Gansu Academy of Agricultural Science, Yuzhong Mature breeding base, potato research institute Lanzhou, Dingxi Weiyuan is late-maturing educates
The Germplasm Resources in kind of base, choose the obvious virus infection such as floral leaf, shrinkage, dwarfing, leaf roll Gansu Province potato 10, Gansu Province potato No. 3 numbers and
LK99 plant, listing mark harvest underground potato piece after plant is withered.
Ribavirin solution dilutes 10 times (concentration 10g/L) by volume, other handling and operation are as follows, and specifying information is shown in
Table 3.
Specific processing method are as follows:
Potato wedge is rinsed well under tap water, is contained in sterilized plate, each potato wedge marking pen is hit exactly in bud eye
Position draws "+" and identifies 5 bud eyes, and number consecutively is 1#-5# since terminal bud, draws "+" mark in potato wedge umbilical region center position,
Number is 6#.Each potato wedge selects 5 milliliters and 1 milliliter of syringe each 6, each syringe successively identifies 1#- on tube wall
6#, the purpose for carrying out reference numeral to bud eye and syringe is that syringe is required to compile in subsequent processes with bud eye or umbilical region
It is corresponded on number, is unable to Confusion Cross use.
Bud eye processing: handling for the first time, had an injection with 5 milliliters of syringes of 1# in potato wedge 1# bud eye "+" center, syringe
It is 90 ° with potato wedge section angle, depth 1.5cm firmly aspirates potato fruit while slowly extraction, then use 1 milliliter of 1#
Syringe first slowly injects 1/2 bulk solution in same pinprick injection 0.3ml Ribavirin dilution, injection, suspends 5min
After be further continued for injecting remaining 1/2 bulk solution；2# bud eye is handled according to the method described above with 2 kinds of 2# syringes, and so on 5 buds
Eye is successively handled；Second of processing is had an injection after first time handles 1.5h at bud eye "+" center 0.5cm, is adjusted
Syringe and potato wedge section angle are 25 ° -35 °, other operation sequences are the same as processing for the first time；Third time processing and the 4th processing
It is handled with second, position of accomplishing to have an injection does not repeat, and by choosing have an injection position and adjustment syringe and potato wedge section folder
Avoid pin hole intersection in angle.
Umbilical region processing: while processing bud eye, with corresponding 6# syringe, with Ribavirin stoste, according to processing bud eye
The step to the 4th time handles umbilical region for the first time, handles depth 2.9cm.
Test result shows (table 4), and in the present embodiment, using method of the invention, stem apex planting percent is by conventional 14.2%
It is increased to 81.3%, viral removal efficiency is increased to 70.9% by conventional 37.7%, shortens detoxification period 52d, and removing stem apex size is
0.3-0.5mm substantially increases detoxification efficiency, material resources and human resources is saved, to reduce cost.
3 Ribavirin of table handles potato wedge information table
4 Ribavirin of table handles potato wedge detoxification efficiency
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention, although
Present invention has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, still can be right
Technical solution documented by foregoing embodiments is modified or equivalent replacement of some of the technical features.It is all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in protection of the invention
Within the scope of.
Claims (5)
1. a kind of method for improving Potyvirus removal efficiency, it is characterised in that: include the following steps:
(1) potato wedge bud eye is handled: after potato wedge is cleaned, first time processing is carried out, with 5 milliliters of syringes in potato wedge bud eye center
It has an injection, depth 1.0-1.5cm firmly aspirates potato fruit, then with 1 milliliter of syringe same while slowly extraction
Pinprick injects 0.2ml-0.3ml Ribavirin dilution, first slow 1/2 bulk solution of injection when injection, suspend after 5min followed by
It is continuous to inject remaining 1/2 bulk solution；Second of processing is after first time handles 1h-1.5h, apart from bud eye center 0.5cm-
It has an injection at 0.7cm, adjustment syringe and potato wedge section angle are 25 ° -35 °, and with handling for the first time, third time is handled for other operations
It is handled with the 4th processing with second, position of accomplishing to have an injection does not repeat, and avoids pin hole intersection；
(2) umbilical region is handled: while processing bud eye, with another set syringe and Ribavirin solution, according to processing bud eye
Method handles umbilical region, wherein processing depth is 2.0cm-3.0cm.
2. according to the method described in claim 1, it is characterized by: in step (1), the concentration of the Ribavirin dilution is
5g/L-10g/L。
3. method according to claim 1 or 2, it is characterised in that: in step (2), the concentration of the Ribavirin solution
For 0.1g/ml.
4. according to the method described in claim 1, it is characterized by: potato wedge removing stem apex size is 0.3 mm-0.5mm.
5. a kind of potato virus-free plantlet is prepared using the described in any item methods of claim 1-4.
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| CN109997641A (en) * | 2019-04-04 | 2019-07-12 | 新疆农业科学院综合试验场 | The method for culturing seedlings for preventing and treating potato soil-borne disease |
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