CN109329026A - A kind of method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling - Google Patents
A kind of method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling Download PDFInfo
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- CN109329026A CN109329026A CN201811453894.4A CN201811453894A CN109329026A CN 109329026 A CN109329026 A CN 109329026A CN 201811453894 A CN201811453894 A CN 201811453894A CN 109329026 A CN109329026 A CN 109329026A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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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 the methods that a kind of high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling, belong to Virus-free Sweetpotato seedling Cultivating techniques field.The method that high temperature of the invention-intermittent warming obtains Sweetpotato Viruses Elimination seedling is to carry out daily 40-45 DEG C of high-temperature process 5h to the sweet potato's seedlings after taking root, later high temperature-intermittent warming of 28 DEG C of processing, continuous processing 30-40d；High temperature of the present invention-intermittent warming obtains the method for Virus-free Sweetpotato seedling to the good passivation effect of virus, and the stem apex length of removing is big, is easy to strip, high-efficient, and adventitious bud growth is fast；The quality of rooting of regrowth is good, seedling is sturdy, high survival rate.
Description
Technical field
The invention belongs to Virus-free Sweetpotato seedling Cultivating techniques fields, and in particular to a kind of high temperature-intermittent warming acquisition sweet potato is de-
The method of malicious seedling.
Background technique
Sweet potato is important grain, feed and raw material of industry processing crop, occupies importantly in agricultural and national economy
Position.Nutrient sweet potato is abundant, also increasingly has been favored by people in recent years with the improvement of people's health awareness,.Sweet potato belongs to
Root crop is bred using trophosome in production process, therefore is easy to be infected by virus, and with improved variety
The extension of Cultivation time, viral density can be higher and higher.Report that the virus for infecting sweet potato has more than 20 at present.What China occurred
Mainly there are Sweet Potato Feathery Mottle Virus (SPFMV), sweet potato cryptovirus (SPLV), sweet potato vein mosaic virus (SPVMV), sweet potato light
Mottle mosaic poison (SPMMV), sweet potato leaf curl virus (SPLCV), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV) (TMV),
Falling ill heavier is SPFMV, SPLV, SPLCV etc..Sweet potato viruses can pass through root tuber, insect mediator, grafting and mechanical inoculation.Disease
Infecting for poison can cause plant shoots growing way is weak, it is slow to return seedling, agglomerate less, potato wedge is small, potato color of the leather is shallow and rough and potato wedge is brown to split
Deng declining yield, quality deteriorates, or even loses commodity value.Virus disease influences the production of China sweet potato especially in recent years, and
And have the tendency that more and more fiery, 90% or more production loss can be caused when serious, or even total crop failure.Currently, in China Guangdong, river
The ground such as Soviet Union, Sichuan, Anhui, Fujian and Shandong have SPVD.
The main method for overcoming virosis to endanger is to produce detoxic seedling using Shoot Tip Culture, because virus is intracorporal in plant
It is unevenly distributed, virus density remoter from separate living tissue is higher, otherwise lower, and the typically no virus of separate living tissue.But
It only strips shoot apical meristem and is cultivated and be often not easy to survive.Therefore, the stem apex with 1-2 phyllopodium is generally stripped to carry out
Culture, but which in turn reduces detoxification efficiencies.The contradiction for overcoming the two, not only having improved regeneration rate but also having improved virus elimination rate becomes Sweetpotato Viruses Elimination
The bottleneck of seedling production.
Detoxic seedling existing research and report, but the stem of previous multi-purpose crop field or greenhouse production are produced using sweet potato stem tip culture
It draws materials on climing and strips stem apex；Also useful potato wedge, which is drawn materials, strips Shoot Tip Culture, but is also vernalization under normal temperature conditions.Choosing
It with these above-mentioned methods, grows and cultivates under normal conditions, because the Material growth for detoxification is slower, metabolism is not strong, strips stem
Point culture detoxification efficiency is poor, and stem apex is not easy to strip, and regeneration rate is low.In addition, old kind serious for some virosis, often
The detoxification efficiency for advising poison-removing method is also not satisfactory.
In addition, seedling grows in solidified MS media before existing sweet potato rooting culture, but culture medium is difficult to clearly when transplanting
It washes, and easy infection bacterium.Easily hurt root when transplanting, detoxic seedling slow seedling is slow.
Summary of the invention
The problem of for existing detoxification technology, the purpose of the present invention is to provide a kind of high temperature-intermittent warmings to obtain
The method for obtaining Sweetpotato Viruses Elimination seedling.
In order to achieve the above object, the technical solution of the present invention is as follows:
A kind of method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling, steps are as follows:
(1) the undamaged sweet potato potato wedge of free from insect pests is chosen, be exposed to the sun 2-3d under the sun；
(2) potato wedge is cleaned with clear water, sand is covered in potato wedge, be put into 28 DEG C of progress vernalization in incubator；
(3) when sprout length to 10cm or more, seedling apical 2-3cm long (band spire) is taken, cuts off macroscopic blade, first
It is rinsed with clear water, then carries out surface sterilization, be inserted into MS culture medium and cultivate after disinfection；
(4) after seedling takes root, daily 40-45 DEG C of high-temperature process 5h is carried out in the incubator, later the high temperature-of 28 DEG C of processing
Intermittent warming, continuous processing 30-40d；
(5) stem apex for being 0.5-0.8mm with phyllopodium length is stripped under stereoscopic anatomical lens, is inoculated into addition 0.1-
On the MS culture medium of 0.2mg/LNAA and 1.0-2.0mg/L BAP, cultivation temperature is 25-27 DEG C, daily illumination in 13 hours,
2000-3000lx is cultivated 3-4 weeks, the differentiation of evoking adventive bud；
(6) when regenerate seedling it is long to 2cm or more when, cut from base portion, be transferred in the culture medium for not adding hormone and obtained
Whole detoxic seedling；
(7) before rooting culture, aseptic seedlings is transferred in MS fluid nutrient medium and are cultivated；
(8) test tube seedling cleans culture medium, divides single plant directly to transplant in the soil of vinyl house, strain spacing after hardening
20-25cm, vinyl house both ends add fly net；Keep humidity 80% or more in canopy in first 1 week.
The advantages of technical solution of the present invention:
High temperature of the present invention-intermittent warming obtains good passivation effect of the method for Virus-free Sweetpotato seedling to virus, the stem apex of removing
Length is big, is easy to strip, high-efficient, and adventitious bud growth is fast；The quality of rooting of regrowth is good, seedling is sturdy, high survival rate.
(" a kind of sweet potato is de- for patent of invention for the preparation method of Virus-free Sweetpotato seedling of the present invention and existing Sweetpotato Viruses Elimination seedling-growing method
The preparation method of the malicious seedling " patent No. 201410291953.8) it compares,
1. existing method is progress high-temperature process (35~37 DEG C) in potato wedge culture, the present invention is that the seedling bud of clip goes out
It is transferred to culture medium after bacterium, then carries out 40-45 DEG C of high-temperature process, if seedling is not transfer to culture medium, directly uses 40-45 DEG C
High-temperature process, seedling are difficult to survive in the sand；And it is more preferable to the passivation effect of virus after temperature raising；
2. more preferable to the passivation effect of virus after the seedling bud high temperature intermittent warming of clip, it is possible to stereoscopic
Bigger stem apex (0.5-0.8m can be increased to) band phyllopodium is stripped under anatomical lens.Big stem apex is easier to strip, more efficient, and
The time for growing adventitious bud is short, and existing method removing difficulty is big, survives difficulty, and slow growth；
That 3. regrowth of the invention is cultivated in liquid medium is better than quality of rooting on solidified MS media, seedling is sturdy,
Culture medium is not easy to hurt root when easily rinsing, transplanting, and seedling is easier to survive.
High temperature-intermittent warming of the invention is to the good passivation effect of virus, and stem apex detoxification efficiency is good, and due to phyllopodium
Internode elongates, and stem apex is easy to strip (because phyllopodium internode elongates, it is readily seen that separate living tissue, stem apex easily strip under anatomical lens),
Regeneration rate is high (because higher temperature growth metabolism is vigorous).The results showed supporting daily high temperature in room in 40-45 DEG C of incubator
5h is handled, after continuous processing 1 month, the growth of seedling of most of sweet potato varieties is had no significant effect.
The stem apex that sweet potato strips under normal conditions is smaller, and the death rate is higher, and planting percent is lower, and virus elimination rate is higher；Conversely,
Although its death rate substantially reduces, but virus elimination rate is lower or the purpose of detoxification is not achieved.It is preferably shelled when detoxic seedling produces at present
The length for taking stem apex is 0.2-0.4mm, but detoxic seedling planting percent is low and slow growth.The present invention by the seedling after high-temperature process,
The length that stem apex is stripped under stereoscope increases to 0.5-0.8mm, is then transferred to callus induction, adventitious shoot regeneration and seedling
Regeneration culture medium, the adventitious bud growth turned out in this way is very fast, and can reach detoxification purpose.
Sweet potato aseptic seedlings are transferred to MS fluid nutrient medium in the present invention, cultivate 15d.This method is taken root than solidified MS media
It is high-quality, seedling is sturdy, culture medium easily rinses, is not easy to hurt root when transplanting, seedling is easier to survive.
Detailed description of the invention
Sweet potato's seedlings (the left side: Beijing 553 of Fig. 1 virus infection；The right: cigarette potato 25)；
Seedling of the Fig. 2 after high temperature-intermittent warming；
Grown on the callus that Fig. 3 is formed by Shoot Tip Culture adventitious bud (left side: seedling pass through high temperature intermittent warming,
And strip the adventitious bud that the stem apex that size is 0.5-0.8mm is grown；The right: seedling strips without high temperature intermittent warming
The adventitious bud that stem apex less than 0.4mm is grown)；
Fig. 4 detoxic seedling is on situation of the taking root (left side: take root feelings of the detoxic seedling in solid medium of solid and fluid nutrient medium
Condition；The right: the situation of taking root of detoxic seedling liquid medium within)；
The indirect ELISA detection (1: positive control of Fig. 5 detoxic seedling；2: susceptible seedling；3: negative control；4-8: pass through
High temperature intermittent warming, and strip the detoxic seedling that the stem apex that size is 0.5-0.8mm generates).
Specific embodiment
Term as used in the present invention generally has those of ordinary skill in the art usual unless otherwise specified
The meaning of understanding.
Combined with specific embodiments below, and referring to the data further detailed description present invention.Following embodiment only be
It illustrates the present invention, rather than limits the scope of the invention in any way.
Embodiment 1
The method that high temperature-intermittent warming of the present invention prepares Sweetpotato Viruses Elimination seedling specifically comprises the following steps:
1. the processing before sweet potato potato wedge vernalization
It is chosen at the heavier sweet potato variety Beijing 553 of field Seedling Stage virosis morbidity, cigarette potato 25, takes nothing after autumn harvest
It damages by worms, undamaged potato wedge, is exposed to the sun under the sun 2-3 days.
2. vernalization
Above-mentioned potato wedge is cleaned with clear water, is put into sand, cladding thickness is the sand of 1-2cm in potato wedge, is put into culture
28 DEG C of progress vernalization in case.Dark culturing 1 week before sprouting, after budding, daily 13h illumination, 1000-2000lx.
3. stem apex is stripped and is cultivated
(1) disinfection of material: when sprout it is long to 10cm or more when, virosis symptom clearly (Fig. 1), takes seedling at this time
Top 2-3cm long (band spire), cuts off macroscopic blade, is first rinsed with clear water, then with 70% in super quiet workbench
Alcohol impregnates 10-20 seconds, then the liquor natrii hypochloritis for being 2% with mass percent impregnates 5-7min and carries out surface sterilization, finally
With rinsed with sterile water 3-5 times, it is inserted into MS culture medium.
(2) after 5-7d seedling takes root, the daily high-temperature process 5h in 40-45 DEG C of culturing room, 28 DEG C of high temperature-change later
Temperature processing, continuous processing 30-40d (Fig. 2).
(3) stem apex of the 0.5-0.8mm with phyllopodium is stripped under stereoscopic anatomical lens, is inoculated on MS culture medium and is cultivated.Training
It supports 1 week or so stem apex and initially forms callus, start to form adventitious bud (Fig. 3) from callus wound after 3-4 weeks.By Fig. 3
As can be seen that with less than 0.4mm stem apex compared with, the stem apex of 0.5-0.8mm size, formed callus on grow not
The normal bud speed of growth is faster.It is small when regenerating after being cultivated 1 month on the MS culture medium of addition 0.2mg/LNAA and 2.0mg/LBAP
Seedling it is long to 2cm or more when, cut from base portion, be transferred in the MS culture medium for do not add hormone and grow up to complete detoxic seedling.
(4) before domestication and transplanting, aseptic seedlings are transferred to MS fluid nutrient medium, after cultivating 15d, promote its (figure of taking root
4).As seen from Figure 4, it compared with solid medium, takes root on seedling liquid medium within more, quality is higher, and cultivates
Base flushing easier than solid medium before transplanting.
4. the viral diagnosis of detoxic seedling
SPVD viral diagnosis is carried out to detoxic seedling using indirect ELISA detection method, testing result is as shown in Figure 5: 2 product
Kind virus elimination rate reaches 100%.
5. the domestication and transplanting of detoxification test tube plantlet
(1) test tube seedling after squamous subculture is opened bottleneck and is tamed, continuous hardening 2-3d.
(2) test tube seedling is cleaned into culture medium, single plant is divided directly to transplant in the soil of vinyl house, strain spacing 20-25cm.
Vinyl house both ends add fly net, keep humidity 80% or more in canopy in first 1 week.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (4)
1. the method that a kind of high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling, it is characterised in that: steps are as follows:
(1) the undamaged sweet potato potato wedge of free from insect pests is chosen, be exposed to the sun 2-3d under the sun；
(2) potato wedge is cleaned with clear water, sand is covered in potato wedge, be put into 28 DEG C of progress vernalization in incubator；
(3) when sprout is long to 10cm or more, seedling apical is taken, macroscopic blade is cut off, is first rinsed with clear water, is then carried out
Surface sterilization is inserted into MS culture medium after disinfection and is cultivated；
(4) after seedling takes root, daily 40-45 DEG C of high-temperature process 5h is carried out in the incubator, later high temperature-alternating temperature of 28 DEG C of processing
Processing, continuous processing 30-40d；
(5) stem apex with phyllopodium is stripped under stereoscopic anatomical lens, is inoculated into addition 0.1-0.2mg/L NAA and 1.0-2.0mg/
On the MS culture medium of L BAP, cultivation temperature is 25-27 DEG C, daily illumination in 13 hours, and 2000-3000lx is cultivated 3-4 weeks, induction
The differentiation of adventitious bud；
(6) when regenerate seedling it is long to 2cm or more when, cut from base portion, be transferred in the culture medium for not adding hormone completely to take off
Malicious seedling；
(7) before rooting culture, aseptic seedlings is transferred in fluid nutrient medium and are cultivated；
(8) test tube seedling cleans culture medium, divides single plant directly to transplant in the soil of vinyl house, strain spacing 20- after hardening
25cm, vinyl house both ends add fly net；Keep humidity 80% or more in canopy in first 1 week.
2. the method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling according to claim 1, it is characterised in that: in step (3)
The seedling apical that takes be to take seedling apical 2-3cm long, band spire.
3. the method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling according to claim 1, it is characterised in that: the step
(5) length for stripping stem apex under stereoscopic anatomical lens is 0.5-0.8mm.
4. the method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling according to claim 1, it is characterised in that: in step (7)
Fluid nutrient medium be MS fluid nutrient medium.
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Cited By (4)
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| CN110367123A (en) * | 2019-08-21 | 2019-10-25 | 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 Resistance Identification method of sweet potato leaf curl viral disease |
| CN111436374A (en) * | 2020-05-08 | 2020-07-24 | 佛山市农业科学研究所(佛山市农业技术推广中心) | Detoxification and rapid propagation method of Gaoming Shuiguan pueraria |
| CN115281089A (en) * | 2022-08-08 | 2022-11-04 | 镇江金豆豆种业有限公司 | Method for improving potato virus disease removal efficiency |
| CN115644063A (en) * | 2022-11-11 | 2023-01-31 | 青岛农业大学 | Rapid propagation method of virus-free raw seedling of sweet potato |
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| CN111436374A (en) * | 2020-05-08 | 2020-07-24 | 佛山市农业科学研究所(佛山市农业技术推广中心) | Detoxification and rapid propagation method of Gaoming Shuiguan pueraria |
| CN115281089A (en) * | 2022-08-08 | 2022-11-04 | 镇江金豆豆种业有限公司 | Method for improving potato virus disease removal efficiency |
| 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 |
| CN115644063A (en) * | 2022-11-11 | 2023-01-31 | 青岛农业大学 | Rapid propagation method of virus-free raw seedling of sweet potato |
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